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An Overview of Severe Acute Respiratory Syndrome–Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

Thanigaimalai Pillaiyar*
Thanigaimalai Pillaiyar
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
*Phone: +49-228-73-2360. E-mail: [email protected]
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,
Manoj Manickam
Manoj Manickam
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, South Korea
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Vigneshwaran Namasivayam
Vigneshwaran Namasivayam
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
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Yoshio Hayashi
Yoshio Hayashi
Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
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Sang-Hun Jung
Sang-Hun Jung
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, South Korea
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Cite this: J. Med. Chem. 2016, 59, 14, 6595–6628

Publication Date (Web):February 15, 2016

https://doi.org/10.1021/acs.jmedchem.5b01461

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Abstract

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 (10–15%) fatalities in 2003. The causative agent of SARS has been identified as a novel human coronavirus (SARS-CoV), and its viral protease, SARS-CoV 3CL^pro, has been shown to be essential for replication and has hence been recognized as a potent drug target for SARS infection. Currently, there is no effective treatment for this epidemic despite the intensive research that has been undertaken since 2003 (over 3500 publications). This perspective focuses on the status of various efficacious anti-SARS-CoV 3CL^pro chemotherapies discovered during the last 12 years (2003–2015) from all sources, including laboratory synthetic methods, natural products, and virtual screening. We describe here mainly peptidomimetic and small molecule inhibitors of SARS-CoV 3CL^pro. Attempts have been made to provide a complete description of the structural features and binding modes of these inhibitors under many conditions.

Note

This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

1. Introduction

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Coronaviruses have been known for more than five decades since the first prototype murine strain, JHM, was reported in 1947. (1,2) Viruses such as porcine transmissible gastroenteritis virus (TGEV), avian infectious bronchitis virus (IBV), and bovine coronavirus (BCoV) severely infect animals. The murine coronavirus mouse hepatitis virus (MHV) was studied as a model for the human disease. Although studies of the mechanism of replication as well as the pathogenesis of several coronaviruses have been very active since 1970s, this family of coronaviruses received much attention when it was recognized that a new human coronavirus was responsible for severe acute respiratory syndrome (SARS), a contagious and fatal illness. (3,4)

Coronaviruses belong to one of two subfamilies of (Coronavirinae and Torovirinae) of the family Coronaviridae, which in turn comprise the order Nidovirales (Figure 1). (5,6) They are classified into four genera (α, β, γ, and δ), and each genus can be further divided into lineage subgroups. SARS-CoV belongs to the Betacoronavirus group (see Figure 1).

Figure 1. Schematic representation of the taxonomy of *Coronaviridae* (according to the International Committee on Taxonomy of Viruses). SARS-CoV belongs to the *Betacoronavirus* family but has a “b” lineage. **Coronaviridae*, along with *Arteriviridae*, *Mesoniviridae*, and *Roniviridae*, are members of this family.

In 2003, a new human coronavirus was identified as an etiological agent of the first global pandemic of the 21st century, severe-acute respiratory syndrome (SARS), and the virus was named SARS-CoV. The first case of “an atypical pneumonia” was reported in China during November 2002. (7) Its rapid and unexpected spread to another 29 countries, mostly in Asia and North America, alarmed both the public and World Health Organization (WHO). Within a few months of this outbreak in 2003, the WHO announced in a cumulative report about its emergence that it had caused 916 deaths among 8422 cases (fatality rate of 10–15%) worldwide, as shown in Table 1. (8) This incidence indicates how rapidly a contagious illness can spread in this highly interconnected society.

Table 1. Summary of SARS Cases by Country or Area, November 1, 2002 to August 7, 2003

	cumulative number of cases				status
country/areas	Fa	Ma	Ta	median age (range)	no. of cases hospitalized	no. of cases recovered	no. of deaths	CFRb (%)	no. of imported cases (%)	no. of HCW affected (%)c	date onset first probable case	date onset last probable case
Australia	4	2	6	15 (1–45)	0	6	0	0	6 (100)	0 (0)	24-Mar-03	1-Apr-03
Brazil	1		1	4	0	1	0	0	1 (100)	0 (0)	3-Apr-03	3-Apr-03
Canada	151	100	251	49 (1–98)	10	200	41	17	5 (2)	108 (43)	23-Feb-03	12-Jun-03
China	P	P	5327	P	29	4949	349	7	NA	1002 (19)	16-Nov-02	25-Jun-03
Hong Kong	977	778	1755	40 (0–100)	7	1448	300	17	NA	386 (22)	15-Feb-03	31-May-03
Macao	0	1	1	28	0	1	0	0	1 (100)	0 (0)	5-May-03	5-May-03
Taiwan	349d	319d	665	46 (2–79)	10	475	180	27	50 (8)	86 (13)	25-Feb-03	15-Jun-03
Colombia	1	0	1	28	0	1	0	0	1 (100)	0 (0)	2-Apr-03	2-Apr-03
Finland	0	1	1	24	0	1	0	0	1 (100)	0 (0)	30-Apr-03	30-Apr-03
France	1	6	7	49 (26–61)	0	6	1	14	7 (100)	2 2 (29)	21-Mar-03	3-May-03
Germany	4	5	9	44 (4–73)	0	9	0	0	9 (100)	1 (11)	9-Mar-03	6-May-03
India	0	3	3	25 (25–30)	0	3	0	0	3 (100)	0 (0)	25-Apr-03	6-May-03
Indonesia	0	2	2	56 (47–65)	0	2	0	0	2 (100)	0 (0)	6-Apr-03	17-Apr-03
Italy	1	3	4	30.5 (25–54)	0	4	0	0	4 (100)	0 (0)	12-Mar-03	20-Apr-03
Kuwait	1	0	1	50	0	1	0	0	1 (100)	0 (0)	9-Apr-03	9-Apr-03
Malaysia	1	4	5	30 (26–84)	0	3	2	40	5 (100)	0 (0)	14-Mar-03	22-Apr-03
Mongolia	8	1	9	32 (17–63)	0	9	0	0	8 (89)	1 (11)	31-Mar-03	6-May-03
New Zealand	1	0	1	67	0	1	0	0	1 (100)		20-Apr-03	20-Apr-03
Philippines	8	6	14	41 (29–73)	0	12	2	14	7 (50)	4 (29)	25-Feb-03	5-May-03
Republic of Ireland	0	1	1	56	0	1	0	0	1 (100)	0 (0)	27-Feb-03	27-Feb-03
Republic of Korea	0	3	3	40 (20–80)	0	3	0	0	3 (100)	0 (0)	25-Apr-03	10-May-03
Romania	0	1	1	52	0	1	0	0	1 (100)	0 (0)	19-Mar-03	19-Mar-03
Russian Federation	0	1	1	25	1	0	0		NA	0 (0)	5-May-03	5-May-03
Singapore	161	77	238	35 (1–90)	0	205	33	14	8 (3)	97 (41)	25-Feb-03	5-May-03
South Africa	0	1	1	62	0	0	1	100	1 (100)	0 (0)	3-Apr-03	3-Apr-03
Spain	0	1	1	33	0	1	0	0	1 (100)	0 (0)	26-Mar-03	26-Mar-03
Sweden	1	2	3	33	0	3	0	0	3 (100)	0 (0)
Switzerland	0	1	1	35	0	1	0	0	1 (100)	0 (0)	9-Mar-03	9-Mar-03
Thailand	5	4	9	42 (2–79)	0	7	2	22	9 (100)	1 2 (11)	11-Mar-03	27-May-03
United Kingdom	2	2	4	59 (28–74)	0	4	0	0	4 (100)	0 (0)	1-Mar-03	1-Apr-03
United States	16	17	33	36 (0–83)	7	26	0	0	31 (94)	1 (3)	9-Jan-03	13-Jul-03
Vietnam	39	24	63	43 (20–76)	0	58	5	8	1 (2)	36 (57)	23-Feb-0	14-Apr-03

^{^a}

Note: F, female; M, male; P, pending; T, total.

^{^b}

Case fatality based on cases with known outcome and irrespective of immediate cause of death.

^{^c}

Health care worker (HCW).

^{^d}

Discarding of three cases, new breakdown by sex pending.

SARS is mainly characterized by a high fever (>38 °C), dyspnea, lymphopenia, headache, and lower respiratory tract infections; (9,10) concurrent gastrointestinal symptoms and diarrhea are also common. (11−13) With the enormous efforts of the WHO and expert scientists from various countries, a novel human coronavirus was identified as the etiological agent for SARS. (4,14) The sequence information on the coronavirus polymerase gene, along with all other previously characterized strains, demonstrated that this was a previously unrecognized coronavirus in humans. (3,15−17) Although the SARS epidemic was successfully controlled in 2003, (18,19) the identification of animal reservoirs for this virus and the recent report of a new virus related to SARS, called Middle East respiratory syndrome (MERS), (20) provide strong motivation for the development of anti-SARS agents to treat this potentially fatal respiratory illness.

The recent outbreak of MERS in South Korea alarmed the public, and the number of patients under quarantine was reported to be 1600. (21) After the first patient was diagnosed with MERS on May 20, 2015, within a period of two months, the total number of cases identified had increased to 186 with 36 fatalities and possible infection of 16700 individuals who were subjected to isolation. (22,23) By the end of August 2015, a total of 1511 patients were infected worldwide with this virus, of which 574 (∼39%) had died after the first case was recorded in June 2012 in Saudi Arabia. (24)

To date, the FDA has not approved an antiviral agent for the treatment of SARS, although the clinical treatments are directed toward symptomatic relief. Therefore, the development of effective antiviral chemotherapy against SARS-CoV is important for future outbreaks. Numerous reports (over 3500 publications) have been published on SARS-CoV since 2002. Recently, a brief review on the progress of anti-SARS chemotherapy was reported. (25) However; no reports have been published about the substrate selectivity, mechanism of action, and SARs of the inhibitors. Therefore, to overcome the drawbacks and to enhance the qualitative understanding of the etiology, pathology, and possible therapeutic targets against this virus, a comprehensive review is currently needed.

This perspective focuses on the status of SARS-CoV 3 chymotrypsin-like protease (3CL^pro) inhibitors discovered during last 12 years from all sources, including laboratory synthetic methods, natural products, virtual screening, and structure-based molecular docking studies. Attempts have been made to provide a complete description of the structural features (SARs) and detailed mechanisms of action of inhibitors. We believe that this perspective will comprise a cumulative source of SARS-CoV 3CL^pro inhibitors for researchers and further the understanding of anti-SARS chemotherapy.

2. SARS-CoV and Structure of 3CL^pro

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Coronaviruses are a family of positive strand, enveloped RNA viruses that can cause acute and chronic respiratory, enteric, and central nervous system diseases in many species of animals, including humans. (26,27) This family features the largest viral genomes (27–31 kb) found to date. (28,29) The genomic RNA is complexed with the basic nucleocapsid (N) protein to form a helical capsid within the membrane. The membrane of all coronaviruses is comprised of a minimum of three viral proteins: (i) a spike protein (S), a type of glycoprotein I, (ii) a membrane protein (M) that spans the membrane, and (iii) an envelope protein (E), a highly hydrophobic protein that covers the entire structure of the coronavirus (Figure 2). (30) The SARS-CoV genome contains two open reading frames, connected by a ribosomal frame shift, which encode two large overlapping replicase polyproteins, pp1a (∼450 kDa) and pp1ab (∼750 kDa), from which the functional proteins are produced by an extensive proteolytic process. (31,32) While other coronaviruses utilize three proteases for proteolytic processing, the SARS-CoV is known to encode only two proteases, which include a papain-like cysteine protease (PL^pro) (33) and a chymotrypsin-like cysteine protease known as 3C-like protease (3CL^pro). (34−39) The 3CL^pro enzyme, also called Main protease (M^pro), is indispensable to the viral replication and infection process, thereby making it an ideal target for antiviral therapy.

Figure 2. Structure of a coronavirus showing proteins used for replication.

The X-ray crystallographic structure of hexapeptidyl chlromethyl ketone (CMK) inhibitor bound to 3CL^pro at different pH values was solved by Yang et al. in 2003 (see Figure 3). (38) It was explained that SARS-CoV 3CL^pro forms as a dimer with the two promoters (denoted as “A” and “B”) oriented almost at right angles to each other (Figure 3A,B). The crystal structure of the SARS-CoV 3CL^pro, similar those of other 3CL^pro, comprises three domains. Domains I (residues 8–101) and II (residues 102–184) contain β-barrels that form the chymotrypsin structure, whereas domain III (residues 201–306) consists mainly of α-helices (Figure 3). (38−40) SARS-CoV 3CL^pro has a Cys-His catalytic dyad, and the substrate or inhibitor binding site is located in a cleft between domain I and II. The substrate-binding subsite S1 specificity in protomer A of a CoV protease confers absolute specificity for the P1-Gln substrate residue on the enzyme. Each N-terminus residue (N-finger) squeezed between domains II and III of the parent monomer and domain II of the other monomer, plays an important role in dimerization and formation of the active site of 3CL^pro. The SARS-CoV 3CL^pro dimer is highly active, while the monomer is principally inactive. (41)

Figure 3. SARS-CoV 3CL^pro dimer structure complexed with a substrate-analogue hexapeptidyl CMK inhibitor (PDB ID 1UK4). (38) (A) SARS-CoV 3CL^pro dimer structure is presented as ribbons, and inhibitor molecules are shown as ball-and-stick models. Protomer A (the catalytically competent enzyme) is shown in red, protomer B (the inactive enzyme) is shown in blue, and the inhibitor molecules are shown in yellow. The N-finger residues of protomer B are shown in green. The molecular surface of the dimer is superimposed. (B) Cartoon diagram illustrating the important role of the N-finger in both the dimerization and maintenance of the active form of the enzyme is shown. Adapted from Yang, H. et al. (permission Copyright (2003) National Academy of Sciences, U.S.A. (38)

3. SARS-CoV 3CL^pro Inhibitors

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In 2004, Kua et al. reported the first preparation of the fully active dimeric SARS-CoV 3CL^pro with the authentic sequence. (42) To screen for inhibitors of SARS-CoV 3CL^pro, they prepared a peptide substrate with a fluorescence quenching pair 4-(4-dimethylaminophenylazo)benzoic acid (Dabcyl) and 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (Edans) at the N- and C-termini, respectively, which resulted extremely sensitive assay and allowed many potent inhibitors of SARS-CoV 3CL^pro to be identified.

3CL^pro are cysteine proteases, which are analogues to the main picornavirus 3C protease, a family of viruses that also cause respiratory illness. The conservation of specificities within the 3CL^pro family of coronaviruses has been reported with the amino acid sequence Leu-Gln-Ser or Leu-Gly-Ala as the preferred P2–P1–P1′ sequence (Table 2). (1) Although the functional similarities of 3CL^pro have “cleavage site-specificity” to that of picornavirus 3C proteases, the structural similarities between the two families are limited. (43) The SARS-CoV 3CL^pro cleaves polyproteins at no less than 11 conserved sites involving the Leu-Gln↓(Ser, Ala, Gly) sequence, which appears to be a conserved pattern of the 3CL^pro of SARS-CoV. (3,37) The active site of SARS-CoV 3CL^pro contains Cys145 and His41, creating a catalytic dyad in which the cysteine functions as a common nucleophile in the proteolytic process (Figure 4). (39,43,44)

Table 2. Predicted Cleavage Sites by SARS-CoV 3CL^pro

P4P3P2P1–P1′P2′P3P4′	proteinsa
AVLQ-SGFR	TM2/3CL^pro
VTFQ-GKFK	3CL^pro/TM3
ATVQ-SKMS	TM3/?
ATLQ-AIAS	?
VKLQ-NNEL	?
VRLQ-AGNA	?/GFL
PLMQ-SADA	GFL/?
TVLG-AVGA	?/RdRp
ATLQ-AENV	RdRp/NTPase, etc.
TRLQ-SLEN	NTPase, etc./exonuclease
PKLQ-ASQA	exonuclease/2′-O-MT

^{^a}

TM, Transmembrane; GFL, growth factor-like domain; RdRp, RNA-dependent RNA polymerase; 2′-O-MT, 2′-O-methyltransferase.

Figure 4. Natural amide substrate hydrolysis by Cys145 and His41 at the active site of 3CL^pro.

The initial step in the process is deprotonation of Cys-thiol (I) and followed by nucleophilic attack of resulting anionic sulfur on the substrate carbonyl carbon(II). In this step, a peptide product is released that has an amine terminus, while histidine is restored its deprotonated form (III). In the next step, the resulting thioester is hydrolyzed (IV) to release a carboxylic acid, and the free enzyme (V) is regenerated in the final step. Therefore, the functional significance of 3CL^pro in the viral life cycle makes this protease an ideal target for the development of drugs against SARS and other coronavirus infections.

In 2003, the first X-ray structure of the SARS-3CL^pro dimer with a peptidic CMK (1; Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK, see Figure 5) inhibitor was elucidated (Yang, H. et al.). (38) The unexpected binding mode of the substrate–analogue 1 provides a structural explanation for the P1-Gln entering into the specific pocket and for the decreased P2-Leu specificity of the SARS enzyme. However, specificities for P2-Leu and P4-Ser have been observed in the structure of 1 bound to TGEV 3CL^pro, (43) whereas P3-Thr is orientated toward bulk solvents. In addition, compound 2 or rupintrivir (AG7088) (43) shown in Figure 5 has already been clinically tested for common cold (targeting rhinovirus 3C protease) binds to human rhinovirus 3C protease in the same orientation as that observed for the CMK inhibitor of TGEV. The X-ray crystal structure of 1 with TGEV 3CL^pro and superimposed 2 (AG7088) with HRV2 3C^pro is depicted in Figure 6.

Figure 5. Chemical structures of inhibitors 1, 2, and 3.

Figure 6. (A) The crystal structure of 1 with TGEV 3CL^pro (PDB ID 1P9U) and superimposed 2 with HRV2 3C^pro (PDB ID 1CQQ). The protein binding pocket is shown in surface representation (pink color). The carbon color of compounds 1 (B), 2 (C), and the binding pocket residues of TGEV 3CL^pro and HRV2 3C^pro are represented in magenta, green, and dark- and light-gray, respectively. Oxygen atoms are colored in red, nitrogen atoms in blue, sulfur atoms in yellow and hydrogen atoms in white.

Because the substrate specificity of picornavirus 3C^pro for the P1–P1′ and P4 sites is very similar to that of coronavirus 3CL^pro, compounds 1 and 2 have been proposed as a starting point in the development of new SARS-CoV 3CL^pro inhibitors (Figure 5). (45−48) In addition, the HIV-1 protease inhibitor 3 (Figure 5) (46,49) was found to have high binding affinity toward SARS-CoV 3CL^pro as well. Using the above three molecules as peptidomimetics, many medicinal chemistry studies have been focused on developing a potent chemotherapy method for SARS.

Drugs designed to treat SARS-CoV 3CL^pro can be broadly classified into two types: (i) peptidic inhibitors, which mimic natural peptide substrates, and (ii) small molecule-based inhibitors, obtained from modifications of existing protease inhibitors, virtual screening, structure-based molecular docking studies, and natural products. Additionally, metal-conjugated inhibitors as well as some miscellaneous SARS-CoV 3CL^pro inhibitors are also discussed in this perspective.

4. Peptidomimetic Inhibitors

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In principle, a good substrate can be converted to a good inhibitor by replacement of a part of the substrate sequence that binds directly to the active site of the protease (reversible or irreversible) with the chemical “warhead” targeting the catalytic mechanism. Peptidic inhibitors were designed by attaching a chemical “warhead” type agent to a peptide that mimics the natural substrate. These warhead groups include Michael acceptors, aldehydes, epoxy ketones, halomethyl ketones, and several others (for example, see Figure 7). Mechanistically, these inhibitors act through a two-step procedure, wherein they first bind and form a noncovalent complex with the enzyme such that the warhead is located in close proximity to the catalytic residue. This is followed by a nucleophilic attack by the catalytic cysteine and covalent bond formation. In this perspective, the discussion of peptidomimetics is focused on the substrate selectivity to each specific site (S1′–S1–S2–S3–S4) of 3CL^pro, mode of action, and SAR studies.

Figure 7. Proposed mechanism of cysteine protease inactivation by inhibitors containing Michael acceptor groups.

4.1. Peptides with a Michael Acceptor

Peptidyl or peptidomimetic derivatives contain Michael acceptors as warheads and are an important class of cysteine protease inhibitors. In general, inhibitor design strategies involve the replacement of a substrate’s scissile amide bond with an appropriate Michael acceptor group. The inactivation of a cysteine protease by a Michael acceptor group is depicted in Figure 7. The cysteine residue undergoes 1,4-addition to the inhibitor at the Michael acceptor warhead group, and the subsequent protonation of the α-carbanion results in the irreversible inhibition of the enzyme.

The SAR study of compound 2 indicated that the inhibitory activity was improved by replacing the following side chain residues: the P1-lactam with a phenyl group (4) and the P2-fluorobenzyl with a benzyl group (5), as shown in Figure 8. (50) It was noted that compound 5 had two P1 and P2-phenylalanine groups and could fit in the S2 and S3 pockets of SARS-CoV 3CL^pro, respectively. In addition, the isoxazole moiety of these analogues adopted a conformation different from that of inhibitor 2 and thus undergoes hydrogen bonding with Gln192 in the S4 pocket. However, the conjugated ester was not accessible (>4.5 Å) to Cys145 to allow a Michael addition for covalent bond (C–S bond) formation. Consequently, this process was achieved by a subsequent strategy using pseudo-C2 symmetric analogues (6–9, Figure 8), (50) thus exhibiting good inhibitory activity against 3CL^pro. In particular, a compound comprised of Phe–Phe dipeptide unsaturated ester and 4-(dimethylaminocinnamic acid) (8) exhibited potent inhibitory activity with an IC₅₀ value of approximately 1.0 μM and a K_i value of 0.52 μM. The cell-based bioassay gave an EC₅₀ = 0.18 μM. The presence of a 4-dimethylamino moiety on the phenyl ring of these cinnamic analogues was found to be an important structural functionality for activity enhancement.

Figure 8. Structural modifications of compound 2 with a Michael acceptor to produce active compounds 4–15.

Another series of compounds were reported based on the modification of compound 2 at the P2 side chain by converting the p-fluorobenzyl group to a smaller benzyl (10) or prenyl group (11). (51) These inhibitors (10 and 11) possess P1/P1′-Michael acceptor groups, which can covalently link to the Cys145 (Figure 8). The resulting analogues are not only potential inhibitors of SARS-CoV 3CL^pro (K_inact values) but are effective in SARS-CoV cell-based bioassays. No toxicity was observed up to 100 μM. In addition, it was observed that compound 12, which contains a hydroxyethylene isostere (12) in place of the ketoethylene of compound 10, was inactive due to the loss of an important hydrogen bond interaction between the backbone amide nitrogen of Glu166 and the carbonyl oxygen of the inhibitor (Figure 8). (51) Further replacement of the P4-isoxazole unit with a Boc-serine and a P2-benzyl, prenyl, or isobutyl (13–15: Figure 7) (52) increased the inhibitory activity against 3CL^pro to several times of that of the lead inhibitor (2) (IC₅₀ = 800 μM), which confirmed both the P4-Boc-serine and P2-isopropyl groups as important structural requirements for greater potency.

Although the activity of the potent analogue 13 was improved to several times that of compound 2 against SARS-CoV 3CL^pro, substrate specificity for each site in 3CL^pro could not be identified because the inhibitory activity was absolutely dependent on the other residues in these peptides. Therefore, the backbone structure of compound 2 was modified in a systematic manner as reported by Yang, S. et al. (53) As a result, a five-member lactam ring was found to be more specific for the P1-site, and leucine was used at the P2-site, which showed much better enzyme activity (>15-fold) than the other residues (Table 3). The strong binding of the five-member ring was evidenced by multiple hydrogen-bonds in the X-ray crystal structure (PDB ID 2GX4). (53) For the P2-site, replacement of phenylalanine or 4-fluorophenylalanine with a leucine group increased the inhibitory activity of the enzyme by 4-fold. This result indicated that the rigid and planar phenyl ring is not favorable for binding to the S2 hydrophobic pocket (16 and 17). A lipophilic tert-butyl group at the P3 site further enhances the binding affinity more than 10-fold (17 and 18). Furthermore, the benzyloxy group was found to be the best replacement moiety for P4-methylisoxazole, resulting in a more than 4-fold increment in enzyme inhibitory activity (2 and 16); this group was found to be the best group for this site. On the basis of the docking study, this benzyloxy group has also been observed in a unique conformation in the X-ray crystal structure (docking study of 18 with PDB ID 2GX4; see Supporting Information (SI), Figure S1). (53)

Table 3. Peptidomimetics with a Michael Acceptor

^{^*}

K_i, binding affinity; IC₅₀, half-maximal inhibitory concentration.

4.2. Peptides with Keto-glutamine

A novel series of keto-glutamine analogues (19–26) with a phthalhydrazido group at the α-position were reported as reversible inhibitors against SARS-CoV 3CL^pro (Figure 9). (54) This discovery originated due to their inhibitory activity against the human hepatitis A virus 3C protease. (55,56) These compounds feature β and β′ functionalities adjacent to the keto group as well as intramolecular hydrogen bonding to the carbonyl, which makes them more electrophilic and susceptible to hemithioacetal formation with Cys145 in the active site of the protease. Compound 25 was recognized as the most potent analogue with an inhibitory value (IC₅₀) of 0.65 μM. SAR studies indicated that both γ-lactam and phthalhydrazide moieties are very important for good inhibition. Specifically, the introduction of the γ-lactam into the inhibitor containing a phthalhydrazide moiety greatly enhanced the inhibitory activity against SARS-CoV 3CL^pro (compare inhibitors 19–22 vs 23–26). This was further supported by molecular modeling studies of the active inhibitors (24–26), which show binding via an extended β-sheet interaction with residues 163–166 of the 3CL^pro and formation of hydrogen bonds between the His163 and the P1 side chain.

Figure 9. Keto-glutamine derivatives with phthalhydrazide (19–27) and thiophene group (28).

A recent report disclosed the X-ray crystal structure of SARS-CoV 3CL^pro complexed with one of the phthalhydrazide (19)-based peptide inhibitors (Figure 10, PDB ID 2Z3C). (57) The inhibitor forms an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme’s catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of the native substrate. (57) Additionally, the data suggest that this structure resembles the proposed tetrahedral intermediate during the deacylation step of normal peptide hydrolysis cleavage. (57) Furthermore, to prove the importance of P1-lactam and phthalhydrazide units in inhibitor 23, a series of analogues modified from P1-lactam to P1-phenyalanine (27) or from phthalhydrazide to thiophene (28) were reported to have only weak activity against SARS-CoV 3CL^pro. (54)

Figure 10. Crystal structure of phthalhydrazide-based inhibitor 19 bound to SARS-CoV 3CL^pro (PDB ID 2Z3C). The protein binding pocket is shown in surface representation and colored in orange. The carbon atoms of the inhibitor 19 and the binding pocket residues are shown in stick model and colored in green and yellow, respectively. The thiiranium ring formed by amino acid Cys145 is colored in magenta.

4.3. Peptides with Nitroanilide

A diverse series of peptide anilides (29–35) were reported based on niclosamide (Figure 11). (58) Unlike typical nitroanilide-based peptides, which are readily hydrolyzed by serine and cysteine protease, (59) these peptides were not efficiently cleaved by SARS-CoV 3CL^pro. Niclosamide showed no inhibitory activity at a concentration of 50 μM. The most potent inhibitor (29) is an anilide derived from 2-chloro-4-nitro aniline, l-phenylalanine, and 4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL^pro with a K_i value of 0.03 μM and showed high selectivity toward SARS-CoV 3CL^pro (IC₅₀ = 0.06 μM) rather than other proteases such as trypsin (IC₅₀ = 110 μM), chymotrypsin (IC₅₀ = 200 μM), and papain (IC₅₀ = 220 μM). Because of the chlorine atom at the o-position, the 2-chloro-4-nitrophenyl ring and amido group cannot be in a coplanar conformation, thus making hydrolysis unfavorable.

Figure 11. Anilide-type peptidomimetics (29–35) and (2S,2S)-aza epoxide (36) and *trans*-aziridine (37) inhibitors.

Modification of compound 29 to a series of analogues resulted in reduced potency (30–35). (58) A docking study (Figure 12, PDB ID 1UK4) showed that the 2-chloro-4-nitroanilide unit of compound 29 occupies the second preferred pocket. Thus, the nitro group was predicted to be hydrogen bonded with Ala46 and His41, providing a possible key interaction with the catalytic dyad. The (dimethylamino)phenyl group fit into the cleft formed by Gln189–Gln192 and Met165–Pro68. (58) A docking study also suggested that anilide 29 has the lowest binding energy (−9.1 kcal/mol) compared to the other derivatives. This experiment supports the observations of the enzymatic assay, which revealed the important roles of 2-chloro-4-nitroaniline and 4-(dimethylamino)benzoic acid residues in effective inhibition. (58)

Figure 12. Docked pose of 29 (green, stick model) is shown with the binding pocket residues (gray, line model) and interacting residues (orange, stick model) with SARS-CoV 3CL^pro (PDB ID 1UK4). The binding pocket of the protein is shown in surface representation and gray in color.

4.4. Aza-epoxide and Aziridine Peptides

It has been reported that some novel classes of aza-peptide epoxides (APEs) act as inhibitors for clan CD cysteine peptidase. (60,61) In the compound library screening, compound 36 (Figure 11) showed prominent activity with irreversible inhibition of SARS-CoV 3CL^pro (K_inact/K_i = 1900 (±400) M^–1 s^–1). (62) From the kinetic data and crystal structure of APEs reported by Lee T-W. et al., the 3CL^pro reacts only with the S,S-diastereomer and not its R,R-diastereomer. In addition, the epoxide C3 atom of APE must be in the S-configuration.

A comprehensive screening of various peptides with electrophilic building block-attached groups (e.g., epoxides and aziridines) identified potential 3CL^pro inhibitors. The data revealed that the aziridine- and oxirane-2-carboxylates are important for the inhibition of 3CL^pro. A trans-configured compound containing Gly-Gly-aziridine peptide 37 (54% inhibition at 100 μM) was selected as a modest active-site directed irreversible SARS-CoV 3CL^pro inhibitor (Figure 11). (63) This study also revealed that epoxide or aziridine building blocks alone, which do not contain an amino acid moiety, are not active.

4.5. Peptide Aldehydes

A series of peptide aldehyde libraries were designed to target the SARS coronavirus, based on the irreversible inhibitor CMK, and were shown to possess very weak inhibitory activity against SARS protease (IC₅₀ > 500 μM). (64) The inhibitor CMK binds in a canonical mode to TGEV 3CL^pro and resulted in a binding mode with P2, P4, and P5 addressing the respective S pockets, while P3 and P6 were exposed to the solvent (Figure 13A). However, in monomer A SARS-CoV 3CL^pro, the CMK inhibitor follows a different side chain orientation (noncannonical binding mode): P2, P4, and P6 residues were not positioned to the respective pockets of the enzyme but remain solvent exposed. Instead, P3-threonine associates with the S2 pocket, and the S4 pocket is occupied by P5-aspargine (Figure 13B).

Figure 13. (A) CMK-canonical binding mode with TGEV 3CL^pro (PDB code 1P9U), (43) CMK-noncanonical binding mode with active monomer A of SARS CoV 3CL^pro (PDB code 1UK4) (B), (38) and (C) the derived inhibitors 38 and 39.

On the basis of these structural findings, it was observed that the sequential variations at the P sites of this initial structure produced potent inhibitors, especially after modifications of the P2 and P5 sites, whereas mutations of the P1 and P3 sites yielded only moderately improved inhibitors. Peptides 38 (AcNSTSQ-H) and 39 (AcESTLQ-H) were found to be more potent, with the best reversible inhibitors having IC₅₀ values in the low micromolar range (7.5 μM) (Figure 13C). Interestingly, these inhibitors are assumed to bind in a noncanonical mode similar to that of CMK with TGEV 3CL^pro (Figure 13B). In addition, the SAR suggested that the substrate specificity of SARS-CoV 3CL^pro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. Moreover, X-ray crystal structures of some pentapeptide aldehydes Ac-ESTLQ-H (40, PDB ID 3SNE), Ac-NSFSQ-H (41, PDB ID 3SNA), Ac-DSFDQ-H (42, PDB ID 3SNB), and Ac-NSTSQ-H (43, PDB ID 3SNC), complexed with SARS-CoV 3CL^pro, revealed that the S2 pocket of the enzyme can accommodate serine and even an aspartic acid side chain in the P2 position (see SI, Figure S2). (65) However, the cleavage efficiency of serine in the P2-position was 160 times lower than the original substrate (P2-Leu), and with aspartic acid, cleavage was not observed at all. Furthermore, the same research group also determined the X-ray crystal structure of SARS-CoV 3CL^pro in complex with Cm-FF-H (44, K_i = 2.24 μM, see Figure 14A). From the complex structure (see SI, Figure S3, PDB ID 3SN8), compound 44 had a P1-phenylalanine residue located in the hydrophilic S1 subsite resulted in hydrophobic interactions with Phe140, Leu141, Asn142, and the P3-cinnamoyl group of Cm-FF-H. This result suggests that the stringent specificity of SARS-CoV 3CL^pro with respect to the P1 and P2 positions can be overcome by the highly electrophilic character of the aldehyde warhead.

Figure 14. (A) Structure of aldehydes 44 and 45 and (B) substrate based inhibitors 46–48.

A novel potent SARS-CoV 3CL^pro peptide–aldehyde inhibitor (45: K_i = 53 nM) was developed as an antiviral agent against SARS-CoV and human coronavirus HCoV 229E replication, which reduced the viral titer by 4.7 log (at 5 μM) for SARS-CoV and 5.2 log (at 1.25 μM) for HCoV 229E (Figure 14A). (53) This inhibitor has distinct functional groups at the P1 to P4 sites compared to those of reference compound 2. This inhibitor was designed to evaluate the issues of cell viability, stability, and drug-like properties based on compound 18. Accordingly, the leucine moiety was replaced with a bulky cyclohexylalanine to improve the cell activity, and the ester group was replaced with an aldehyde to avoid hydrolysis by esterase. As a result, compound 45 (TG-0205221) (53) displayed a very stable profile in mouse, rat, and human plasma (Table 4). The X-ray crystal structure of 45 (PDB ID 2GX4) revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions (see SI, Figure S4). (53)

Table 4. In Vivo Evaluation of Compound 45 for Stabilitya

^{^a}

The drug was added to 90% rat, mouse, or human plasma and incubated for 0, 30, and 120 min in respective wells.

In the course of studies on the SARS-CoV 3CL^pro and its inhibitors, (66) it was found that the mature SARS-CoV 3CL^pro is very sensitive to degradation at the Arg188/Gln189 site, which causes a loss of catalytic activity. The stability of the SARS-CoV 3CL^pro is dramatically increased by mutating the Arg at position 188 to Ile. The enzymatic efficiency of the R188I mutant was increased by a factor of more than 1 × 10⁶. The potency of the mutant protease makes it possible to quantitatively evaluate substrate-based peptide–aldehyde inhibitors using conventional high-performance liquid chromatography (HPLC). A P-site pentapeptide sequence, Ac-Ser-Ala-Val-Leu-NHCH-(CH₂CH₂CON(CH₃)₂)-CHO (46: Figure 14B), inhibits the catalytic activity of the SARS-CoV 3CL^pro with an IC₅₀ value of 37 μM. The side chain structures, especially at sites P1, P2, and P4, were then optimized step by step based on X-ray crystallographic analyses of the inhibitor–protease complex to provide potent tetra peptide aldehyde inhibitors (47 and 48) (Figure 14B). (67)

4.6. Peptides with Halomethyl Ketone or Electrophilic Substituents

A new series of N,N′-dimethyl glutaminyl (49–53) or aspartic acid (54) inhibitors with fluoromethyl a ketone warhead were reported as SARS-CoV 3CL^pro inhibitors (Table 5). (68) These inhibitors were designed based on their caspase inhibitory activities. (69,70) Antiviral activity assessed by cytopathic effect (CPE) inhibition in SARS-CoV infected Vero cultures revealed that compounds effectively inhibit both FFM1 and 6109 strains of SARS-CoV replication.

Table 5. Inhibitory Values of Analogues 49–54

^{^a}

Concentration of compound inhibiting cytopathic effect to 50% of untreated cells. Values represent the mean (standard deviation) from three independent experiments.

^{^b}

Incubation of confluent CaCo2 or Vero cell layers with different concentrations of all the dipeptides for 3 days.

^{^c}

CC₅₀, 50% cytotoxic concentration.

Among these inhibitors, compound 49 exhibited promising activity with low toxicity in cells, protecting the cells with an EC₅₀ value of 2.5 μM and exhibiting a selectivity index >40. (68) In addition, compound 49 showed low toxicity in mice. From the SAR studies, P1-glutamine, a residue that has been identified as a conservative recognition site in SARS-CoV 3CL^pro, can be replaced by N,N′-dimethyl glutamine (see 49–51). However, compound 54, a potent caspase inhibitor with P1-aspartic acid, abolished activity in this series. (68) Furthermore, the P2-leucine can also be replaced by isoleucine (50) and valine (51). The active compounds 49–51 were found to be inactive against rhinovirus type-2 in a cell-based assay suggested that compounds 49–51 are specific against SARS-CoV. Compound 51 was found to have low toxicity in mice after administration of a single dose at 25, 50, and 100 mg/kg. No weight loss or behavioral changes nor any gross pathology of the major organs was observed at the tested doses. This study suggested that compound 51 could be a promising candidate for animal efficacy studies (68)

Abeles et al. proposed that trifluoromethyl ketones (FMK) (71) can also be used as protease inhibitors. (72) An interesting feature of these inhibitors is the formation of thermodynamically stable hemiketal or hemithioketal that occurs upon nucleophilic attack by the Ser-hydroxyl or Cys-thiol groups present in the serine or cysteine protease, respectively. On the basis of this observation, Hayashi et al. reported Gln-derived CF₃^– ketones 55 and 56 as SARS-CoV 3CL^pro inhibitors (Figure 15). (73) Compounds 55 and 56 showed modest inhibitory activity due to the formation of typical cyclic structures that are not expected to interact effectively with the active site. (69) To avoid this problem, the side chain at the P1 site was modified in order to block cyclization. (74−77) As shown in Figure 15, compounds 57 (74) and 58 (75) showed excellent activities and further optimization provided compounds 59–60, (76,77) which showed low nanomolar inhibition of SARS-CoV 3CL^pro.

Figure 15. Inhibitors with halomethyl ketones and their derivatives 55–60.

While continuing to explore the SARs based on FMK inhibitors, a series of trifluoro methyl ketones 61–68 were developed, mainly focusing on the P1 and P2–P4 positions (Table 6). (78) Three different amino acids were demonstrated as variable residues at positions P1–P4. The inhibitory activities were observed to range from 10 to 50 μM. The potent inhibitor, compound 61, which possesses the same moiety as the substrate sequence of the peptide at the P1–P4 sites, exhibited comparable activity to other compounds. As shown in Table 6, replacement of the P1-benzyl (62) with a methyl group (64) or hydrogen (66) resulted in a loss of activity. Inhibitor 61 showed time-dependent inhibition, with a K_i value of 0.3 μM after a 4 h incubation. (78)

Table 6. Inhibitory Activity of Halomethyl Ketonesa

compd	R	X	IC₅₀ (μM)
61	see above structure	see above structure	10
62	Bn	Cbz-Leu	15
63	Bn	Cbz-Phe	20
64	Me	Boc-Leu	40
65	H	Boc-γ-Glu(OtBu)-Ala	40
66	H	γ-Glu-Ala	50
67	Bn	CH₃(CH₂)₈CO-Leu	50
68	Bn	CH₃(CH₂)₇CO-Leu	>50

^{^a}

IC₅₀, half-maximal inhibitory concentration; Bn, benzyl; Me, methyl.

4.7. Symmetric Peptides

It was previously proposed that HIV protease inhibitors could serve as good starting points for the development of SARS-CoV 3CL^pro inhibitors. In general, reversible inhibitors produce fewer side effects than suicide inhibitors and are thus more suitable for drug development. Recently, compound 69, a noncovalent HIV protease inhibitor (K_i = 1.5 nM), was used as a lead structure and optimized using computational analysis for the development of SARS-CoV 3CL^pro inhibitors. (79) As shown in the Figure 16, introduction of peripheral Val-Ala residues in place of the Cbz groups or introduction of 3-indolyl groups in place of the phenyl groups in compound 69 led to the formation of inhibitors (70 and 71) that were potent SARS-CoV 3CL^pro inhibitors with K_i values of 0.34 and 0.073 μM, respectively. In addition, compound 71 is highly selective for the 3CL^pro, with no inhibition observed against HIV protease at 100 μM.

Figure 16. Symmetric peptide diols 69–71.

5. Small Molecule Inhibitors of SARS-CoV 3CL^pro

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The other category of inhibitors against SARS-CoV 3CL^pro includes nonpeptidic small molecules. In general, small molecules have been found to be noncovalent or reversible covalent inhibitors, which have advantages regarding side effects and toxicity which often arise with covalent inhibitors. These inhibitors were discovered by high throughput screening of synthetic compounds and natural products.

5.1. Etacrynic Acid Derivatives

An HPLC-based screen of electrophilic compounds revealed etacrynic acid derivatives 73 (75% inhibition at 100 μM and K_i = 45.8 μM) and 74 (88% inhibition at 100 μM and K_i = 35.3 μM) as effective inhibitors of SARS-CoV 3CL^pro. (80) These inhibitors were obtained from the sequential modifications of an etacrynic acid (72), a well-known diuretic drug, (81) and also showed activity toward the cysteine proteases such as papain protease (K_i = 375 μM). (82) Ester 73 showed more potency toward papain protease (K_i = 3.2 μM) than SARS-CoV 3CL^pro (K_i = 45.8 μM). However, etacrynic acid amide (74, K_i = 35.3 μM) was found to have more affinity toward SARS-CoV 3CL^pro. The SAR studies revealed that chloro substituents on the phenyl moiety were necessary for SARS-CoV 3CL^pro inhibition (Figure 17). Compounds with an unsubstituted phenyl ring or methyl substituent were inactive at 100 μM. (80) In addition, it is quite promising that only esters or amides display 3CL^pro inhibition. (80)

Figure 17. Structural features of etacrynic acids produce their inhibitory activity against SARS-CoV 3CL^pro.

5.2. Isatin (2,3-Dioxindole) Inhibitors

It has been established that certain isatin (2,3-dioxindole) compounds are potent inhibitors of rhinovirus 3C^pro. (83) Because the proteases of SARS-CoV and rhinovirus share similar active sites and catalytic residues, (15) isatin derivatives may also be good candidates for anti-SARS drug development. Accordingly, a series of synthetic isatin derivatives (75–81) were reported as noncovalent SARS protease inhibitors, (84,85) unlike rhinovirus 3C^pro, which has a covalent bond binding mode (Table 7). These isatin derivatives inhibited SARS-CoV 3CL^pro in the low micromolar range, and inhibitors 78 and 80 were found to be the most potent. SAR studies revealed that the inhibitory potency heavily depended on the hydrophobicity and electron affinity of the substituents on the isatin core. Moreover, computational analysis (docking studies of 78 with PDB ID 1UK4, see SI, Figure S5) of both active compounds showed that they fit very well into the active pocket of SARS-CoV 3CL^pro. The two carbonyl groups on isatin could form hydrogen bond interactions with the NH groups on Gly143, Ser144, Cys145, and the His41 side chain. In addition, compounds 78 and 80 (86) exhibited better selectivity for SARS than for other proteases including papain (103, 87.24 μM), chymotrypsin (1 mM, 10.4 μM) and trypsin (362, 243 μM).

Table 7. Inhibitory Activities of Isatin Derivatives

5.3. Flavonoid and Biflavonoid Derivatives

Chemotherapeutic agents that target viral entry are an important class of antiviral therapy as they can block the propagation of the virus at an early stage, thus minimizing the chance for the virus to evolve and acquire drug resistance. Screening of Chinese herbal medicine-based molecules resulted in the discovery of luteolin (82) as inhibitor of wild-type SARS-CoV activity with an effective concentration (EC₅₀) of 10.6 μM (Figure 18). (87) Compound 82 was identified as active using a two-step screening method consisting of frontal affinity chromatography–mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. This flavone analogue binds with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus into the host cells. However, the related flavone quercetin (83) and its derivatives exhibited modest inhibitory activity against the SARS virus (Figure 18).

Figure 18. Flavonoids and biflavonoid derivatives.

Quercetin-3-β-galactoside (84) was identified as a potential inhibitor of SARS-CoV and showed inhibitory activity with an IC₅₀ of 42.79 ± 4.97 μM in a SPR/FRET-based enzymatic inhibition assay. (88) The docking study of 84 with SARS-CoV 3CL^pro suggested that the residue Gln189 (Q189) plays a key role in the binding interaction. To confirm this prediction, the binding mode of 84 was compared between the wild-type SARS-CoV 3CL^pro and its mutated SARS-CoV 3CL^pro Q189A. This comparative study was consistent with the docking prediction and the inhibitory potency of 84 on SARS-CoV 3CL^pro Q189A was significantly decreased to 127.89 ± 10.06 μM. Besides, the experimental evidence showed that the enzymatic activity of SARS-CoV 3CL^pro was not affected by the Q189A mutation. The l-fucose derivative (85) exhibited 2-fold potent inhibitory activity compared to 84. The SAR and molecular docking studies of these new derivatives revealed that four hydroxy groups on the quercetin moiety are key determinants for its potential biological activity.

As part of ongoing investigation of bioflavonoids from medicinal plants as potential SARS-CoV 3CL^pro inhibitors, a series of inhibitors (86–90) were reported from the leaves of Torreya nucifera (Figure 18). (89) Among the isolated compounds, biflavone amentoflavone (86) was recognized as a potent noncompetitive inhibitor, exhibiting an IC₅₀ value of 8.3 μM. An SAR study demonstrated the three authentic flavones, apigenin (90), luteolin (82), and quercetin (83), showed inhibitory activities (IC₅₀) of 280.8, 20.2, and 23.8 μM, respectively. The activity of amentoflavone (86) was consistent with the binding interactions (docking studies of 86 with PDB ID 2Z3E, see SI, Figure S6), with Val186 and Gln192 as one of the key binding modes with the target site. Moreover, the binding energy difference between apigenin (90; −7.79 kcal/mol) and amentoflavone (86; −11.42 kcal/mol) are consistent with a 30-fold lower IC₅₀ value of 86 toward SARS-CoV 3CL^pro than apigenin (90).

5.4. Terpenoid Derivatives

A series of diterpenoids (91–93) from Torreya nucifera were evaluated for their anti-SARS activity (Figure 19). (89) However, these terpenoids exhibited very low activity compared to biflavonoids against SARS-CoV 3CL^pro at concentrations up to 100 μM. One exception was ferruginol (91, IC₅₀ = 49.6 μM), which exhibited significantly greater activity. Moreover, the quinone-methide triterpenoids celastrol (94), pritimererin (95), tingenone (96), and iguesterin (97) were isolated from the methanol (95%) extracts of Tripterygium regelii (Celastraceae) and showed moderate inhibitory activities with IC₅₀ values of 2.6, 9.9, 5.5, and 10.3 μM, respectively, whereas the corresponding a semisynthetic analogue dihydrocelastrol (98: IC₅₀ = 21.7 μM) reduced the inhibitory potency (Figure 19). (90) A SAR study suggested that the quinone–methide moiety in the A ring and the more hydrophobic E-ring assist in producing the potent inhibitory activity. The compounds mentioned above (91–98) have been proven to be competitive inhibitors using kinetic analysis.

Figure 19. Terpenoid derivatives with inhibitory activity against SARS-CoV 3CL^pro.

Furthermore, abietane-type diterpenoids and lignoids exhibit a strong anti-SARS-CoV effect. (91) In particular, betulinic acid 99 and savinin 100 were shown to act as competitive inhibitors against SARS-CoV 3CL^pro with the K_i values of 8.2 and 9.1 μM, respectively (Figure 19). (91) On the basis of molecular modeling analysis, it was observed that the competitive inhibition of 99 and 100 on SARS-CoV 3CL^pro activity was consistent with the formation of multiple hydrogen bond interactions between the compound and specific amino acid residues located at the active site of the pocket of the protease enzyme.

5.5. Sulfone, Dihydroimidazole, and N-Phenyl-2-(2-pyrimidinylthio)acetamide Type Analogues

Structure-based virtual screening of a chemical database containing 58855 compounds for SARS-CoV 3CL^pro inhibition produced two hits, sulfone (101) and dihydroimidazole (102) (Figure 20). (92) The core structures of these two hits, defined by a molecular docking study, were used for further searches of analogues.

Figure 20. Sulfone, dihydroimidazole, and N-phenyl-2-(2-pyrimidinylthio)acetamide-type analogues.

Accordingly, 21 analogues derived from these two hits exhibited IC₅₀ values below 50 μM, and the two most potent compounds (103 and 104) obtained from each hit show IC₅₀ values of 0.3 and 3 μM, respectively. (92) Furthermore, a combination of structure-based virtual screening and three-dimensional quantitative structure–activity relationship (3D-QSAR) studies of compound databases of 59363 compounds led to the identification of compounds 105–110, which exhibited modest inhibition with IC₅₀ values of 3, 10, 11, 12, 14, and 15 μM, respectively (Figure 20). (93) On the basis of the structure–functional analysis, a common core structure, N-phenyl-2-(2-pyrimidinylthio)acetamide, was identified. A potential binding mode of compound 105 was predicted by the molecular modeling study (docking study of 107 with PDB ID 1UK4, see SI, Figure S7); the strong interaction of benzene and thiazole units with Glu166, Leu167, Pro168, and Gln192 at the SARS-CoV 3CL^pro active site could explain its increase in potency.

5.6. Active Heterocyclic Ester Analogues

Wong and co-workers (94) reported a novel class of mechanism-based irreversible inhibitors with activity in the nanomolar range, using combinatorial synthesis in microtiter plates followed by in situ screening. (95−97) Instead of the expected amide reaction products, a series of benzotriazole esters (111–114) were isolated. Surprisingly, the inhibitory activity of these analogues was much higher than that of the other small molecules or peptidomimetics. Further SAR optimization yielded analogues 115–118 with nanomolar inhibitory activities (Figure 21). An interesting point was found that the esters derived from the benzoic acid-containing electron withdrawing substituents, e.g., NO₂, CN and CF₃ were susceptible to hydrolysis, whereas esters 111–114 and those with electron-donating substituents were relatively stable in pH 5.0–8.0 solutions over 24 h at room temperature. Compound 116 (K_i = 7.5 nM) was the most potent among the benzotriazole esters. (94) The possible mode of action could be acylation of Cys145 at the active site assisted by the catalytic dyad; this irreversible enzyme acylation was verified by electrospray ionization mass spectrometry of the inhibited enzyme with the compound 112 (Figure 22).

Figure 21. Active heterocyclic ester analogues and their inhibitory activities against SARS-CoV 3CL^pro.

Figure 22. Mechanism of covalent bond formation of inhibitors **112** and **120** with the active site cysteine residue of SARS-CoV 3CL^pro.

In addition, the recent X-ray crystal structure of the SARS-CoV 3CL^pro complex with the benzotriazole ester also confirmed that the active-site cysteine is acylated by the ester ligand which acts as a suicide inhibitor. (98) It should be noted that the formation of N-hydroxybenzotriazole is a very potent inhibitor of CYP450 enzymes. Heteroaromatic ester 119 (IC₅₀ = 0.5 μM) was also identified as a potent inhibitor of the SARS coronavirus. (99) The 5-chloropyridine moiety in compound 119 proved to be the key unit for activity against SARS-CoV 3CL^pro. Continuing SAR studies provided the very potent inhibitors 120–123, with inhibitory activities spanning from the micromolar to nanomolar range.

The structural biology analysis suggested, in addition to the halopyridyl unit, the other aromatic rings are also key factors for potent inhibition (Figure 21). (100,101) A covalent bond formation mechanism for the enzyme–inhibitor complex (120) has been proposed on the basis of electrospray mass spectrometry investigation (Figure 22).

However, another strategy was demonstrated by combining key parts of the previously mentioned mechanism-based inhibitors (116 and 119) to produce a novel series of 5-chloropyridinyl indolecarboxylate inhibitors (124–128) with enzymatic potency in the submicromolar range (Figure 23). (102) The SAR study suggested that the positions of the carboxylic acid ester and free indole hydrogen (NH) are critical for activity. Indole carboxylate 124 with carboxylate functionality at position 4 was the most potent inhibitor with an enzyme inhibitory activity (IC₅₀) of 30 nM and an antiviral EC₅₀ value of 6.9 μM.

Figure 23. Active 5-chloropyridine ester analogues and their inhibitory activity against SARS 3CL^pro.

5.7. Aryl Methylene Ketones and Fluoro Methylene Ketones

5-Halopyridinyl-3-aromatic esters, as described in a previous section 5.6, act as highly potent inhibitors of SARS-CoV 3CL^pro with IC₅₀ values in the low nanomolar range. They initially bind competitively and strongly to the active site but are then hydrolyzed by the enzyme as substrates and released. Despite their potent inhibition of SARS-CoV 3CL^pro and relatively long half-life in buffer at neutral pH values, they are likely to be problematic as drug candidates due to their propensity to be rapidly hydrolyzed by lipase, esterase, and other enzyme in the mammalian cells. Moreover, these compounds can also potentially react nonspecifically with other thiols or nucleophiles in mammalian cells, thereby leading to toxicity. Therefore, to develop stable and noncovalent inhibitors based on pyridinyl esters, a group of methylene ketones and corresponding mono and difluorinated methylene ketones were reported as SARS-CoV 3CL^pro inhibitors by Zhang. J. et al. (Figure 24). (103) Compounds 129, 131, and 132 showed the best inhibition, and specifically, inhibitor 129 was the most potent among these analogues. The molecular modeling study of these active ketone analogues predicts a binding conformation similar to that of corresponding pyridinyl esters. (100,101) A SAR study suggested that fluorination decreases inhibition despite enhancing the electrophilicity of the carbonyl carbon. Enzymatic analysis and ESI-MS studies indicate that these inhibitors utilize a noncovalent, reversible mechanism of action.

Figure 24. Halomethyl pyridyl ketones and their inhibition potential against SARS-CoV 3CL^pro.

5.8. Pyrazolone and Pyrimidines

High throughput screening identified 3,3-dihydropyrazolidine 133 (104) and tetrasubstituted pyrazole 134, (105) which displayed 1,3,5-triaryl substitution patterns, as SARS-CoV 3CL^pro inhibitors. Further exploration of SAR produced a series of pyrazolones that demonstrated inhibitory activities against SARS-CoV 3CL^pro (Figure 25). (106) Among them, compounds 135–137 exhibited potent inhibitory activities with the IC₅₀ values of 5.5, 6.8, and 8.4 μM, respectively.

Figure 25. Pyrazolones and pyrimidines and their inhibition potential against SARS-CoV 3CL^pro.

Structure–functionality analysis indicated that the 4-carboxylbenzylidine-aryl ring attached to C4-of pyrazolone accompanied by electron withdrawing groups, such as CN, NO₂, and F, favors inhibitory activity. Molecular modeling studies of the active compound 137 predicted that the N1-phenyl group located in the S1 pocket and the carboxyl benzylidene group in the S3 pocket of 3CL^pro is crucial for its inhibitory activity. Pyrimidine derivatives (138–140) were designed, and their anti-SARS activity was reported (Figure 25). (107,108) Compound 140 was the most potent inhibitor that showed enzyme inhibitory activity (IC₅₀ = 6.1 μM) against SARS-CoV 3CL^pro. SAR studies revealed that the presence of nitro functionality at position 4 on the benzylidene ring was more important for activity enhancement. This potent activity was consistent with a molecular docking study (docking study of 140 with PDB ID 1UK4, see SI, Figure S8); (38) the oxygen of the nitro group formed a hydrogen bond with side chains of Gly143 and Cys145. In addition, the 4-chloro phenyl ring was predicted to fit into the S2 pocket due to hydrophobic inter actions.

5.9. Decahydroisoquinoline Derivatives

Starting from the peptide inhibitor 47 (see section 4.5), (67) a novel nonpeptide decahydroisoquinoline inhibitor was designed and synthesized based on the cleavage site interactions at the S1, and hydrophobic interaction at the S2 sites of SARS SARS-CoV 3CL^pro. (109) The decahydroisoquinoline inhibitors (141–144, Figure 26) showed weak inhibitory activities for SARS-CoV 3CL^pro, which confirmed that the fused ring structure of the decahydroisoquinolin scaffold can be accommodated in the active site of SARS-CoV 3CL^pro. From the X-ray crystallographic studies (PDB ID 4TWW), it was confirmed that the decahydroisoquinoline inhibitors were at the active site cleft of 3CL^pro, as observed in peptide–aldehyde inhibitors. The decahydroisoquinoline scaffold was inserted into a large S2 pocket and occupied most of the pocket. The P1 site imidazole was inserted into the S1 pocket as expected. These interactions were effective in holding the terminal aldehyde tightly inside the active site cleft, which resulted in the compact fitting of the novel scaffold to SARS-CoV 3CL^pro.

Figure 26. Novel decahydroisoquinoline derivatives as SARS-CoV 3CL^pro inhibitors.

5.10. 3-Pyridyl and Benzotriazole-Based SARS-CoV 3CL^pro Inhibitors

Jacobs et al. conducted a high-throughput screening of NIH molecular libraries (∼293000 compounds) by evaluating the inhibition of 3CL^pro mediated peptide cleavage using a novel FRET-based substrate. (110,111) In this screen, a dipeptide class, represented by 3-pyridyl-based hit 145 (Figure 27) was identified.

Figure 27. Primary SAR study at hit furyl amide **145** and schematic representation of enzyme pockets occupied by **146** and 11.

Optimization study based on derivatives (Ugi library) structurally related to hit compound 145 resulted in a series of 3-pyridyl-based inhibitors among which the two compounds, 146 and 147 (Figure 27), were shown to be active against SARS-CoV 3CL^pro.

The X-ray crystal structure of 146 bound to SARS-CoV 3CL^pro (Figure 28) demonstrated that the binding orientation of 146 was similar to that of known covalent peptidomimetic inhibitors (for example compound 11) and preferentially occupies the S3–S1′ subpockets of SARS-CoV 3CL^pro enzyme as R-enantiomer. The tert-butyl amide occupies the S3-pocket, the tert-butylanilido group occupies the deep S2-pocket, and the 3-pyridyl moiety occupies the S1; the furyl amide acts as a P1′ group. Inhibitor 146 lacks a reactive warhead.

Figure 28. X-ray crystal structure of **146** bound to the binding pocket SARS-CoV 3CL^pro (PDB ID 3V3M). The pockets S1′–S3 are highlighted, and the compound **146** is represented in stick model and colored in cyan.

On the basis of the SAR for 146 and related analogues, first a chemical library focusing exclusively on the P1′ group was synthesized while holding the P1–P3 groups constant. This resulted in a series of inhibitors. (110) The SAR study around P1′ of 146 showed that the five-membered π-excessive heterocycles proved the most successful 148–153 (Figure 29A). Especially, compound bearing imidazole (150) and 5-chlorofuran (152) analogue exhibited equipotent to 146 with IC₅₀ values of 6.0 and 5.2 μM, respectively. Next, the P1 3-pyridyl unit in 146 was replaced with its isosteres in order to identify alternate hydrogen bond acceptor groups. This effort led to identify another set of compounds (154–156, Figure 29B). Among them, only pyridazine (154) and pyrazine (155) were tolerated, although no improvement was found around the pyridyl ring over 146. Both 2-and 4-pyridyl (156) analogues were not tolerable and reduced the potency. (110)

Figure 29. SAR studies at the P1′ (A) and P1 sites (B) of **146** and chiral separation of **146**-(R,S) (C) to **146**-(R) and **146**-(S) enantiomers.

In a continuing study, the racemic compound 146 was purified by chiral supercritical fluid chromatography to separate 146-(R) (ML188) (110) and 146-(S) enantiomers (Figure 29C). The evaluation of a compound 146-(R) exhibited inhibitory activity with an IC₅₀ of 1.5 ± 0.3 μM against SARS-CoV 3CL^pro, while the other enantiomer 146-(S) was inactive. The mechanism of inhibition of SARS-CoV 3CL^pro by 146-(R) was determined to be competitive (K_i, 1.6 ± 0.26 μM) with noncovalent inhibition. Owing to the excellent 3CL^pro inhibition and antiviral activity (12.9 ± 0.7 μM) against mock-infected and SARS-CoV infected Vero E6 Cells, 146-(R) was elected as a first in class probe candidate from the furyl amide.

Following the identification of probe compound 146-(R), the same research group continued their further efforts to develop potent, noncovalent SARS-CoV 3CL^pro inhibitors based upon a chemical class of benzotriazoles from MLPCN screening. (112) This resulted in a hit compound 157 (Figure 30A) demonstrating a SARS-CoV 3CL^pro IC₅₀ of 6.2 μM and good selectivity versus PL^pro (IC₅₀ > 60 μM).

Figure 30. (A) SAR studies at the P1, (B) P2–P1′, and (C) P3-truncation of hit **157** to inhibitors (**158**–**167**).

The X-ray crystal structure of 157 bound to SARS-CoV 3CL^pro shows the diamide 157 binds into an induced-fit binding site that is formed by a rearrangement of the Gln189 and Met49 residue side chains (PDB ID 4MDS, Figure 31). This induced fit site accommodates the syn-N-methyl pyrrole and anilido acetamide moieties of the inhibitors within subpockets that can be characterized as S2–S4 and S2–S1′ subpockets, respectively. Figure 30A schematically illustrates the inhibitor-active site interactions oriented in a similar manner as depicted in Figure 31.

Figure 31. X-ray crystal structure of **157** bound to SARS-CoV 3CL^pro (PDB ID: 4MDS) is represented in surface model. The compound **157** (green) is shown in stick model, and the interacting residues (magenta) and the binding pocket residues (gray) are shown in line model.

To improve the activity, first, the SAR study focusing on benzotriazole replacements in 157 for alternate hydrogen bond acceptor functionality was demonstrated. This resulted the replacement of benzotriazole with 4-phenyl 1,2,3-trizole 158 (IC₅₀ of 11 μM, Figure 30A) was tolerable.

Second, the acetamide modification (P2–P1′ region) with a series of cyclic and acyclic congeners yielded many inhibitors which show activities below 10 μM (159–162, Figure 30B), specifically, the branched i-propyl derivative (159) and cyclobutylamide (160) having the greatest activity below 5 μM.

Third, the researchers turned to P3-truncation for minimum pharmacophore to reduce overall molecular weight. This effort led to a series of analogues and SAR proved that truncated amides (163–167, Figure 30C) have comparable activity versus the elaborated amides; for example, compare 163–167 Vs 159–162. The compound 167 represented the first sub-100 nM inhibitor for the series and one of the most potent nonwarhead based SARS-CoV 3CL^pro inhibitors to date.

From the above compounds, one of the potent inhibitors, 165 (ML300) (112) was selected for probe declaration. (113) The biological profiles of inhibitors 146-(R), 160, and 165 are indicated in Figure 32. Relative to probe 146-(R) and the equipotent diamide 160, the compound 165 proved to offer progresses in several areas. Inhibitor 165 is ∼100 amu lower MW (MW = 431) relative to 160 with moderate ligand efficiency (LE). (114) Moderate cLogP value of 165 (cLogP = 3.2) greatly improves ligand efficiency-dependent lipophilicity (LELP) (114) versus 146-(R) and 160. When both probe 146-(R) and 165 tested in an in-house in vitro DMPK panel including plasma protein binding, P450 enzyme inhibition, and intrinsic clearance using liver microsomes, both 146-(R) and 165 possess good free fraction. However, intrinsic clearance indicates both 146-(R) and 165 are predicted to be highly cleared. 146-(R) and 165 possess modest P450 enzyme inhibition, with 165 maintaining 5–10 μM activity across four major CYP enzymes (see Figure 32). Probe 165 was found to be highly selective in a Eurofins lead-profiling screen, (115) with only modest activity (10 μM) for melatonin MT1 receptor in a radioligand binding assay.

Figure 32. Profiles of SARS-CoV 3CL^pro inhibitors **146**-(R), **160**, and **165**.

6. Metal Conjugated SARS-CoV 3CL^pro Inhibitors

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Metal ions have been shown to inhibit many viral proteases such as 3CL^pro of noroviruses, papain-like protease (PLP2) of SARS-CoV, human cytomegalovirus (hCMV) protease, and hepatitis C virus (HCV) NS3 protease. (116−120) The screening of 960 metal conjugated compounds allowed inhibitors with potent inhibitory activity against SARS-CoV 3CL^pro to be identified. These include competitive inhibitors phenyl mercuric acetate (168, K_i = 0.7 μM), thimerosal (169, K_i = 2.4 μM), and phenyl mercuric nitrate (170, K_i = 0.3 μM) (Figure 33). (121,122) However, inhibition was more pronounced using zinc-conjugated compounds (171–174), i.e., 1-hydroxypyridine-2-thione zinc (171, K_i = 0.17 μM) compared to Zn²⁺ ions alone (K_i = 1.1 μM).

Figure 33. Metal-conjugated inhibitors and their inhibition potential against SARS-CoV 3CL^pro.

The X-ray crystal structure of SARS-CoV 3CL^pro–168 (PDB ID 1Z1I) revealed that phenyl-bound mercury occupied the S3 pocket, which is responsible for its enzymatic activity. Hg(II) ions are known to cause toxic effects because the affinity of Hg²⁺ ions to thiol groups in proteins leads to nonspecific inhibition of cellular enzymes. (123) However, regarding the structures of zinc-centered complexes, the zinc ion plays a key role in targeting the catalytic residues via binding to the His41–Cys145 catalytic dyad to yield a zinc central tetrahedral geometry. This type of inhibition was similar to the zinc-mediated serine protease inhibitor keto-BABIM-Zn²⁺ for trypsin in that a zinc ion was coordinated to the two chelating nitrogen atoms of bis(5-amidino-2-benimidazilyl)methane (BABIM) and the two catalytic residues (His-Ser) of trypsin in the tetrahedral geometry. (124) The safety of zinc-containing compounds for human use has been indicated by the fact that zinc acetate and zinc sulfate are added as supplements to drugs for the treatment of Wilson’s disease and Behcet’s disease, respectively. (125,126) Moreover, the possibility of zinc complexes incorporated into cells through the cell membrane was also demonstrated by studies on type-2 diabetic treatment. (127)

Analysis of the active site cavity of this SARS–cysteine protease reveals the presence of a subsite contains a cluster of serine residues (Ser139, Ser144, and Ser147) and is an attractive target for the design of high affinity small molecule inhibitors. This cluster is conserved in all known coronavirus proteases. In particular, Ser139 and Ser147 are conserved in all known coronavirus. Because of the known potential reactivity of boronic acid compounds with the hydroxyl group of the serine residue, a series of bifunctional boronic acid-conjugated compounds (175–177) have been reported against SARS-CoV 3CL^pro enzyme (Figure 33). (128) The greatest improvement in affinity was achieved with an amide type compound (177) with a K_i of 40 nM. Isothermal titration microcalorimetric experiments indicated that these inhibitors bind reversibly to SARS-CoV 3CL^pro in an enthalpically favorable manner, implying that they establish strong interactions with the protease molecule.

7. Miscellaneous SARS-CoV 3CL^pro Inhibitors

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Over the past decade, in silico virtual screening (VS), in particular structure-based virtual screening (SBVS), has emerged as a reliable, cost-effective, and time-saving technique for the discovery of lead compounds as an alternative to high throughput screening (HTPS). (129) The application of VS to the discovery of new enzyme inhibitors involves docking, computational fitting of the compound structure to the active site of an enzyme, and scoring and ranking of each compound. (130) On the basis of the structural information, 361413 structurally diverse small molecules were screened by a “genome-to-drug-lead” approach. Compound 178 showed modest activity against targeted human SARS-CoV 3CL^pro Toronto-2-strain with an EC₅₀ of 23 μM (Figure 34). Virtual screening of 50240 structurally diverse small molecules allowed 104 compounds with anti-SARS-CoV activities to be identified. (131) Inhibitor 179 showed potent inhibitory activity with an IC₅₀ value of 2.5 μM and an EC₅₀ of 7 μM in a Vero cell-based SARS-CoV plaque reduction assay (Figure 34). Another group of researchers, using a quenched fluorescence resonance energy transfer assay, screened 50000 drug-like molecules, resulting in 572 hits. (99) After applying a series of virtual and experimental filters, five structurally novel molecules were identified that showed potent inhibitory activity (IC₅₀ = 0.5–7 μM) against SARS-CoV 3CL^pro.

Figure 34. Miscellaneous SAR–CoV 3CL^pro inhibitors.

Among them, compounds 180 (IC₅₀ = 4.3 μM) and 181 (IC₅₀ = 4.3 μM) (Figure 34) showed good inhibitory activity of SARS-CoV 3CL^pro and exhibited interesting selectivity with no inhibition against other proteases tested (HAV 3C^pro, NS3^pro, chymotrypsin, and papain). (99)

The elucidation of the crystal structure of SARS-CoV 3CL^pro provided enormous opportunities for the discovery of inhibitors through rational drug design. As part of an effort to discover small molecule inhibitors of SARS-CoV 3CL^pro, structure-based virtual screening of 32000 small molecules was screened against the SARS-CoV 3CL^pro enzyme. (47) Use of knowledge-based filters yielded 27 molecules for follow-up. A biological evaluation of the inhibitors in the low micromolar range found two compounds, 182 and 183, with IC₅₀ values of 18.2 and 17.2, respectively (Figure 34). It has been reported that several nucleoside derivatives have 6-chloropurine as a nucleobase showed potent antiviral activity against some types of viruses. (132,133) Because 6-chloropurine analogues are known to inhibit bacterial RNA polymerases, a series of nucleoside analogues with 6-chloropurines were evaluated for anti-SARS-CoV activity by a plaque reduction activity. (134) Among them, two compounds, 184 and 185, exhibited modest anti-SARS-CoV activity (IC₅₀ values of 48.7 and 14.5 μM, respectively) that was comparable to those of mizoribine and ribavirin (Figure 34). This study revealed several SAR trends such as a 6-chloropurine moiety, 5′-hydroxy, and protected (benzylated)-5′-hydroxy group are responsible for the potent inhibitory activity.

Ribavirin, a broad-spectrum of inhibitor of RNA and DNA viruses, was used for the treatment of SARS affected patients (135) but it does not inhibit viral growth at concentrations attainable in human serum. In contrast, interferon (IFN)-α showed an in vitro inhibitory effect at concentrations of 1000 IU/mL. (136) Interestingly, the combination of ribavirin and IFN-β synergistically inhibited SARS-CoV replication. The HIV protease inhibitor nelfinavir (137) and the antimalarial agent chloroquine (138) showed strong inhibitory activity against SARS-CoV replication. However, no cytoprotective effect was found for nelfinavir in an independent study. (139,140) Structure-based virtual screening of compounds was conducted to identify novel SARS-CoV 3CL^pro inhibitors. (141) The top-ranked 1468 compounds with free binding energy ranging from −14.0 to −17.09 kcal mol^–1 were selected to evaluate the hydrogen bond interactions in the active site of SARS-CoV 3CL^pro. Among them, 53 compounds were selected for their inhibitory activity toward SARS-CoV 3CL^pro from Escherichia coli. Two of the compounds (186 and 187) were demonstrated to be competitive inhibitors of 3CL^pro with K_i values of 9.11 and 9.93 μM, respectively (Figure 34). (141) A detailed docking simulation analyses suggested that these inhibitors could be stabilized by the formation of hydrogen bonds with catalytic residues and the establishment of hydrophobic contacts at the opposite region of the active site. In particular, for the potent compound 187, the nitrophenyl group was likely to be very crucial in the SARS-CoV 3CL^pro inhibitory activity through its formation of H-bonds with Cys145 and Gly143, as well as its hydrophobic interactions with His41 and Cys145.

Recently, the combination of virtual screening (VS) and high-throughput screening (HTS) techniques were applied to screen 41000 compounds from structurally diverse libraries have allowed novel, nonpeptidic small molecule inhibitors (188, IC₅₀ = 13.9 μM) and (189, IC₅₀ = 18.2 μM) against human SARS-CoV 3CL^pro to be identified (Figure 34). (142) Because the newly identified compounds are of low molecular weight, they were examined for selectivity against three proteases, namely SARS-CoV PL^pro (a cysteine protease), human UCH-L1 (a cysteine protease), and hepatitis C virus NS3/4A (a serine protease), and two nonproteolytic enzymes, Bacillus anthracis dihydroorotase and Streptococcus pneumoniae PurC. Compound 189 displayed good selectivity for SARS-CoV 3CL^pro and did not show inhibitory activity (>200 μM) against other five enzymes, whereas compound 188 showed 20-fold selectivity against the two SARS cysteine proteases, 3CL^pro and PL^pro, over other enzymes. Because low molecular weight compounds typically lack high specificity, lack of inhibition of compound 188 for other enzymes, especially the UCH-L1 cysteine protease, is particularly noteworthy.

8. Conclusion and Perspectives

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The emergence of SARS and the identification of a coronavirus as the causative agent of the disease astounded the coronavirus community, as it was the first definitive association of a coronavirus with a severe disease in humans. Because the first crystal structure of the SARS-CoV 3CL^pro dimer with a peptidic CMK inhibitor covalently bound was elucidated in 2003, over 20 crystal structures of the enzyme have been reported. Structure-based design and virtual screens have provided both peptidomimetic and nonpeptidomimetic inhibitors with potency in the micromolar to nanomolar range. Yet, to date, there is no effective therapy for the treatment of SARS in humans, and to our knowledge, no CoV 3CL^pro inhibitor has been taken into clinical development. In this perspective, we have described the SAR for several classes of inhibitors, highlighting their structural features and binding modes. Both peptidomimetic and small molecule SARS-CoV 3CL^pro are largely based on a warhead-based design strategy. So far, only a few inhibitors have been described that exhibit good enzymatic and cellular potency, and the majority of these inhibitors have not been followed up with additional studies (such as antiviral activity or in vivo evaluation), likely due to their unattractive structures and/or their nonideal physiochemical properties.

The reactive warhead groups used in peptidomimetic inhibitors for SARS-CoV 3CL^pro include Michael acceptors, aldehydes, epoxy ketones, electrophilic ketones such as halomethyl ketones, and trifluoromethyl ketones. Although these peptidomimetics are covalent inhibitors with the potential for toxicity, significant improvements have been made in enzymatic and cellular potency.

Of the many peptidomimetics inhibitors described in the literature, those highlighted in Figure 35 appear to be the most promising for further optimization efforts. Compound 2 (Figure 5) is an example of an inhibitor incorporating a Michael acceptor. It was developed by Pfizer as an inhibitor of human rhinovirus 3C protease for common cold (targeted rhinovirus 3C-protease). Although 2 was not active against SARS-CoV in cell culture, it served as a good starting point for anti-SARS drug design, leading to inhibitors 8 and 18 (see section 4.1), which are the two most potent inhibitors against SARS-CoV 3CL^pro incorporating a Michael acceptor warhead. Specifically, compound 8 exhibited excellent cellular potency with an EC₅₀ value of 0.18 μM and it is a nontoxic anti-SARS agent. However, further in vivo studies for compound 8 have not been reported in the literature.

Figure 35. Profile of representative peptidic SARS-CoV 3CL^pro inhibitors highlighting reactive warhead groups (red).

Peptidic aldehydes are promising enzymatic inhibitors, but they are unlikely to be effective as therapeutic agents due to their rapid in vivo metabolism and low oral bioavailability. In contrast, the peptide aldehyde thrombin inhibitor efegatran was well tolerated in a phase I clinical trial. (143,144) Inhibitor 45, a potent peptide aldehyde, showed remarkable activity against SARS-CoV and human coronavirus (HCoV) 229E replications, reducing the viral titer by 4.7 log (at 5 μM) for SARS-CoV and 5.2 log (at 1.25 μM) for HCoV 229E. This inhibitor also displayed a stable profile in mouse, rat, and human plasma (see section 4.5) and may represent a starting point for the development of an anti-SARS agent.

Inhibitor 51 is one of the potent inhibitors in the halomethyl series, exhibiting low toxicity in mice after a single ip dose at 25, 50, and 100 mg/kg, no weight loss, behavioral changes, or gross pathology of major organs was observed at the tested doses (see section 4.6). The low molecular weight of 51 is a potential advantage. Because peptidyl monofluoromethyl ketones have been shown to be effective in vivo, (145−147) the inhibitor 51 may be a suitable candidate for further in vivo efficacy and toxicology studies.

Numerous small molecules were also discussed in this perspective. The majority of efforts to develop nonpeptide SARS-CoV 3CL^pro inhibitors have also relied on warhead-based design strategy, and several of these nonpeptide inhibitors achieved nanomolar potency. The most interesting inhibitors (78, 116, 119, 124, 129, 146, 160, 165, and 186) are illustrated in Figure 36. In the case of pyridyl esters, the potent mechanism-based enzyme inactivator 124 (see section 5.6) achieved cell-based inhibition below 10 μM in SARS-CoV infected Vero E6 cells. Compounds 146-(R), 160, and 165 are promising examples of noncovalent SARS-CoV 3CL^pro inhibitors of moderate molecular weights and good enzymatic and antiviral activity (see section 5.10). These inhibitors are potential starting points for the design of more potent 3CL^pro inhibitors with a noncovalent mechanism of action. However, further in vivo studies for above-mentioned small molecules have not reported so far.

Figure 36. Profile of representative nonpeptidic SARS-CoV 3CL^pro inhibitors highlighting reactive warhead groups (red).

Although many structural and nonstructural proteins are known to be potential targets for anticoronavirus therapy, none of them are well-conserved due to their possible role in the viral life cycle, thus limiting the potential success of wide-spectrum inhibitors. In contrast, the coronavirus 3CL^pro is highly conserved among coronaviruses, making it an attractive target for broad-spectrum inhibitors (see SI, Table S1). (148) The proteases share 40–60% sequence identity and 60–100% sequence similarity. Therefore, targeting SARS-CoV 3CL^pro is an important approach for the development of antiviral therapy that can be applied for broad viral infections. Recent reports have revealed that many SARS-CoV 3CL^pro inhibitors showed potential activity against the recent outbreak of MERS-CoV. (149)

A feasible and rapid advancement in the drug discovery for the development of effective chemotherapeutics against SARS-CoV might be achieved by repurposing existing and clinically approved drugs. It was recently reported that screening a library of drugs either clinically developed or with a well-defined cellular pathway from different classes of therapeutics produced a series of compounds with good activity against SARS-CoV. (149) Drugs that inhibit CoV included neurotransmitter inhibitors, estrogen receptor antagonists, kinase signaling inhibitors, protein-processing inhibitors, inhibitors of lipid or sterol metabolism, and inhibitors of DNA synthesis or pair. However, the inhibitors (peptidomimetics or nonpeptidomimetics) that target other serine proteases (e.g., HCV protease, thrombin) and cysteine proteases (e.g., calpain, cathepsin K, caspases) have not been tested against 3CL^pro. For examples, ketoamides (such as A-705253 for calpain), (150) nitriles (such as odanacatib/MK-0822 and vildagliptin/LAF237 for cathepsin K and dipeptidyl peptidase-4 (DPP4)), (151,152) phenyloxymethyl ketones (such as VX-166 for caspases), (153) fused triazole derivatives (such as sitagliptin/MK-0431 for DPP4), (154) nonpeptides (such as apixaban/BMS-562247-01 for factor Xa), (155) and beta lactams for penicillin binding proteins such as penicillin. (156) Therefore, these structural types should be considered in future for the development of anti-SARS therapy.

N-Finger residues (N-finger) of SARS 3CL^pro play an important role in enzyme dimerization, and therefore peptides with N-terminal amino acid sequences may act as inhibitors of 3CL^pro dimerization, similar HIV protease, and other viral enzymes. (157−163) In 2006, Wei et al. reported that N-terminal octapeptide N8 (K_i of 2.20 mM) was the first example of inhibitor targeting the dimeric interface of SARS 3CL^pro, (164) providing a novel strategy for drug design against SARS and other coronaviruses. However, no peptidomimetic or small molecule inhibitor has yet been reported in the literature. Although it would be a great challenge to explore new inhibitors of dimerization, with the current development of computational approaches, the structure-based design of novel inhibitors may be successful.

In conclusion, although huge efforts have been taken by both academia and pharmaceutical industries, no coronavirus protease inhibitor has yet successfully completed a preclinical development program. We hope that this perspective will be useful to medicinal chemists targeting 3CL^pro to identify novel anti-SARS CoV inhibitors with drug-like properties and that effective therapy for coronaviruses will be discovered.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01461.

Docking figures of compounds 18, 41–44, 45, 46, 83, 92, 112, and 155; sequence comparison analysis of 3CL^pro of coronaviruses to the SARS-CoV 3CL^pro; list of X-ray structures of ligands with 3C^pro and 3CL^pro (PDF)

jm5b01461_si_001.pdf (1.04 MB)

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Author Information

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Corresponding Author
- Thanigaimalai Pillaiyar - Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany; Email: [email protected]
Authors
- Manoj Manickam - College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, South Korea
- Vigneshwaran Namasivayam - Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
- Yoshio Hayashi - Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
- Sang-Hun Jung - College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, South Korea
Notes
The authors declare no competing financial interest.

Biographies

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Thanigaimalai Pillaiyar

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Thanigaimalai Pillaiyar received his Master’s degree in Chemistry in 2006 from Bharathiar University, India. Prior to his doctoral study, he worked as a Research Executive at Orchid Chemicals and Pharmaceuticals Limited, India. He received his Doctoral degree in Medicinal Chemistry in 2011 under the supervision of Prof. Dr. Sang-Hun Jung at Chungnam National University, South Korea. In 2011, he won a “Japanese Society for the Promotion of Science Postdoctoral fellowship” for two years with Prof. Dr. Yoshio Hayashi at Tokyo University of Pharmacy and Life sciences, Japan. He was awarded an Alexander von Humboldt Postdoctoral fellowship” in 2013 for two years with Prof. Dr. Christa E. Müller at University of Bonn, Germany. He has been working on various therapeutic targets, focusing on infective and inflammatory diseases.

Manoj Manickam

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Manoj Manickam received his Ph.D. in 2010 from Bharathiar University under the supervision of Prof. Dr. K. J. Rajendra Prasad, Coimbatore, India. He continued to work as a Research Associate at Orchid Chemicals and Pharmaceuticals Ltd. Then he moved to Chungam National University, South Korea, for continuing his research. Currently, he is a Senior Research Scientist at the Department of Pharmacy and Institute of Drug Research and Development, Chungnam National University, working with Professor Sang-Hun Jung.

Vigneshwaran Namasivayam

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Vigneshwaran Namasivayam is a Scientific Staff at Pharmaceutical Institute, University of Bonn, Germany (since 2010), and involved in the field of cheminformatics, computational chemistry, and molecular modelling. He gained his Master of Technology in Bioinformatics from SASTRA University, India (2004), and Doctoral degree under the supervision of Prof. Dr. Hans-Jörg Hofmann from Leipzig University, Germany (2009). He carried out his postdoctoral research at Technical University of Munich, Germany (2010). Prior to his doctoral studies in Germany, he worked as a Research Executive (2004–2006) at Orchid Chemical and Pharmaceutical Limited, Chennai, India.

Yoshio Hayashi

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Yoshio Hayashi earned his Ph.D. in 1990 in the Faculty of Pharmaceutical Science, Kyoto University, under the guidance of Emeritus Prof. Haruaki Yajima and Prof. Nobutaka Fujii. After spending two years at Calpis Food Industry Co., Ltd. and three years at Nippon Steel Corporation (NSC) as a researcher, he was promoted to senior researcher at the Life Science Research Center of the NSC, where he stayed for another eight years. In 1999, he joined Prof. Yoshiaki Kiso’s group in the Department of Medicinal Chemistry of Kyoto Pharmaceutical University as a lecturer and, in 2001, was appointed as an associate professor. In 2007, he moved to Tokyo University of Pharmacy and Life Sciences as a full professor. His research interests include peptide chemistry, peptidomimetics, and medicinal chemistry.

Sang-Hun Jung

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Sang-Hun Jung received his M.S. degree from the College of Pharmacy of the Seoul National University in 1976. He received his Ph.D. from the Chemistry Department at the University of Houston, USA, in 1984. He served as a postdoctoral fellow at the University of Pittsburgh until 1985 and as a Principle investigator of LG life Science from 1985 to 1989. He has been a professor at the College of Pharmacy, Chungnam National University, South Korea, since 1989. He has served as a Department Chairman (1993–2000), Dean of the College of Pharmacy (2003–2004), and President of Institute of Drug Research and Development of Chungnam National University (2007–2009). His research interests include antimicrotubule-based anticancer agents, novel inotropes with selective activation of cardiac myosin, and melanogenesis inhibitors.

Acknowledgments

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T.P. thanks the Japanese Society for the Promotion of Science (JSPS) foundation for a support for postdoctoral study in Japan. We thank Proceedings of the National Academy of Sciences (PNAS) for the permission to use Figure 3.

Abbreviations Used

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hCoV	human coronavirus
SARS	severe acute respiratory syndrome
Pros	proteases
TGEV	transmissible gastroenteritis virus
IBV	infectious bronchitis virus
BCoV	bovine coronavirus
hCMV	human cytomegalovirus
HCV	hepatitis C virus
MHV	murine coronavirus mouse hepatitis virus
WHO	World Health Organization
MERS	Middle East respiratory syndrome
FDA	Food and Drug Administration
RNA	ribonucleic acid
DNA	DNA
PLP	papain-like cysteine protease
3CL^pro	chymotrypsin-like cysteine protease
M^pro	main protease
APEs	aza-peptide epoxides
HPLC	high performance liquid chromatography
HIV	human immunodeficiency virus
HCoV-229E	human coronavirus 229E
SAR	structure–activity relationship
QSAR	quantitative structure–activity relationship
Cys	cysteine
His	histidine
Ser	serine
S	spike protein
M	membrane protein
N	nuleocapsid
E	envelope
BABIM	bis(5-amidino-2-benimidazilyl)methane
SBVS	structure-based virtual screening
HTPS	high throughput screening
IFN	interferon
ORF	open reading frame
MW	molecular weight
LELP	ligand efficiency-dependent lipophilicity
LE	ligand efficiency
DPP4	dipeptidyl peptidase-4

References

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This article references 164 other publications.

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Cheever, F. S.; Daniels, J. B.; Pappenheimer, A. M.; Baily, O. T. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin: I. Isolation and biological properties of the virus. J. Exp. Med. 1949, 90, 181– 210, DOI: 10.1084/jem.90.3.181
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A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin
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The Journal of experimental medicine (1949), 90 (3), 181-210 ISSN:0022-1007.
The isolation of a murine virus causing disseminated encephalomyelitis accompanied by extensive destruction of myelin in the central nervous system, and focal necrosis of the liver has been described. Young mice can be infected by a number of parenteral routes. Both encephalitic and paralytic signs can be observed. After intracerebral inoculation the virus has been isolated from brain, spinal cord, liver, lung, spleen, and kidney, but not from blood or from intestinal walls and contents. Hamsters, cotton rats, and Hisaw rats can be infected by the intracerebral route. Guinea pigs and rabbits appear to be insusceptible. Attempts to infect chick embryos have so far met with failure. Under proper conditions the agent can pass through the usual bacterial filters. No inclusion bodies have been seen. No serological relationship to other neurotropic viruses has been demonstrated as yet.
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A MURINE VIRUS (JHM) CAUSING DISSEMINATED ENCEPHALOMYELITIS WITH EXTENSIVE DESTRUCTION OF MYELIN : II. PATHOLOGY
Bailey O T; Pappenheimer A M; Cheever F S; Daniels J B
The Journal of experimental medicine (1949), 90 (3), 195-212 ISSN:0022-1007.
A description has been given of the pathologic changes produced experimentally in animals by the inoculation of a virus material obtained from a mouse with spontaneous encephalomyelitis. The most distinctive feature of the lesions in the central nervous system is the widespread destruction of myelin. Giant cells derived from a variety of tissue elements characterize the early lesions. The liver in the majority of cases is the seat of focal necrosis. In some mice, infected with large doses by the intravenous route, there is produced massive necrosis of the liver, with fat infiltration and calcification. Giant cells are occasionally found in lymphatic tissue, but no significant changes were noted in other organs. Inclusions or elementary bodies were not demonstrated in the lesions. Similar lesions were produced by the inoculation of mouse virus into hamsters. In rats, the lesions were of a more chronic character. The relation of this disease to other demyelinating diseases of man and animals is discussed.
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Rota, P. A.; Oberste, M. S.; Monroe, S. S.; Nix, W. A.; Campagnoli, R.; Icenogle, J. P.; Penaranda, S.; Bankamp, B.; Maher, K.; Chen, M. H.; Tong, S.; Tamin, A.; Lowe, L.; Frace, M.; DeRisi, J. L.; Chen, Q.; Wang, D.; Erdman, D. D.; Peret, T. C.; Burns, C.; Ksiazek, T. G.; Rollin, P. E.; Sanchez, A.; Liffick, S.; Holloway, B.; Limor, J.; McCaustland, K.; Olsen-Rasmussen, M.; Fouchier, R.; Gunther, S.; Osterhaus, A. D.; Drosten, C.; Pallansch, M. A.; Anderson, L. J.; Bellini, W. J. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 2003, 300, 1394– 1399, DOI: 10.1126/science.1085952
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3
Characterization of a novel coronavirus associated with severe acute respiratory syndrome
Rota, Paul A.; Oberste, M. Steven; Monroe, Stephan S.; Nix, W. Allan; Campagnoli, Ray; Icenogle, Joseph P.; Penaranda, Silvia; Bankamp, Bettina; Maher, Kaija; Chen, Min-hsin; Tong, Suxiong; Tamin, Azaibi; Lowe, Luis; Frace, Michael; DeRisi, Joseph L.; Chen, Qi; Wang, David; Erdman, Dean D.; Peret, Teresa C. T.; Burns, Cara; Ksiazek, Thomas G.; Rollin, Pierre E.; Sanchez, Anthony; Liffick, Stephanie; Holloway, Brian; Limor, Josef; McCaustland, Karen; Olsen-Rasmussen, Melissa; Fouchier, Ron; Guenther, Stephan; Osterhaus, Albert D. M. E.; Drosten, Christian; Pallansch, Mark A.; Anderson, Larry J.; Bellini, William J.
Science (Washington, DC, United States) (2003), 300 (5624), 1394-1399CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
In Mar. 2003, a novel coronavirus (SARS-CoV) was discovered in assocn. with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was detd., and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
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Drosten, C.; Gunther, S.; Preiser, W.; van der Werf, S.; Brodt, H. R.; Becker, S.; Rabenau, H.; Panning, M.; Kolesnikova, L.; Fouchier, R. A.; Berger, A.; Burguiere, A. M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.; Kramme, S.; Manuguerra, J. C.; Muller, S.; Rickerts, V.; Sturmer, M.; Vieth, S.; Klenk, H. D.; Osterhaus, A. D.; Schmitz, H.; Doerr, H. W. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 2003, 348, 1967– 1976, DOI: 10.1056/NEJMoa030747
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Identification of a novel coronavirus in patients with severe acute respiratory syndrome
Drosten, Christian; Guenther, Stephan; Preiser, Wolfgang; van der Werf, Sylvie; Brodt, Hans-Reinhard; Becker, Stephan; Rabenau, Holger; Panning, Marcus; Kolesnikova, Larissa; Fouchier, Ron A. M.; Berger, Annemarie; Burguiere, Ana-Maria; Cinatl, Jindrich; Eickmann, Markus; Escriou, Nicolas; Grywna, Klaus; Kramme, Stefanie; Manuguerra, Jean-Claude; Mueller, Stefanie; Rickerts, Volker; Stuermer, Martin; Vieth, Simon; Klenk, Hans-Dieter; Osterhaus, Albert D. M. E.; Schmitz, Herbert; Doerr, Hans Wilheim
New England Journal of Medicine (2003), 348 (20), 1967-1976CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
The severe acute respiratory syndrome (SARS) has recently been identified as a new clin. entity. SARS is thought to be caused by an unknown infectious agent. Clin. specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and mol. techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a PCR (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60% of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clin. specimens from patients with SARS but not in controls. High concns. of viral RNA of up to 100 million mols. per mL were found in sputum. Viral RNA was also detected at extremely low concns. in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS.
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de Groot, R. J.; Baker, S. C.; Baric. R.; Enjuanes, L.; Gorbalenya, A. E.; Holmes, K. V.; Perlman, S.; Poon, L.; Rottier, P. J. M.; Talbot, P. J.; Woo, P. C. Y.; Ziebuhr, J. Family Coronaviridae. In Ninth Report of the International Committee on Taxonomy of Viruses; King, A. M. Q., Lefkowitz, E., Adams, M. J., Carstens, E. B., Eds.; Elsevier: Oxford, 2011; pp 806– 828.
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ICTV Master Species List 2009; International Committee on Taxonomy of Viruses, August 24, 2010; vol. 10.
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Parry, J. WHO investigates China’s fall in SARS cases. Br. Med. J. 2003, 326, 1285-c, DOI: 10.1136/bmj.326.7402.1285-c
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Communicable Disease Surveillance and Response; World Health Organization: Geneva, May 7, 2003; http://www.who.int/csr/sars/archive/2003_05_07a/en and http://www.who.int/csr/sars/country/en/country2003_08_15.pdf (August 15, 2003).
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Nie, Q. H.; Luo, X. D.; Zhang, J. Z.; Su, Q. Current status of severe acute respiratory syndrome in China. World J. Gastroenterol. 2003, 9, 1635– 1645, DOI: 10.3748/wjg.v9.i8.1635
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Current status of severe acute respiratory syndrome in China
Nie Qing-He; Luo Xin-Dong; Zhang Jian-Zhong; Su Qin
World journal of gastroenterology (2003), 9 (8), 1635-45 ISSN:1007-9327.
Severe acute respiratory syndrome (SARS), also called infectious atypical pneumonia, is an emerging infectious disease caused by a novel variant of coronavirus (SARS-associated coronavirus, SARS-CoV). It is mainly characterized by pulmonary infection with a high infectivity and fatality. SARS is swept across almost all the continents of the globe, and has currently involved 33 countries and regions, including the mainland China, Hong Kong, Taiwan, North America and Europe. On June 30, 2003, an accumulative total reached 8450 cases with 810 deaths. SARS epidemic was very rampant in March, April and May 2003 in the mainland of China and Hong Kong. Chinese scientists and healthcare workers cooperated closely with other scientists from all over the world to fight the disease. On April 16, 2003, World Health Organization (WHO) formally declared that SARS-CoV was an etiological agent of SARS. Currently, there is no specific and effective therapy and prevention method for SARS. The main treatments include corticosteroid therapy, anti-viral agents, anti-infection, mechanical ventilation and isolation. This disease can be prevented and controlled, and it is also curable. Under the endeavor of the Chinese Government, medical staffs and other related professionals, SARS has been under control in China, and Chinese scientists have also made a great contribution to SARS research. Other studies in developing new detection assays and therapies, and discovering new drugs and vaccines are in progress. In this paper, we briefly review the current status of SARS in China.
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Tsui, P. T.; Kwok, M. L.; Yuen, H.; Lai, S. T. Severe acute respiratory syndrome: clinical outcome and prognostic correlates. Emerging Infect. Dis. 2003, 9, 1064– 1069, DOI: 10.3201/eid0909.030362
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Severe acute respiratory syndrome: clinical outcome and prognostic correlates
Tsui Ping Tim; Kwok Man Leung; Yuen Hon; Lai Sik To
Emerging infectious diseases (2003), 9 (9), 1064-9 ISSN:1080-6040.
Severe acute respiratory syndrome (SARS) poses a major threat to the health of people worldwide. We performed a retrospective case series analysis to assess clinical outcome and identify pretreatment prognostic correlates of SARS, managed under a standardized treatment protocol. We studied 127 male and 196 female patients with a mean age of 41+14 (range 18-83). All patients, except two, received ribavirin and steroid combination therapy. In 115 (36%) patients, the course of disease was limited. Pneumonitis progressed rapidly in the remaining patients. Sixty-seven (21%) patients required intensive care, and 42 (13%) required ventilator support. Advanced age, high admission neutrophil count, and high initial lactate dehydrogenase level were independent correlates of an adverse clinical outcome. SARS-associated coronavirus caused severe illnesses in most patients, despite early treatment with ribavirin and steroid. This study has identified three independent pretreatment prognostic correlates.
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Leung, W. K.; To, K. F.; Chan, P. K.; Chan, H. L.; Wu, A. K.; Lee, N.; Yuen, K. Y.; Sung, J. J. Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology 2003, 125, 1011– 1017, DOI: 10.1016/S0016-5085(03)01215-0
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Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection
Leung Wai K; To Ka-Fai; Chan Paul K S; Chan Henry L Y; Wu Alan K L; Lee Nelson; Yuen Kwok Y; Sung Joseph J Y
Gastroenterology (2003), 125 (4), 1011-7 ISSN:0016-5085.
BACKGROUND AND AIMS: Severe acute respiratory syndrome (SARS) is a recently emerged infection from a novel coronavirus (CoV). Apart from fever and respiratory complications, gastrointestinal symptoms are frequently observed in patients with SARS but the significance remains undetermined. Herein, we describe the clinical, pathologic, and virologic features of the intestinal involvement of this new viral infection. METHODS: A retrospective analysis of the gastrointestinal symptoms and other clinical parameters of the first 138 patients with confirmed SARS admitted for a major outbreak in Hong Kong in March 2003 was performed. Intestinal specimens were obtained by colonoscopy or postmortem examination to detect the presence of coronavirus by electron microscopy, virus culture, and reverse-transcription polymerase chain reaction. RESULTS: Among these 138 patients with SARS, 28 (20.3%) presented with watery diarrhea and up to 38.4% of patients had symptoms of diarrhea during the course of illness. Diarrhea was more frequently observed during the first week of illness. The mean number of days with diarrhea was 3.7 +/- 2.7, and most diarrhea was self-limiting. Intestinal biopsy specimens obtained by colonoscopy or autopsy showed minimal architectural disruption but the presence of active viral replication within both the small and large intestine. Coronavirus was also isolated by culture from these specimens, and SARS-CoV RNA can be detected in the stool of patients for more than 10 weeks after symptom onset. CONCLUSIONS: Diarrhea is a common presenting symptom of SARS. The intestinal tropism of the SARS-CoV has major implications on clinical presentation and viral transmission.
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Lee, N.; Hui, D.; Wu, A.; Chan, P.; Cameron, P.; Joynt, G. M.; Ahuja, A.; Yung, M. Y.; Leung, C. B.; To, K. F.; Lui, S. F.; Szeto, C. C.; Chung, S.; Sung, J. J. A major outbreak of severe acute respiratory syndrome in Hong Kong. N. Engl. J. Med. 2003, 348, 1986– 1994, DOI: 10.1056/NEJMoa030685
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A major outbreak of severe acute respiratory syndrome in Hong Kong
Lee Nelson; Hui David; Wu Alan; Chan Paul; Cameron Peter; Joynt Gavin M; Ahuja Anil; Yung Man Yee; Leung C B; To K F; Lui S F; Szeto C C; Chung Sydney; Sung Joseph J Y
The New England journal of medicine (2003), 348 (20), 1986-94 ISSN:.
BACKGROUND: There has been an outbreak of the severe acute respiratory syndrome (SARS) worldwide. We report the clinical, laboratory, and radiologic features of 138 cases of suspected SARS during a hospital outbreak in Hong Kong. METHODS: From March 11 to 25, 2003, all patients with suspected SARS after exposure to an index patient or ward were admitted to the isolation wards of the Prince of Wales Hospital. Their demographic, clinical, laboratory, and radiologic characteristics were analyzed. Clinical end points included the need for intensive care and death. Univariate and multivariate analyses were performed. RESULTS: There were 66 male patients and 72 female patients in this cohort, 69 of whom were health care workers. The most common symptoms included fever (in 100 percent of the patients); chills, rigors, or both (73.2 percent); and myalgia (60.9 percent). Cough and headache were also reported in more than 50 percent of the patients. Other common findings were lymphopenia (in 69.6 percent), thrombocytopenia (44.8 percent), and elevated lactate dehydrogenase and creatine kinase levels (71.0 percent and 32.1 percent, respectively). Peripheral air-space consolidation was commonly observed on thoracic computed tomographic scanning. A total of 32 patients (23.2 percent) were admitted to the intensive care unit; 5 patients died, all of whom had coexisting conditions. In a multivariate analysis, the independent predictors of an adverse outcome were advanced age (odds ratio per decade of life, 1.80; 95 percent confidence interval, 1.16 to 2.81; P=0.009), a high peak lactate dehydrogenase level (odds ratio per 100 U per liter, 2.09; 95 percent confidence interval, 1.28 to 3.42; P=0.003), and an absolute neutrophil count that exceeded the upper limit of the normal range on presentation (odds ratio, 1.60; 95 percent confidence interval, 1.03 to 2.50; P=0.04). CONCLUSIONS: SARS is a serious respiratory illness that led to significant morbidity and mortality in our cohort.
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Booth, C. M.; Matukas, L. M.; Tomlinson, G. A.; Rachlis, A. R.; Rose, D. B.; Dwosh, H. A.; Walmsley, S. L.; Mazzulli, T.; Avendano, M.; Derkach, P.; Ephtimios, I. E.; Kitai, I.; Mederski, B. D.; Shadowitz, S. B.; Gold, W. L.; Hawryluck, L. A.; Rea, E.; Chenkin, J. S.; Cescon, D. W.; Poutanen, S. M.; Detsky, A. S. Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. JAMA 2003, 289, 2801– 2809, DOI: 10.1001/jama.289.21.JOC30885
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Clinical features and short-term outcomes of 144 patients with SARS in the Greater Toronto area
Booth, Christopher M.; Matukas, Larissa M.; Tomlinson, George A.; Rachlis, Anita R.; Rose, David B.; Dwosh, Hy A.; Walmsley, Sharon L.; Mazzulli, Tony; Avendano, Monica; Derkach, Peter; Ephtimios, Issa E.; Kitai, Ian; Mederski, Barbara D.; Shadowitz, Steven B.; Gold, Wayne L.; Hawryluck, Laura A.; Rea, Elizabeth; Chenkin, Jordan S.; Cescon, David W.; Poutanen, Susan M.; Detsky, Allan S.
JAMA, the Journal of the American Medical Association (2003), 289 (21), 2801-2809CODEN: JAMAAP; ISSN:0098-7484. (American Medical Association)
Context: Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in China in Nov. 2002 and has subsequently spread worldwide. Objectives: To describe the clin. characteristics and short-term outcomes of SARS in the first large group of patients in North America; to describe how these patients were treated and the variables assocd. with poor outcome. Design, Setting, and Patients: Retrospective case series involving 144 adult patients admitted to 10 academic and community hospitals in the greater Toronto, Ontario, area between Mar. 7 and Apr. 10, 2003, with a diagnosis of suspected or probable SARS. Patients were included if they had fever, a known exposure to SARS, and respiratory symptoms or infiltrates obsd. on chest radiograph. Patients were excluded if an alternative diagnosis was detd. Main Outcome Measures: Location of exposure to SARS; features of the history, phys. examn., and lab. tests at admission to the hospital; and 21-day outcomes such as death or intensive care unit (ICU) admission with or without mech. ventilation. Results: Of the 144 patients, 111 (77%) were exposed to SARS in the hospital setting. Features of the clin. examn. most commonly found in these patients at admission were self-reported fever (99%), documented elevated temp. (85%), nonproductive cough (69%), myalgia (49%), and dyspnea (42%). Common lab. features included elevated lactate dehydrogenase (87%), hypocalcemia (60%), and lymphopenia (54%). Only 2% of patients had rhinorrhea. A total of 126 patients (88%) were treated with ribavirin, although its use was assocd. with significant toxicity, including hemolysis (in 76%) and decrease in Hb of 2 g/dL (in 49%). Twenty-nine patients (20%) were admitted to the ICU with or without mech. ventilation, and 8 patients died (21-day mortality, 6.5%; 95% confidence interval [CI], 1.9%-11.8%). Multivariable anal. showed that the presence of diabetes (relative risk [RR], 3.1; 95% CI, 1.4-7.2) or other comorbid conditions (RR, 2.5; 95% CI, 1.1-5.8) were independently assocd. with poor outcome (death, ICU admission, or mech. ventilation). Conclusions: The majority of cases in the SARS outbreak in the greater Toronto area were related to hospital exposure. In the event that contact history becomes unreliable, several features of the clin. presentation will be useful in raising the suspicion of SARS. Although SARS is assocd. with significant morbidity and mortality, esp. in patients with diabetes or other comorbid conditions, the vast majority (93.5%) of patients in the authors' cohort survived.
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Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S.; Zaki, S. R.; Peret, T.; Emery, S.; Tong, S.; Urbani, C.; Comer, J. A.; Lim, W.; Rollin, P. E.; Dowell, S. F.; Ling, A. E.; Humphrey, C. D.; Shieh, W. J.; Guarner, J.; Paddock, C. D.; Rota, P.; Fields, B.; DeRisi, J.; Yang, J. Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson, L. J. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 2003, 348, 1953– 1966, DOI: 10.1056/NEJMoa030781
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A novel coronavirus associated with severe acute respiratory syndrome
Ksiazek, Thomas G.; Erdman, Dean; Goldsmith, Cynthia S.; Zaki, Sherif R.; Peret, Teresa; Emery, Shannon; Tong, Suxiang; Urbani, Carlo; Comer, James A.; Lim, Wilina; Rollin, Pierre E.; Dowell, Scott F.; Ling, Ai-Ee; Humphrey, Charles D.; Shieh, Wan-Ju; Guarner, Jeannette; Paddock, Christopher D.; Rota, Paul; Fields, Barry; DeRisi, Joseph; Yang, Jyh-Yuan; Cox, Nancy; Hughes, James M.; LeDuc, James W.; Bellini, William J.; Anderson, Larry J.; Cannon, A. D. L.; Curtis, M.; Farrar, B.; Morgan, L.; Pezzanite, L.; Sanchez, A. J.; Slaughter, K. A.; Stevens, T. L.; Stockton, P. C.; Wagoner, K. D.; Sanchez, A.; Nichol, S.; Vincent, M.; Osborne, J.; Honig, J.; Brickson, B. R.; Holloway, B.; McCaustland, K.; Lingappa, J.; Lowe, L.; Scott, S.; Lu, X.; Villamarzo, Y.; Cook, B.; Chen, Q.; Birge, C.; Shu, B.; Pallansch, M.; Tatti, K. M.; Morken, T.; Smith, C.; Greer, P.; White, E.; McGlothen, T.; Bhatnagar, J.; Patel, M.; Bartlett, J.; Montague, J.; Lee, W.; Packard, M.; Thompson, H. A.; Moen, A.; Fukuda, K.; Uyeki, T.; Harper, S.; Klimov, A.; Lindstrom, S.; Benson, R.; Carlone, G.; Facklam, R.; Fields, P.; Levett, P.; Mayer, L.; Talkington, D.; Thacker, W. L.; Tondella, M. L. C.; Whitney, C.; Robertson, B.; Warnock, D.; Brooks, T.; Schrag, S.; Rosenstein, N.; Arthur, R.; Ganem, D.; Poutanen, S. M.; Chen, T.-J.; Hsiao, C.-H.; Wai-Fu, N. G.; Ho, M.; Keung, T.-K.; Nghiem, K. H.; Nguyen, H. K. L.; Le, M. Q.; Nguyen, H. H. T.; Hoang, L. T.; Vu, T. H.; Vu, H. Q.; Chunsuttiwat, S.
New England Journal of Medicine (2003), 348 (20), 1953-1966CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
A worldwide outbreak of severe acute respiratory syndrome (SARS) was assocd. with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiol. agent of this outbreak. We received clin. specimens from patients in 7 countries and tested them, using virus-isolation techniques, electron-microscopical and histol. studies, and mol. and serol. assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathol. features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examn. revealed ultrastructural features characteristic of coronaviruses. Immunohistochem. and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers the authors identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate were used to demonstrate a virus-specific serol. response. This virus may never before have circulated in the U.S. population. Conclusions: A novel coronavirus is assocd. with this outbreak, and the evidence indicates that this virus has an etiol. role in SARS. Because of the death of Dr. Carlo Urbani, the authors propose that this first isolate be named the Urbani strain of SARS-assocd. coronavirus.
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Snijder, E. J.; Bredenbeek, P. J.; Dobbe, J. C.; Thiel, V.; Ziebuhr, J.; Poon, L. L.; Guan, Y.; Rozanov, M.; Spaan, W. J.; Gorbalenya, A. E. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 2003, 331, 991– 1004, DOI: 10.1016/S0022-2836(03)00865-9
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Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage
Snijder, Eric J.; Bredenbeek, Peter J.; Dobbe, Jessika C.; Thiel, Volker; Ziebuhr, John; Poon, Leo L. M.; Guan, Yi; Rozanov, Mikhail; Spaan, Willy J. M.; Gorbalenya, Alexander E.
Journal of Molecular Biology (2003), 331 (5), 991-1004CODEN: JMOBAK; ISSN:0022-2836. (Elsevier)
The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed exptl. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total no. of SARS-CoV proteins to 28). A phylogenetic anal. of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a no. of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.
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Kuiken, T.; Fouchier, R. A. M.; Schutten, M.; Rimmelzwaan, G. F.; van Amerongen, G.; Van Riel, D.; Laman, J. D.; de Jong, T.; Van Doornum, G.; Lim, W.; Ling, A. E.; Chan, P. K. S.; Tam, J. S.; Zambon, M. C.; Gopal, R.; Drosten, C.; Van der Werf, S.; Escriou, N.; Manuguerra, J. C.; Stohr, K.; Peiris, J. S. M.; Osterhaus, A. D. M. E. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 2003, 362, 263– 270, DOI: 10.1016/S0140-6736(03)13967-0
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Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome
Kuiken, Thijs; Fouchier, Ron A. M.; Schutten, Martin; Rimmelzwaan, Guus F.; van Amerongen, Geert; van Riel, Debby; Laman, Jon D.; de Jong, Ton; van Doornum, Gerard; Lim, Wilina; Ling, Ai Ee; Chan, Paul K. S.; Tam, John S.; Zambon, Maria C.; Gopal, Robin; Drosten, Christian; van der Werf, Sylvie; Escriou, Nicolas; Manuguerra, Jean-Claude; Stohr, Klaus; Peiris, J. S. Malik; Osterhaus, Albert D. M. E.
Lancet (2003), 362 (9380), 263-270CODEN: LANCAO; ISSN:0140-6736. (Elsevier Science Ltd.)
Background: The worldwide outbreak of severe acute respiratory syndrome (SARS) is assocd. with a newly discovered coronavirus, SARS-assocd. coronavirus (SARS-CoV). We did clin. and exptl. studies to assess the role of this virus in the cause of SARS. Methods: We tested clin. and postmortem samples from 436 SARS patients in six countries for infection with SARS-CoV, human metapneumovirus, and other respiratory pathogens. We infected four cynomolgus macaques (Macaca fascicularis) with SARS-CoV in an attempt to replicate SARS and did necropsies on day 6 after infection. Findings: SARS-CoV infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of SARS; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. SARS-CoV was, therefore, the most likely causal agent of SARS. The four SARS-CoV-infected macaques excreted SARS-CoV from nose, mouth, and pharynx from 2 days after infection. Three of four macaques developed diffuse alveolar damage, similar to that in SARS patients, and characterized by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. SARS-CoV was detected in pneumonic areas by virus isolation and RT-PCR, and was localized to alveolar epithelial cells and syncytia by immunohistochem. and TEM. Interpretation: Replication in SARS-CoV-infected macaques of pneumonia similar to that in human beings with SARS, combined with the high prevalence of SARS-CoV infection in SARS patients, fulfill the criteria required to prove that SARS-CoV is the primary cause of SARS.
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Peiris, J. S. M.; Lai, T. L.; Poon, L. M.; Guan, Y.; Yam, L. Y. C.; Lim, W.; Nicholls, J.; Yee, W. K. S.; Yan, W. W.; Cheung, M. T.; Cheng, V. C. C.; Chan, K. H.; Tsang, D. N. C.; Yung, R. W. H.; Ng, T. K.; Yuen, K. Y. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003, 361, 1319– 1325, DOI: 10.1016/S0140-6736(03)13077-2
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Coronavirus as a possible cause of severe acute respiratory syndrome
Peiris J S M; Lai S T; Poon L L M; Guan Y; Yam L Y C; Lim W; Nicholls J; Yee W K S; Yan W W; Cheung M T; Cheng V C C; Chan K H; Tsang D N C; Yung R W H; Ng T K; Yuen K Y
Lancet (London, England) (2003), 361 (9366), 1319-25 ISSN:0140-6736.
BACKGROUND: An outbreak of severe acute respiratory syndrome (SARS) has been reported in Hong Kong. We investigated the viral cause and clinical presentation among 50 patients. METHODS: We analysed case notes and microbiological findings for 50 patients with SARS, representing more than five separate epidemiologically linked transmission clusters. We defined the clinical presentation and risk factors associated with severe disease and investigated the causal agents by chest radiography and laboratory testing of nasopharyngeal aspirates and sera samples. We compared the laboratory findings with those submitted for microbiological investigation of other diseases from patients whose identity was masked. FINDINGS: Patients' age ranged from 23 to 74 years. Fever, chills, myalgia, and cough were the most frequent complaints. When compared with chest radiographic changes, respiratory symptoms and auscultatory findings were disproportionally mild. Patients who were household contacts of other infected people and had older age, lymphopenia, and liver dysfunction were associated with severe disease. A virus belonging to the family Coronaviridae was isolated from two patients. By use of serological and reverse-transcriptase PCR specific for this virus, 45 of 50 patients with SARS, but no controls, had evidence of infection with this virus. INTERPRETATION: A coronavirus was isolated from patients with SARS that might be the primary agent associated with this disease. Serological and molecular tests specific for the virus permitted a definitive laboratory diagnosis to be made and allowed further investigation to define whether other cofactors play a part in disease progression.
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Fleck, F. WHO says SARS outbreak is over, but fight should go on. Br. Med. J. 2003, 327, 70-c, DOI: 10.1136/bmj.327.7406.70-c
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Ashraf, H. WHO declares Beijing to be free of SARS. Lancet 2003, 361, 2212, DOI: 10.1016/S0140-6736(03)13790-7
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WHO declares Beijing to be free of SARS
Ashraf Haroon
Lancet (London, England) (2003), 361 (9376), 2212 ISSN:.
There is no expanded citation for this reference.
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New 'Sars-like' Coronavirus Identified by UK Officials. BBC News, September 24, 2012; http://www.bbc.co.uk/news/health-19698335.
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21
South Korea Declares 'DDe Facto End' to MERS Virus. BBC News, July 28, 2015; http://www.bbc.com/news/world-asia-33684981.
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22
S. Korea Reports 23 New Cases of MERS, Bringing Total to 87. Yonhap News Agency, June 8, 2015.
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23
South Korea MERS Outbreak Began with a Cough; The Wall Street Journal 8 June 2015.
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Rapid Risk Assessment: Severe Respiratory Disease Associated with MiddleEast Respiratory Syndrome Coronavirus (MERS-CoV); European Centre for Disease Prevention and Control, August 28, 2015; http://ecdc.europa.eu/en/publications/Publications/MERS-CoV-rapid-risk-assessment-August-2015.pdf.
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Ghosh, A. K.; Xi, K.; Johnson, M. E.; Baker, S. C.; Mesecar, A. D. Progress in anti-sars coronavirus chemistry, biology and chemotherapy. Annu. Rep. Med. Chem. 2006, 41, 183– 196, DOI: 10.1016/S0065-7743(06)41011-3
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Progress in anti-SARS coronavirus chemistry, biology and chemotherapy
Ghosh, Arun K.; Xi, Kai; Johnson, Michael E.; Baker, Susan C.; Mesecar, Andrew D.
Annual Reports in Medicinal Chemistry (2006), 41 (), 183-196CODEN: ARMCBI; ISSN:0065-7743. (Elsevier)
A review.
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McIntosh, K. Coronaviruses: A comparative review. Curr. Top. Microbiol. Immunol. 1974, 63, 85– 129, DOI: 10.1007/978-3-642-65775-7_3
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Marra, M. A.; Jones, S. J.; Astell, C. R.; Holt, R. A.; Brooks-Wilson, A.; Butterfield, Y. S.; Khattra, J.; Asano, J. K.; Barber, S. A.; Chan, S. Y.; Cloutier, A.; Coughlin, S. M.; Freeman, D.; Girn, N.; Griffith, O. L.; Leach, S. R.; Mayo, M.; McDonald, H.; Montgomery, S. B.; Pandoh, P. K.; Petrescu, A. S.; Robertson, A. G.; Schein, J. E.; Siddiqui, A.; Smailus, D. E.; Stott, J. M.; Yang, G. S.; Plummer, F.; Andonov, A.; Artsob, H.; Bastien, N.; Bernard, K.; Booth, T. F.; Bowness, D.; Czub, M.; Drebot, M.; Fernando, L.; Flick, R.; Garbutt, M.; Gray, M.; Grolla, A.; Jones, S.; Feldmann, H.; Meyers, A.; Kabani, A.; Li, Y.; Normand, S.; Stroher, U.; Tipples, G. A.; Tyler, S.; Vogrig, R.; Ward, D.; Watson, B.; Brunham, R. C.; Krajden, M.; Petric, M.; Skowronski, D. M.; Upton, C.; Roper, R. L. The genome sequence of the SARS-associated coronavirus. Science 2003, 300, 1399– 1404, DOI: 10.1126/science.1085953
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The genome sequence of the SARS-associated coronavirus
Marra, Marco A.; Jones, Steven J. M.; Astell, Caroline R.; Holt, Robert A.; Brooks-Wilson, Angela; Butterfield, Yaron S. N.; Khattra, Jaswinder; Asano, Jennifer K.; Barber, Sarah A.; Chan, Susanna Y.; Cloutier, Alison; Coughlin, Shaun M.; Freeman, Doug; Girn, Noreen; Griffith, Obi L.; Leach, Stephen R.; Mayo, Michael; McDonald, Helen; Montgomery, Stephen B.; Pandoh, Pawan K.; Petrescu, Anca S.; Robertson, A. Gordon; Schein, Jacqueline E.; Siddiqui, Asim; Smailus, Duane E.; Stott, Jeff M.; Yang, George S.; Plummer, Francis; Andonov, Anton; Artsob, Harvey; Bastien, Nathalie; Bernard, Kathy; Booth, Timothy F.; Bowness, Donnie; Czub, Martin; Drebot, Michael; Fernando, Lisa; Flick, Ramon; Garbutt, Michael; Gray, Michael; Grolla, Allen; Jones, Steven; Feldmann, Heinz; Meyers, Adrienne; Kabani, Amin; Li, Yan; Normand, Susan; Stroher, Ute; Tipples, Graham A.; Tyler, Shaun; Vogrig, Robert; Ward, Diane; Watson, Brynn; Brunham, Robert C.; Krajden, Mel; Petric, Martin; Skowronski, Danuta M.; Upton, Chris; Roper, Rachel L.
Science (Washington, DC, United States) (2003), 300 (5624), 1399-1404CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-assocd. coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic anal. of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunol. tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
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Lomniczi, B. J. Biological properties of avian coronavirus RNA. J. Gen. Virol. 1977, 36, 531– 533, DOI: 10.1099/0022-1317-36-3-531
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Biological properties of avian coronavirus RNA
Lomniczi B
The Journal of general virology (1977), 36 (3), 531-3 ISSN:0022-1317.
RNA with a sedimentation coefficient of 64S was isolated from infectious bronchitis virus, an avian coronavirus. The SNA contained a polyadenylic acid tract and was found to be infectious.
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Lee, H. J.; Shieh, C. K.; Gorbalenya, A. E.; Koonin, E. V.; La Monica, N.; Tuler, J.; Bagdzhadzhyan, A.; Lai, M. M. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 1991, 180, 567– 582, DOI: 10.1016/0042-6822(91)90071-I
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The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase
Lee, Han Jung; Shieh, Chien Kou; Gorbalenya, Alexander E.; Koonin, Eugene V.; La Monica, Nicola; Tuler, Jeremy; Bagdzhadzhyan, Anush; Lai, Michael M. C.
Virology (1991), 180 (2), 567-82CODEN: VIRLAX; ISSN:0042-6822.
The 5'-most gene, gene 1, of the genome of murine coronavirus, mouse hepatitis virus (MHV), is presumed to encode the viral RNA-dependent RNA polymerase. The complete sequence of this gene of the JHM strain was detd. by cDNA cloning and sequencing. The total length of this gene is 21,798 nucleotides long, which includes 2 overlapping, large open reading frames. The first open reading frame, ORF 1A, is 4488 amino acids long. The second open reading frame, ORF 1b, overlaps ORF 1a for 75 nucleotides, and is 2731 amino acids long. The overlapping region may fold into a pseudoknot RNA structure, similar to the corresponding region of the RNA of avian coronavirus, infectious bronchitis virus (IBV). The in vitro transcription and translation studies of this region indicated that these 2 ORFs were most likely translated into one polyprotein by a ribosomal frameshifting mechanism. Thus, the predicted mol. wt. of the gene 1 product is more than 800,000 Da. The sequence of ORF 1b is very similar to the corresponding ORF of IBV. In contrast, the ORF 1a of these 2 viruses differ in size and have a high degree of divergence. The amino acid sequence anal. suggested that ORF 1a contains several functional domains, including 2 hydrophobic, membrane-anchoring domains, and 3 cysteine-rich domains. It also contains a picornaviral 3C-like protease domain and 2 papain-like protease domains. The presence of these protease domains suggests that the polyprotein is most likely processed into multiple protein products. In contrast, the ORF 1b contains polymerase, helicase, and zinc-finger motifs. These sequence studies suggested that the MHV gene 1 product is involved in RNA synthesis, and that this product is processed autoproteolytically after translation. This study completes the sequence of the MHV genome, which is 31 kb long, and constitutes the largest viral RNA known.
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Bond, C. W.; Leibowitz, J. L.; Robb, J. A. Pathogenic murine coronaviruses. II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V. Virology 1979, 94, 371– 384, DOI: 10.1016/0042-6822(79)90468-9
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Pathogenic murine coronaviruses. II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V
Bond, Clifford W.; Leibowitz, Julian L.; Robb, James A.
Virology (1979), 94 (2), 371-84CODEN: VIRLAX; ISSN:0042-6822.
Nine intracellular virus-specific proteins in cells infected with JHM or A59 viruses were identified. Seven virus-specific proteins were detected by Na dodecylsulfate polyacrylamide gel electrophoresis and 2 addnl. virus-specific proteins were detected by 2-dimensional gel electrophoresis. The A59V- and JHMV-specific proteins differ slightly in mol. wt. Four of the 9 are structural proteins. Synthesis of the 9 virus-specific proteins is noncoordinate with respect to time.
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Ziebuhr, J.; Heusipp, G.; Siddell, S. G. Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase. J. Virol. 1997, 71, 3992– 3997
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Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase
Ziebuhr, John; Heusipp, Gerhard; Siddell, Stuart G.
Journal of Virology (1997), 71 (5), 3992-3997CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. This report describes the biosynthesis of human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, single amino acid substitutions and C-terminal deletions were introduced into the recombinant protein and the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate was detd.. This approach identified residues Cys-3109 and His-3006 as being indispensable for catalytic activity. The results also support the involvement of His-3127 in substrate recognition, and confirm the requirement of the C-terminal extension found in coronavirus 3C-like proteinases for enzymic activity. These data provide exptl. evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has addnl., unique properties.
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Dougherty, W. G.; Semler, B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol. Rev. 1993, 57, 781– 822
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Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes
Dougherty, William G.; Semler, Bert L.
Microbiological Reviews (1993), 57 (4), 781-822CODEN: MBRED3; ISSN:0146-0749.
A review with 396 refs.
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Ratia, K.; Saikatendu, K. S.; Santarsiero, B. D.; Barretto, N.; Baker, S. C.; Stevens, R. C.; Mesecar, A. D. Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5717– 5722, DOI: 10.1073/pnas.0510851103
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Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme
Ratia, Kiira; Saikatendu, Kumar Singh; Santarsiero, Bernard D.; Barretto, Naina; Baker, Susan C.; Stevens, Raymond C.; Mesecar, Andrew D.
Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (15), 5717-5722CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality assocd. with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes (DUBs). Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitin-like (Ubl) N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin-aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.
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Chen, S.; Chen, L.; Tan, J.; Chen, J.; Du, L.; Sun, T.; Shen, J.; Chen, K.; Jiang, H.; Shen, X. Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization. Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations. J. Biol. Chem. 2005, 280, 164– 173, DOI: 10.1074/jbc.M408211200
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Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization: Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations
Chen, Shuai; Chen, Lili; Tan, Jinzhi; Chen, Jing; Du, Li; Sun, Tao; Shen, Jianhua; Chen, Kaixian; Jiang, Hualiang; Shen, Xu
Journal of Biological Chemistry (2005), 280 (1), 164-173CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quant. characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CLpros by using glutaraldehyde crosslinking SDS-PAGE, size-exclusion chromatog., and isothermal titrn. calorimeter techniques. Glutaraldehyde crosslinking SDS-PAGE and size-exclusion chromatog. results show that similar to the full-length SARS 3CLpro, the N-terminal deleted SARS 3CLpro still remains a dimer/monomer mixt. within a wide range of protein concns. Isothermal titrn. calorimeter detns. indicate that the equil. dissocn. const. (Kd) of the N-terminal deleted proteinase dimer (262 μM) is very similar to that of the full-length proteinase dimer (227 μM). Enzymic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymic activity for SARS 3CLpro. Mol. dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor detn., indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymic activity.
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Huang, C.; Wei, P.; Fan, K.; Liu, Y.; Lai, L. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism. Biochemistry 2004, 43, 4568– 4574, DOI: 10.1021/bi036022q
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3C-like Proteinase from SARS Coronavirus Catalyzes Substrate Hydrolysis by a General Base Mechanism
Huang, Changkang; Wei, Ping; Fan, Keqiang; Liu, Ying; Lai, Luhua
Biochemistry (2004), 43 (15), 4568-4574CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to det. whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be crit. for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pKa's for His41 and Cys145 in the wild-type enzyme were estd. to be 6.38 and 8.34, while the pKa's for His41 and Ser145 in the C145S mutant were estd. to be 6.15 and 9.09, resp. The C145S mutant has a normal isotope effect in D2O for general base catalysis, i.e., reacts slower in D2O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pKa values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.
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Shi, J.; Wei, Z.; Song, J. Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors. J. Biol. Chem. 2004, 279, 24765– 24773, DOI: 10.1074/jbc.M311744200
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Dissection Study on the Severe Acute Respiratory Syndrome 3C-like Protease Reveals the Critical Role of the Extra Domain in Dimerization of the Enzyme: Defining the extra domain as a new target for design of highly specific protease inhibitors
Shi, Jiahai; Wei, Zheng; Song, Jianxing
Journal of Biological Chemistry (2004), 279 (23), 24765-24773CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold contg. the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzyme-substrate interactions by use of the dynamic light scattering, CD, and NMR spectroscopy, we (1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and (2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; (1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and (2) although the chymotrypsin fold remained a monomer within a wide range of protein concns., the extra domain existed as a stable dimer even at a very low concn. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the detn. of the preferred soln. conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.
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Fan, K.; Wei, P.; Feng, Q.; Chen, S.; Huang, C.; Ma, L.; Lai, B.; Pei, J.; Liu, Y.; Chen, J.; Lai, L. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase. J. Biol. Chem. 2004, 279, 1637– 1642, DOI: 10.1074/jbc.M310875200
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Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase
Fan, Keqiang; Wei, Ping; Feng, Qian; Chen, Sidi; Huang, Changkang; Ma, Liang; Lai, Bing; Pei, Jianfeng; Liu, Ying; Chen, Jianguo; Lai, Luhua
Journal of Biological Chemistry (2004), 279 (3), 1637-1642CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixt. of monomer and dimer at a protein concn. of 4 mg/mL and mostly monomer at 0.2 mg/mL, which correspond to the concn. used in the enzyme assays. The linear decrease of the enzymic-specific activity with the decrease of enzyme concn. revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liq. chromatog. assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more β-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.
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Yang, H.; Yang, M.; Ding, Y.; Liu, Y.; Lou, Z.; Zhou, Z.; Sun, L.; Mo, L.; Ye, S.; Pang, H.; Gao, G. F.; Anand, K.; Bartlam, M.; Hilgenfeld, R.; Rao, Z. The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13190– 13195, DOI: 10.1073/pnas.1835675100
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The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor
Yang, Haitao; Yang, Maojun; Ding, Yi; Liu, Yiwei; Lou, Zhiyong; Zhou, Zhe; Sun, Lei; Mo, Lijuan; Ye, Sheng; Pang, Hai; Gao, George F.; Anand, Kanchan; Bartlam, Mark; Hilgenfeld, Rolf; Rao, Zihe
Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (23), 13190-13195CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiol. agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, the authors report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to the authors' knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.
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Hsu, M. F.; Kuo, C. J.; Chang, K. T.; Chang, H. C.; Chou, C. C.; Ko, T. P.; Shr, H. L.; Chang, G. G.; Wang, H.; Liang, P. H. Mechanism of the maturation process of SARS-CoV 3CL protease. J. Biol. Chem. 2005, 280, 31257– 31266, DOI: 10.1074/jbc.M502577200
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Mechanism of the Maturation Process of SARS-CoV 3CL Protease
Hsu, Min-Feng; Kuo, Chih-Jung; Chang, Kai-Ti; Chang, Hui-Chuan; Chou, Chia-Cheng; Ko, Tzu-Ping; Shr, Hui-Lin; Chang, Gu-Gang; Wang, Andrew H.-J.; Liang, Po-Huang
Journal of Biological Chemistry (2005), 280 (35), 31257-31266CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. We report here that the 3CLpro contg. addnl. N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asym. unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln-1-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Anal. ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (Kd = 0.35 nM) than for the mutant (C145A) contg. 10 extra N-terminal (Kd = 17.2 nM) or C-terminal amino acids (Kd = 5.6 nM). The data indicate that immature 3CLpro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CLpro from the polyprotein and design of new structure-based inhibitors.
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Hegyi, A.; Ziebuhr, J. Conservation of substrate specificities among coronavirus main proteases. J. Gen. Virol. 2002, 83, 595– 599, DOI: 10.1099/0022-1317-83-3-595
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40
Conservation of substrate specificities among coronavirus main proteases
Hegyi Annette; Ziebuhr John
The Journal of general virology (2002), 83 (Pt 3), 595-599 ISSN:0022-1317.
The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.
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Grum-Tokars, V.; Ratia, K.; Begaye, A.; Baker, S. C.; Mesecar, A. D. Evaluating the 3C-like protease activity of SARS-coronavirus: Recommendations for standardized assays for drug discovery. Virus Res. 2008, 133, 63– 73, DOI: 10.1016/j.virusres.2007.02.015
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Evaluating the 3C-like protease activity of SARS-Coronavirus: Recommendations for standardized assays for drug discovery
Grum-Tokars, Valerie; Ratia, Kiira; Begaye, Adrian; Baker, Susan C.; Mesecar, Andrew D.
Virus Research (2008), 133 (1), 63-73CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a no. of high resoln. X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymic activity. Moreover, we provide exptl. evidence showing that the activity of 3CLpro enzyme is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concns. below the equil. dissocn. const. of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine anal. and high-throughput screening, and show that kinetic consts. detd. from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.
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Kuo, C. J.; Chi, Y. H.; Hsu, J. T.; Liang, P. H. Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate. Biochem. Biophys. Res. Commun. 2004, 318, 862– 867, DOI: 10.1016/j.bbrc.2004.04.098
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Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate
Kuo, Chih-Jung; Chi, Ya-Hui; Hsu, John T.-A.; Liang, Po-Huang
Biochemical and Biophysical Research Communications (2004), 318 (4), 862-867CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Science)
SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissocd. into inactive monomer and the deduced Kd of the dimer was 100 μM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concn. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepd. a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat=1.9 s-1 and Km=17 μM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissocg. into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.
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Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Coronavirus main proteinase (3CLpro) structure: Basis for design of anti-SARS drugs. Science 2003, 300, 1763– 1767, DOI: 10.1126/science.1085658
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43
Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs
Anand, Kanchan; Ziebuhr, John; Wadhwani, Parvesh; Mesters, Jeroen R.; Hilgenfeld, Rolf
Science (Washington, DC, United States) (2003), 300 (5626), 1763-1767CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. The authors detd. crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and the authors constructed a homol. model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Mol. modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
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Chou, K.; Wei, D.; Zhong, W. Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS. Biochem. Biophys. Res. Commun. 2003, 308, 148– 151, DOI: 10.1016/S0006-291X(03)01342-1
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44
Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS
Chou, Kuo-Chen; Wei, Dong-Qing; Zhong, Wei-Zhu
Biochemical and Biophysical Research Communications (2003), 308 (1), 148-151CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Science)
In order to stimulate the development of drugs against severe acute respiratory syndrome (SARS), based on the at. coordinates of the SARS coronavirus main proteinase detd. recently [Science 13 (May) (2003) (online)], studies of docking KZ7088 (a deriv. of AG7088) and the AVLQSGFR octapeptide to the enzyme were conducted. It has been obsd. that both the above compds. interact with the active site of the SARS enzyme through six hydrogen bonds. Also, a clear definition of the binding pocket for KZ7088 has been presented. These findings may provide a solid basis for subsite anal. and mutagenesis relative to rational design of highly selective inhibitors for therapeutic application. Meanwhile, the idea of how to develop inhibitors of the SARS enzyme based on the knowledge of its own peptide substrates (the so-called "distorted key" approach) was also briefly elucidated.
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Clarke, T. Nature (Science Update): SARS’ Achilles’ Heel Revealed. Science, May 15, 2003; DOI: DOI: 10.1038/news030512-11 .
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There is no corresponding record for this reference.
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Jenwitheesuk, E.; Samudrala, R. Identifying inhibitors of the SARS coronavirus proteinase. Bioorg. Med. Chem. Lett. 2003, 13, 3989– 3992, DOI: 10.1016/j.bmcl.2003.08.066
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Identifying inhibitors of the SARS coronavirus proteinase
Jenwitheesuk, Ekachai; Samudrala, Ram
Bioorganic & Medicinal Chemistry Letters (2003), 13 (22), 3989-3992CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
The Severe Acute Respiratory Syndrome (SARS) is a serious respiratory illness that has recently been reported in parts of Asia and Canada. In this study, we use mol. dynamics (MD) simulations and docking techniques to screen 29 approved and exptl. drugs against the theor. model of the SARS CoV proteinase as well as the exptl. structure of the transmissible gastroenteritis virus (TGEV) proteinase. Our predictions indicate that existing HIV-1 protease inhibitors, L-700,417 for instance, have high binding affinities and may provide good starting points for designing SARS CoV proteinase inhibitors.
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Mukherjee, P.; Desai, P.; Ross, L.; White, E. L.; Avery, M. A. Structure-based virtual screening against SARS-3CL(pro) to identify novel non-peptidic hits. Bioorg. Med. Chem. 2008, 16, 4138– 4149, DOI: 10.1016/j.bmc.2008.01.011
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47
Structure-based virtual screening against SARS-3CLpro to identify novel non-peptidic hits
Mukherjee, Prasenjit; Desai, Prashant; Ross, Larry; White, E. Lucile; Avery, Mitchell A.
Bioorganic & Medicinal Chemistry (2008), 16 (7), 4138-4149CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
Severe acute respiratory syndrome is a highly infectious upper respiratory tract disease caused by SARS-CoV, a previously unidentified human coronavirus. SARS-3CLpro is a viral cysteine protease crit. to the pathogen's life cycle and hence a therapeutic target of importance. The recently elucidated crystal structures of this enzyme provide an opportunity for the discovery of inhibitors through rational drug design. In the current study, Gold docking program was utilized to conduct extensive docking studies against the target crystal structure to develop a robust and predictive docking protocol. The validated docking protocol was used to conduct a structure-based virtual screening of the Asinex Platinum collection. Biol. evaluation of a screened selection of compds. was carried out to identify novel inhibitors of the viral protease.
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Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota, P. A.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D. Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors. J. Med. Chem. 2005, 48, 6767– 6771, DOI: 10.1021/jm050548m
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48
Design and Synthesis of Peptidomimetic Severe Acute Respiratory Syndrome Chymotrypsin-like Protease Inhibitors
Ghosh, Arun K.; Xi, Kai; Ratia, Kiira; Santarsiero, Bernard D.; Fu, Wentao; Harcourt, Brian H.; Rota, Paul A.; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D.
Journal of Medicinal Chemistry (2005), 48 (22), 6767-6771CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Design, synthesis, and biol. evaluation of peptidomimetics as inhibitors for severe acute respiratory syndrome chymotrypsin-like protease (SARS-3CLpro) for SARS coronavirus are described. These inhibitors exhibited antiviral activity against SARS coronavirus in infected cells in the micromolar range. An x-ray crystal structure of the lead inhibitor peptidomimetic I bound to SARS-3CLpro provided important drug-design templates for the design of small-mol. inhibitors.
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Bone, R.; Vacca, J. P.; Anderson, P. S.; Holloway, M. K. X-ray crystal structure of the hiv protease complex with l-700,417, an inhibitor with pseudo C2 symmetry. J. Am. Chem. Soc. 1991, 113, 9382– 9384, DOI: 10.1021/ja00024a061
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X-ray crystal structure of the HIV protease complex with L-700,417, an inhibitor with pseudo C2 symmetry
Bone, Roger; Vacca, Joseph P.; Anderson, Paul S.; Holloway, M. Katharine
Journal of the American Chemical Society (1991), 113 (24), 9382-4CODEN: JACSAT; ISSN:0002-7863.
The structure of the complex formed between the human immunodeficiency virus type-1 protease and L-700,417 (I) was detd. by x-ray crystallog. to a resoln. of 2.1 Å. The inhibitor is approx. C2 sym. about the central 4-hydroxyl group, yet cocrystallizes with the protease in a space group in which the protease dimer is the asym. unit of the crystal (P21212). The central hydroxyl group of the inhibitor interacts with Asp 25 from each subunit of the dimer, the Ph rings pack into the P1/P1' pockets and the novel indanolamide groups straddle the carbonyl oxygens of Gly 27 on each subunit while projecting into the P2/P2' pockets. Electron d. maps do not distinguish between alternate orientations of the inhibitor (rotation of the inhibitor by 180° approx. about the central hydroxyl group) with respect to the asym. active site. The inhibitory complex is stabilized by 8 intermol. hydrogen bonds, by removal of hydrophobic surface area from solvent (792 Å2) and by interactions with the protein though 3 waters trapped between the inhibitor and the protease.
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Shie, J.-J.; Fang, J.-M.; Kuo, T.-H.; Kuo, C.-J.; Liang, P.-H.; Huang, H.-J.; Wu, Y.-T.; Jan, J.-T.; Cheng, Y.-S. E.; Wong, C.-H. Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha, beta-unsaturated esters. Bioorg. Med. Chem. 2005, 13, 5240– 5252, DOI: 10.1016/j.bmc.2005.05.065
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50
Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters
Shie, Jiun-Jie; Fang, Jim-Min; Kuo, Tun-Hsun; Kuo, Chih-Jung; Liang, Po-Huang; Huang, Hung-Jyun; Wu, Yin-Ta; Jan, Jia-Tsrong; Cheng, Yih-Shyun E.; Wong, Chi-Huey
Bioorganic & Medicinal Chemistry (2005), 13 (17), 5240-5252CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide α,β-unsatd. esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 μM), the ketomethylene isosteres and tripeptide α,β-unsatd. esters contg. both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 μM). The Phe-Phe dipeptide inhibitors are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor (I) with an inhibition const. of 0.52 μM is obtained by condensation of the Phe-Phe dipeptide α,β-unsatd. ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that I is a nontoxic anti-SARS agent with an EC50 value of 0.18 μM.
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Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota, P. A.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D. Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors. J. Med. Chem. 2005, 48, 6767– 6770, DOI: 10.1021/jm050548m
[ACS Full Text ], [CAS], Google Scholar
51
Design and Synthesis of Peptidomimetic Severe Acute Respiratory Syndrome Chymotrypsin-like Protease Inhibitors
Ghosh, Arun K.; Xi, Kai; Ratia, Kiira; Santarsiero, Bernard D.; Fu, Wentao; Harcourt, Brian H.; Rota, Paul A.; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D.
Journal of Medicinal Chemistry (2005), 48 (22), 6767-6771CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Design, synthesis, and biol. evaluation of peptidomimetics as inhibitors for severe acute respiratory syndrome chymotrypsin-like protease (SARS-3CLpro) for SARS coronavirus are described. These inhibitors exhibited antiviral activity against SARS coronavirus in infected cells in the micromolar range. An x-ray crystal structure of the lead inhibitor peptidomimetic I bound to SARS-3CLpro provided important drug-design templates for the design of small-mol. inhibitors.
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Ghosh, A. K.; Xi, K.; Grum-Tokars, V.; Xu, X.; Ratia, K.; Fu, W.; Houser, K. V.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D. Structure-based design, synthesis, and biological evaluation of peptidomimetic SARS-CoV 3CL^pro inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 5876– 5880, DOI: 10.1016/j.bmcl.2007.08.031
[Crossref], [PubMed], [CAS], Google Scholar
52
Structure-based design, synthesis, and biological evaluation of peptidomimetic SARS-CoV 3CLpro inhibitors
Ghosh, Arun K.; Xi, Kai; Grum-Tokars, Valerie; Xu, Xiaoming; Ratia, Kiira; Fu, Wentao; Houser, Katherine V.; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D.
Bioorganic & Medicinal Chemistry Letters (2007), 17 (21), 5876-5880CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
Structure-based design, synthesis, and biol. evaluation of a series of peptidomimetic severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors are described. These inhibitors were designed and synthesized based upon our X-ray crystal structure of inhibitor (I) (Boc = tert-butoxycarbonyl) bound to SARS-CoV 3CLpro. Incorporation of Boc-Ser as the P4-ligand resulted in enhanced SARS-CoV 3CLpro inhibitory activity. Structural anal. of the inhibitor-bound X-ray structure revealed high binding affinity toward the enzyme.
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Yang, S.; Chen, S.- J.; Hsu, M.-F.; Wu, J.-D.; Tseng, C.-T. K.; Liu, Y.-F.; Chen, H.-C.; Kuo, C.-W.; Wu, C.-S.; Chang, L.-W.; Chen, W.-C.; Liao, S.-Y.; Chang, T.-Y.; Hung, H.-H.; Shr, H.-L.; Liu, C.-Y.; Huang, Y.-A.; Chang, L.-Y.; Hsu, J.-C.; Peters, C. J.; Wang, A. H.-J.; Hsu, M.-C. Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor. J. Med. Chem. 2006, 49, 4971– 4980, DOI: 10.1021/jm0603926
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Synthesis, Crystal Structure, Structure-Activity Relationships, and Antiviral Activity of a Potent SARS Coronavirus 3CL Protease Inhibitor
Yang, Syaulan; Chen, Shu-Jen; Hsu, Min-Feng; Wu, Jen-Dar; Tseng, Chien-Te K.; Liu, Yu-Fan; Chen, Hua-Chien; Kuo, Chun-Wei; Wu, Chi-Shen; Chang, Li-Wen; Chen, Wen-Chang; Liao, Shao-Ying; Chang, Teng-Yuan; Hung, Hsin-Hui; Shr, Hui-Lin; Liu, Cheng-Yuan; Huang, Yu-An; Chang, Ling-Yin; Hsu, Jen-Chi; Peters, Clarence J.; Wang, Andrew H.-J.; Hsu, Ming-Chu
Journal of Medicinal Chemistry (2006), 49 (16), 4971-4980CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 μM) for SARS CoV and 5.2 log (at 1.25 μM) for HCoV 229E. The crystal structure of TG-0205221 (resoln. = 1.93 Å) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.
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Jain, R. P.; Pettersson, H. I.; Zhang, J.; Aull, K. D.; Fortin, P. D.; Huitema, C.; Eltis, L. D.; Parrish, J. C.; James, M. N. G.; Wishart, D. S.; Vederas, J. C. Synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome 3CLpro. J. Med. Chem. 2004, 47, 6113– 6116, DOI: 10.1021/jm0494873
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Synthesis and Evaluation of Keto-Glutamine Analogues as Potent Inhibitors of Severe Acute Respiratory Syndrome 3CLpro
Jain, Rajendra P.; Pettersson, Hanna I.; Zhang, Jianmin; Aull, Katherine D.; Fortin, Pascal D.; Huitema, Carly; Eltis, Lindsay D.; Parrish, Jonathan C.; James, Michael N. G.; Wishart, David S.; Vederas, John C.
Journal of Medicinal Chemistry (2004), 47 (25), 6113-6116CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome (SARS) coronavirus is a key target for structure-based drug design against this viral infection. The enzyme recognizes peptide substrates with a glutamine residue at the P1 site. A series of keto-glutamine analogs with a phthalhydrazido group at the α-position were synthesized and tested as reversible inhibitors against SARS 3CLpro. Attachment of tripeptide (Ac-Val-Thr-Leu) to these glutamine-based "warheads" generated significantly better inhibitors peptides I and II (R1 = R2 = H, R3 = CH2Ph; R1 = R2 = R3 = H; R1 = H, R2 = NO2, R3 = CH2Ph; R1 = R3 = H, R2 = NO2) with IC50 values ranging from 0.60 to 70 μM.
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Jain, R. P.; Vederas, J. C. Structural variations in keto-glutamines for improved inhibition against hepatitis A virus 3C proteinase. Bioorg. Med. Chem. Lett. 2004, 14, 3655– 3658, DOI: 10.1016/j.bmcl.2004.05.021
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Structural variations in keto-glutamines for improved inhibition against hepatitis A virus 3C proteinase
Jain, Rajendra P.; Vederas, John C.
Bioorganic & Medicinal Chemistry Letters (2004), 14 (14), 3655-3658CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
A series of keto-glutamine tetrapeptide analogs contg. a 2-oxo-pyrrolidine ring as a glutamine side chain mimic were synthesized with both R and S configuration at the β-carbon. Compds. bearing a phthalhydrazide moiety show improved reversible inhibition of hepatitis A virus 3C proteinase in the low micromolar range.
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Ramtohul, Y. K.; James, M. N. G.; Vederas, J. C. Synthesis and evaluation of keto-glutamine analogues as inhibitors of hepatitis A virus 3C proteinase. J. Org. Chem. 2002, 67, 3169– 3178, DOI: 10.1021/jo0157831
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Synthesis and Evaluation of Keto-Glutamine Analogues as Inhibitors of Hepatitis A Virus 3C Proteinase
Ramtohul, Yeeman K.; James, Michael N. G.; Vederas, John C.
Journal of Organic Chemistry (2002), 67 (10), 3169-3178CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
Hepatitis A virus (HAV) 3C enzyme is a picornaviral cysteine proteinase involved in the processing of the initially synthesized viral polyprotein and is therefore important for viral maturation and infectivity. Although it is a cysteine proteinase, this enzyme has a topol. similar to those of the chymotrypsin-like serine proteinases. Since the enzyme recognizes peptide substrates with a glutamine residue at the P1 site, a no. of ketone-contg. glutamine compds. analogous to nanomolar inhibitors of cathepsin K were synthesized and tested for inhibition against HAV 3C proteinase. For example, an azetidinone scaffold was incorporated into the glutamine fragment, as in azetidinone I, but this resulted in only modest inhibition. However, introduction of a phthalhydrazido group α to the ketone moiety, i.e., phthalhydrazides II (R = Boc, R1 = R2 = H; R = Boc, R1 = H, R2 = NO2; R = Boc, R1 = NO2, R2 = H) produced significantly better inhibitors with IC50 values ranging from 13 to 164 μM, presumably due to the effect of intramol. hydrogen bonding to the ketone. In addn., the tetrapeptide phthalhydrazide II (R = MeCO-Leu-Ala-Ala, R1 = R2 = H) was found to be a competitive reversible inhibitor (Ki = 9 × 10-6 M) and it showed no loss of inhibitory potency in the presence of dithiothreitol.
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Yin, J.; Niu, C.; Cherney, M. M.; Zhang, J.; Huitema, C.; Eltis, L. D.; Vederas, J. C.; James, M. N. G. A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase. J. Mol. Biol. 2007, 371, 1060– 1074, DOI: 10.1016/j.jmb.2007.06.001
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A Mechanistic View of Enzyme Inhibition and Peptide Hydrolysis in the Active Site of the SARS-CoV 3C-like Peptidase
Yin, Jiang; Niu, Chunying; Cherney, Maia M.; Zhang, Jianmin; Huitema, Carly; Eltis, Lindsay D.; Vederas, John C.; James, Michael N. G.
Journal of Molecular Biology (2007), 371 (4), 1060-1074CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
The 3C-like main peptidase 3CLpro is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CLpro and the structurally related 3Cpro viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CLpro in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallog. data are consistent with a scenario in which a water mol., possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.
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Shie, J. J.; Fang, J.-M.; Kuo, C.-J.; Kuo, T.-H.; Liang, P.-H.; Huang, H.-J.; Yang, W.-B.; Lin, C.-H.; Chen, J.-L.; Wu, Y.-T.; Wong, C.-H. Discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3CL protease. J. Med. Chem. 2005, 48, 4469– 4473, DOI: 10.1021/jm050184y
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Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory Syndrome 3CL Protease
Shie, Jiun-Jie; Fang, Jim-Min; Kuo, Chih-Jung; Kuo, Tun-Hsun; Liang, Po-Huang; Huang, Hung-Jyun; Yang, Wen-Bin; Lin, Chun-Hung; Chen, Jiun-Ling; Wu, Yin-Ta; Wong, Chi-Huey
Journal of Medicinal Chemistry (2005), 48 (13), 4469-4473CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
A diversified library of peptide anilides was prepd., and the inhibition activity of these compds. against the SARS-CoV (severe acute respiratory syndrome coronavirus) 3CL protease was examd. by a fluorogenic tetradecapeptide substrate. The most potent inhibitor is an anilide I, derived from 2-chloro-4-nitroaniline, L-phenylalanine and 4-(dimethylamino)benzoic acid. I is a competitive inhibitor of SARS-CoV 3CL protease with Ki = 0.03 μM. The mol. docking expt. indicates that the P1 residue of I is distant from the nucleophilic SH of Cys145 in the active site.
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Zhang, R.; Malcolm, B. A.; Beyer, B. M.; Njoroge, F. G.; Durkin, J. P.; Windsor, W. T. Peptide Substrates for Hepatitis C Virus NS3 Protease Assays. U.S. Patent, US 6,251,583 B1, 2001; 21 pp.
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Asgian, J. L.; James, K. E.; Li, Z. Z.; Carter, W.; Barrett, A. J.; Mikolajczyk, J.; Salvesen, G. S.; Powers, J. C. Aza-peptide epoxides: A new class of inhibitors selective for clan CD cysteine proteases. J. Med. Chem. 2002, 45, 4958– 4960, DOI: 10.1021/jm025581c
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Aza-Peptide Epoxides: A New Class of Inhibitors Selective for Clan CD Cysteine Proteases
Asgian, Juliana L.; James, Karen Ellis; Li, Zhao Zhao; Carter, Wendy; Barrett, Alan J.; Mikolajczyk, Jowita; Salvesen, Guy S.; Powers, James C.
Journal of Medicinal Chemistry (2002), 45 (23), 4958-4960CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Aza-peptide epoxides, a new class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. The inhibitors have second-order rate consts. ≤105 M-1 s-1, with the most potent epoxides having the S,S stereochem. The aza-Asn derivs. are effective legumain inhibitors, while the aza-Asp epoxides were specific for caspases. The inhibitors have little or no inhibition with other proteases such as chymotrypsin, papain, or cathepsin B.
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Lee, T. W.; Cherney, M. M.; Huitema, C.; Liu, J.; James, K. E.; Powers, J. C.; Eltis, L. D.; James, M. N. G. Crystal structures of the main peptidase from the SARS coronavirus inhibited by a substrate-like aza-peptide epoxide. J. Mol. Biol. 2005, 353, 1137– 1151, DOI: 10.1016/j.jmb.2005.09.004
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61
Crystal Structures of the Main Peptidase from the SARS Coronavirus Inhibited by a Substrate-like Aza-peptide Epoxide
Lee, Ting-Wai; Cherney, Maia M.; Huitema, Carly; Liu, Jie; James, Karen Ellis; Powers, James C.; Eltis, Lindsay D.; James, Michael N. G.
Journal of Molecular Biology (2005), 353 (5), 1137-1151CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
The main peptidase (Mpro) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive mol. targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV Mpro by an aza-peptide epoxide (APE; kinact/Ki=1900(±400) M-1 s-1). The crystal structures of the Mpro:APE complex in the space groups C2 and P212121 revealed the formation of a covalent bond between the catalytic Cys145 Sγ atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of Mpro in a substrate-like manner, with excellent structural and chem. complementarity. In addn., the crystal structure of unbound Mpro in the space group C2 revealed that the "N-fingers" (N-terminal residues 1 to 7) of both protomers of Mpro are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystn. pH of 6.5, contrary to the previously detd. crystal structures of unbound Mpro in the space group P21.
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Lee, T. W.; Cherney, M. M.; Liu, J.; James, K. E.; Powers, J. C.; Eltis, L. D.; James, M. N. G. Crystal structures reveal an induced-fit binding of a substrate-like aza-peptide epoxide to SARS coronavirus main peptidase. J. Mol. Biol. 2007, 366, 916– 932, DOI: 10.1016/j.jmb.2006.11.078
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Crystal Structures Reveal an Induced-fit Binding of a Substrate-like Aza-peptide Epoxide to SARS Coronavirus Main Peptidase
Lee, Ting-Wai; Cherney, Maia M.; Liu, Jie; James, Karen Ellis; Powers, James C.; Eltis, Lindsay D.; James, Michael N. G.
Journal of Molecular Biology (2007), 366 (3), 916-932CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
The SARS coronavirus main peptidase (SARS-CoV Mpro) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of Mpro at a resoln. of 1.82 Å, in space group P21 at pH 6.0. In contrast to the previously reported structure of Mpro in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of Mpro reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of Mpro having an addnl. Ala at the N terminus of each protomer (Mpro+A(-1)), both at a resoln. of 2.00 Å, in space group P43212: One unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of Mpro+A(-1) are obsd. in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The obsd. conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of Mpro exist in dynamic equil. The structural data further suggest that the binding of APE to Mpro follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.
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Martina, E.; Stiefl, N.; Degel, B.; Schulz, F.; Breuning, A.; Schiller, M.; Vicik, R.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor. Bioorg. Med. Chem. Lett. 2005, 15, 5365– 5369, DOI: 10.1016/j.bmcl.2005.09.012
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63
Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor
Martina, Erika; Stiefl, Nikolaus; Degel, Bjoern; Schulz, Franziska; Breuning, Alexander; Schiller, Markus; Vicik, Radim; Baumann, Knut; Ziebuhr, John; Schirmeister, Tanja
Bioorganic & Medicinal Chemistry Letters (2005), 15 (24), 5365-5369CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
The coronavirus main protease, Mpro, is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compds. were performed to identify potential Mpro inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-contg. peptide was found to be the most potent inhibitor.
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Al-Gharabli, S. I.; Shah, S. T.; Weik, S.; Schmidt, M. F.; Mesters, J. R.; Kuhn, D.; Klebe, G.; Hilgenfeld, R.; Rademann, J. An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors. ChemBioChem 2006, 7, 1048– 1055, DOI: 10.1002/cbic.200500533
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An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors
Al-Gharabli, Samer I.; Shah, Syed T. Ali; Weik, Steffen; Schmidt, Marco F.; Mesters, Jeroen R.; Kuhn, Daniel; Klebe, Gerhard; Hilgenfeld, Rolf; Rademann, Joerg
ChemBioChem (2006), 7 (7), 1048-1055CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidn. of amino alcs. with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV Mpro (also known as 3CLpro), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepd. by this method and investigated in inhibition assays against SARS-CoV Mpro. Several potent inhibitors were found with IC50 values in the low micromolar range. An IC50 of 7.5μM was found for Ac-NSTSQ-H and Ac-ESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV Mpro in a noncanonical binding mode.
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Zhu, L.; George, S.; Schmidt, M. F.; Al-Gharabli, S. I.; Rademann, J.; Hilgenfeld, R. Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease. Antiviral Res. 2011, 92, 204– 212, DOI: 10.1016/j.antiviral.2011.08.001
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Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease
Zhu, Lili; George, Shyla; Schmidt, Marco F.; Al-Gharabli, Samer I.; Rademann, Joerg; Hilgenfeld, Rolf
Antiviral Research (2011), 92 (2), 204-212CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
SARS coronavirus main protease (SARS-CoV Mpro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV Mpro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV Mpro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We detd. four crystal structures of SARS-CoV Mpro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV Mpro, with Ki values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also detd. a crystal structure of SARS-CoV Mpro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV Mpro, with Ki = 2.24 ± 0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV Mpro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the obsd. cleavage specificity of the target protease.
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Akaji, K.; Konno, H.; Onozuka, M.; Makino, A.; Saito, H.; Nosaka, K. Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant. Bioorg. Med. Chem. 2008, 16, 9400– 9408, DOI: 10.1016/j.bmc.2008.09.057
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Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant
Akaji, Kenichi; Konno, Hiroyuki; Onozuka, Mari; Makino, Ayumi; Saito, Hiroyuki; Nosaka, Kazuto
Bioorganic & Medicinal Chemistry (2008), 16 (21), 9400-9408CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degrdn. at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K m of 33.8 μM and k cat of 4753 s-1. Compared with the value reported for the mature protease contg. a C-terminal His-tag, the relative activity of the mutant was nearly 106. Novel peptide-aldehyde derivs. contg. a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivs.
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Akaji, K.; Konno, H.; Mitsui, H.; Teruya, K.; Shimamoto, Y.; Hattori, Y.; Ozaki, T.; Kusunoki, M.; Sanjoh, A. Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors. J. Med. Chem. 2011, 54, 7962– 73, DOI: 10.1021/jm200870n
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Structure-Based Design, Synthesis, and Evaluation of Peptide-Mimetic SARS 3CL Protease Inhibitors
Akaji, Kenichi; Konno, Hiroyuki; Mitsui, Hironori; Teruya, Kenta; Shimamoto, Yasuhiro; Hattori, Yasunao; Ozaki, Takeshi; Kusunoki, Masami; Sanjoh, Akira
Journal of Medicinal Chemistry (2011), 54 (23), 7962-7973CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The design and evaluation of low mol. wt. peptide-based severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL) protease inhibitors are described. A substrate-based peptide aldehyde was selected as a starting compd., and optimum side-chain structures were detd., based on a comparison of inhibitory activities with Michael type inhibitors. For the efficient screening of peptide aldehydes contg. a specific C-terminal residue, a new approach employing thioacetal to aldehyde conversion mediated by N-bromosuccinimide was devised. Structural optimization was carried out based on x-ray crystallog. analyses of the R188I SARS 3CL protease in a complex with each inhibitor to provide a tetrapeptide aldehyde with an IC50 = 98 nM. The resulting compd. carried no substrate sequence, except for a P3 site directed toward the outside of the protease. X-ray crystallog. provided insights into the protein-ligand interactions.
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Zhang, H.- Z.; Zhang, H.; Kemnitzer, W.; Tseng, B.; Cinatl, J., Jr.; Michaelis, M.; Doerr, H. W.; Cai, S. X. Design and synthesis of dipeptidyl glutaminyl fluoromethyl ketones as potent severe acute respiratory syndrome coronavirus (SARS-CoV) inhibitors. J. Med. Chem. 2006, 49, 1198– 1201, DOI: 10.1021/jm0507678
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68
Design and Synthesis of Dipeptidyl Glutaminyl Fluoromethyl Ketones as Potent Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Inhibitors
Zhang, Han-Zhong; Zhang, Hong; Kemnitzer, William; Tseng, Ben; Cinatl, Jindrich, Jr.; Michaelis, Martin; Doerr, Hans Wilhelm; Cai, Sui Xiong
Journal of Medicinal Chemistry (2006), 49 (3), 1198-1201CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
This paper describes the design and synthesis of dipeptidyl N,N-di-Me glutaminyl fluoromethyl ketones (fmk) as severe acute respiratory syndrome coronavirus (SARS-CoV) inhibitors. The compds. were tested against SARS-CoV-induced cell death in Vero or CaCo2 cells as a measurement of the inhibiting effects of the compds. on the replication of the virus. PhCH2O2C-Leu-Gln(NMe2)-fmk was found to be a potent inhibitor with low toxicity in cells, protecting cells with an EC50 value of 2.5 μM and exhibiting a selectivity index of >40.
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69
Yang, W.; Guastella, J.; Huang, J.-C.; Wang, Y.; Zhang, L.; Xue, D.; Tran, M.; Woodward, R.; Kasibhatla, S.; Tseng, B.; Drewe, J.; Cai, S. X. MX1013, a dipeptide caspase inhibitor with potent in vivo antiapoptotic activity. Br. J. Pharmacol. 2003, 140, 402– 412, DOI: 10.1038/sj.bjp.0705450
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69
MX1013, a dipeptide caspase inhibitor with potent in vivo antiapoptotic activity
Yang, Wu; Guastella, John; Huang, Jin-Cheng; Wang, Yan; Zhang, Li; Xue, Dong; Tran, Minhtam; Woodward, Richard; Kasibhatla, Shailaja; Tseng, Ben; Drewe, John; Cai, Sui Xiong
British Journal of Pharmacology (2003), 140 (2), 402-412CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
Caspases play a crit. role in apoptosis, and are considered to be key targets for the design of cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor. MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC50 values ranging from 5 to 20 nM. MX1013 is selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I, or Factor Xa (IC50 values > 10 μM). In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and DNA fragmentation, MX1013 is more active than tetrapeptide- and tripeptide-based caspase inhibitors, and blocked apoptosis at concns. as low as 0.5 μM. MX1013 is more aq. sol. than tripeptide-based caspase inhibitors such as Z-VAD-fmk. At a dose of 1 mg kg-1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure. At a dose of 20 mg kg-1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical damage by approx. 50% in a model of brain ischemia/reperfusion injury. At a dose of 20 mg kg-1 (i.v. bolus) followed by i.v. infusion for 12h, MX1013 reduced heart damage by approx. 50% in a model of acute myocardial infarction. Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of apoptotic insults, and is systemically active in three animal models of apoptosis, including brain ischemia.
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70
Wang, Y.; Huang, J.-C.; Zhou, Z.-L.; Yang, W.; Guastella, J.; Drewe, J.; Cai, S. X. Dipeptidyl aspartyl fluoromethylketones as potent caspase-3 inhibitors: SAR of the P2 amino acid. Bioorg. Med. Chem. Lett. 2004, 14, 1269– 1272, DOI: 10.1016/j.bmcl.2003.12.065
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70
Dipeptidyl aspartyl fluoromethylketones as potent caspase-3 inhibitors: SAR of the P2 amino acid
Wang, Yan; Huang, Jin-Chen; Zhou, Zhang-lin; Yang, Wu; Guastella, John; Drewe, John; Cai, Sui Xiong
Bioorganic & Medicinal Chemistry Letters (2004), 14 (5), 1269-1272CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
This work describes the synthesis and biol. evaluation of a series of dipeptidyl aspartyl fluoromethylketones as caspase-3 inhibitors. Structure-activity relationship (SAR) studies showed that valine is the best P2 amino acid for caspase-3 inhibition. The SAR studies also showed that aspartyl free carboxylic acid in P1 is important for caspase-inhibiting activities, as well as for selectivity over other proteases.
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71
Begue, J.-P.; Bonnet-Delpon, D. Preparation of trifluoromethyl ketones and related fluorinated ketones. Tetrahedron 1991, 47, 3207– 3258, DOI: 10.1016/S0040-4020(01)86391-2
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71
Preparation of trifluoromethyl ketones and related fluorinated ketones
Begue, Jean Pierre; Bonnet-Delpon, Daniele
Tetrahedron (1991), 47 (20-21), 3207-58CODEN: TETRAB; ISSN:0040-4020.
A review with 245 refs. on the synthesis and properties of trifluoromethyl, hemiperfluoroalkyl, α,α-difluoro, and α-functionalized fluoromethyl ketones.
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Gelb, M. H.; Svaren, J. P.; Abeles, R. H. Fluoro ketone inhibitors of hydrolytic enzymes. Biochemistry 1985, 24, 1813– 1817, DOI: 10.1021/bi00329a001
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72
Fluoro ketone inhibitors of hydrolytic enzymes
Gelb, Michael H.; Svaren, John P.; Abeles, Robert H.
Biochemistry (1985), 24 (8), 1813-17CODEN: BICHAW; ISSN:0006-2960.
The use of fluoro ketones as inhibitors of hydrolytic enzymes was investigated. The acetylcholine analogs 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone were inhibitors of acetylcholinesterase with Ki values of 16 × 10-9 and 1.6 × 10-9M, resp. These fluoro ketones were 104-105-fold better as inhibitors than the corresponding Me ketones. Since nucleophiles readily add to fluoro ketones, it is likely that these compds. inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogs were also inhibitors of Zn-metalloproteases and aspartic proteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid was an inhibitor of carboxypeptidase A (Ki = 2 × 10-7M). Trifluoromethyl ketone dipeptide analogs were good inhibitors of angiotensin-converting enzyme. An analog of pepstatin that contains a difluorostatone residue in place of statine was prepd. and found to be an extremely potent inhibitor of pepsin (Ki = 6 × 10-11M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.
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73
Sydnes, M. O.; Hayashi, Y.; Sharma, V. K.; Hamada, T.; Bacha, U.; Barrila, J.; Freire, E.; Kiso, Y. Synthesis of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group as SARS-CoV 3CL protease inhibitors. Tetrahedron 2006, 62, 8601– 8609, DOI: 10.1016/j.tet.2006.06.052
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73
Synthesis of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group as SARS-CoV 3CL protease inhibitors
Sydnes, Magne O.; Hayashi, Yoshio; Sharma, Vinay K.; Hamada, Takashi; Bacha, Usman; Barrila, Jennifer; Freire, Ernesto; Kiso, Yoshiaki
Tetrahedron (2006), 62 (36), 8601-8609CODEN: TETRAB; ISSN:0040-4020. (Elsevier B.V.)
Trifluoromethyl-β-amino alc. I was synthesized in five steps starting from Cbz-L-Glu-OH where the key step involved the introduction of the trifluoromethyl (CF3) group to oxazolidinone II (Cbz = benzyloxycarbonyl), resulting in the formation of silyl ether III. Compd. I was then converted into four tri- and tetraglutamic acid and glutamine peptides possessing a CF3-ketone group that exhibited inhibitory activity against severe acute respiratory syndrome coronavirus protease (SARS-CoV 3CLpro).
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74
Regnier, T.; Sarma, D.; Hidaka, K.; Bacha, U.; Freire, E.; Hayashi, Y.; Kiso, Y. New developments for the design, synthesis and biological evaluation of potent SARS-CoV 3CL(pro) inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 2722– 2727, DOI: 10.1016/j.bmcl.2009.03.118
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74
New developments for the design, synthesis and biological evaluation of potent SARS-CoV 3CLpro inhibitors
Regnier, Thomas; Sarma, Diganta; Hidaka, Koushi; Bacha, Usman; Freire, Ernesto; Hayashi, Yoshio; Kiso, Yoshiaki
Bioorganic & Medicinal Chemistry Letters (2009), 19 (10), 2722-2727CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
A series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-contg. peptidic compds. as SARS-CoV 3CLpro protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Three candidates had encouraging results for the development of new anti-SARS compds.
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75
Konno, S.; Thanigaimalai, P.; Yamamoto, T.; Nakada, K.; Kakiuchi, R.; Takayama, K.; Yamazaki, Y.; Yakushiji, F.; Akaji, K.; Kiso, Y.; Kawasaki, Y.; Chen, S. E.; Freire, E.; Hayashi, Y. Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety. Bioorg. Med. Chem. 2013, 21, 412– 424, DOI: 10.1016/j.bmc.2012.11.017
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75
Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety
Konno, Sho; Thanigaimalai, Pillaiyar; Yamamoto, Takehito; Nakada, Kiyohiko; Kakiuchi, Rie; Takayama, Kentaro; Yamazaki, Yuri; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Freire, Ernesto; Hayashi, Yoshio
Bioorganic & Medicinal Chemistry (2013), 21 (2), 412-424CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
We describe here the design, synthesis and biol. evaluation of a series of mols. toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CLpro. A docking study involving binding between the initial lead compd. (I) and the SARS-CoV 3CLpro motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1' site. This modification led to the identification of more potent derivs., including (II) (R1 = R2 = H; R1 = OMe, R2 = H; R1 = H, R2 = OMe; R1 = NMe2, H2 = H; R1 = H, R2 = NMe2), with IC50 or Ki values in the submicromolar to nanomolar range. In particular, compds. II (R1 = R2 = H; R1 = H, R2 = NMe2) exhibited the most potent inhibitory activities, with Ki values of 4.1 and 3.1 nM, resp. The peptidomimetic compds. identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CLpro may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1'-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochem. and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.
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76
Thanigaimalai, P.; Konno, S.; Yamamoto, T.; Koiwai, Y.; Taguchi, A.; Takayama, K.; Yakushiji, F.; Akaji, K.; Chen, S. E.; Naser-Tavakolian, A.; Schön, A.; Freire, E.; Hayashi, Y. Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: design, synthesis, biological evaluation, and docking studies. Eur. J. Med. Chem. 2013, 68, 372– 384, DOI: 10.1016/j.ejmech.2013.07.037
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76
Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: Design, synthesis, biological evaluation, and docking studies
Thanigaimalai, Pillaiyar; Konno, Sho; Yamamoto, Takehito; Koiwai, Yuji; Taguchi, Akihiro; Takayama, Kentaro; Yakushiji, Fumika; Akaji, Kenichi; Chen, Shen-En; Naser-Tavakolian, Aurash; Schon, Arne; Freire, Ernesto; Hayashi, Yoshio
European Journal of Medicinal Chemistry (2013), 68 (), 372-384CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
We report the design and synthesis of a series of dipeptide-type inhibitors with novel P3 scaffolds that display potent inhibitory activity against SARS-CoV 3CLpro. A docking study involving binding between the dipeptidic lead compd. (I) and 3CLpro suggested the modification of a structurally flexible P3 N-(3-methoxyphenyl)glycine with various rigid P3 moieties in I. The modifications led to the identification of several potent derivs. with the inhibitory activities (Ki or IC50) in the submicromolar to nanomolar range. Compd. (II) , in particular, displayed the most potent inhibitory activity, with a Ki value of 0.006 μM. This potency was 65-fold higher than the potency of the lead compd. I (Ki = 0.39 μM). In addn., the Ki value of II was in very good agreement with the binding affinity (16 nM) obsd. in isothermal titrn. calorimetry (ITC). A SAR study around the P3 group in the lead 4 led to the identification of a rigid indole-2-carbonyl unit as one of the best P3 moieties (III). Further optimization showed that a methoxy substitution at the 4-position on the indole unit was highly favorable for enhancing the inhibitory potency.
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77
Thanigaimalai, P.; Konno, S.; Yamamoto, T.; Koiwai, Y.; Taguchi, A.; Takayama, K.; Yakushiji, F.; Akaji, K.; Kiso, Y.; Kawasaki, Y.; Chen, S. E.; Naser-Tavakolian, A.; Schön, A.; Freire, E.; Hayashi, Y. Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study. Eur. J. Med. Chem. 2013, 65, 436– 447, DOI: 10.1016/j.ejmech.2013.05.005
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77
Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure-activity relationship study
Thanigaimalai, Pillaiyar; Konno, Sho; Yamamoto, Takehito; Koiwai, Yuji; Taguchi, Akihiro; Takayama, Kentaro; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Naser-Tavakolian, Aurash; Schon, Arne; Freire, Ernesto; Hayashi, Yoshio
European Journal of Medicinal Chemistry (2013), 65 (), 436-447CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
This work describes the design, synthesis, and evaluation of low-mol. wt. peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (I) (Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CLpro. In particular, compds. (II) (R1 = OMe, R2 = H and R1 = H, R2 = OMe) exhibited good inhibitory activities with Ki values of 0.39 and 0.33 μM, resp. These low-mol. wt. compds. are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compd. II (R1 = OMe, R2 = H) with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compds. with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.
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78
Shao, Y.-M.; Yang, W.-B.; Kuo, T.-H.; Tsai, K.-C.; Lin, C.-H.; Yang, A.-S.; Liang, P.-H.; Wong, C.-H. Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease. Bioorg. Med. Chem. 2008, 16, 4652– 4660, DOI: 10.1016/j.bmc.2008.02.040
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78
Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease
Shao, Yi-Ming; Yang, Wen-Bin; Kuo, Tun-Hsun; Tsai, Keng-Chang; Lin, Chun-Hung; Yang, An-Suei; Liang, Po-Huang; Wong, Chi-Huey
Bioorganic & Medicinal Chemistry (2008), 16 (8), 4652-4660CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
A series of trifluoromethyl ketones as SARS-Co-V 3CL protease inhibitors was developed. The inhibitors were synthesized in four steps from com. available compds. Three different amino acids were explored in the P1-position and in the P2-P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a K i value of 0.3 μM after 4 h incubation.
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79
Shao, Y.-M.; Yang, W.-B.; Peng, H.-P.; Hsu, M.-F.; Tsai, K.-C.; Kuo, T.-H.; Wang, A. H.-J.; Liang, P.-H.; Lin, C.-H.; Yang, A.-S.; Wong, C.-H. Structure-based design and synthesis of highly potent SARS-CoV 3CL protease inhibitors. ChemBioChem 2007, 8, 1654– 1657, DOI: 10.1002/cbic.200700254
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79
Structure-based design and synthesis of highly potent SARS-CoV 3CL protease inhibitors
Shao, Yi-Ming; Yang, Wen-Bin; Peng, Hung-Pin; Hsu, Min-Feng; Tsai, Keng-Chang; Kuo, Tun-Hsun; Wang, Andrew H.-J.; Liang, Po-Huang; Lin, Chun-Hung; Yang, An-Suei; Wong, Chi-Huey
ChemBioChem (2007), 8 (14), 1654-1657CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
The structure-based design and synthesis of highly potent coronavirus protease inhibitors is described, for potential use in treatment of coronavirus-assocd. acute respiratory syndrome.
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80
Kaeppler, U.; Stiefl, N.; Schiller, M.; Vicik, R.; Breuning, A.; Schmitz, W.; Rupprecht, D.; Schmuck, C.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods. J. Med. Chem. 2005, 48, 6832– 6842, DOI: 10.1021/jm0501782
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80
A New Lead for Nonpeptidic Active-Site-Directed Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus Main Protease Discovered by a Combination of Screening and Docking Methods
Kaeppler, Ulrich; Stiefl, Nikolaus; Schiller, Markus; Vicik, Radim; Breuning, Alexander; Schmitz, Werner; Rupprecht, Daniel; Schmuck, Carsten; Baumann, Knut; Ziebuhr, John; Schirmeister, Tanja
Journal of Medicinal Chemistry (2005), 48 (22), 6832-6842CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compds. that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (I) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the Ph ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-Bu moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (II) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compd. II showed a Ki value of 35.3 μM. Since the novel lead compd. does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compd. II.
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82
Kaeppler, U.; Schirmeister, T. New non-peptidic inhibitors of papain derived from etacrynic acid. Med. Chem. 2005, 1, 361– 370, DOI: 10.2174/1573406054368701
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New non-peptidic inhibitors of papain derived from etacrynic acid
Kaeppler, U.; Schirmeister, T.
Medicinal Chemistry (2005), 1 (4), 361-370CODEN: MCEHAJ; ISSN:1573-4064. (Bentham Science Publishers Ltd.)
Cysteine proteases are connected to various viral and parasitic infections, as well as to other severe diseases like arthritis, stroke and cancer. Due to its α,β-unsatd. carbonyl moiety etacrynic acid, a well known diuretic, can inhibit cysteine proteases in a Michael-type reaction by reaction with the nucleophilic cysteine residue of the active site. For first structure-activity-relation studies modifications at various positions of the etacrynic acid structure have been investigated concerning inhibition potency against the CAC1 protease papain: length of the side chain, substitution pattern of the arom. ring as well as influence and necessity of acidic groups, esters or amides. Addnl., the effect of the arom. ring was evaluated by replacement with a cyclohexyl moiety.
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83
Webber, S. E.; Tikhe, J.; Worland, S. T.; Fuhrman, S. A.; Hendrickson, T. F.; Matthews, D. A.; Love, R. A.; Patick, A. K.; Meador, J. W.; Ferre, R. A.; Brown, E. L.; DeLisle, D. M.; Ford, C. E.; Binford, S. L. Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease. J. Med. Chem. 1996, 39, 5072– 5082, DOI: 10.1021/jm960603e
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83
Design, Synthesis, and Evaluation of Nonpeptidic Inhibitors of Human Rhinovirus 3C Protease
Webber, Stephen E.; Tikhe, Jayashree; Worland, Stephen T.; Fuhrman, Shella A.; Hendrickson, Thomas F.; Matthews, David A.; Love, Robert A.; Patick, Amy K.; Meador, James W.; et al.
Journal of Medicinal Chemistry (1996), 39 (26), 5072-5082CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The design, synthesis, and biol. evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, mol. modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Mol.-modeling expts. using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed the authors to design recognition features into the P1 and P2 subsites, resp., from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addn., a series of ab initio calcns. were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepd. by general synthetic methods, starting with com. available 5-substituted isatins in nearly every case. All compds. were tested for inhibition of purified HRV-14 3CP. Compds. I, II, and III were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compds. were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 Å cocrystal structure of deriv. III covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.
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84
Chen, L. R.; Wang, Y. C.; Lin, Y. W.; Chou, S. Y.; Chen, S. F.; Liu, L. T.; Wu, Y. T.; Kuo, C. J.; Chen, T. S. S.; Juang, S. H. Synthesis and evaluation of isatin derivatives as effective SARS coronavirus 3CL protease inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3058– 3062, DOI: 10.1016/j.bmcl.2005.04.027
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84
Synthesis and evaluation of isatin derivatives as effective SARS coronavirus 3CL protease inhibitors
Chen, Li-Rung; Wang, Yu-Chin; Lin, Yi Wen; Chou, Shan-Yen; Chen, Shyh-Fong; Liu, Lee Tai; Wu, Ying-Ta; Kuo, Chih-Jung; Chen, Tom Shieh-Shung; Juang, Shin-Hun
Bioorganic & Medicinal Chemistry Letters (2005), 15 (12), 3058-3062CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
N-Substituted isatin derivs., e.g., I, were prepd. from the reaction of isatin and various bromides via two steps. Bioactivity assay results (in vitro tests) demonstrated that some of these compds. were potent and selective inhibitors against SARS coronavirus 3CL protease with IC50 values ranging from 0.95 to 17.50 μM. Addnl., I exhibited more potent inhibition for SARS coronavirus protease than for other proteases including papain, chymotrypsin, and trypsin.
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85
Liu, W.; Zhu, H.-M.; Niu, G.-J.; Shi, E.-Z.; Chen, J.; Sun, B.; Chen, W.-Q.; Zhou, H.-G.; Yang, C. Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors. Bioorg. Med. Chem. 2014, 22, 292– 302, DOI: 10.1016/j.bmc.2013.11.028
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85
Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors
Liu, Wei; Zhu, He-Min; Niu, Guo-Jun; Shi, En-Zhi; Chen, Jie; Sun, Bo; Chen, Wei-Qiang; Zhou, Hong-Gang; Yang, Cheng
Bioorganic & Medicinal Chemistry (2014), 22 (1), 292-302CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
The Severe Acute Respiratory Syndrome (SARS) is a serious life-threatening and strikingly mortal respiratory illness caused by SARS-CoV. SARS-CoV which contains a chymotrypsin-like main protease analogous to that of the main picornavirus protease, 3CLpro. 3CLpro plays a pivotal role in the viral replication cycle and is a potential target for SARS inhibitor development. A series of isatin derivs. as possible SARS-CoV 3CLpro inhibitors was designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide, in which several showed potent inhibition against the 3CLpro. Structure-activity relationship was analyzed, and possible binding interaction modes were proposed by mol. docking studies. Among all compds., 8k1 showed most potent inhibitory activity against 3CLpro (IC50 = 1.04 μM). These results indicated that these inhibitors could be potentially developed into anti-SARS drugs.
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Güngör, T.; Chen, Y.; Golla, R.; Ma, Z.; Corte, J. R.; Northrop, J. P.; Bin, B.; Disckson, J. K.; Stouch, T.; Zhou, R.; Johnson, S. E.; Seethala, R.; Feyen, J. H. M. Synthesis and characterization of 3-arylquinazolinone and 3-arylquinazolinethione derivatives as selective estrogen receptor beta modulators. J. Med. Chem. 2006, 49, 2440– 2455, DOI: 10.1021/jm0509389
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86
Synthesis and characterization of 3-arylquinazolinone and 3-arylquinazolinethione derivatives as selective estrogen receptor beta modulators
Gungor Timur; Chen Ying; Golla Rajasree; Ma Zhengping; Corte James R; Northrop John P; Bin Bin; Dickson John K; Stouch Terry; Zhou Rong; Johnson Susan E; Seethala Ramakrishna; Feyen Jean H M
Journal of medicinal chemistry (2006), 49 (8), 2440-55 ISSN:0022-2623.
On the basis of the stucture of genistein, a new series of 3-arylquinazolines was prepared and tested for their estrogen receptor (ER) alpha and beta affinities. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-4(3H)-quinazolinone (1aa) acts as an agonist on both ER subtypes. It has 62-fold higher binding affinity [IC(50)(ERbeta) = 179 nM] and 38-fold higher functional potency in a transcription assay [EC(50)(ERbeta) = 76 nM] with ERbeta than with ERalpha, thus improving upon the selectivity of genistein. All of the analogues showed preferential binding affinity for ERbeta. Many are also more potent in activating transcription by ERbeta than by ERalpha. Transformation of the C=O functionality at position 4 into a C=S group provided 5,7-dihydroxy-3-(4-hydroxyphenyl)-4(3H)-quinazolinethione (1ba), which acts as an agonist on both ER subtypes but has 56-fold higher binding affinity for ERbeta over ERalpha [IC(50)(ERbeta) = 47 nM] and 215-fold higher potency in the transcription assay [EC(50)(ERbeta) = 13 nM]. These ERbeta-selective compounds may represent valuable tools in understanding the differences in structure and biological function of ERbeta and ERalpha.
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Yi, L.; Li, Z.; Yuan, K.; Qu, X.; Chen, J.; Wang, G.; Zhang, H.; Luo, H.; Zhu, L.; Jiang, P.; Chen, L.; Shen, Y.; Luo, M.; Zuo, G.; Hu, J.; Duan, D.; Nie, Y.; Shi, X.; Wang, W.; Han, Y.; Li, T.; Liu, Y.; Ding, M.; Deng, H.; Xu, X. Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells. J. Virol. 2004, 78, 11334– 11339, DOI: 10.1128/JVI.78.20.11334-11339.2004
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87
Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells
Yi, Ling; Li, Zhengquan; Yuan, Kehu; Qu, Xiuxia; Chen, Jian; Wang, Guangwen; Zhang, Hong; Luo, Hongpeng; Zhu, Lili; Jiang, Pengfei; Chen, Lirong; Shen, Yan; Luo, Min; Zuo, Guoying; Hu, Jianhe; Duan, Deliang; Nie, Yuchun; Shi, Xuanling; Wang, Wei; Han, Yang; Li, Taisheng; Liu, Yuqing; Ding, Mingxiao; Deng, Hongkui; Xu, Xiaojie
Journal of Virology (2004), 78 (20), 11334-11339CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small mols. that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatog.-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small mols., tetra-O-galloyl-β-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concn. of 4.5 μM and a selective index of 240.0. The two-step screening method described here yielded several small mols. that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.
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88
Chen, L.; Li, J.; Luo, C.; Liu, H.; Xu, W.; Chen, G.; Liew, O. W.; Zhu, W.; Puah, C. M.; Shen, X.; Jiang, H. Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): Structure-activity relationship studies reveal salient pharmacophore features. Bioorg. Med. Chem. 2006, 14, 8295– 8306, DOI: 10.1016/j.bmc.2006.09.014
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88
Binding interaction of quercetin-3-β-galactoside and its synthetic derivatives with SARS-CoV 3CLpro: Structure-activity relationship studies reveal salient pharmacophore features
Chen, Lili; Li, Jian; Luo, Cheng; Liu, Hong; Xu, Weijun; Chen, Gang; Liew, Oi Wah; Zhu, Weiliang; Puah, Chum Mok; Shen, Xu; Jiang, Hualiang
Bioorganic & Medicinal Chemistry (2006), 14 (24), 8295-8306CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
The 3C-like protease (3CLpro) of severe acute respiratory syndrome-assocd. coronavirus (SARS-Co-V) is one of the most promising targets for discovery of drugs against SARS, because of its crit. role in the viral life cycle. In this study, a natural compd. called quercetin-3-β-galactoside was identified as an inhibitor of the protease by mol. docking, SPR/FRET-based bioassays, and mutagenesis studies. Both mol. modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, exptl. evidence showed that the secondary structure and enzymic activity of SARS-Co-V 3CLpro were not affected by the Q189A mutation. With the help of mol. modeling, eight new derivs. of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure-activity relationship of the new compds.: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivs.; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compds. as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.
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89
Ryu, Y. B.; Jeong, H. J.; Kim, J. H.; Kim, Y. M.; Park, J.-Y.; Kim, D.; Naguyen, T. T. H.; Park, S.-J.; Chang, J. S.; Park, K. H. Biflavonoids from Torreya nucifera displaying SARS-CoV 3CLpro inhibition. Bioorg. Med. Chem. 2010, 18, 7940– 7947, DOI: 10.1016/j.bmc.2010.09.035
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89
Biflavonoids from Torreya nucifera displaying SARS-CoV 3CLpro inhibition
Ryu, Young Bae; Jeong, Hyung Jae; Kim, Jang Hoon; Kim, Young Min; Park, Ji-Young; Kim, Doman; Naguyen, Thi Thanh Hanh; Park, Su-Jin; Chang, Jong Sun; Park, Ki Hun; Rho, Mun-Chual; Lee, Woo Song
Bioorganic & Medicinal Chemistry (2010), 18 (22), 7940-7947CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
As part of our search for botanical sources of SARS-CoV 3CLpro inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol ext. of T. nucifera leaves exhibited good SARS-CoV 3CLpro inhibitory activity (62% at 100 μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CLpro inhibition using fluorescence resonance energy transfer anal. Of these compds., the biflavone amentoflavone (9) (IC50 = 8.3 μM) showed most potent 3CLpro inhibitory effect. Three addnl. authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CLpro activity with IC50 values of 280.8, 20.2, and 23.8 μM, resp. Values of binding energy obtained in a mol. docking study supported the results of enzymic assays. More potent activity appeared to be assocd. with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CLpro inhibitory activity.
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90
Ryu, Y. B.; Park, S.-J.; Kim, Y. M.; Lee, J.-Y.; Seo, W. D.; Chang, J. S.; Park, K. H.; Rho, M.-C.; Lee, W. S. SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii. Bioorg. Med. Chem. Lett. 2010, 20, 1873– 1876, DOI: 10.1016/j.bmcl.2010.01.152
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90
SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii
Ryu, Young Bae; Park, Su-Jin; Kim, Young Min; Lee, Ju-Yeon; Seo, Woo Duck; Chang, Jong Sun; Park, Ki Hun; Rho, Mun-Chual; Lee, Woo Song
Bioorganic & Medicinal Chemistry Letters (2010), 20 (6), 1873-1876CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
Quinone-methide triterpenes, celastrol (1), pristimerin (2), tingenone (3), and iguesterin (4) were isolated from Triterygium regelii and dihydrocelastrol (5) was synthesized by hydrogenation under palladium catalyst. Isolated quinone-methide triterpenes (1-4) and 5 were evaluated for SARS-CoV 3CLpro inhibitory activities and showed potent inhibitory activities with IC50 values of 10.3, 5.5, 9.9, and 2.6 μM, resp., whereas the corresponding 5 having phenol moiety was obsd. in low activity (IC50 = 21.7 μM). As a result, quinone-methide moiety in A-ring and more hydrophobic E-ring assist to exhibit potent activity. Also, all quinone-methide triterpenes 1-4 have proven to be competitive by the kinetic anal.
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91
Wen, C.-C.; Kuo, Y.-H.; Jan, J.-T.; Liang, P.-H.; Wang, S.-Y.; Liu, H.-G.; Lee, C.-K.; Chang, S.-T.; Kuo, C.-J.; Lee, S.-S.; Hou, C.-C.; Hsiao, P.-W.; Chien, S.-C.; Shyur, L.-F.; Yang, N.-S. Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus. J. Med. Chem. 2007, 50, 4087– 4095, DOI: 10.1021/jm070295s
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91
Specific Plant Terpenoids and Lignoids Possess Potent Antiviral Activities against Severe Acute Respiratory Syndrome Coronavirus
Wen, Chih-Chun; Kuo, Yueh-Hsiung; Jan, Jia-Tsrong; Liang, Po-Huang; Wang, Sheng-Yang; Liu, Hong-Gi; Lee, Ching-Kuo; Chang, Shang-Tzen; Kuo, Chih-Jung; Lee, Shoei-Sheng; Hou, Chia-Chung; Hsiao, Pei-Wen; Chien, Shih-Chang; Shyur, Lie-Fen; Yang, Ning-Sun
Journal of Medicinal Chemistry (2007), 50 (17), 4087-4095CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
In this study, 221 phytocompounds were evaluated for activity against anti-severe acute respiratory syndrome assocd. coronavirus (SARS-CoV) activities using a cell-based assay measuring SARS-CoV-induced cytopathogenic effect on Vero E6 cells. Ten diterpenoids (1-10), two sesquiterpenoids (11 and 12), two triterpenoids (13 and 14), five lignoids (15-19), curcumin (20), and ref. controls niclosamide (21) and valinomycin (22) were potent inhibitors at concns. between 3.3 and 10 μM. The concns. of the 22 compds. to inhibit 50% of Vero E6 cell proliferation (CC50) and viral replication (EC50) were measured. The selective index values (SI = CC50/EC50) of the most potent compds. 1, 5, 6, 8, 14, and 16 were 58, >510, 111, 193, 180, and >667, resp. Betulinic acid (13) and savinin (16) were competitive inhibitors of SARS-CoV 3CL protease with Ki values = 8.2±0.7 and 9.1±2.4 μM, resp. Our findings suggest that specific abietane-type diterpenoids and lignoids exhibit strong anti-SARS-CoV effects.
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92
Lu, I.-L.; Mahindroo, N.; Liang, P.-H.; Peng, Y.-H.; Kuo, C.-J.; Tsai, K.-C.; Hsieh, H.-P.; Chao, Y.-S.; Wu, S.-Y. Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease. J. Med. Chem. 2006, 49, 5154– 5161, DOI: 10.1021/jm060207o
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92
Structure-Based Drug Design and Structural Biology Study of Novel Nonpeptide Inhibitors of Severe Acute Respiratory Syndrome Coronavirus Main Protease
Lu, I-Lin; Mahindroo, Neeraj; Liang, Po-Huang; Peng, Yi-Hui; Kuo, Chih-Jung; Tsai, Keng-Chang; Hsieh, Hsing-Pang; Chao, Yu-Sheng; Wu, Su-Ying
Journal of Medicinal Chemistry (2006), 49 (17), 5154-5161CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (Mpro), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chem. database contg. 58 855 compds. followed by the testing of potential compds. for SARS-CoV Mpro inhibition leads to two hit compds. The core structures of these two hits, defined by the docking study, are used for further analog search. Twenty-one analogs derived from these two hits exhibited IC50 values below 50 μM, with the most potent one showing 0.3 μM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV Mpro were solved by x-ray crystallog. They bind to the protein in a distinct manner compared to all published SARS-CoV Mpro complex structures. They inhibit SARS-CoV Mpro activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.
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93
Tsai, K.-C.; Chen, S.-Y.; Liang, P.-H.; Lu, I.-L.; Mahindroo, N.; Hsieh, H.-P.; Chao, Y.-S.; Liu, L.; Liu, D.; Lien, W.; Lin, T.-H.; Wu, S.-Y. Discovery of a novel family of SARS-CoV protease inhibitors by virtual screening and 3D-QSAR studies. J. Med. Chem. 2006, 49, 3485– 3495, DOI: 10.1021/jm050852f
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93
Discovery of a Novel Family of SARS-CoV Protease Inhibitors by Virtual Screening and 3D-QSAR Studies
Tsai, Keng-Chang; Chen, Shih-Yuan; Liang, Po-Huang; Lu, I-Lin; Mahindroo, Neeraj; Hsieh, Hsing-Pang; Chao, Yu-Sheng; Liu, Lincoln; Liu, Donald; Lien, Wei; Lin, Thy-Hou; Wu, Su-Ying
Journal of Medicinal Chemistry (2006), 49 (12), 3485-3495CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The severe acute respiratory syndrome-assocd. coronavirus (SARS-Co-V) 3C-like protease (3CLpro or Mpro) is an attractive target for the development of anti-SARS drugs because of its crucial role in the viral life cycle. In this study, a compd. database was screened by the structure-based virtual screening approach to identify initial hits as inhibitors of SARS-Co-V 3CLpro. Out of the 59 363 compds. docked, 93 were selected for the inhibition assay, and 21 showed inhibition against SARS-Co-V 3CLpro (IC50 ≤ 30 μM), with three of them having common substructures. Furthermore, a search for analogs with common substructure in the Maybridge, ChemBridge, and SPECS_SC databases led to the identification of another 25 compds. that exhibited inhibition against SARS-Co-V 3CLpro (IC50 = 3-1000 μM). These compds., 28 in total, were subjected to 3D-QSAR studies to elucidate the pharmacophore of SARS-Co-V 3CLpro.
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94
Wu, C.-Y.; King, K.-Y.; Kuo, C.-J.; Fang, J.-M.; Wu, Y.-T.; Ho, M.-Y.; Liao, C.-L.; Shie, J.-J.; Liang, P.-H.; Wong, C.-H. Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease. Chem. Biol. 2006, 13, 261– 268, DOI: 10.1016/j.chembiol.2005.12.008
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94
Stable Benzotriazole Esters as Mechanism-Based Inactivators of the Severe Acute Respiratory Syndrome 3CL Protease
Wu, Chung-Yi; King, Ke-Yung; Kuo, Chih-Jung; Fang, Jim-Min; Wu, Ying-Ta; Ho, Ming-Yi; Liao, Chung-Lin; Shie, Jiun-Jie; Liang, Po-Huang; Wong, Chi-Huey
Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (3), 261-268CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected >8000 individuals and resulted in >800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CLpro has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CLpro, and the most potent inactivator exhibited a kinact of 0.0011 s-1 and a Ki of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was obsd. with the use of the C145A mutant. In addn., a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.
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95
Brik, A.; Lin, Y.-C.; Elder, J.; Wong, C.-H. A quick diversity-oriented amide-forming reaction to optimize P-subsite residues of HIV protease inhibitors. Chem. Biol. 2002, 9, 891– 896, DOI: 10.1016/S1074-5521(02)00184-9
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95
A Quick Diversity-Oriented Amide-Forming Reaction to Optimize P-Subsite Residues of HIV Protease Inhibitors
Brik, Ashraf; Lin, Ying-Chuan; Elder, John; Wong, Chi-Huey
Chemistry & Biology (2002), 9 (8), 891-896CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
We report a new simple method that allows rapid prepn. in soln. of a library of compds. for in situ high-throughput screening to identify new inhibitors of HIV-1 protease. The method is based on the amide-forming reaction of a C2-sym. diamino diol core with various carboxylic acids, followed by a direct assay of the inhibition activity without product isolation. Sixty-two compds. were made and screened in less than 1 h. The utility of this method is demonstrated by the identification of new P3-P3' residues that convert a transition state analog core from a poor binding mol. (1, Ki > 2 μM) to a potent inhibitor (AB1, Ki = 2 nM) against the wild-type, and the inhibition activities against resistant mutants are better than those of two existing drugs. This method reduces the time required for synthesis and testing of a large no. of characterized inhibitors and should find useful applications in other enzyme systems.
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96
Wu, C.-Y.; Chang, C.-F.; Chen, J. S.-Y.; Wong, C.-H.; Lin, C.- H. Rapid diversity-oriented synthesis in microtiter plates for in situ screening: discovery of potent and selective alpha-fucosidase inhibitors. Angew. Chem., Int. Ed. 2003, 42, 4661– 4664, DOI: 10.1002/anie.200351823
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96
Rapid diversity-oriented synthesis in microtiter plates for in situ screening: Discovery of potent and selective α-fucosidase inhibitors
Wu, Chung-Yi; Chang, Chuan-Fa; Chen, Jenny Szu-Yu; Wong, Chi-Huey; Lin, Chun-Hung
Angewandte Chemie, International Edition (2003), 42 (38), 4661-4664CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
The authors report the generation of a library based on fuconojirimycin (FNJ) in a microtiter plate, followed by the direct in situ evaluation of these reaction mixts. as fucosidase inhibitors without product isolation. This approach has led to the discovery of the most potent inhibitors of α-fucosidases from bovine kidney and Corynebacterium sp. The inhibitors may be used a probes for the study of fucosidases with regard to their functions and for the development of potential therapeutic agents. The method described here represents the first rapid search for an optimal group attached to the core of a common transition-state mimic to bind the hydrophobic site of a glycosyltransfer enzyme using a simple amide-bond forming reaction followed by screening in situ without product isolation.
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Chang, C.-F.; Ho, C.-W.; Wu, C.-Y.; Chao, T.-A.; Wong, C.-H.; Lin, C.-H. Discovery of picomolar slow tight-binding inhibitors of alpha-fucosidase. Chem. Biol. 2004, 11, 1301– 1306, DOI: 10.1016/j.chembiol.2004.07.009
[Crossref], [PubMed], [CAS], Google Scholar
97
Discovery of Picomolar Slow Tight-Binding Inhibitors of α-Fucosidase
Chang, Chuan-Fa; Ho, Chin-Wen; Wu, Chung-Yi; Chao, Tsrong-An; Wong, Chi-Huey; Lin, Chun-Hung
Chemistry & Biology (2004), 11 (9), 1301-1306CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
Glycosidase inhibitors have shown great medicinal and pharmaceutical values as exemplified by the therapeutic treatment of influenza virus and non-insulin-dependent diabetes. We herein report the discovery of picomolar slow tight-binding inhibitors 2-5 against the α-fucosidase from Corynebacterium sp. by a rapid screening for an optimal aglycon attached to 1-aminomethyl fuconojirimycin (1). The time-dependent inhibition displays the progressive tightening of enzyme-inhibitor complex from a low nanomolar Ki* to picomolar Ki* value. Particularly compd. 2 with a Ki* of 0.46 pM represents the most potent glycosidase inhibitor to date. The effect of compd. 3 on the intrinsic fluorescence of α-fucosidase is both time- and concn.-dependent in a satn.-type manner, which is consistent with the initial formation of a rapid equil. complex of enzyme and inhibitor (E·I), followed by the slower formation of a tightly bound enzyme-inhibitor complex (E·I*). The binding affinity increases 3.5 × 104-fold from 1 (Ki = 16.3 nM) to 2 (Ki = 0.46 pM). This work clearly demonstrates the effectiveness of our combinatorial approach leading to the rapid discovery of potent inhibitors.
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98
Verschueren, K. H. G.; Pumpor, K.; Anemüller, S.; Chen, S.; Mesters, J. R.; Hilgenfeld, R. A structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters. Chem. Biol. 2008, 15, 597– 606, DOI: 10.1016/j.chembiol.2008.04.011
[Crossref], [PubMed], [CAS], Google Scholar
98
A Structural View of the Inactivation of the SARS Coronavirus Main Proteinase by Benzotriazole Esters
Verschueren, Koen H. G.; Pumpor, Ksenia; Anemueller, Stefan; Chen, Shuai; Mesters, Jeroen R.; Hilgenfeld, Rolf
Chemistry & Biology (Cambridge, MA, United States) (2008), 15 (6), 597-606CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
Summary: The main proteinase (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus is a principal target for the design of anticoronaviral compds. Benzotriazole esters have been reported as potent nonpeptidic inhibitors of the enzyme, but their exact mechanism of action remains unclear. Here we present crystal structures of SARS-CoV Mpro, the active-site cysteine of which has been acylated by benzotriazole esters that act as suicide inhibitors. In one of the structures, the thioester product has been hydrolyzed and benzoic acid is obsd. to bind to the hydrophobic S2 pocket. This structure also features the enzyme with a shortened N-terminal segment ("amputated N finger"). The results further the understanding of the important role of the N finger for catalysis as well as the design of benzotriazole inhibitors with improved specificity.
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Blanchard, J. E.; Elowe, N. H.; Huitema, C.; Fortin, P. D.; Cechetto, J. D.; Eltis, L. D.; Brown, E. D. High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase. Chem. Biol. 2004, 11, 1445– 1453, DOI: 10.1016/j.chembiol.2004.08.011
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99
High-Throughput Screening Identifies Inhibitors of the SARS Coronavirus Main Proteinase
Blanchard, Jan E.; Elowe, Nadine H.; Huitema, Carly; Fortin, Pascal D.; Cechetto, Jonathan D.; Eltis, Lindsay D.; Brown, Eric D.
Chemistry & Biology (2004), 11 (10), 1445-1453CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small mols. on a fully automated system. The primary screen identified 572 hits; through a series of virtual and exptl. filters, this no. was reduced to five novel small mols. that show potent inhibitory activity (IC50 = 0.5-7 μM) toward SARS-CoV 3CLpro.
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100
Zhang, J.; Pettersson, H. I.; Huitema, C.; Niu, C.; Yin, J.; James, M. N.; Eltis, L. D.; Vederas, J. C. Design, synthesis, and evaluation of inhibitors for severe acute respiratory syndrome 3C-like protease based on phthalhydrazide ketones or heteroaromatic esters. J. Med. Chem. 2007, 50, 1850– 1864, DOI: 10.1021/jm061425k
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100
Design, synthesis, and evaluation of inhibitors for severe acute respiratory syndrome 3C-Like protease based on phthalhydrazide ketones or heteroaromatic esters
Zhang, Jianmin; Pettersson, Hanna I.; Huitema, Carly; Niu, Chunying; Yin, Jiang; James, Michael N. G.; Eltis, Lindsay D.; Vederas, John C.
Journal of Medicinal Chemistry (2007), 50 (8), 1850-1864CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The 3C-like protease (3CLpro), which controls the severe acute respiratory syndrome (SARS) coronavirus replication, has been identified as a potential target for drug design in the treatment of SARS. A series of tetrapeptide phthalhydrazide ketones, pyridinyl esters, and their analogs have been designed, synthesized, and evaluated as potential SARS 3CLpro inhibitors. Some pyridinyl esters, e.g., I, were identified as very potent inhibitors, with IC50 values in the nanomolar range (50-65 nM). Electrospray mass spectrometry indicated a mechanism involving acylation of the active site cysteine thiol for this class of inhibitors.
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Niu, C.; Yin, J.; Zhang, J.; Vederas, J. C.; James, M. N. Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV Mpro. Bioorg. Med. Chem. 2008, 16, 293– 302, DOI: 10.1016/j.bmc.2007.09.034
[Crossref], [PubMed], [CAS], Google Scholar
101
Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV Mpro
Niu, Chunying; Yin, Jiang; Zhang, Jianmin; Vederas, John C.; James, Michael N. G.
Bioorganic & Medicinal Chemistry (2008), 16 (1), 293-302CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-Co-V Mpro, is widely considered to be a major drug target for the development of anti-SARS treatment. Based on the chem. structure of a lead compd. from a previous screening, the authors have designed and synthesized a no. of nonpeptidyl inhibitors, some of which have shown significantly improved inhibitory activity against SARS-Co-V Mpro with IC50 values of ∼60 nM. In the absence of SARS-Co-V Mpro crystal structures in complex with these synthetic inhibitors, mol. docking tools have been employed to study possible interactions between these inhibitors and SARS-Co-V Mpro. The docking results suggest two major modes for the initial binding of these inhibitors to the active site of SARS-Co-V Mpro. They also establish a structural basis for the 'core design' of these inhibitors by showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket. In addn., intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting that this structural plasticity can be further exploited for optimizing inhibitor-enzyme interactions that should promote a tighter binding mode. Most importantly, the results provide the structural basis for rational design of wide-spectrum antiviral drugs targeting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.
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102
Ghosh, A. K.; Gong, G.; Grum-Tokars, V.; Mulhearn, D. C.; Baker, S. C.; Coughlin, M.; Prabhakar, B. S.; Sleeman, K.; Johnson, M. E.; Mesecar, A. D. Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 5684– 5688, DOI: 10.1016/j.bmcl.2008.08.082
[Crossref], [PubMed], [CAS], Google Scholar
102
Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors
Ghosh, Arun K.; Gong, Gangli; Grum-Tokars, Valerie; Mulhearn, Debbie C.; Baker, Susan C.; Coughlin, Melissa; Prabhakar, Bellur S.; Sleeman, Katrina; Johnson, Michael E.; Mesecar, Andrew D.
Bioorganic & Medicinal Chemistry Letters (2008), 18 (20), 5684-5688CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
Design, synthesis and biol. evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is crit. to potency. Inhibitor I is the most potent inhibitor with a SARS-CoV 3CLpro IC50 value of 30 nM and an antiviral EC50 value of 6.9 μM. Mol. docking studies have provided possible binding modes of these inhibitors.
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103
Zhang, J.; Huitema, C.; Niu, C.; Yin, J.; James, M. N.G.; Eltis, L. D.; Vederas, J. C. Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase. Bioorg. Chem. 2008, 36, 229– 240, DOI: 10.1016/j.bioorg.2008.01.001
[Crossref], [PubMed], [CAS], Google Scholar
103
Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase
Zhang, Jianmin; Huitema, Carly; Niu, Chunying; Yin, Jiang; James, Michael N. G.; Eltis, Lindsay D.; Vederas, John C.
Bioorganic Chemistry (2008), 36 (5), 229-240CODEN: BOCMBM; ISSN:0045-2068. (Elsevier Inc.)
The severe acute respiratory syndrome (SARS) virus depends on a chymotrypsin-like cysteine proteinase (3CLpro) to process the translated polyproteins to functional viral proteins. This enzyme is a target for the design of potential anti-SARS drugs. A series of ketones and corresponding mono- and di-fluoro ketones having two or three arom. rings were synthesized as possible reversible inhibitors of SARS 3CLpro. The design was based on previously established potent inhibition of the enzyme by oxa analogs (esters), which also act as substrates. Structure-activity relationships and modeling studies indicate that three arom. rings, including a 5-bromopyridin-3-yl moiety, are key features for good inhibition of SARS 3CLpro. 2-(5-Bromopyridin-3-yl)-1-(5-(4-chlorophenyl)furan-2-yl)ethanone and its α-monofluorinated analog, gave the best reversible inhibition with IC50 values of 13 μM and 28 μM, resp. In contrast to inhibitors having two arom. rings, α-fluorination of compds. with three rings unexpectedly decreased the inhibitory activity.
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Chen, L.; Chen, S.; Gui, C.; Shen, J.; Shen, X.; Jiang, H. Discovering severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays. J. Biomol. Screening 2006, 11, 915– 921, DOI: 10.1177/1087057106293295
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104
Discovering severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays
Chen, Lili; Chen, Shuai; Gui, Chunshan; Shen, Jianhua; Shen, Xu; Jiang, Hualiang
Journal of Biomolecular Screening (2006), 11 (8), 915-921CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CLpro) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small mol. compds. against SPECS database by virtual screening; 256 compds. were subsequently selected for biol. evaluation. Through SPR technol.-based assay, 52 from these 256 compds. were discovered to show binding to SARS-CoV 3CLpro. The enzymic inhibition activities of these 52 SARS-CoV 3CLpro binders were further applied to FRET-based assay, and IC50 values were detd. Based on this integrated assay platform, 8 new SARS-CoV 3CLpro inhibitors were discovered. The fact that the obtained IC50 values for the inhibitors are in good accordance with the discovered dissocn. equil. consts. (KDs) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CLpro inhibitors, and the detd. inhibitors could be used as possible lead compds. for further research.
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Kuo, C. J.; Liu, H. G.; Lo, Y. K.; Seong, C. M.; Lee, K. I.; Jung, Y. S.; Liang, P. H. Individual and common inhibitors of coronavirus and picornavirus main proteases. FEBS Lett. 2009, 583, 549– 555, DOI: 10.1016/j.febslet.2008.12.059
[Crossref], [PubMed], [CAS], Google Scholar
105
Individual and common inhibitors of coronavirus and picornavirus main proteases
Kuo, Chih-Jung; Liu, Hun-Ge; Lo, Yueh-Kuei; Seong, Churl-Min; Lee, Kee-In; Jung, Young-Sik; Liang, Po-Huang
FEBS Letters (2009), 583 (3), 549-555CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
Picornaviruses (PV) and coronaviruses (CoV) are pos.-stranded RNA viruses which infect millions of people worldwide each year, resulting in a wide range of clin. outcomes. As reported in this study, using high throughput screening against ∼6800 small mols., we have identified several novel inhibitors of SARS-CoV 3CLpro with IC50 of low μM. Interestingly, one of them equally inhibited both 3Cpro and 3CLpro from PV and CoV, resp. Using computer modeling, the structural features of these compds. as individual and common protease inhibitors were elucidated to enhance our knowledge for developing antiviral agents against PV and CoV.
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Ramajayam, R.; Tan, K.-P.; Liu, H.-G.; Liang, P.-H. Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors. Bioorg. Med. Chem. 2010, 18, 7849– 7854, DOI: 10.1016/j.bmc.2010.09.050
[Crossref], [PubMed], [CAS], Google Scholar
106
Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors
Ramajayam, R.; Tan, Kian-Pin; Liu, Hun-Ge; Liang, Po-Huang
Bioorganic & Medicinal Chemistry (2010), 18 (22), 7849-7854CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
A series of pyrazolone compds. as possible SARS-CoV 3CL protease inhibitors were designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors, I, was also active against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-coronaviral and anti-picornaviral agents.
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107
Ahn, T. Y.; Kuo, C. J.; Liu, H. G.; Ha, D. C.; Liang, P. H.; Jung, Y. S. Synthesis and evaluation of benzoquinolinone derivatives as sars-cov 3cl protease inhibitors. Bull. Korean Chem. Soc. 2010, 31, 87– 91, DOI: 10.5012/bkcs.2010.31.01.087
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107
Synthesis and evaluation of benzoquinolinone derivatives as SARS-CoV 3CL protease inhibitors
Ahn, Tae-Young; Kuo, Chih-Jung; Liu, Hun-Ge; Ha, Deok-Chan; Liang, Po-Huang; Jung, Young-Sik
Bulletin of the Korean Chemical Society (2010), 31 (1), 87-91CODEN: BKCSDE; ISSN:0253-2964. (Korean Chemical Society)
For the discovery of new antivirals against Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV), several benzoquinolines were prepd. and evaluated as 3C-like protease (3CLpro) inhibitors. Based on the computer modeling that each of the 2 rigid benzoquinolinone and N-phenoltetrazole moieties of compd. I [R = R1 = H, X = Cl (II)] is bound to the S1 and S2 sites, resp., of the SARS protease by forming H-bonds and hydrophobic interactions, benzoquinolines alkylated at both the OH groups were synthesized. Compd. I (R = Bn, R1 = Me, X = H) showed a 5 times higher inhibiting activity against 3CLpro compared to II.
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108
Ramajayam, R.; Tan, K.-P.; Liu, H.-G.; Liang, P.-H. Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease. Bioorg. Med. Chem. Lett. 2010, 20, 3569– 3572, DOI: 10.1016/j.bmcl.2010.04.118
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108
Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease
Ramajayam, R.; Tan, Kian-Pin; Liu, Hun-Ge; Liang, Po-Huang
Bioorganic & Medicinal Chemistry Letters (2010), 20 (12), 3569-3572CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
A series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates had encouraging results for the development of new anti-SARS compds.
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Shimamoto, Y.; Hattori, Y.; Kobayashi, K.; Teruya, K.; Sanjoh, A.; Nakagawa, A.; Yamashita, K.; Akaji, K. Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors. Bioorg. Med. Chem. 2015, 23, 876– 890, DOI: 10.1016/j.bmc.2014.12.028
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109
Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors
Shimamoto, Yasuhiro; Hattori, Yasunao; Kobayashi, Kazuya; Teruya, Kenta; Sanjoh, Akira; Nakagawa, Atsushi; Yamashita, Eiki; Akaji, Kenichi
Bioorganic & Medicinal Chemistry (2015), 23 (4), 876-890CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CLpro) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resoln., trans-decahydroisoquinolin derivs. were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CLpro, which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CLpro inhibitor. X-ray crystallog. analyses of the SARS 3CLpro in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as addnl. interactions at the N-substituent of the inhibitor.
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Jacobs, J.; Grum-Tokars, V.; Zhou, Y.; Turlington, M.; Saldanha, S. A.; Chase, P.; Eggler, A.; Dawson, E. S.; Baez-Santos, Y. M.; Tomar, S.; Mielech, A. M.; Baker, S. C.; Lindsley, C. W.; Hodder, P.; Mesecar, A.; Stauffer, S. R. Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease. J. Med. Chem. 2013, 56, 534– 546, DOI: 10.1021/jm301580n
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110
Discovery, Synthesis, And Structure-Based Optimization of a Series of N-(tert-Butyl)-2-(N-arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease
Jacobs, Jon; Grum-Tokars, Valerie; Zhou, Ya; Turlington, Mark; Saldanha, S. Adrian; Chase, Peter; Eggler, Aimee; Dawson, Eric S.; Baez-Santos, Yahira M.; Tomar, Sakshi; Mielech, Anna M.; Baker, Susan C.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew; Stauffer, Shaun R.
Journal of Medicinal Chemistry (2013), 56 (2), 534-546CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
A high-throughput screen of the NIH mol. libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compd. ML188 (I), [(R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844]. Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, I is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S1', S1, and S2 enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with I was instrumental in guiding subsequent rounds of chem. optimization. I provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.
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111
Barretto, N.; Jukneliene, D.; Ratia, K.; Chen, Z.; Mesecar, A. D.; Baker, S. C. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J. Virol. 2005, 79, 15189– 15198, DOI: 10.1128/JVI.79.24.15189-15198.2005
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111
The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity
Barretto, Naina; Jukneliene, Dalia; Ratia, Kiira; Chen, Zhongbin; Mesecar, Andrew D.; Baker, Susan C.
Journal of Virology (2005), 79 (24), 15189-15198CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics anal. on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Mol. modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approx. 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the crit. determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.
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112
Turlington, M.; Chun, A.; Tomar, S.; Eggler, A.; Grum-Tokars, V.; Jacobs, J.; Daniels, J. S.; Dawson, E.; Saldanha, A.; Chase, P.; Baez-Santos, Y. M.; Lindsley, C. W.; Hodder, P.; Mesecar, A. D.; Stauffer, S. R. Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding. Bioorg. Med. Chem. Lett. 2013, 23, 6172– 6177, DOI: 10.1016/j.bmcl.2013.08.112
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112
Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-((benzyl)acetamido)phenyl carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: Identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding
Turlington, Mark; Chun, Aspen; Tomar, Sakshi; Eggler, Aimee; Grum-Tokars, Valerie; Jacobs, Jon; Daniels, J. Scott; Dawson, Eric; Saldanha, Adrian; Chase, Peter; Baez-Santos, Yahira M.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew D.; Stauffer, Shaun R.
Bioorganic & Medicinal Chemistry Letters (2013), 23 (22), 6172-6177CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Mol. Libraries Probe Prodn. Centers Network (MLPCN). In addn. to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The x-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.
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Turlington, M.; Chun, A.; Jacobs, J.; Dawson, E.; Daniels, J. S.; Saldanha, A.; Chase, P.; Hodder, P.; Eggler, A.; Tokars, V.; Mesecar, A.; Lindsley, C. W.; Stauffer, S. R. Noncovalent triazole-based inhibitors of the SARS main proteinase 3CLpro. Probe Reports from the NIH Molecular Libraries Program; National Center for Biotechnology Information: Bethesda, MD, 2012; http://www.ncbi.nlm.nih.gov/books/NBK143547/.
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Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430– 431, DOI: 10.1016/S1359-6446(04)03069-7
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114
Ligand efficiency: a useful metric for lead selection
Hopkins Andrew L; Groom Colin R; Alex Alexander
Drug discovery today (2004), 9 (10), 430-1 ISSN:1359-6446.
There is no expanded citation for this reference.
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115
For information on MLPCN’s probe compound ancillary screen, see Eurofins LeadProfilingScreen: www.eurofinspanlabs.com. May 18, 2004.
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116
Someya, Y.; Takeda, N.; Miyamura, T. Characterization of the norovirus 3C-like protease. Virus Res. 2005, 110, 91– 97, DOI: 10.1016/j.virusres.2005.02.002
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116
Characterization of the norovirus 3C-like protease
Someya, Yuichi; Takeda, Naokazu; Miyamura, Tatsuo
Virus Research (2005), 110 (1-2), 91-97CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temp., salt contents, and SH reagents on its proteolytic activity. The optimal pH and temp. of the 3C-like protease for the proteolytic activity were 8.6 and 37 °C, resp. Increased concn. (∼100 mM) of monovalent cations such as Na+ and K+ was inhibitory to the activity. Hg2+ and Zn2+ remarkably inhibited the protease activity, while Mg2+ and Ca2+ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, Me methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.
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Han, Y. S.; Chang, G. G.; Juo, C. G.; Lee, H. J.; Yeh, S. H.; Hsu, J. T.; Chen, X. Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. Biochemistry 2005, 44, 10349– 10359, DOI: 10.1021/bi0504761
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117
Papain-Like Protease 2 (PLP2) from Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV): Expression, Purification, Characterization, and Inhibition
Han, Yu-San; Chang, Gu-Gang; Juo, Chiun-Gung; Lee, Hong-Jen; Yeh, Shiou-Hwei; Hsu, John Tsu-An; Chen, Xin
Biochemistry (2005), 44 (30), 10349-10359CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as detd. by reversed-phase high-performance liq. chromatog. anal. coupled with mass spectrometry. This protease is esp. selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S-/(His)-Im+H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymic activity of SARS-CoV PLP2. In addn., we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochem. properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.
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Dhanak, D.; Burton, G.; Christmann, L. T.; Darcy, M. G.; Elrod, K. C.; Kaura, A.; Keenan, R. M.; Link, J. O.; Peishoff, C. E.; Shah, D. H. Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease. Bioorg. Med. Chem. Lett. 2000, 10, 2279– 2282, DOI: 10.1016/S0960-894X(00)00462-5
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118
Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease
Dhanak, D.; Burton, G.; Christmann, L. T.; Darcy, M. G.; Elrod, K. C.; Kaura, A.; Keenan, R. M.; Link, J. O.; Peishoff, C. E.; Shah, D. H.
Bioorganic & Medicinal Chemistry Letters (2000), 10 (20), 2279-2282CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
A versatile synthetic route to a novel series of diimidazolemethanes designed to inhibit the hCMV protease was developed and a series of potential metal-binding inhibitors was identified. In selectivity assays, the compds. were highly specific for CMV protease and showed no inhibition (IC50 > 100 μM) of other prototypical serine proteases such as trypsin, elastase, and chymotrypsin. Although the presence of free Zn ions was found to be an abs. requirement for the in-vitro biol. activity of this class of inhibitor, the potency of the inhibitors could not be improved beyond the micromolar level.
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Tong, L.; Qian, C.; Massariol, M.-J.; Bonneau, P. R.; Cordingley, M. G.; Lagace, L. A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease. Nature 1996, 383, 272– 275, DOI: 10.1038/383272a0
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119
A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease
Tong, Liang; Qian, Chungeng; Massariol, Marie-Josee; Bonneau, Pierre R.; Cordingley, Michael G.; Lagace, Lisette
Nature (London) (1996), 383 (6597), 272-275CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
Human cytomegalovirus (HCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. HCMV protease is essential for the prodn. of mature infections virions, as it performs proteolytic processing near the C-terminus (M-site) of the viral assembly protein precursor. HCMV protease is a serine protease, although it has little homol. to other clans of serine proteases. Here, the crystal structure of HCMV protease at 2.0 Å resoln. is reported, and it is shown to possess a new polypeptide backbone fold. Ser-132 and His-63 are found in close proximity in the active site, confirming earlier biochem. and mutagenesis studies. The structure suggests that the third member of the triad is probably His-157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.
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120
Yeung, K. S.; Meanwell, N. A.; Qiu, Z.; Hernandez, D.; Zhang, S.; McPhee, F.; Weinheimer, S.; Clark, J. M.; Janc, J. W. Structure-activity relationship studies of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of HCV NS3 serine protease. Bioorg. Med. Chem. Lett. 2001, 11, 2355– 2359, DOI: 10.1016/S0960-894X(01)00457-7
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120
Structure-activity relationship studies of a bisbenzimidazole-based, Zn2+-dependent inhibitor of HCV NS3 serine protease
Yeung, K.-S.; Meanwell, N. A.; Qiu, Z.; Hernandez, D.; Zhang, S.; McPhee, F.; Weinheimer, S.; Clark, J. M.; Janc, J. W.
Bioorganic & Medicinal Chemistry Letters (2001), 11 (17), 2355-2359CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn2+-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compds. with extended side chains. However, Zn2+-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examd. but dependent on the presence of neg. charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn2+, with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.
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121
Hsu, J. T.-A.; Kuo, C.-J.; Hsieh, H.-P.; Wang, Y.-C.; Huang, K.-K.; Lin, C. P.-C.; Huang, P.-F.; Chen, X.; Liang, P.-H. Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-CoV. FEBS Lett. 2004, 574, 116– 120, DOI: 10.1016/j.febslet.2004.08.015
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121
Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-CoV
Hsu, John T.-A.; Kuo, Chih-Jung; Hsieh, Hsing-Pang; Wang, Yeau-Ching; Huang, Kuo-Kuei; Lin, Coney P.-C.; Huang, Ping-Fang; Chen, Xin; Liang, Po-Huang
FEBS Letters (2004), 574 (1-3), 116-120CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
3C-like (3CL) protease is essential for the life cycle of severe acute respiratory syndrome-coronavirus (SARS-CoV) and therefore represents a key anti-viral target. A compd. library consisting of 960 com. available drugs and biol. active substances was screened for inhibition of SARS-CoV 3CL protease. Potent inhibition was achieved using the mercury-contg. compds. thimerosal and phenylmercuric acetate, as well as hexachlorophene. As well, 1 - 10 μM of each compd. inhibited viral replication in Vero E6 cell culture. Detailed mechanism studies using a fluorescence-based protease assay demonstrated that the three compds. acted as competitive inhibitors (Ki = 0.7, 2.4, and 13.7 μM for phenylmercuric acetate, thimerosal, and hexachlorophene, resp.). A panel of metal ions including Zn2+ and its conjugates were then evaluated for their anti-3CL protease activities. Inhibition was more pronounced using a zinc-conjugated compd. (1-hydroxypyridine-2-thione zinc; Ki = 0.17 μM) than using the ion alone (Ki = 1.1 μM).
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Lee, C.-C.; Kuo, C.-J.; Hsu, M.-F.; Liang, P.-H.; Fang, J.-M.; Shie, J.-J.; Wang, A. H.-J. Structural basis of mercury- and zinc-conjugated complexes as SARS-CoV 3C-like protease inhibitors. FEBS Lett. 2007, 581, 5454– 5458, DOI: 10.1016/j.febslet.2007.10.048
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122
Structural basis of mercury- and zinc-conjugated complexes as SARS-CoV 3C-like protease inhibitors
Lee, Cheng-Chung; Kuo, Chih-Jung; Hsu, Min-Feng; Liang, Po-Huang; Fang, Jim-Min; Shie, Jiun-Jie; Wang, Andrew H.-J.
FEBS Letters (2007), 581 (28), 5454-5458CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease of severe acute respiratory syndrome (SARS)-assocd. coronavirus were analyzed crystallog. The complex structures reveal two major inhibition modes: Hg2+-PMA is coordinated to C44, M49 and Y54 with a square planar geometry at the S3 pocket, whereas each Zn2+ of the four zinc-inhibitors is tetrahedrally coordinated to the H41-C145 catalytic dyad. For anti-SARS drug design, this Zn2+-centered coordination pattern would serve as a starting platform for inhibitor optimization.
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Katz, B. A.; Clark, J. M.; Finer-Moore, J. S.; Jenkins, T. E.; Johnson, C. R.; Ross, M. J.; Luong, C.; Moore, W. R.; Stroud, R. M. Design of potent selective zinc-mediated serine protease inhibitors. Nature 1998, 391, 608– 612, DOI: 10.1038/35422
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123
Design of potent selective zinc-mediated serine protease inhibitors
Katz, Bradley A.; Clark, James M.; Finer-Moore, Janet S.; Jenkins, Thomas E.; Johnson, Charles R.; Ross, Michael J.; Luong, Christine; Moore, William R.; Stroud, Robert M.
Nature (London) (1998), 391 (6667), 608-612CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
Many serine proteases are targets for therapeutic intervention because they often play key roles in disease. Small mol. inhibitors of serine proteases with high affinity are esp. interesting as they could be used as scaffolds from which to develop drugs selective for protease targets. One such inhibitor is bis(5-amidino-2-benzimidazolyl)methane (BABIM), standing out as the best inhibitor of trypsin (by a factor of over 100) in over 60 relatively closely related analogs. By probing the structural basis of inhibition, the authors discovered, using crystallog. methods, a new mode of high-affinity binding in which a Zn2+ ion is tetrahedrally coordinated between two chelating nitrogens of BABIM and two active site residues, His 57 and Ser 195, Zn2+, at sub-physiol. levels, enhances inhibition by over 103-fold. The distinct Zn2+ coordination geometry implies a strong dependence of affinity on substituents. This unique structural paradigm has enabled development of potent, highly selective, Zn2+-dependent inhibitors of several therapeutically important serine proteases, using a physiol. ubiquitous metal ion.
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Christianson, D. W.; Lipscomb, W. N. X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 7568– 7572, DOI: 10.1073/pnas.83.20.7568
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124
X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature
Christianson, David W.; Lipscomb, William N.
Proceedings of the National Academy of Sciences of the United States of America (1986), 83 (20), 7568-72CODEN: PNASA6; ISSN:0027-8424.
A high-resoln. x-ray crystallog. investigation of the complex between carboxypeptidase A (EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was carried out at -9°. Although this enzyme-substrate complex has been the subject of earlier crystallog. investigation, a higher resoln. electron-d. map of the complex with greater occupancy of the substrate was desired. All crystal chem. (i.e., crystal soaking and x-ray data collection) was performed in a diffractometer-mounted flow cell in which the crystal was immobilized. The x-ray data to 1.6-Å resoln. yielded a well-resolved structure in which the Zn2+ of the active site is 5-coordinate: 3 enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl O and N-terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important Zn2+-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected N-terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.
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125
Brewer, G. J.; Johnson, V. D.; Dick, R. D.; Hedera, P.; Fink, J. K.; Kluin, K. J. Treatment of Wilson’s disease with zinc. XVII: Treatment during pregnancy. Hepatology 2000, 31, 364– 370, DOI: 10.1002/hep.510310216
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125
Treatment of wilson's disease with zinc. XVII: treatment during pregnancy
Brewer, George J.; Johnson, Virginia D.; Dick, Robert D.; Hedera, Peter; Fink, John K.; Kluin, Karen J.
Hepatology (Philadelphia) (2000), 31 (2), 364-370CODEN: HPTLD9; ISSN:0270-9139. (W. B. Saunders Co.)
Therapy of Wilson's disease continues to evolve. In 1997, zinc acetate was added to the list of drugs approved by the Food and Drug Administration, which includes penicillamine and trientine. The mechanism of zinc's anticopper action is unique. It induces intestinal cell metallothionein, which binds copper and prevents its transfer into blood. As intestinal cells die and slough, the contained copper is eliminated in the stool. Thus, zinc prevents the intestinal absorption of copper. It is universally agreed that pregnant Wilson's disease patients should remain on anticopper therapy during pregnancy. There are numerous reports of such patients stopping penicillamine therapy to protect their fetus from teratogenicity, only to undergo serious deterioration and even death from renewed copper toxicity. Penicillamine and trientine have teratogenic effects in animals, and penicillamine has known teratogenic effects in humans. In this report we discuss the results of 26 pregnancies in 19 women who were on zinc therapy throughout their pregnancy. The evidence is good that zinc protects the health of the mother during pregnancy. Fetal outcomes were generally quite good, although one baby had a surgically correctable heart defect and one had microcephaly.
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126
Sharquie, K. E.; Najim, R. A.; Al-Dori, W. S.; Al-Hayani, R. K. Oral zinc sulfate in the treatment of Behcet’s disease: a double blind cross-over study. J. Dermatol. 2006, 33, 541– 546, DOI: 10.1111/j.1346-8138.2006.00128.x
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126
Oral zinc sulfate in the treatment of Behcet's disease: a double blind cross-over study
Sharquie, Khalifa E.; Najim, Rafid A.; Al-Dori, Wisam S.; Al-Hayani, Raafa K.
Journal of Dermatology (2006), 33 (8), 541-546CODEN: JDMYAG; ISSN:0385-2407. (Blackwell Publishing Asia Pty Ltd.)
This was a randomized, controlled, double-blind trial of zinc sulfate in the treatment of Behcet's disease. Patients with Behcet's disease were recruited in this study between Nov. 2001 and Feb. 2003. A clin. manifestations index (CMI) was calcd. for each patient. Serum zinc was estd. in all patients both at the beginning and monthly throughout the trial. Serum zinc levels were estd. from 30 healthy normal subjects matched for age and sex as a control group. Patients were randomly allocated to receive either 100 mg zinc sulfate or identical placebo tablet three times daily in a double-blind manner. After 3 mo of starting treatment, patients were crossed over, i.e., patients on placebo received zinc sulfate and vice versa. Mean serum zinc level in Behcet's disease patients was statistically significantly lower than mean serum zinc levels in healthy the control. In group A (started with zinc sulfate), the mean CMI started to decline directly after the first month of therapy with zinc sulfate to significantly lower levels. After shifting to placebo treatment in the fourth month, the mean of CMI started to rise again gradually but remained significantly lower than levels before therapy for the fourth and fifth months. In group B (started with placebo), the mean of CMI remained high for the first 3 mo. After crossing over to zinc sulfate in the fourth month, the mean of CMI started to decrease after the fourth month. An inverse correlation between CMI and serum zinc level was found. No side-effects were seen in either group. In conclusion, zinc sulfate was found to be a good option in the treatment of Behcet's disease.
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Sakurai, H.; Adachi, Y. The pharmacology of the insulinomimetic effect of zinc complexes. BioMetals 2005, 18, 319– 323, DOI: 10.1007/s10534-005-3688-8
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127
The Pharmacology of the Insulinomimetic Effect of Zinc Complexes
Sakurai, Hiromu; Adachi, Yusuke
BioMetals (2005), 18 (4), 319-323CODEN: BOMEEH; ISSN:0966-0844. (Springer)
A review. In developing new insulinomimetic zinc(II) complexes with different coordination structures and with a blood glucose-lowering effect to treat type 2 diabetic animals, we found a potent bis(maltolato)zinc(ll) complex, Zn(mal)2. Using the complex as the leading compd., we examd. the in vitro and in vivo structure-activity relationships of Zn(mal)2 and its related complexes in respect to the inhibition of free fatty acids (FFA) release and the enhancement of glucose uptake in isolated rat adipocytes treated with epinephrine (adrenaline), and hypoglycemic activity. Among the compds. tested, a new Zn(II) complex with allixin that was isolated from garlic, bis(allixinato)Zn(II), Zn(alx)2, was found to exhibit the highest insulin-mimetic and hypoglycemic activities in type 2 KK-Ay diabetic mice. On the basis of the results, Zn(alx)2, complex was proposed to be a potent candidate for the treatment of type 2 diabetes.
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128
Bacha, U.; Barrila, J.; Velazquez-Campoy, A.; Leavitt, S. A.; Freire, E. Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro. Biochemistry 2004, 43, 4906– 4912, DOI: 10.1021/bi0361766
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128
Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro
Bacha, Usman; Barrila, Jennifer; Velazquez-Campoy, Adrian; Leavitt, Stephanie A.; Freire, Ernesto
Biochemistry (2004), 43 (17), 4906-4912CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CLpro (also termed SARS-CoV 3CLpro). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CLpro as well as a truncated form contg. only the catalytic domains. The recombinant proteins have been characterized enzymically using a fluorescently labeled substrate; their structural stability in soln. has been detd. by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the α-helical domain. Differential scanning calorimetry indicates that the α-helical domain does not contribute to the structural stability of the catalytic domains. Anal. of the active site cavity reveals the presence of subsites that can be targeted with specific chem. functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compds. contg. boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compds. were particularly effective at inhibiting the protease, with inhibition consts. as strong as 40 nM. Isothermal titrn. microcalorimetric expts. indicate that these inhibitors bind reversibly to 3CLpro in an enthalpically favorable fashion, implying that they establish strong interactions with the protease mol., thus defining attractive mol. scaffolds for further optimization.
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Hou, T. J.; Xu, X. J. Recent development and application of virtual screening in drug discovery: an overview. Curr. Pharm. Des. 2004, 10, 1011– 1033, DOI: 10.2174/1381612043452721
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129
Recent development and application of virtual screening in drug discovery: an overview
Hou, Tingjun; Xu, Xiaojie
Current Pharmaceutical Design (2004), 10 (9), 1011-1033CODEN: CPDEFP; ISSN:1381-6128. (Bentham Science Publishers Ltd.)
A review. Virtual screening, esp. the structure-based virtual screening, has emerged as a reliable, cost-effective and time-saving technique for the discovery of lead compds. Here, the basic ideas and computational tools for virtual screening have been briefly introduced, and emphasis is placed on aspects of recent development of docking-based virtual screening, scoring functions in mol. docking and ADME/Tox-based virtual screening in the past three years (2000 to 2003). Moreover, successful examples are provided to further demonstrate the effectiveness of virtual screening in drug discovery.
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Andricopulo, A. D.; Guido, R. V. C.; Oliva, G. Virtual screening and its integration with modern drug design technologies. Curr. Med. Chem. 2008, 15, 37– 46, DOI: 10.2174/092986708783330683
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130
Virtual screening and its integration with modern drug design technologies
Guido, Rafael V. C.; Oliva, Glaucius; Andricopulo, Adriano D.
Current Medicinal Chemistry (2008), 15 (1), 37-46CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
A review. Drug discovery is a highly complex and costly process, which demands integrated efforts in several relevant aspects involving innovation, knowledge, information, technologies, expertise, R&D investments and management skills. The shift from traditional to genomics- and proteomics-based drug research has fundamentally transformed key R&D strategies in the pharmaceutical industry addressed to the design of new chem. entities as drug candidates against a variety of biol. targets. Therefore, drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. The combination of available knowledge of several 3D protein structures with hundreds of thousands of small-mols. have attracted the attention of scientists from all over the world for the application of structure- and ligand-based drug design approaches. In this context, virtual screening technologies have largely enhanced the impact of computational methods applied to chem. and biol. and the goal of applying such methods is to reduce large compd. databases and to select a limited no. of promising candidates for drug design. This review provides a perspective of the utility of virtual screening in drug design and its integration with other important drug discovery technologies such as high-throughput screening (HTS) and QSAR, highlighting the present challenges, limitations, and future perspectives in medicinal chem.
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131
Kao, R. Y.; Tsui, W. H. W.; Lee, T. S. W.; Tanner, J. A.; Watt, R. M.; Huang, J. D.; Hu, L. H.; Chen, G. H.; Chen, Z. W.; Zhang, L. Q.; He, T.; Chan, K. H.; Tse, H.; To, A. P. C.; Ng, L. W. Y.; Wong, B. C. W.; Tsoi, H. W.; Yang, D.; Ho, D. D.; Yuen, K. Y. Identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics. Chem. Biol. 2004, 11, 1293– 1299, DOI: 10.1016/j.chembiol.2004.07.013
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131
Identification of Novel Small-Molecule Inhibitors of Severe Acute Respiratory Syndrome-Associated Coronavirus by Chemical Genetics
Kao, Richard Y.; Tsui, Wayne H. W.; Lee, Terri S. W.; Tanner, Julian A.; Watt, Rory M.; Huang, Jian-Dong; Hu, Lihong; Chen, Guanhua; Chen, Zhiwei; Zhang, Linqi; He, Tian; Chan, Kwok-Hung; Tse, Herman; To, Amanda P. C.; Ng, Louisa W. Y.; Wong, Bonnie C. W.; Tsoi, Hoi-Wah; Yang, Dan; Ho, David D.; Yuen, Kwok-Yung
Chemistry & Biology (2004), 11 (9), 1293-1299CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
The severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) infected more than 8,000 people across 29 countries and caused more than 900 fatalities. Based on the concept of chem. genetics, we screened 50,240 structurally diverse small mols. from which we identified 104 compds. with anti-SARS-CoV activity. Of these 104 compds., 2 target the SARS-CoV main protease (Mpro), 7 target helicase (Hel), and 18 target spike (S) protein-angiotensin-converting enzyme 2 (ACE2)-mediated viral entry. The EC50 of the majority of the 104 compds. detd. by SARS-CoV plaque redn. assay were found to be at low micromolar range. Three selected compds., MP576, HE602, and VE607, validated to be inhibitors of SARS-CoV Mpro, Hel, and viral entry, resp., exhibited potent antiviral activity (EC50 < 10 μM) and comparable inhibitory activities in target-specific in vitro assays.
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132
Maruyama, T.; Sato, Y.; Oto, Y.; Takahashi, Y.; Snoeck, R.; Andrei, G.; Witvrouw, M.; De Clercq, E. Synthesis and antiviral activity of 6-chloropurine arabinoside and its 2′-deoxy-2′-fluoro derivative. Chem. Pharm. Bull. 1996, 44, 2331– 2334, DOI: 10.1248/cpb.44.2331
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132
Synthesis and antiviral activity of 6-chloropurine arabinoside and its 2'-deoxy-2'-fluoro derivative
Maruyama, Tokumi; Sato, Yoshiko; Oto, Yuri; Takahashi, Yuka; Snoeck, Robert; Andrei, Graciela; Witvrouw, Myriam; De Clercq, Erik
Chemical & Pharmaceutical Bulletin (1996), 44 (12), 2331-2334CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)
Title nucleosides I [R = OH, (II) F] were prepd. as virucides. The antiviral effects of I were assayed against several DNA and RNA viruses. Only II displayed potent activity against varicella-zoster virus (VZV). This antiviral activity was dependent on phosphorylation by the VZV-induced thymidine kinase (TK). Compd. II showed moderate activity against other DNA viruses, herpes simplex type 1 (HSV-1) and type 2 (HSV-2), and vaccinia virus. They were equally active against TK- and TK+ strains of HSV-1, which suggests that the HSV-1-encoded TK does not play a role in the anti-HSV-1 activity. No activity was noted with any of the compds. against various RNA viruses, including human immunodeficiency virus, at subtoxic concns.
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133
Honjo, M.; Maruyama, T.; Horikawa, M.; Balzarini, J.; De Clercq, E. Synthesis and biological evaluation of phosphonopyrimidine and phosphonopurine ribonucleosides. Chem. Pharm. Bull. 1987, 35, 3227– 3234, DOI: 10.1248/cpb.35.3227
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133
Synthesis and biological evaluation of phosphonopyrimidine and phosphonopurine ribonucleosides
Honjo, Mikio; Maruyama, Tokumi; Horikawa, Mitzuyo; Balzarini, Jan; De Clercq, Erik
Chemical & Pharmaceutical Bulletin (1987), 35 (8), 3227-34CODEN: CPBTAL; ISSN:0009-2363.
Treatment of lithiated 2',3',5'-tri-O-protected uridine and 6-chloropurine ribonucleoside with ClP(O)(OEt)2, followed by deblocking, amination, and hydrolysis, provided 5- (I) and 6-phosphonouridine (II), and 8-phosphonoadenosine (III), resp. Arbuzov reaction of 2',3',5'-tri-O-protected 4-chloro-2(1H)-pyrimidinone ribonucleoside and P(OEt)3 afforded the 4-phosphonate deriv. I-III and their di-Et and monoethyl esters were inactive in vitro as antiviral and cytostatic agents, but di-Et 6-chloro-9-(β-D-ribofuranosyl)purine-8-phosphonate showed some antiviral and cytostatic activities, which were comparable in all respects to those of the unphosphonylated compd.
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134
Ikejiri, M.; Saijo, M.; Morikawa, S.; Fukushi, S.; Mizutani, T.; Kurane, I.; Maruyama, T. Synthesis and biological evaluation of nucleoside analogues having 6-chloropurine as anti-SARS-CoV agents. Bioorg. Med. Chem. Lett. 2007, 17, 2470– 2473, DOI: 10.1016/j.bmcl.2007.02.026
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134
Synthesis and biological evaluation of nucleoside analogs having 6-chloropurine as anti-SARS-CoV agents
Ikejiri, Masahiro; Saijo, Masayuki; Morikawa, Shigeru; Fukushi, Shuetsu; Mizutani, Tetsuya; Kurane, Ichiro; Maruyama, Tokumi
Bioorganic & Medicinal Chemistry Letters (2007), 17 (9), 2470-2473CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
Nucleoside analogs that have 6-chloropurine as the nucleobase were synthesized and evaluated for anti-SARS-CoV activity by plaque redn. and yield redn. assays in order to develop novel anti-SARS-CoV agents. Among these analogs, two compds., namely, 1 and 11, exhibited promising anti-SARS-CoV activity that was comparable to those of mizoribine and ribavirin, which are known anti-SARS-CoV agents. Moreover, we obsd. several SAR trends such as the antiviral effects of the 6-chloropurine moiety, unprotected 5'-hydroxyl group and benzoylated 5'-hydroxyl group.
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135
Koren, G.; King, S.; Knowles, S.; Phillips, E. Ribavirin in the treatment of SARS: A new trick for an old drug?. Can. Med. Assoc. J. 2003, 168, 1289– 1292
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135
Ribavirin in the treatment of SARS: A new trick for an old drug?
Koren Gideon; King Susan; Knowles Sandra; Phillips Elizabeth
CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne (2003), 168 (10), 1289-92 ISSN:0820-3946.
There is no expanded citation for this reference.
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136
Stroher, U.; DiCaro, A.; Li, Y.; Strong, J. E.; Aoki, F.; Plummer, F.; Jones, S. M.; Feldmann, H. Severe acute respiratory syndrome-related coronavirus is inhibited by interferon- alpha. J. Infect. Dis. 2004, 189, 1164– 1167, DOI: 10.1086/382597
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136
Severe acute respiratory syndrome-related coronavirus is inhibited by interferon- alpha
Stroher Ute; DiCaro Antonino; Li Yan; Strong James E; Aoki Fred; Plummer Frank; Jones Steven M; Feldmann Heinz
The Journal of infectious diseases (2004), 189 (7), 1164-7 ISSN:0022-1899.
Current treatment schemes for severe acute respiratory syndrome (SARS) include broad-spectrum antibiotics, glucocorticoids, and ribavirin. We evaluated the susceptibility of the SARS-related coronavirus (SARS CoV) to ribavirin and interferon (IFN)- alpha in vitro by use of cytopathic effect, plaque assay, and immunoblot analysis. Ribavirin did not inhibit viral growth at concentrations attainable in human serum. In contrast, IFN- alpha showed an in vitro inhibitory effect starting at concentrations of 1000 IU/mL. In conclusion, ribavirin alone is unlikely to be beneficial in the prophylaxis or treatment of SARS CoV infections. Clinical trials with IFN- alpha might be justified to determine a beneficial effect on the outcome of SARS.
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137
Yamamoto, N.; Yang, R.; Yoshinaka, Y.; Amari, S.; Nakano, T.; Cinatl, J.; Rabenau, H.; Doerr, H. W.; Hunsmann, G.; Otaka, A.; Tamamura, H.; Fujii, N.; Yamamoto, N. HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus. Biochem. Biophys. Res. Commun. 2004, 318, 719– 725, DOI: 10.1016/j.bbrc.2004.04.083
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137
HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus
Yamamoto, Norio; Yang, Rongge; Yoshinaka, Yoshiyuki; Amari, Shinji; Nakano, Tatsuya; Cinatl, Jindrich; Rabenau, Holger; Doerr, Hans Wilhelm; Hunsmann, Gerhard; Otaka, Akira; Tamamura, Hirokazu; Fujii, Nobutaka; Yamamoto, Naoki
Biochemical and Biophysical Research Communications (2004), 318 (3), 719-725CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Science)
A novel coronavirus has been identified as an etiol. agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clin. use, we screened a set of compds. that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quant. RT-PCR anal. showed that nelfinavir could decrease the prodn. of virions from Vero cells. Expts. with various timings of drug addn. revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examd. clin. for the treatment of SARS and has potential as a good lead compd. for designing anti-SARS drugs.
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Keyaerts, E.; Vijgen, L.; Maes, P.; Neyts, J.; Ranst, M. V. In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine. Biochem. Biophys. Res. Commun. 2004, 323, 264– 268, DOI: 10.1016/j.bbrc.2004.08.085
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138
In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine
Keyaerts, Els; Vijgen, Leen; Maes, Piet; Neyts, Johan; Van Ranst, Marc
Biochemical and Biophysical Research Communications (2004), 323 (1), 264-268CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clin. approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8±1.2 μM) was significantly lower than its cytostatic activity; CC50 (261.3 ± 14.5 μM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concns. of chloroquine reached during treatment of acute malaria. Addn. of chloroquine to infected cultures could be delayed for up to 5 h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.
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Tan, E. L. C.; Ooi, E. E.; Lin, C. Y.; Tan, H. C.; Ling, A. E.; Lim, B.; Stanton, L. W. Inhibition of SARS coronavirus infection in vitro with clinically approved antiviral drugs. Emerging Infect. Dis. 2004, 10, 581– 586, DOI: 10.3201/eid1004.030458
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139
Inhibition of SARS coronavirus infection in vitro with clinically approved antiviral drugs
Tan, Emily L. C.; Ooi, Eng Eong; Lin, Chin-Yo; Tan, Hwee Cheng; Ling, Ai Ee; Lim, Bing; Stanton, Lawrence W.
Emerging Infectious Diseases (2004), 10 (4), 581-586CODEN: EIDIFA; ISSN:1080-6040. (National Center for Infectious Diseases, Centers for Disease Control and Prevention)
Severe acute respiratory syndrome (SARS) is an infectious disease caused by a newly identified human coronavirus (SARS-CoV). Currently, no effective drug exists to treat SARS-CoV infection. In this study, we investigated whether a panel of com. available antiviral drugs exhibit in vitro anti-SARS-CoV activity. A drug-screening assay that scores for virus-induced cytopathic effects on cultured cells was used. Tested were 19 clin. approved compds. from several major antiviral pharmacol. classes: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors, and neuraminidase inhibitors. Complete inhibition of cytopathic effects of SARS-CoV in culture was obsd. for interferon subtypes, β-1b, α-n1, α-n3, and human leukocyte interferon a. These findings support clin. testing of approved interferons for the treatment of SARS.
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140
Liu, Y. C.; Huang, V.; Chao, T. C.; Hsiao, C. D.; Lin, A.; Chang, M. F.; Chow, L. P. Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease. Biochem. Biophys. Res. Commun. 2005, 333, 194– 199, DOI: 10.1016/j.bbrc.2005.05.095
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140
Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease
Liu, Yu-Chih; Huang, Vicky; Chao, Ti-Chun; Hsiao, Chwan-Deng; Lin, Atsui; Chang, Ming-Fu; Chow, Lu-Ping
Biochemical and Biophysical Research Communications (2005), 333 (1), 194-199CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examd., hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concns. (K i = 4 μM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biol. evaluation of various hexachlorophene analogs were described. These analogs gave optimal inhibitory activity against SARS-CoV 3CL protease with IC50 values ranging from 7.6 to 84.5 μM. Optimization of hexachlorophene analogs was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.
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141
Nguyen, T. T. H.; Ryu, H.-J.; Lee, S.-H.; Hwang, S. W.; Breton, V.; Rhee, J. H.; Kim, D. Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation. Bioorg. Med. Chem. Lett. 2011, 21, 3088– 3091, DOI: 10.1016/j.bmcl.2011.03.034
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141
Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation
Nguyen, Thi Thanh Hanh; Ryu, Hwa-Ja; Lee, Se-Hoon; Hwang, Soonwook; Breton, Vincent; Rhee, Joon Haeng; Kim, Doman
Bioorganic & Medicinal Chemistry Letters (2011), 21 (10), 3088-3091CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
The 3C-like protease (3CLpro) of severe acute respiratory syndrome assocd. coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chem. compds. was performed using the computation tool Autodock 3.0.5 on a WISDOM Prodn. Environment. The top 1468 ranked compds. with free binding energy ranging from -14.0 to -17.09 kcal mol-1 were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CLpro. Fifty-three compds. from 35 main groups were tested in an in vitro assay for inhibition of 3CLpro expressed by Escherichia coli. Seven of the 53 compds. were selected; their IC50 ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CLpro inhibitors were further identified as competitive inhibitors of 3CLpro with K i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compd. with amino acid residues in the active site of 3CLpro were also identified.
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Lee, H.; Mittal, A.; Patel, K.; Gatuz, J. L.; Truong, L.; Torres, J.; Mulhearn, D. C.; Johnson, M. E. Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings. Bioorg. Med. Chem. 2014, 22, 167– 177, DOI: 10.1016/j.bmc.2013.11.041
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142
Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings
Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L.; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C.; Johnson, Michael E.
Bioorganic & Medicinal Chemistry (2014), 22 (1), 167-177CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small mol. inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compds. from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymic HTS assay was developed and optimized to exptl. screen approx. 41,000 compds. from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding anal. using surface plasmon resonance (SPR). The campaign identified a reversible small mol. inhibitor exhibiting mixed-type inhibition with a Ki value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chem. libraries, and the newly identified compd. reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.
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Steinmetzer, T.; Hauptmann, J.; Sturzebecher, J. Advances in the development of thrombin inhibitors. Expert Opin. Invest. Drugs 2001, 10, 845– 864, DOI: 10.1517/13543784.10.5.845
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143
Advances in the development of thrombin inhibitors
Steinmetzer, Torsten; Hauptmann, Jorg; Sturzebecher, Jorg
Expert Opinion on Investigational Drugs (2001), 10 (5), 845-864CODEN: EOIDER; ISSN:1354-3784. (Ashley Publications Ltd.)
A review with 122 refs. Thromboembolic diseases are a major cause of morbidity and mortality, particularly in the Western world, which has stimulated enormous research efforts by the pharmaceutical industry to introduce new antithrombotic therapies. One strategy is the development of direct inhibitors of the serine protease thrombin, which holds a central position in the final steps of the blood coagulation cascade and in platelet activation. At present these is only limited clin. use of some parenteral prepns. of thrombin inhibitors in acute situations, esp. when the common antithrombotic drugs heparin, warfarin and aspirin are ineffective or assocd. with side effects. However, for use in prophylaxis of thrombotic diseases such inhibitors should be orally available, must be safe to avoid bleeding complications and should have an appropriate half-life, allowing once or twice daily dosing to maintain adequate antithrombotically effective blood levels. Details of several new and potent thrombin inhibitors have been published during the last years. For some of them oral bioavailability is claimed and they are effective in in vitro coagulation assays. However, most of them showed only limited efficacy in animal studies with respect to the doses administered. For that reason, effort is concd. on the evaluation and optimization of the overall physicochem. characteristics of the inhibitors in order to improve the pharmacokinetics and, thus, the development of promising drug candidates. Nevertheless, only careful clin. studies can give clear answers about the true therapeutical benefit of new developments in this field. This review summarizes the current status of direct thrombin inhibitors which are already in clin. use and clin. development and gives an overview on recently published and promising new compds.
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Klootwijk, P.; Lenderink, T.; Meij, S.; Boersma, H.; Melkert, R.; Umans, V. A.; Stibbe, J.; Müller, E. J.; Poortermans, K. J.; Deckers, J. W.; Simoons, M. L. Anticoagulant properties, clinical efficacy and safety of efegatran, a direct thrombin inhibitor, in patients with unstable angina. Eur. Heart J. 1999, 20, 1101– 1111, DOI: 10.1053/euhj.1999.1477
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144
Anticoagulant properties, clinical efficacy and safety of efegatran, a direct thrombin inhibitor, in patients with unstable angina
Klootwijk, P.; Lenderink, T.; Meij, S.; Boersma, H.; Melkert, R.; Umans, V. A. W.; Stibbe, J.; Muller, E. J.; Poortermans, K. J.; Deckers, J. W.; Simoons, M. L.
European Heart Journal (1999), 20 (15), 1101-1111CODEN: EHJODF; ISSN:0195-668X. (W. B. Saunders Co. Ltd.)
Aims Thrombin plays a key role in the clin. syndrome of unstable angina. We investigated the safety and efficacy of five dose levels of efegatran sulfate, a direct thrombin inhibitor, compared to heparin in patients with unstable angina. Methods Four hundred and thirty-two patients with unstable angina were enrolled. Five dose levels of efegatran were studied sequentially, ranging from 0.105 mg • kg-1 • h-1 to 1.2 mg • kg-1 • h-1 over 48 h. Safety was assessed clin., with ref. to bleeding and by measuring clin. lab. parameters. Efficacy was assessed by the no. of patients experiencing any episode of recurrent ischemia as measured by computer-assisted continuous ECG ischemia monitoring. Clin. end-points were: episodes of recurrent angina, myocardial infarction, coronary intervention (PTCA or CABG), and death. Results Efegatran demonstrated dose dependent ex-vivo anticoagulant activity with the highest dose level of 1.2 mg • kg-1 • h-1 resulting in steady state mean activated partial thromboplastin time values of approx. three times baseline. Thrombin time was also increased. Neither of the efegatran doses studied were able to suppress myocardial ischemia during continuous ECG ischemia monitoring to a greater extent than that seen with heparin. There were no statistically significant differences in clin. outcome or major bleeding between the efegatran and heparin groups. Minor bleeding and thrombophlebitis occurred more frequently in the efegatran treated patients. Conclusion Administration of efegatran sulfate at levels of at least 0.63 mg • kg-1 • h-1 provided an anti-thrombotic effect which is at least comparable to an activated partial thromboplastin time adjusted heparin infusion. There was no excess of major bleeding. The level of thrombin inhibition by efegatran, as measured by activated partial thromboplastin time, appeared to be more stable than with heparin. Thus, like other thrombin inhibitors, efegatran sulfate is easier to administer than heparin. However, no clin. benefits of efegatran over heparin were apparent.
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145
Esser, R. E.; Angelo, R. A.; Murphey, M. D.; Watts, L. M.; Thornburg, L. P.; Palmer, J. T.; Talhouk, J. W.; Smith, R. E. Cysteine proteinase inhibitors decrease articular cartilage and bone destruction in chronic inflammatory arthritis. Arthritis Rheum. 1994, 37, 236– 247, DOI: 10.1002/art.1780370213
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145
Cysteine proteinase inhibitors decrease articular cartilage and bone destruction in chronic inflammatory arthritis
Esser, Ronald E.; Angelo, Richard A.; Murphey, Mark D.; Watts, Lynnetta M.; Thornburg, Larry P.; Palmer, James T.; Talhouk, Jamil W.; Smith, Robert E.
Arthritis & Rheumatism (1994), 37 (2), 236-47CODEN: ARHEAW; ISSN:0004-3591.
The effects of peptidyl fluoromethyl ketones were detd. on the in vitro activity of purified cathepsins B and L, on tissue cysteine proteinase activity, and on cartilage and bone destruction in exptl. arthritis. The effects of the fluoroketones on cathepsins B and L in vitro and the effects of oral administration of fluoroketones on ex vivo cysteine proteinase activity in tissue homogenates were detd. by measuring the inhibition of fluorogenic substrate cleavage. To det. the effects on arthritis, animals were injected with adjuvant or type II collagen, treated orally with the fluoroketones, and the severity of arthritis was assessed by clin., histol., and radiol. methods. All of the fluoroketones tested were potent inhibitors of purified cathepsins B and L activity. Oral administration of the fluoroketones reduced tissue cysteine proteinase activity by up to 77%. In addn., fluoroketone treatment significantly reduced the severity of clin. joint disease and decreased the destruction of articular cartilage and bone. Quant. anal. of radiog. images indicated that treatment significantly reduced soft tissue changes, periosteal proliferation, and bone erosion, but only partially reduced juxtaarticular osteoporosis. These studies suggest that cysteine proteinase inhibitors may limit tissue destruction in diseases such as rheumatoid arthritis.
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146
McGrath, M. E.; Eakin, A. E.; Engel, J. C.; McKerrow, J. H.; Craik, C. S.; Fletterick, R. J. The crystal structure of cruzain: A therapeutic target for Chagas’ disease. J. Mol. Biol. 1995, 247, 251– 259, DOI: 10.1006/jmbi.1994.0137
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146
The crystal structure of cruzain: a therapeutic target for Chagas' disease
McGrath, Mary E.; Eakin, Ann E.; Engel, Juan C.; McKerrow, James H.; Craik, Charles C.; Fletterick, Robert J.
Journal of Molecular Biology (1995), 247 (2), 251-9CODEN: JMOBAK; ISSN:0022-2836. (Academic)
Trypanosoma cruzi, a protozoan parasite, is the etiol. agent of American trypanosomiasis or Chagas' disease. Chagas disease afflicts >24 million individuals in South and Central America producing a debilitating life-long disease. It is the leading cause of heart failure in many Latin American countries. Currently, there is no satisfactory treatment for this parasitic infection. Cruzain (also known as cruzipain; gp 57/51), the major cysteine protease present in T. cruzi, is crit. for the development and survival of the parasite within the host cells, making this enzyme a target for potential trypanocidal drugs. Here, the x-ray crystal structure of cruzain complexed with the potent inhibitor, Z-Phe-Ala-fluoromethyl ketone, is reported. The structure was detd. at 2.35 Å (Rcryst = 0.15) by mol. replacement using a modified papain as the search model. The refined structure was compared to papain. Features which distinguish cruzain from papain were discussed since they may aid in the design of specificity inhibitors. Fluorescence microscopy showed that a biotinylated form of the bound inhibitor did not effectively reach the host protease in their lysosomal compartment, but was selectively taken up by the parasite. The inhibitor greatly reduced parasitemia in a cell culture system, without adverse effects to mammalian cells. This biol. selectivity could be exploited, in conjunction with unique active site features revealed by the crystal structure, to develop chemotherapy for Chagas' disease.
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147
Richer, J. K.; Hunt, W. G.; Sakanari, J. A.; Grieve, R. B. Dirofilaria immitis: Effect of fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt. Exp. Parasitol. 1993, 76, 221– 231, DOI: 10.1006/expr.1993.1027
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147
Dirofilaria immitis: effect of fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt
Richer, Jennifer K.; Hunt, W. Garrett; Sakanari, Judy A.; Grieve, Robert B.
Experimental Parasitology (1993), 76 (3), 221-31CODEN: EXPAAA; ISSN:0014-4894.
D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl-Phe-Ala-CH2F (Z-Phe-Ala-CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concns. of inhibitor more larvae had initiated, but not completed, the molt. In addn. to Z-Phe-Ala-CH2F, four other fluoromethyl ketone derivs., Z-Phe-Arg-CH2F, amorpholine urea-(Mu)-Leu-Phe-CH2F, Mu-Tyr-Phe-CH2F, and Mu-Phe-Phe-CH2F, were tested to det. their effects on L3 in culture. All fluoromethyl ketones tested except Z-Phe-Arg-CH2F inhibited molting. Larvae cultured in inhibitors were detd. to be alive as judged qual. by motility and quant. by redn. of 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone deriv. had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone deriv. that did not inhibit molting, Z-Phe-Arg-CH2F, was a slightly less effective inhibitor of larval ext.-initiated hydrolysis of the synthetic peptide substrate, Z-Val-Leu-Arg-7-amino-4-methylcoumarin. L3 were also cultured through the molt in media contg. the synthetic peptide substrate Z-Val-Leu-Arg-4-methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fluorescence as seen on Days 0-4 during the molting process was first obsd. on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle.
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148
Barrila, J. A. Dimerization of the SARS coronavirus 3CL protease is controlled through long-range interactions. The Johns Hopkins University, ProQuest, UMI Dissertations Publishing, 2009, 3339678.
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149
Dyall, J.; Coleman, C. M.; Hart, B. J.; Venkataraman, T.; Holbrook, M. R.; Kindrachuk, J.; Johnson, R. F.; Olinger, G. G., Jr.; Jahrling, P. B.; Laidlaw, M.; Johansen, L. M.; Lear-Rooney, C. M.; Glass, P. J.; Hensley, L. E.; Frieman, M. B. Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob. Agents Chemother. 2014, 58, 4885– 4893, DOI: 10.1128/AAC.03036-14
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149
Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection
Dyall, Julie; Coleman, Christopher M.; Hart, Brit J.; Venkataraman, Thiagarajan; Holbrook, Michael R.; Kindrachuk, Jason; Johnson, Reed F.; Olinger, Gene G.; Jahrling, Peter B.; Laidlaw, Monique; Johansen, Lisa M.; Lear-Rooney, Calli M.; Glass, Pamela J.; Hensley, Lisa E.; Frieman, Matthew B.
Antimicrobial Agents and Chemotherapy (2014), 58 (8), 4885-4893, 10 pp.CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)
Outbreaks of emerging infections present health professionals with the unique challenge of trying to select appropriate pharmacol. treatments in the clinic with little time available for drug testing and development. Typically, clinicians are left with general supportive care and often untested convalescent-phase plasma as available treatment options. Repurposing of approved pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies, drug developers and funding agencies. Given the development times and manufg. requirements for new products, repurposing of existing drugs is likely the only soln. for outbreaks due to emerging viruses. In the studies described here, a library of 290 compds. was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compds. for inclusion in the library was dependent on current or previous FDA approval or advanced clin. development. Some drugs that had a well-defined cellular pathway as target were included. In total, 27 compds. with activity against both MERS-CoV and SARS-CoV were identified. The compds. belong to 13 different classes of pharmaceuticals, including inhibitors of estrogen receptors used for cancer treatment and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo studies as well as incorporation into ongoing clin. studies.
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(b) Pillaiyar, T.; Manickam, M.; Jung, S. H. Middle East respiratory syndrome coronavirus (MERS-CoV): An updated overview and pharmacotherapeutics. Med. Chem. 2015, 5, 361– 372, DOI: 10.4172/2161-0444.1000287
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150
Medeiros, R.; Kitazawa, M.; Chabrier, M. A.; Cheng, D.; Baglietto-Vargas, D.; Kling, A.; Moeller, A.; Green, K. N.; LaFerla, F. M. Calpain inhibitor A-705253 mitigates Alzheimer’s disease-like pathology and cognitive decline in aged 3xTgAD mice. Am. J. Pathol. 2012, 181, 616– 625, DOI: 10.1016/j.ajpath.2012.04.020
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150
Calpain inhibitor A-705253 mitigates Alzheimer's disease-like pathology and cognitive decline in aged 3xTgAD mice
Medeiros, Rodrigo; Kitazawa, Masashi; Chabrier, Meredith A.; Cheng, David; Baglietto-Vargas, David; Kling, Andreas; Moeller, Achim; Green, Kim N.; LaFerla, Frank M.
American Journal of Pathology (2012), 181 (2), 616-625CODEN: AJPAA4; ISSN:0002-9440. (Elsevier)
Calpains are cysteine proteinases that selectively cleave proteins in response to calcium signals. Exacerbated activation of calpain has been implicated as a major component in the signaling cascade that leads to β-amylold (Aβ) prodn. and tau hyperphosphorylation in Alzheimer's disease (AD). In this study, we analyzed the potential therapeutic efficacy of inhibiting the activation of calpain by a novel calpain inhibitor in aged 3xTgAD mice with well-established cognitive impairment, plaques, and tangles. The administration of a novel inhibitor of calpain, A-705253, attenuated cognitive impairment and synaptic dysfunction in a dose-dependent manner in 3xTgAD mice. Inhibition of calpain lowered Aβ40 and Aβ42 levels in both detergent-sol. and detergent-insol. fractions and also reduced the total no. and size of thioflavin S-pos. fibrillar Aβ deposits. Mechanistically, these effects were, in part, explained by a down-regulation of β-secretase 1 (BACE1) and an up-regulation of ATP-binding cassette transporter A1 (ABCA1) expression, which, in turn, contributed to reduced prodn. and increased clearance of Aβ, resp. Moreover, A-705253 decreased the activation of cyclin-dependent kinase 5 (CDK5) and thereby diminished the hyperphosphorylation of tau. Finally, blockage of calpain activation reduced the astrocytic and microglial responses assocd. with AD-like pathol. characteristics in aged 3xTgAD mice. Our data provide relevant functional and mol. insights into the beneficial therapeutic effects of inhibiting calpain activation for the management of AD.
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151
Gauthier, J. Y.; Chauret, N.; Cromlish, W.; Desmarais, S.; Duong, L. T.; Falgueyret, J. P.; Kimmel, D. B.; Lamontagne, S.; Léger, S.; LeRiche, T.; Li, C. S.; Massé, F.; McKay, D. J.; Nicoll-Griffith, D. A.; Oballa, R. M.; Palmer, J. T.; Percival, M. D.; Riendeau, D.; Robichaud, J.; Rodan, G. A.; Rodan, S. B.; Seto, C.; Thérien, M.; Truong, V. L.; Venuti, M. C.; Wesolowski, G.; Young, R. N.; Zamboni, R.; Black, W. C. The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K. Bioorg. Med. Chem. Lett. 2008, 18, 923– 928, DOI: 10.1016/j.bmcl.2007.12.047
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151
The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K
Gauthier, Jacques Yves; Chauret, Nathalie; Cromlish, Wanda; Desmarais, Sylvie; Duong, Le T.; Falgueyret, Jean-Pierre; Kimmel, Donald B.; Lamontagne, Sonia; Leger, Serge; LeRiche, Tammy; Li, Chun Sing; Masse, Frederic; McKay, Daniel J.; Nicoll-Griffith, Deborah A.; Oballa, Renata M.; Palmer, James T.; Percival, M. David; Riendeau, Denis; Robichaud, Joel; Rodan, Gideon A.; Rodan, Sevgi B.; Seto, Carmai; Therien, Michel; Truong, Vouy-Linh; Venuti, Michael C.; Wesolowski, Gregg; Young, Robert N.; Zamboni, Robert; Black, W. Cameron
Bioorganic & Medicinal Chemistry Letters (2008), 18 (3), 923-928CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclin. species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.
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152
Ahrén, B.; Landin-Olsson, M.; Jansson, P. A.; Svensson, M.; Holmes, D.; Schweizer, A. Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels, and reduces glucagon levels in type 2 diabetes. J. Clin. Endocrinol. Metab. 2004, 89, 2078– 2084, DOI: 10.1210/jc.2003-031907
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152
Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels, and reduces glucagon levels in type 2 diabetes
Ahren, Bo; Landin-Olsson, Mona; Jansson, Per-Anders; Svensson, Maria; Holmes, David; Schweizer, Anja
Journal of Clinical Endocrinology and Metabolism (2004), 89 (5), 2078-2084CODEN: JCEMAZ; ISSN:0021-972X. (Endocrine Society)
The stimulation of insulin vs. inhibition of glucagon secretion in relation to the antidiabetic action of glucagon-like peptide-1 (GLP-1) is not established. Here, the influence of a 4-wk increase in circulating GLP-1 by inhibition of dipeptidyl peptidase-4 (DPP-4) on 24-h glucose and insulin and glucagon responses to breakfast was studied in subjects with dietary controlled diabetes [age: 65±8 yr (SD), body mass index: 27.3±3.3 kg/m2, fasting plasma glucose: 9.0±1.3 mmol/L]. Compared with placebo (n = 19), a specific DPP-4 inhibitor [(1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine) (LAF237); 100 mg daily, n = 18] reduced fasting glucose by 0.70 mmol/L (P = 0.037), 4-h prandial glucose excursion by 1.45 mmol/L (P < 0.001), and mean 24-h glucose by 0.93 mmol/L (P < 0.001). Baseline and postprandial active GLP-1 were increased by LAF237. The glucagon response to breakfast was reduced by LAF237 (glucagon levels at 60 min were 88±8 pg/mL before treatment vs. 77±5 pg/mL after; P = 0.001). In contrast, the overall insulin levels were not altered. The 4-wk redn. in glucagon correlated with the redn. in 2-h glucose (r = 0.61; P = 0.008). No such assocn. was obsd. for insulin. Thus, improved metabolic control by DPP-4 inhibition in type 2 diabetes is seen in assocn. with reduced glucagon levels and, despite the lower glycemia, unaltered insulin levels.
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153
Weber, P.; Wang, P.; Maddens, S.; Wang, P. S. H.; Wu, R.; Miksa, M.; Dong, W.; Mortimore, M.; Golec, J. M. C.; Charlton, P. VX-166: a novel potent small molecule caspase inhibitor as a potential therapy for sepsis. Crit. Care 2009, 13, R146, DOI: 10.1186/cc8041
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154
Herman, G. A.; Stevens, C.; Van Dyck, K.; Bergman, A.; Yi, B.; De Smet, M.; Snyder, K.; Hilliard, D.; Tanen, M.; Tanaka, W.; Wang, A. Q.; Zeng, W.; Musson, D.; Winchell, G.; Davies, M. J.; Ramael, S.; Gottesdiener, K. M.; Wagner, J. A. Pharmacokinetics and pharmacodynamics of sitagliptin, an inhibitor of dipeptidyl peptidase IV, in healthy subjects: results from two randomized, double-blind, placebo-controlled studies with single oral doses. Clin. Pharmacol. Ther. 2005, 78, 675– 688, DOI: 10.1016/j.clpt.2005.09.002
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154
Pharmacokinetics and pharmacodynamics of sitagliptin, an inhibitor of dipeptidyl peptidase IV, in healthy subjects: Results from two randomized, double-blind, placebo-controlled studies with single oral doses
Herman, Gary A.; Stevens, Cathy; Van Dyck, Kristien; Bergman, Arthur; Yi, Bingming; De Smet, Marina; Snyder, Karen; Hilliard, Deborah; Tanen, Michael; Tanaka, Wesley; Wang, Amy Q.; Zeng, Wei; Musson, Donald; Winchell, Gregory; Davies, Michael J.; Ramael, Steven; Gottesdiener, Keith M.; Wagner, John A.
Clinical Pharmacology & Therapeutics (New York, NY, United States) (2005), 78 (6), 675-688CODEN: CLPTAT; ISSN:0009-9236. (Elsevier)
Background: Sitagliptin (MK-0431 [(2R)-4-oxo-4-(3-[trifluoromethyl]-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7[8H]-yl)-1-(2,4,5-trifluorophenyl)butan-2-amine]) is an orally active, potent, and selective inhibitor of dipeptidyl peptidase IV (DPP-IV) currently in phase III development for the treatment of type 2 diabetes. Methods: Two double-blind, randomized, placebo-controlled, alternating-panel studies evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of sitagliptin (1.5-600 mg) in healthy male volunteers. Results: Sitagliptin was well absorbed (approx. 80% excreted unchanged in the urine) with an apparent terminal half-life ranging from 8 to 14 h. Renal clearance of sitagliptin averaged 388 mL/min and was largely uninfluenced by the dose administered. The area under the plasma concn.-time curve for sitagliptin increased in an approx. dose-dependent manner and was not meaningfully influenced by food. Single doses of sitagliptin markedly and dose-dependently inhibited plasma DPP-IV activity, with approx. 80% or greater inhibition of DPP-IV activity occurring at 50 mg or greater over a 12-h period and at 100 mg or greater over a 24-h period. Compared with placebo, sitagliptin produced an approx. 2-fold increase in postmeal active glucagon-like peptide 1 levels. Sitagliptin was well tolerated and was not assocd. with hypoglycemia. Conclusions: This study provides proof of pharmacol. characteristics for sitagliptin in humans. By inhibiting plasma DPP-IV activity, sitagliptin increases the postprandial rise in active glucagon-like peptide 1 concns. without causing hypoglycemia in normoglycemic healthy male volunteers. Sitagliptin possesses pharmacokinetic and pharmacodynamic characteristics that support a once-daily dosing regimen.
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Nutescu, E. Apixaban: A novel oral inhibitor of factor Xa. Am. J. Health-Syst. Pharm. 2012, 69, 1113– 1126, DOI: 10.2146/ajhp110418
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155
Apixaban: a novel oral inhibitor of factor Xa
Nutescu, Edith
American Journal of Health-System Pharmacy (2012), 69 (13), 1113-1126CODEN: AHSPEK; ISSN:1079-2082. (American Society of Health-System Pharmacists)
A review. Purpose. The pharmacol., pharmacokinetics, efficacy, and safety of apixaban are reviewed. Summary. Apixaban is an oral, direct, selective factor Xa inhibitor with a rapid onset of action. It has a plasma elimination half-life of 12 h and has been administered in a twice-daily dosing regimen in clin. trials without the need for anticoagulation monitoring or dosage adjustment. Apixaban has multiple elimination pathways, and its pharmacokinetics is not substantially altered by patient age, sex, race, or ethnicity. The results of three Phase III trials indicated that apixaban was similar to or more effective than enoxaparin for preventing venous thromboembolism (VTE) in patients undergoing total hip or knee replacement, with similar or lower rates of bleeding. Two Phase III trials found that apixaban was more effective for stroke prevention than either aspirin or warfarin in patients with atrial fibrillation (AF), with a similar (vs. aspirin) or improved (vs. warfarin) safety profile. A Phase III trial evaluating apixaban plus antiplatelet monotherapy or dual-antiplatelet therapy in patients with acute coronary syndrome ended early due to clear evidence of a clin. important increase in bleeding among patients randomized to apixaban without any meaningful redn. in ischemic events. The adverse-event profiles for apixaban and comparators have been similar in studies conducted to date. Conclusion. Apixaban, a new anticoagulant, appears to offer an efficacy and safety profile comparable with that of enoxaparin for preventing VTE after orthopedic surgery, with the advantage of oral administration. In patients with AF, apixaban is more effective than either warfarin or aspirin for stroke prevention, with an acceptable safety profile.
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156
Zervosen, A.; Sauvage, E.; Frère, J. M.; Charlier, P.; Luxen, A. Development of new drugs for an old target: the penicillin binding proteins. Molecules 2012, 17, 12478– 12505, DOI: 10.3390/molecules171112478
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156
Development of new drugs for an old target - the penicillin binding proteins
Zervosen, Astrid; Sauvage, Eric; Frere, Jean-Marie; Charlier, Paulette; Luxen, Andre
Molecules (2012), 17 (), 12478-12505CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
A review. The widespread use of ss-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the prodn. of β-lactamases, sometimes accompanied by a decrease of outer membrane permeability, and the prodn. of low-affinity, drug resistant Penicillin Binding Proteins (PBPs). PBPs remain attractive targets for developing new antibiotic agents because they catalyze the last steps of the biosynthesis of peptidoglycan, which is unique to bacteria, and lies outside the cytoplasmic membrane. Here we summarize the "current state of the art" of non-β-lactam inhibitors of PBPs, which have being developed in an attempt to counter the emergence of β-lactam resistance. These mols. are not susceptible to hydrolysis by β-lactamases and thus present a real alternative to β-lactams. We present transition state analogs such as boronic acids, which can covalently bind to the active serine residue in the catalytic site. Mols. contg. ring structures different from the β-lactam-ring like lactivicin are able to acylate the active serine residue. High throughput screening methods, in combination with virtual screening methods and structure based design, have allowed the development of new mols. Some of these novel inhibitors are active against major pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and thus open avenues new for the discovery of novel antibiotics.
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157
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The active human immunodeficiency virus type 1 (HIV-1) protease has a homodimeric structure, the subunits are connected by an 'interface' β-sheet formed by the NH2- and COOH-terminal amino acid segments. Short peptides derived from these segments are able to inhibit the protease activity in the range of micromolar IC50 values. We have further improved the inhibitory power of such peptides by computer modeling. The best inhibitor, the palmitoyl-blocked peptide Pam-Thr-Val-Ser-Tyr-Glu-Leu, has an IC50 value of less than 1 μM. Some of the peptides also showed very good inhibition of the HIV-2 protease. The C-terminal segment of the HIV-1 matrix protein, Acetyl-Gln-Val-Ser-Gln-Asn-Tyr, also inhibits HIV-1 protease. Kinetic studies confirmed the 'dissociative' mechanism of inhibition by the peptides. Depending on the peptide structure and ionic strength, both dimerization inhibition and competitive inhibition were obsd., as well as synergistic effects between competitive inhibitors and interface peptides.
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Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reprodn. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging, since they may be improved by various means.
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A review. By targeting the highly conserved antiparallel β-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance obsd. with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.
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A novel strategy was used to irreversibly inhibit HIV-1 protease. The inhibitor was designed to form a disulfide bond with Cys95, present at the dimerization interface of HIV-1 protease. The inhibitor was shown to be active against HIV-1 protease with Kinact=3.7 μM and Vinact=0.012 min-1.
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A review with 31 refs. Protein-protein assocn. is found throughout mechanisms of cellular growth and differentiation, and viral replication. Inhibiting the assembly of protein complexes, therefore, presents itself as a novel means of inhibition for a wide variety of cellular and viral events. Peptides and small mols. that modify the overall quaternary structure of a selection of receptor-ligand interactions and oligomeric viral enzymes have been developed recently.
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The crystal structures of the proteases (PRs) encoded by the Rous sarcoma virus (RSV) and the human immunodeficiency virus (HIV) have been compared. The crystallog. monomer of HIV PR superimposes on the 2 crystallog. independent subunits of the RSV PR dimer with root mean square deviations of 1.45 and 1.55 Å for 86 and 88 common Cα atoms, resp. There is a conserved structural core consisting of 7 β-strands forming 2 perpendicular layers, a helix, and the N- and C--terminal β-strands. PRs from related retroviruses fold into similar structures with surface turns of variable length between the β-strands. Both HIV and RSV PR dimers have significant subunit-subunit interactions in 3 regions: the firemen's grip at the active site; the salt bridges involving arginine-8 (Arg8), aspartate-29, and Arg87 of HIV PR; and the termini of the 2 subunits, which form a 4-stranded antiparallel β-sheet. The specific interactions of the termini differ in the two PRs. The C-termini, residues 96-99 of HIV PR and residues 119-124 of RSV PR, contribute ∼50% of the intersubunit ionic and H bond interactions and ∼45% of the buried surface area involved in dimer formation.
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Journal of Biological Chemistry (1991), 266 (24), 15591-4CODEN: JBCHA3; ISSN:0021-9258.
Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical promoters linked by a four-stranded antiparallel β-sheet consisting of the NH2- and COOH-terminal segments. Kinetic anal. of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1M sodium acetate, 1M NaCl, 1 mM EDTA buffer, 37°, the equil. dissocn. const., Kd = 3.6 nM. The tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic anal. shows that the inhibitor binds to the inactive promoters and prevents their assocn. into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equil. expts. in which the apparent mol. wt. of the enzyme was obsd. to be 20,800 and 12,100, in the absence and presence of the COOH-terminal tetrapeptide, resp. The dissocn. const. of the protomer-inhibitor complex is Ki = 45.1 μM. This is the first kinetic anal. and direct exptl. demonstration of noncovalent and dissociative inhibition.
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Wei, P.; Fan, K.; Chen, H.; Ma, L.; Huang, C.; Tan, L.; Xi, D.; Li, C.; Liu, Y.; Cao, A.; Lai, L. The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase. Biochem. Biophys. Res. Commun. 2006, 339, 865– 872, DOI: 10.1016/j.bbrc.2005.11.102
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The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase
Wei, Ping; Fan, Keqiang; Chen, Hao; Ma, Liang; Huang, Changkang; Tan, Lei; Xi, Dong; Li, Chunmei; Liu, Ying; Cao, Aoneng; Lai, Luhua
Biochemical and Biophysical Research Communications (2006), 339 (3), 865-872CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate detn. of the dimer dissocn. const. and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissocn. const. of the wild-type protein was detd. to be 14.0 μM by anal. ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and anal. ultracentrifugation. A single mutation of M6A was found to be crit. to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K i of 2.20 mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.
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An Overview of Severe Acute Respiratory Syndrome–Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

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Abstract

Note

1. Introduction

Figure 1

2. SARS-CoV and Structure of 3CLpro

Figure 2

Figure 3

3. SARS-CoV 3CLpro Inhibitors

Figure 4

Figure 5

Figure 6

4. Peptidomimetic Inhibitors

Figure 7

4.1. Peptides with a Michael Acceptor

Figure 8

4.2. Peptides with Keto-glutamine

Figure 9

Figure 10

4.3. Peptides with Nitroanilide

Figure 11

Figure 12

4.4. Aza-epoxide and Aziridine Peptides

4.5. Peptide Aldehydes

Figure 13

Figure 14

4.6. Peptides with Halomethyl Ketone or Electrophilic Substituents

Figure 15

4.7. Symmetric Peptides

Figure 16

5. Small Molecule Inhibitors of SARS-CoV 3CLpro

5.1. Etacrynic Acid Derivatives

Figure 17

5.2. Isatin (2,3-Dioxindole) Inhibitors

5.3. Flavonoid and Biflavonoid Derivatives

Figure 18

5.4. Terpenoid Derivatives

Figure 19

5.5. Sulfone, Dihydroimidazole, and N-Phenyl-2-(2-pyrimidinylthio)acetamide Type Analogues

Figure 20

5.6. Active Heterocyclic Ester Analogues

Figure 21

Figure 22

Figure 23

5.7. Aryl Methylene Ketones and Fluoro Methylene Ketones

Figure 24

5.8. Pyrazolone and Pyrimidines

Figure 25

5.9. Decahydroisoquinoline Derivatives

Figure 26

5.10. 3-Pyridyl and Benzotriazole-Based SARS-CoV 3CLpro Inhibitors

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

6. Metal Conjugated SARS-CoV 3CLpro Inhibitors

Figure 33

7. Miscellaneous SARS-CoV 3CLpro Inhibitors

Figure 34

8. Conclusion and Perspectives

Figure 35

Figure 36

Supporting Information

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Author Information

Biographies

Thanigaimalai Pillaiyar

Manoj Manickam

Vigneshwaran Namasivayam

Yoshio Hayashi

Sang-Hun Jung

Acknowledgments

Abbreviations Used

References

2. SARS-CoV and Structure of 3CL^pro

3. SARS-CoV 3CL^pro Inhibitors

5. Small Molecule Inhibitors of SARS-CoV 3CL^pro

5.10. 3-Pyridyl and Benzotriazole-Based SARS-CoV 3CL^pro Inhibitors

6. Metal Conjugated SARS-CoV 3CL^pro Inhibitors

7. Miscellaneous SARS-CoV 3CL^pro Inhibitors