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Identification of Potent, Broad-Spectrum Coronavirus Main Protease Inhibitors for Pandemic Preparedness
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Identification of Potent, Broad-Spectrum Coronavirus Main Protease Inhibitors for Pandemic Preparedness
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  • David T. Barkan
    David T. Barkan
    Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
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  • Keira Garland
    Keira Garland
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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  • Lei Zhang
    Lei Zhang
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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  • Richard T. Eastman
    Richard T. Eastman
    Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Orcidhttps://orcid.org/0000-0003-3580-380X
  • Matthew Hesse
    Matthew Hesse
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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  • Mark Knapp
    Mark Knapp
    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Elizabeth Ornelas
    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Jenny Tang
    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Wilian Augusto Cortopassi
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Yu Wang
    Discovery Sciences, Novartis Biomedical Research, La Jolla, California 92121, United States
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    Frederick King
    Discovery Sciences, Novartis Biomedical Research, La Jolla, California 92121, United States
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    Weiping Jia
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Zachary Nguyen
    Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
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    Andreas O. Frank
    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Ryan Chan
    Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Eric Fang
    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
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    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Dustin Dovala
    Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Stephanie Moquin
    Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
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    Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2024, 67, 19, 17454–17471
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https://doi.org/10.1021/acs.jmedchem.4c01404
Published September 27, 2024

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Abstract

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The COVID-19 pandemic highlights the ongoing risk of zoonotic transmission of coronaviruses to global health. To prepare for future pandemics, it is essential to develop effective antivirals targeting a broad range of coronaviruses. Targeting the essential and clinically validated coronavirus main protease (Mpro), we constructed a structurally diverse Mpro panel by clustering all known coronavirus sequences by Mpro active site sequence similarity. Through screening, we identified a potent covalent inhibitor that engaged the catalytic cysteine of SARS-CoV-2 Mpro and used structure-based medicinal chemistry to develop compounds in the pyrazolopyrimidine sulfone series that exhibit submicromolar activity against multiple Mpro homologues. Additionally, we solved the first X-ray cocrystal structure of Mpro from the human-infecting OC43 coronavirus, providing insights into potency differences among compound–target pairs. Overall, the chemical compounds described in this study serve as starting points for the development of antivirals with broad-spectrum activity, enhancing our preparedness for emerging human-infecting coronaviruses.

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Introduction

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The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1) pandemic is the third global health crisis caused by a novel coronavirus in just the last twenty-two years, following the SARS-CoV-1 epidemic in 2002 (2) and the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemic in 2012. (3) Recent examination of clinical evidence has also suggested that another human-infecting coronavirus, OC43, may have been the true etiologic agent for the “Russian Flu” pandemic in 1889–1891, which resulted in around a million deaths. (4)
Zoonotic emergence of novel coronaviruses represents a significant and ever-present threat to global health and the global economy. Cross-species transmission appears to be more prevalent in RNA viruses such as coronaviruses, (5) and opportunities for spillover events may become more common with a rise in global poverty and as humans continue to encroach on new habitats and interact with nature in new ways. (6,7) Examples of these situations include the following: incorporation of bat guano in fertilizer exposes guano miners, farmers, and livestock to reservoirs of α- and β-coronaviruses; (8,9) trade of exotic pets and wildlife expose populations to potential spillover events at population-dense ports of entry, (7,10) and the continued livestock intensification and the broad use of anti-infectives facilitate disease transmission as well as the emergence of treatment-resistant variants. (11) Aside from potentially nucleating a pandemic event, the spread of new diseases through agricultural routes has the secondary effect of exacerbating food shortages and global poverty. (12) Overall, there has been a steady increase in the number of emerging infectious diseases each decade going back to 1940, likely driven by socioeconomic, environmental, and ecological factors. (13)
The staggering costs to human life, well-being, and the global economy demand pre-emptive action to curtail the spread of future zoonotic coronaviruses before they become pandemic. One strategy is the creation of direct-acting broad-spectrum coronavirus antivirals (14) which, when optimized against currently circulating coronaviruses, may be efficacious against related emerging coronaviruses and thus either stop the spread entirely or reduce the health burden sufficiently to allow time for the development of new vaccines.
While it is impossible to know the structure of, and thus design drugs against, a viral target for a virus that is not yet known, there are ways we can increase our chances of success against future coronavirus outbreaks. First is the selection of validated, well-conserved, and essential viral targets. The nonstructural protein 5 (nsp5), also known as the main protease or Mpro, is one of the best-characterized drug targets in coronaviruses. (15−25) Mpro protease activity is necessary to process the large viral polyproteins pp1a and pp1ab into individual proteins required for replication. (26,27) Mpro is attractive for several other reasons, one of which is the preferred residue at the P1 site is glutamine, a substrate specificity that is not shared by any known human proteases (15) and thus decreases the chance of nonspecific inhibition of host proteases by Mpro inhibitors. Additionally, Mpro is a cysteine protease, which utilizes a catalytic histidine-cysteine dyad. The presence of a conserved reactive cysteine nucleophile in the active site enables the discovery of covalent inhibitors with pharmacological advantages such as increased potency, selectivity, and duration of inhibition. (28) Inhibition of Mpro may also protect against direct protease-induced cell toxicity. (29)
While work on Mpro inhibitors, such as nirmatrelvir, the antiviral component of Paxlovid, (19) has mainly focused on activity against human coronaviruses, activity against a broader set of coronaviruses has not been investigated. To that end, we developed a computational protocol to identify a set of Mpro homologues across all available Coronaviridae sequences that maximize structural diversity in the active site across a tractable number of proteins. We used this set to build a panel of RapidFire mass spectrometry (RFMS)-based biochemical enzymatic activity assays to drive medicinal chemistry optimization of our high-throughput screening hits against SARS-CoV-2 Mpro toward broad-spectrum activity across the viral family.

Results and Discussion

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We constructed a pipeline to compile unique Mpro active site residue compositions across the 46,701 Coronaviridae family isolate sequences deposited in GenBank in preparation for clustering (Figure 1). These isolate sequences are available in the NCBI Nucleotide database (30) where the majority have the positions of pp1ab annotated, but do not further identify the boundaries of the post-translationally processed proteins such as Mpro. As such, we compiled a data set of 35 diverse reference Mpro sequences from RefSeq and BV-BRC (31) (Supporting Information, Table S1) and used them to search for and extract isolate Mpro sequences with the tBLASTn algorithm (32) (Figure 1A(i–ii)). The resulting data set totaled 4533 viral isolate Mpro sequences as most deposited isolates did not span the full genome; quality control filtering and removal of identical sequences further reduced this number to 1192.

Figure 1

Figure 1. Mpro clustering procedure and results. (A) Active site curation flowchart; Mpro sequences are extracted from the NCBI Coronavirus Nucleotide database and active site residues are inferred through transitive structure alignment using SARS-CoV-2 as a reference. Dashed lines indicate sequence or structure alignments. Blue spheres are active site pocket residues. (B) Number of sequences following each step of the curation process. (C) Clustering results; trees rooted below the red-dashed line (distance cutoff of 8) represent the final clusters.

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Next, we identified the active site pocket residues of these isolate Mpro sequences. To establish a reference set of these residues, we visually inspected multiple solved structures of SARS-CoV-2 Mpro in complex with various active site binders (21,33,34) to enumerate the 38 amino acid positions comprising the active site pocket (Supporting Information, Figure S1). As the sequence identity between the isolate sequences and the SARS-CoV-2 sequence was too low to infer equivalent active site positions in isolates following a sequence alignment, we devised a strategy to make this assignment using transitive equivalence of positions when aligning isolates to their closest structure. First, all available crystal structures of coronavirus family Mpro proteins in the PDB (12 species total; Supporting Information, Table S2) were structurally aligned to the SARS-CoV-2 structure to identify with high-confidence equivalent active site positions in those solved structures. Then BLASTp was used to align the isolate Mpro sequences to these coronavirus structures to identify the closest structure to each isolate, followed by assigning active site residues based on the sequence alignment. The distribution of sequence identities between isolate Mpro sequences and their closest structure skews much higher than the identities of isolate sequences to SARS-CoV-2 Mpro (Supporting Information, Figure S2), thus allowing us to annotate active site residues in isolates with high confidence using these structures as intermediates. This process resulted in 222 unique active site residue sequences (Figure 1A(iii–iv),1B), each composed of 38 discontinuous residues that define the active site.
Next, a simple all-vs-all pairwise identity matrix was calculated across these 222 unique active site residue sequences and used as input to a hierarchical clustering method (Figure 1C). When constructing these clusters, one critical decision was the choice of distance cutoff to apply; a restrictive cutoff results in higher similarity between cluster members, while a more permissive cutoff has lower similarity but fewer clusters, making creating and testing an Mpro diversity panel more feasible. Here we empirically assigned a cutoff of eight residue differences, resulting in a final set of 19 clusters, which was considered an appropriate trade-off between intracluster diversity and experimental tractability. Notably, the median intercluster distance between any pair of cluster centroids was below 50%, while the mean intracluster distance between a centroid and other members of its cluster was at least 89% for all clusters (Supporting Information, Figure S3).
From each cluster we selected a representative member (Table 1). These representatives were typically the centroids of the cluster, defined as the sequences with the lowest average number of active site residue differences to all other members of the cluster. Some clusters had multiple identical centroids, in which case we prioritized sequences present in the RefSeq database or that were otherwise well-annotated. In other clusters we prioritized Mpro sequences from viruses that infect humans due to being more therapeutically relevant. These representatives were then considered for final Mpro diversity panel selection, protein production and enzymatic assay development. Here, because we were focused on covering diversity in the α- and β-coronaviruses, delta- and γ-coronavirus clusters 1, 7, and 12 were not considered. MERS-CoV Mpro was initially chosen as the representative for cluster 6; however, we observed compound-induced activation of this homologue in the enzymatic assay, which has previously been reported in the literature. (35) Because this phenomenon results in inaccurate IC50 curves, MERS-CoV Mpro was replaced with HKU4 Mpro from the same cluster for panel inclusion. We also observed compound-induced activation for HKU9 Mpro (cluster 9) and thus this representative was excluded from the panel. Additionally, clusters 13–19 were omitted because they were represented by a single sequence; these were deprioritized to reduce the total volume of experimental work required. Finally, assays were developed for the other three human-infecting coronaviruses, HKU1 (cluster 3), SARS-CoV-1 (cluster 5), and NL63 (cluster 8), thus leading to multiple members in the panel for these clusters (Table 1). Full details, including annotation and cluster assignment, on these panel members, as well as all isolates, are available in the Supporting Information (Table S3 and Datasheet S1).
Table 1. Mpro Cluster and Panel Composition
clustersizerepresentativein panelgenushost speciesother panel members
1353IBV (36,37)noγchicken 
2244PEDV (38,39)yesαpig 
3147HCoV-OC43 (40)yesβhumanHCoV-HKU1 (41,42)
4107UU23 (43)yesαcat 
581SARS-CoV-2 (1,44)yesβhumanSARS-CoV-1 (2,45)
671HKU4 (46)yesβbat 
769HKU15 (47,48)noδpig 
857HCoV-229E (49,50)yesαhumanHCoV-NL63 (51,52)
931HKU9 (53)noβbat 
1015Lucheng rat CoV (54)yesαrat 
117Wencheng Shrew CoV (55)yesαAsian musk shrew 
123SW1 (48,56)noγBeluga whale 
131Guangdong Chinese Water Skink CoV (57)noN/Askink 
141Pacific salmon nidovirus (58)no*salmon 
151Kanakana letovirus (59)no*lamprey 
161Hipposideros bat CoVnoN/Abat 
171HKU19 (47)noδnight heron 
181Shrew CoV (60)noαcommon shrew 
191Bat CoV GCCDC1 (61)noβbat 
With the panel in hand, we successfully completed a high-throughput screen (HTS) of 1.9 million compounds against SARS-CoV-2 Mpro. We used an enzymatic cleavage assay with a Rhodamine-110 fluorescence intensity readout at a 10 μM single point, (50% inhibition cutoff) which resulted in ∼57,000 positive compounds (∼3% hit rate). In silico triaging based on physicochemical properties, structural flags for Pan-Assay INterference compoundS (PAINS), and promiscuous binders reduced this initial set down to 16,600 compounds which were then taken into an 8-point dose response assay. Approximately 8000 of these compounds were confirmed with an IC50 of <30 μM. The 8000 compound hit list was then manually triaged based on data quality, desirability of chemical matter and in silico projections of likely solubility, permeability, and covalent reactivity. This process led to the identification of ∼2000 compounds, which were classified as either covalent or noncovalent based on substructure flags to take forward into confirmation in a series of orthogonal assays. To remove fluorescence interference compounds, the hits were put through a SARS-CoV-2 Mpro RFMS cleavage inhibition assay. Most compounds (∼90%) were reconfirmed in this assay. Next, a surface plasmon resonance (SPR) binding assay was used to identify genuine Mpro binders in either a covalent or noncovalent binding mode. A relatively low percentage (0.66%) of compounds were SPR-positive; however, these compounds represented high-confidence hits.
To identify any possible false negative compounds arising from SPR artifacts, SPR-negative compounds were assayed using two additional orthogonal binding experiments. First, native mass spectrometry (nMS) was used to identify specific binders to Mpro (either as the monomer or active dimer), with active site binding being determined by competition with a known active site engager. Positive compounds were then taken into a 13C-labeled Mpro NMR binding experiment, using a known Mpro inhibitor as a positive control to define the fingerprint of chemical shift perturbations consistent with genuine active site engagement.
The hits from both the high-confidence SPR-positive and SPR-negative rescue workflows were then resynthesized. Their chemical structures were established, and their activity was confirmed in the SARS-CoV-2 Mpro RFMS cleavage inhibition assay. Reassaying the resynthesized hits resulted in further attrition to give only a select handful of compounds which were ultimately confirmed as active site binders through generation of X-ray cocrystal structures. Of these new hits, a scaffold bearing a dihydropyrazolo[1,5-a]pyrimidine core was prioritized based on its initial promising SAR and physiochemical property space (Compound 1, Figure 2, Table 1).

Figure 2

Figure 2. Cocrystal structure of 1 covalently bound to the catalytic C145 of SARS-CoV-2 Mpro (PDB ID 9C8O).

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The cocomplex structure revealed that the fluorophenyl group of 1 occupies the S1 pocket and interacts with the side chain of H163 via fluorine hydrogen bonding. Hydrogen bonding between the carboxylic acid and the backbone nitrogen of C145 was observed, along with hydrogen bonding between the nitrogen of the pyrazole core and the backbone nitrogen of E166. Interestingly, the ligand was discovered to be covalently bound in the active site forming a C–S bond with the catalytic C145. The second fluorophenyl group, which is linked to the core via an amide bond, occupies the S2 region of the active site. The carbonyl of the amide linker participates in a water-mediated hydrogen bonding interaction with the backbone carbonyl of H41.
The enantiomers of 1 were separated to yield compound 2 which exhibited similar potency of 3.4 μM against SARS-CoV-2 Mpro (Table 2). As the enantiomer of compound 2 was inactive (>50 μM), compound 2 was assigned as (R)-7-(2-fluorophenyl)-3-((2-fluorophenyl)carbamoyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acid based on the cocrystal structure of compound 1 bound to SARS-CoV-2 Mpro (Figure 2). Exploration of the S1 pocket was carried out with the goal of optimizing the hydrogen bonding interaction between the aryl fluorine and H163–monitoring of lipophilic efficiency (LipE) (62) was utilized to confirm that potency changes were not attributable to changes in lipophilicity alone. Heterocycles bearing hydrogen bond acceptors such as pyridine (3) showed comparable potency to the monofluoro phenyl group. However, a small improvement in potency was seen with hydrogen bond donor containing heterocycles such as pyridone (4), with a corresponding increase in lipophilic efficiency (LipE). No potency improvements were gained with dihalogenated phenyl groups such as compound 5. Replacements for the amide linker present in the S2 region were then explored; as the hydrogen bond donor was not engaging in any direct interactions with the protein, it was hypothesized that this linker could be removed entirely. Indeed, compounds 6–11 demonstrate that the amide linker at this position is not required in order to maintain biochemical potency, and LipE analysis indicates potency improvements are primarily driven by lipophilicity - as expected given the lipophilic nature of the S2 pocket.
Table 2. SARS-CoV-2 Mpro Biochemical Potency of Compounds with P2 Modifications
a

Shown are mean IC50 values derived from a minimum of three replicates;

b

cLogD was used to calculate LipE.

One of the main challenges that was faced while exploring SAR within this series was chemical instability, which resulted in many compounds being difficult to isolate or obtain in sufficient purity. This instability was thought to be caused by the high reactivity of the α-β unsaturation present in the core. However, the inherent reactivity of the core was also hypothesized to be responsible for the observed covalent mode of binding. Thus, although compounds synthesized with the aromatized (pyrazolopyrimidine) core showed improved chemical stability, this change caused a significant loss in SARS-CoV-2 activity (i.e., compound 12, Figure 3). As the carboxylic acid group of compounds 3 and 12 occupy a polar area of the active site adjacent to the catalytic residues (Figure 2), 3D-modeling hypothesized that inclusion of a sulfone group could facilitate hydrogen bonding interactions with residues located in the oxyanion hole and activate the core for covalent interaction with C145 via a SNAr mechanism. Indeed, replacing the carboxylic acid with a sulfone moiety rescued the potency of the aromatized (pyrazolopyrimidine) series, and even resulted in a dramatic improvement in potency (i.e., compound 13, Figure 3).

Figure 3

Figure 3. Aromatization of the core leads to potency gains after replacement of the carboxylic acid moiety with a sulfone.

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A cocrystal structure of a close analog (14) bound to SARS-CoV-2 Mpro confirmed this hypothetical binding mode, showing covalent binding to C145 (Figure 4). As also expected, the pyridine nitrogen in the S1 pocket interacts with the H163 side chain, as seen in previous series. The cyclohexyl group replaces the amide linked fluorophenyl group and occupies the lipophilic S2 pocket.

Figure 4

Figure 4. Co-structure of 14 covalently bound to the catalytic C145 of SARS-CoV-2 Mpro (PDB ID 9C8Q).

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The more stable pyrazolopyrimidine core enabled further SAR exploration of the S1 region. Numerous compounds bearing a sulfone group were synthesized and tested for broad-spectrum activity in the full Mpro biochemical panel, with submicromolar potency being observed across multiple coronavirus species (Table 3). Substituted pyridines (13, 14, 16, 17, 20) were observed to be optimal in the S1 pocket and led to similar potency outcomes. However, replacement of pyridine with lactams 18 and 19 resulted in up to 100-fold loss in potency across the Mpro diversity panel. Bromination on the 2-position of the core (15) was also tolerated. Although various alternative potential covalent warheads (e.g., thioether, cyano, chloro) were not tolerated, sulfoxides (20) were active against SARS-CoV-2 Mpro- however work was focused on sulfones as they were consistently more potent than sulfoxides. Intriguingly, although structural isomers 21 and 22 lost activity against SARS-CoV-2 Mpro, good potency was noted against HCoV-OC43 Mpro. Structural isomer 23 was also potent against HCoV-OC43 Mpro but showed less activity against some of the other Mpro strains. To gain further insight into how compounds such as 21–23 bind to HCoV-OC43 Mpro, an X-ray cocrystal structure was obtained with 21 (Figure 5). As a result, we were able to determine that 21 is selective toward HCoV-OC43 Mpro over SARS-CoV-2 Mpro due to its large leaving group which results in a different binding orientation; steric hindrance prevents the 2-(methylsulfonyl)propane group from approaching close enough to catalytic C145 to form a covalent adduct in SARS-CoV-2 Mpro. However, it can form an activated complex at C142, which is uniquely present in HCoV-OC43 Mpro (the corresponding residue in SARS-CoV-2 Mpro, N142, is unreactive). Although compound 21 partially fills the lipophilic S2 region of HCoV-OC43 Mpro with the difluorocyclohexyl moiety, the pyridine moiety is not in the correct position to occupy the S1 pocket and as such is unable to make a hydrogen bonding interaction with H163 (as in SARS-CoV-2 Mpro). To the best of our knowledge, this work also represents the first disclosure of the structure of HCoV-OC43 Mpro.

Figure 5

Figure 5. Cocrystal structure of 21 (green) bound to HCoV-OC43 Mpro (rainbow). Compound’s pyrimidine moiety covalently interacts with OC43 at residue C142, which is N142 in wt SARS-CoV-2 Mpro (salmon). (PDB ID 9C7W).

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Table 3. Broad-spectrum Biochemical Activity of Pyrazolopyrimidine Sulfones in the Mpro Diversity Panel
a

Shown are mean IC50 values derived from a minimum of three replicates.

Although the pyrazolopyrimidine series displayed promising broad-spectrum activity, it was difficult to achieve desirable ADME properties while maintaining good potency (Table 4). This series generally displayed high liver microsomal clearance and was also highly reactive to glutathione (GSH). To improve GSH half-life, the sulfone methyl group was replaced with larger groups (ethyl, isopropyl) to sterically encumber the reactive site. Although this strategy was indeed successful, with longer GSH half-lives observed, there was a corresponding potency loss, particularly for tert-butyl sulfone analogs (compounds 24–27) and microsomal stability was not improved. Further modifications to the scaffold in the S2 and S1 areas were viewed as a plausible path forward for improving liver microsomal stability, but this generally came at a further loss in potency (compounds 28–31). As we were unable to achieve potent compounds with balanced properties in the pyrazolopyrimidine series, further development, including profiling in cellular assays, was not pursued. This novel series serves as proof-of-concept for the potential for the discovery of broad-spectrum Mpro inhibitors, as well as inspiration and novel structural insight into targeting other coronaviruses such as HCoV-OC43.
Table 4. SARS-CoV-2 Mpro Biochemical Potency and In Vitro ADME Data for Representative Compounds
a

Shown are mean IC50 values derived from a minimum of three replicates.

Synthetic access to analogues bearing the dihydropyrazolo[1,5-a]pyrimidine core generally followed the synthetic strategy of compound 1 (Scheme 1). (63,64) 2-Fluoroaniline (32) was acylated to form 33 in 94% yield, then condensed with DMF-DMA, and cyclized with hydroxylamine to furnish the key aminopyrazole intermediate 35 (22% over two steps). Coupling 35 with (E)-4-(2-fluorophenyl)-2-oxobut-3-enoic acid readily constructed the core and final product in 33% yield. This modular strategy provided efficient access to a variety of analogues including compounds 3 to 11.

Scheme 1

Scheme 1. Synthesis of Compound 1a
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aReagents and conditions: (a) 2-cyanoacetic acid (1.0 equiv), EDCI·HCl (1.2 equiv), CH2Cl2, 25 °C, 94%; (b) DMF-DMA (2.0 equiv), toluene, 80 °C; (c) NH2NH2·H2O (2.0 equiv), EtOH, 25–80 °C, 22% over two steps; (d) (E)-4-(2-fluorophenyl)-2-oxobut-3-enoic acid (1.0 equiv), HOAc, 25–80 °C, 33%.

The route to access compound 13 originally involved further derivatization of intermediate 35 (Scheme 2). Condensation with diethyl malonate (88%) followed by treatment with POCl3 provided the dihalogenated core 37 in 71% yield. A regioselective Stille reaction with the corresponding pyridyl stannane afforded 38 with a yield of 8%. SNAr with NaSMe (5%) followed by oxidation furnished the sulfone, accessing 13 (34%).

Scheme 2

Scheme 2. Synthesis of Compound 13a
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aReagents and conditions: (a) diethyl malonate (1.5 equiv), NaOEt (2.5 equiv), EtOH, 25–80 °C, 88%; (b) POCl3, 25–80 °C, 71%; (c) 3-methyl-2-(tributylstannyl)pyridine (0.8 equiv), Pd(PPh3)2Cl2 (0.1 equiv), CuI (0.1), dioxane, 130 °C, 8%; (d) NaSMe (1.1 equiv), THF/H2O = 1:1, 20 °C, 5%; (e) m-CPBA (3.5 equiv), CH2Cl2, 20 °C, 34%.

Compound 21 was synthesized from 1-(pyrimidin-2-yl)ethan-1-one (40) (Scheme 3). Condensation of 40 with CS2 followed by alkylation with i-PrI afforded 41 in 20% yield. Coupling of 41 with an aminopyrazole afforded the core 42 with the desired regioselectivity (30%), and oxidation of 42 readily afforded 21 in 36% yield.

Scheme 3

Scheme 3. Synthesis of 21a
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aReagents and conditions: (a) CS2 (1.0 equiv), i-PrI (2.2 equiv), NaH (2.5 equiv), THF, 25 °C, 20%; (b) 4-(4,4-difluorocyclohexyl)-1H-pyrazol-5-amine (1.0 equiv), piperidine (0.1 equiv), HOAc/H2O = 3/1, 25–110 °C, 30%; (c) m-CPBA (2.0 + 0.5 + 0.5 equiv), CH2Cl2, 0–25 °C, 36%.

A second generation synthesis of analogues of 13 was adapted from the synthesis of 21 and further elaborated to access compounds such as 24 (Scheme 4). (65,66) Condensing 43 with CS2 followed by alkylation with methyl iodide afforded the dialkylated compound 44 in 75% yield. This intermediate reacted with the corresponding aminopyrazole selectively to form regioisomer 45 in 19% yield. Subsequent oxidation afforded methyl sulfone 27 (78%) which readily underwent a two-step sequence of an SNAr with a thiolate and oxidation (35% over two steps) to form 24.

Scheme 4

Scheme 4. Synthesis of 24a
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aReagents and conditions: (a) CS2 (1.0 equiv), MeI (2.2 equiv), NaH (2.5 equiv), THF, 25 °C, 75%; (b) 4-cyclohexyl-1H-pyrazol-5-amine (1.0 equiv), piperidine (0.1 equiv), HOAc/H2O = 3/1, 25–110 °C, 19%; (c) Oxone (3.0 eq +1.0 equiv), MeOH/H2O = 2/1, 25 °C, 78%; (d) t-BuSNa (3.0 equiv), t-BuOH, 25 °C; (e) Oxone (2.9 equiv), MeOH/H2O = 2/1, 25 °C, 35% over two steps.

Conclusions

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A primary challenge in developing broad-spectrum antivirals is the amount of sequence diversity in the target binding site that can be present within and across viral species. (67) Residues that contribute to compound engagement in one species may be different in another species and lead to reduced or abolished compound binding. It thus becomes critical to assess activity against multiple species to determine the degree of coverage for a set of compounds. This is clearly not feasible when the number of viral species under consideration is in the thousands. We therefore developed a strategy to cluster Mpro active site residues by similarity and select a centroid representative from each cluster, with the assumption being that if a compound is active against the representative, it is also likely to be active against most of its other cluster members. This heuristic dramatically reduces the total experimental workload and makes the drug discovery process much more tractable.
One important parameter in our approach was the choice of residue distance cutoff below which we separated species in our hierarchical clustering approach. We empirically evaluated how different cutoffs affected both the number of clusters as well as the degree of similarity within each cluster. A cutoff of eight differences was deemed optimal, corresponding to a maximum panel of 19 members, which was considered to be experimentally tractable in our antiviral program.
After identifying a hit from high-throughput screening, medicinal chemistry efforts produced compounds that showed submicromolar biochemical activity against Mpro from multiple coronaviruses. When considering both these compounds as well as availability of developed assays for these diverse coronaviruses, this work serves as an inspiration for further investigation, and provides a foundation for the discovery of future generations of broad-spectrum Mpro inhibitors.

Experimental Section

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General Chemistry

All materials and reagents used were of the commercially available grade and used without further purification. Reaction progress was monitored by either TLC (Merck, 0.2 mm silica gel 60 F254 on glass plates) or LCMS systems (Shimadzu 20AD XR or Waters Acquity). Normal-phase column chromatography was carried out using prepacked silica gel cartridges on a Combiflash Rf separation system by Teledyne ISCO. All microwave reactions were conducted in a Biotage Initiator, irradiating at 0–400 W from a magnetron at 2.45 GH. 1H NMR, 19F, and 2D-spectra were determined on a Varian 400 or Bruker 300 or 400 and 500 MHz NMR spectrometers. Chemical shifts (d-values) are reported in ppm downfield from tetramethylsilane. The following abbreviations are used: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, bs = broad singlet. Mass spectra were acquired on LCMS systems using electrospray, chemical and electron impact ionization methods with a range of instruments of the following configurations: Agilent 1260 and 6125B, Shimadzu LC-20AD XR&MS 2020, Agilent 1200 and 6120B. [M+1]+ refers to the protonated molecular ion of the chemical species. Preparative HPLC was performed on Waters Prep HPLC systems or Gilson 281 Semipreparative HPLC systems using C18 reversed-phase columns Waters Xbridge Prep OBD C18, Phenomenex luna C18, eluting with gradient mixtures of water/acetonitrile containing a modifier 0.05% trifluoroacetic acid or formic acid. Enantiomers were purified by supercritical fluid chromatography (SFC) on a Waters Preparative SFC-100-MS system with ABSYS update, with a Waters 2998 Photodiode Array Detector and a Waters MS Single Quadrupole Detector using a DAICEL CHIRALPAK AD column. All compounds are >95% pure by HPLC (with the exception of compound 4, which was determined to be 93% purity).

2-Cyano-N-(2-fluorophenyl)acetamide (33)

Two batches were carried out in parallel. To a mixture of 2-fluoroaniline (32) (10.0 g, 89.99 mmol, 1.0 equiv) in CH2Cl2 (180 mL) was added 2-cyanoacetic acid (7.7 g, 89.99 mmol, 1.0 equiv) and EDCI·HCl (20.7 g, 107.99 mmol, 1.2 equiv) at 25 °C. Then the mixture was stirred at 25 °C for 1 h under Ar. TLC (Petroleum ether/Ethyl acetate = 3/1, Rf = 0.30) indicated the starting material was consumed completely and one new spot was formed. Two parallel reactions were worked up together and concentrated. The residue was diluted with CH2Cl2 (50 mL) and aq. HCl solution (0.1 M, 100 mL). The mixture was extracted with CH2Cl2 (50 mL × 2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound (30 g, 94%) as a white solid was used for the next step directly. 1H NMR (400 MHz, DMSO-d6) δ = 10.14 (br s, 1H), 7.98–7.78 (m, 1H), 7.32–7.23 (m, 1H), 7.23–7.13 (m, 2H), 3.99 (s, 2H).

(E)-2-Cyano-3-(dimethylamino)-N-(2-fluorophenyl)acrylamide (34)

Two batches were carried out in parallel. A mixture of 2-cyano-N-(2-fluorophenyl)acetamide (33) (10.0 g, 56.13 mmol, 1.0 equiv) and DMF-DMA (13.4 g, 112.25 mmol, 2.0 equiv) in toluene (180 mL) was stirred at 80 °C for 3 h under N2. TLC (Petroleum ether/Ethyl acetate = 1/1, Rf = 0.30) indicated the starting material was consumed completely and one new spot was formed. Two parallel reactions were worked up together. The reaction mixture was concentrated to give the title compound (26 g, crude) as a yellow solid which would be used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ = 8.69–8.63 (m, 1H), 7.84 (s, 1H), 7.76–7.64 (m, 1H), 7.28–7.18 (m, 1H), 7.18–7.09 (m, 2H), 3.29 (s, 3H), 3.22 (s, 3H).

5-Amino-N-(2-fluorophenyl)-1H-pyrazole-4-carboxamide (35)

Three parallel reactions were carried out. To the mixture of (E)-2-cyano-3-(dimethylamino)-N-(2-fluorophenyl)acrylamide (34) (11.3 g, 48.45 mmol, 1.0 equiv) in EtOH (100 mL) was added NH2NH2·H2O (4.8 g, 96.89 mmol, 2.0 equiv) at 25 °C. The mixture was stirred at 80 °C for 3 h under N2. TLC (Ethyl acetate, Rf = 0.20) indicated the starting material was consumed completely and one new spot was formed. Three parallel reactions were worked up together. The reaction mixture was concentrated to give the crude product. The crude product was purified by RP-MPLC (neutral) to give the title compound (7.0 g, 22%) as a white solid. LCMS (ESI): m/z = 221.0 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 11.86 (br s, 1H), 9.31 (s, 1H), 7.99 (s, 1H), 7.69–7.54 (m, 1H), 7.28–7.14 (m, 3H), 5.82 (br s, 2H).

7-(2-Fluorophenyl)-3-((2-fluorophenyl)carbamoyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acid (1)

To a solution of 5-amino-N-(2-fluorophenyl)-1H-pyrazole-4-carboxamide (35) (200 mg, 0.91 mmol, 1.0 equiv) in AcOH (3 mL) was added (E)-4-(2-fluorophenyl)-2-oxobut-3-enoic acid (211 mg, 0.91 mmol, 1.0 equiv) at 25 °C. The solution was stirred at 80 °C for 16 h under N2. LCMS showed the starting material was consumed completely and the desired product was detected. The reaction mixture was quenched by H2O (5 mL) and then the solid was precipitated and collected by filtration. The crude material was purified by prep-HPLC (column: Phenomenex Luna C18 100 × 30 mm × 3 μm; mobile phase: [A: H2O (0.04% HCl); B: ACN]; B%: 40.00%- 70.00%, 8.00 min) to afford the title compound (70 mg, 33%) as a white solid. LCMS (ESI): m/z = 397.1 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 9.72 (s, 1H), 8.42 (d, J = 1.5 Hz, 1H), 8.08 (s, 1H), 7.63 (dt, J = 2.1, 7.7 Hz, 1H), 7.41–7.35 (m, 1H), 7.32–7.28 (m, 1H), 7.27–7.23 (m, 2H), 7.22 (d, J = 1.8 Hz, 1H), 7.19 (s, 1H), 7.18–7.16 (m, 1H), 7.15 (br d, J = 1.5 Hz, 1H), 6.47 (d, J = 4.1 Hz, 1H), 5.72 (br s, 1H).

N-(2-Fluorophenyl)-5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxamide (36)

To the mixture of 5-amino-N-(2-fluorophenyl)-1H-pyrazole-4-carboxamide (35) (20.0 g, 90.82 mmol, 1.0 equiv) in EtOH (200 mL) was added diethyl malonate (21.8 g, 136.82 mmol, 1.5 equiv) and NaOEt (77.3 g, 227.06 mmol, 2.5 equiv) at 25 °C. The mixture was stirred at 80 °C under N2 for 16 h. LCMS showed the starting material was consumed completely and one main peak with desired mass was detected. The mixture was diluted with water (400 mL), adjusted to pH = 4 with 2 M HCl at 0 °C. The reaction mixture was filtered and the filter cake was washed with H2O (100 mL × 3). The filter cake was dried under vacuum to give the title compound (23 g, 88%) as a white solid. LCMS (ESI): m/z = 288.9 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 11.86–10.78 (m, 1H), 9.96 (br s, 1H), 8.45 (br s, 1H), 7.67 (br s, 1H), 7.44–7.03 (m, 4H), 5.11 (br s, 1H).

5,7-Dichloro-N-(2-fluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (37)

Three batches were carried out in parallel. N-(2-fluorophenyl)-5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxamide (36) (1.0 g, 3.47 mmol, 1.0 equiv) and POCl3 (4 mL) were combined at 25 °C under N2. The mixture was stirred at 80 °C for 16 h under N2. LCMS showed the starting material was consumed completely and one main peak with desired mass was detected. Three batches were worked up together. The reaction mixture was quenched with aq. NaHCO3 (50 mL) and filtered. The filter cake was washed by H2O (10 mL × 3) and dried under vacuum to give the title compound (2.4 g, 71%) as a white solid. LCMS (ESI): m/z = 324.9 [M + H]+.

5-Chloro-N-(2-fluorophenyl)-7-(3-methylpyridin-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (38)

To a solution of 5,7-dichloro-N-(2-fluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (37) (240 mg, 0.06 mmol, 1.0 equiv), 3-methyl-2-(tributylstannyl)pyridine (225 mg, 0.59 mmol, 0.8 equiv) and CuI (14 mg, 0.01 mmol, 0.1 equiv) in dioxane (3 mL) was added Pd(PPh3)2Cl2 (52 mg, 0.01 mmol, 0.1 equiv). Then the mixture was stirred at 130 °C for 2 h under N2. TLC (Petroleum ether/Ethyl acetate = 1/1, Rf-P1 = 0.30) indicated the starting material was consumed completely and three new spots were shown on TLC. The reaction mixture was quenched by H2O (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (12 g SepaFlash Silica Flash Column, Eluent of 0–30% Ethyl acetate/Petroleum ether gradient @ 80 mL/min). The crude product was further purified by prep-HPLC (column: 3_Phenomenex Luna C18 75 × 30 mm × 3 μm; mobile phase: [A: H2O (0.2% FA), B: ACN], B%: 40.00–70.00%, 8.00 min) to give the title compound (22 mg, 8%) as a white solid. LCMS (ESI): m/z = 382.1 [M + H]+;1H NMR (400 MHz, DMSO-d6) δ = 9.91 (d, J = 2.5 Hz, 1H), 8.75 (s, 1H), 8.62 (d, J = 4.1 Hz, 1H), 8.38 (dt, J = 1.4, 8.0 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.61 (dd, J = 4.7, 7.8 Hz, 1H), 7.37 (ddd, J = 1.2, 8.2, 11.2 Hz, 1H), 7.29–7.22 (m, 1H), 7.21–7.14 (m, 1H), 2.16 (s, 3H).

N-(2-Fluorophenyl)-7-(3-methylpyridin-2-yl)-5-(methylthio)pyrazolo[1,5-a]pyrimidine-3-carboxamide (39)

To a stirred solution of 5-chloro-N-(2-fluorophenyl)-7-(3-methylpyridin-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (38) (200 mg, 0.52 mmol, 1.0 equiv) in THF (2 mL) and H2O (2 mL) was added NaSMe (202 mg, 20%, 0.14 mmol, 1.1 equiv). The reaction was stirred at 20 °C for 12 h. LCMS showed the reagent was consumed completely and desired mass was detected. The reaction mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (10 mL × 3). The filter cake was dried under vacuum. The crude product was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*3um; mobile phase: [A: H2O (0.2% FA); B: ACN]; B%: 30.00–60.00%, 8.00 min) to afford the title compound (10 mg, 28%) as a yellow solid. LCMS (ESI): m/z = 394.1 [M + H]+;1H NMR (400 MHz, DMSO-d6) δ = 9.96 (br d, J = 2.0 Hz, 1H), 8.59 (br d, J = 4.4 Hz, 1H), 8.56 (s, 1H), 8.49 (t, J = 7.8 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.57 (dd, J = 4.8, 7.7 Hz, 1H), 7.43 (s, 1H), 7.38 (dd, J = 8.9, 11.3 Hz, 1H), 7.29–7.21 (m, 1H), 7.20–7.11 (m, 1H), 2.80 (s, 3H), 2.14 (s, 3H).

N-(2-Fluorophenyl)-7-(3-methylpyridin-2-yl)-5-(methylsulfonyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (13)

To a stirred solution of N-(2-fluorophenyl)-7-(3-methylpyridin-2-yl)-5-(methylthio)pyrazolo[1,5-a]pyrimidine-3-carboxamide (39) (80 mg, 0.20 mmol, 1.0 equiv) in CH2Cl2 (2 mL) was added m-CPBA (144 mg, 85%, 0.71 mmol, 3.5 equiv). The reaction was stirred at 20 °C for 1 h. LCMS showed the starting material was consumed completely and ∼59% of desired mass was detected. The reaction mixture was quenched by Na2SO3 aq. (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (column: Phenomenex luna C18 100 × 40 mm × 5 μm; mobile phase: [A: H2O (0.2% FA); B: ACN]; B%: 25.00–65.00%, 8.00 min) to afford the title compound (29 mg, 33%) as a yellow solid. LCMS (ESI): m/z = 426.0 [M + H]+;1H NMR (400 MHz, DMSO-d6) δ = 9.91 (d, J = 2.4 Hz, 1H), 8.97 (s, 1H), 8.65 (d, J = 3.9 Hz, 1H), 8.44–8.36 (m, 1H), 7.99–7.89 (m, 2H), 7.64 (dd, J = 4.7, 7.8 Hz, 1H), 7.40 (dt, J = 1.1, 9.7 Hz, 1H), 7.31–7.24 (m, 1H), 7.23–7.15 (m, 1H), 3.63 (s, 3H), 2.17 (s, 3H).

(Z)-3-(Isopropylthio)-3-mercapto-1-(pyrimidin-2-yl)prop-2-en-1-one (41)

To the mixture of NaH (2.5 g, 61.41 mmol, 2.5 equiv) in THF (30 mL) was added 1-(pyrimidin-2-yl)ethan-1-one (40) (3.0 g, 24.56 mmol, 1.0 equiv) at 25 °C under N2. The reaction mixture was stirred at 25 °C for 0.5 h. CS2 (1.9 g, 24.56 mmol, 1.0 equiv) was added to the reaction mixture over 0.5 h followed by addition of i-PrI (9.2 g, 54.04 mmol, 2.2 equiv) and then the mixture was stirred for 15 h at 25 °C. TLC (Petroleum ether/Ethyl acetate = 1/1, Rf-p1 = 0.30) indicated the starting material was consumed completely and three new spots were shown on TLC. The reaction mixture was quenched by aq. NH4Cl (40 mL). The reaction mixture was extracted with CH2Cl2 (40 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (120 g SepaFlash Silica Flash Column, Eluent of 30–80% Ethyl acetate/Petroleum ether gradient @ 60 mL/min) to give the title compound (1.4 g, 20%) as a brown solid. LCMS (ESI): m/z = 241.0 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 8.98 (s, 1H), 8.97 (s, 1H), 7.64 (t, J = 4.9 Hz, 1H), 7.51 (s, 1H), 4.00–3.94 (m, 1H), 1.40 (s, 3H), 1.38 (s, 3H).

3-(4,4-Difluorocyclohexyl)-7-(isopropylthio)-5-(pyrimidin-2-yl)pyrazolo[1,5-a]pyrimidine (42)

A solution of (Z)-3-(isopropylthio)-3-mercapto-1-(pyrimidin-2-yl)prop-2-en-1-one (41) (250 mg, 1.04 mmol, 1.0 equiv) in HOAc/H2O (2.8 mL, 3/1) was added 4-(4,4-difluorocyclohexyl)-1H-pyrazol-5-amine (209 mg, 1.04 mmol, 1.0 equiv) and piperidine (9 mg, 0.10 mmol, 0.1 equiv) under N2 at 25 °C. Then the mixture was stirred at 110 °C for 16 h. TLC (Petroleum ether/Ethyl acetate = 2:1, Rf-P1 = 0.50) indicated the starting material was consumed completely and several new spots were detected. The reaction mixture was quenched by aq. NaHCO3 (4 mL) and extracted with CH2Cl2 (4 mL × 3). The combined organic layers were washed with brine (3 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (10 g SepaFlash Silica Flash Column, Eluent of 25–60% Ethyl acetate/Petroleum ether gradient @20 mL/min) to give the title compound (120 mg, 30%) as a yellow solid. LCMS (ESI): m/z = 390.2 [M + H]+;1H NMR (400 MHz, DMSO-d6) δ = 9.06 (d, J = 4.9 Hz, 2H), 8.26 (s, 1H), 7.92 (s, 1H), 7.65 (t, J = 4.9 Hz, 1H), 4.05–3.97 (m, 1H), 3.24–3.14 (m, 1H), 214–2.09 (m, 4H), 1.98 (s, 2H), 1.91–1.82 (m, 2H), 1.51 (s, 3H), 1.50 (s, 3H).

3-(4,4-Difluorocyclohexyl)-7-(isopropylsulfonyl)-5-(pyrimidin-2-yl)pyrazolo[1,5-a]pyrimidine (21)

To the mixture of 3-(4,4-difluorocyclohexyl)-7-(isopropylthio)-5-(pyrimidin-2-yl)pyrazolo[1,5-a]pyrimidine (42) (100 mg, 0.26 mmol, 1.0 equiv) in CH2Cl2 (4 mL) was added m-CPBA (104 mg, 0.51 mmol, 2.0 equiv) at 0 °C. The reaction was stirred at 25 °C for 0.5 h under N2. Then the m-CPBA (26 mg, 0.13 mmol, 0.5 equiv) was added to the mixture at 0 °C and stirred at 25 °C for 0.5 h. Then the m-CPBA (26 mg, 0.13 mmol, 0.5 equiv) was added to the mixture at 0 °C stirred at 25 °C for 0.5 h. LCMS showed the starting material was consumed completely and 29% with desired mass was detected. The reaction solution was quenched by aq. Na2SO3 (5 mL) and extracted with CH2Cl2 (5 mL × 3). The combined organic layers were washed with brine (2 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by prep-HPLC (column: Waters Xbridge BEH C 18 100 × 30 mm × 10 μm; mobile phase: [A: H2O (10 mM NH4HCO3); B: ACN]; B%: 35.00–65.00%, 8.00 min) to give the title compound (38.5 mg, 36%) as yellow solid. LCMS (ESI): m/z = 358.1 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 9.11 (d, J = 4.8 Hz, 2H), 8.52 (s, 1H), 8.50 (s, 1H), 7.70 (t, J = 4.8 Hz, 1H), 4.43–4.33 (m, 1H), 3.30–3.23 (m, 1H), 2.18–2.11 (m, 5H), 2.07 (br d, J = 14.2 Hz, 1H), 1.97–1.87 (m, 2H), 1.31 (d, J = 6.8 Hz, 6H).

1-(3-Methoxypyridin-2-yl)-3,3-bis(methylthio)prop-2-en-1-one (44)

To a solution of NaH (860 mg, 21.50 mmol, 2.5 equiv) in THF (20 mL) was added slowly 1-(3-methoxypyridin-2-yl)ethan-1-one (43) (1.3 g, 8.60 mmol, 1.0 equiv) at 25 °C under N2, and the reaction mixture was stirred at 25 °C for 0.5 h. CS2 (655 mg, 8.60 mmol, 1.0 equiv) was added to the reaction mixture over 0.5 h followed by addition of MeI (2.7 g, 18.92 mmol, 2.2 equiv). The mixture was stirred for 12 h at 25 °C. TLC (Ethyl acetate, Rf-P1 = 0.35) indicated the starting material was consumed completely and two new spots were shown on TLC. The reaction mixture was quenched by aq. NH4Cl (30 mL) and extracted with CH2Cl2 (40 mL × 3). The combined organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (25 g SepaFlash Silica Flash Column, Eluent of 0–70% Ethyl acetate/Petroleum ether gradient @ 80 mL/min) to the title compound (1.7 g, 75%) as a yellow solid. LCMS (ESI): m/z = 256.1 [M + H]+; 1H NMR (400 MHz, CDCl3) δ = 8.22 (dd, J = 1.6, 4.0 Hz, 1H), 7.38–7.29 (m, 2H), 7.01 (s, 1H), 3.90 (s, 3H), 2.52 (s, 6H).

3-Cyclohexyl-7-(3-methoxypyridin-2-yl)-5-(methylthio)pyrazolo[1,5-a]pyrimidine (45)

A solution of 1-(3-methoxypyridin-2-yl)-3,3-bis(methylthio)prop-2-en-1-one (44) (100 mg, 0.39 mmol, 1.0 equiv) in HOAc (0.9 mL) and H2O (0.3 mL) was added dropwise into a solution of 4-cyclohexyl-1H-pyrazol-5-amine (65 mg, 0.39 mmol, 1.0 equiv) and piperidine (3 mg, 0.04 mmol, 0.1 equiv) at 25 °C under N2. The mixture was stirred at 110 °C for 12 h. LCMS showed the starting material was consumed completely and one main peak with desired mass was detected. The mixture was diluted with water (10 mL), adjusted to pH = 7 with saturated aqueous NaHCO3 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (15 mL × 3), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by prep-HPLC (column: Phenomenex Luna C18 75*30 mm*3um; mobile phase: [A: H2O (0.2% FA); B: ACN]; B%: 50.00–90.00%, 8.00 min) to afford the title compound (26 mg, 19%) as a white solid. LCMS (ESI): m/z = 355.1 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 8.29 (dd, J = 1.1, 4.6 Hz, 1H), 7.87 (s, 1H), 7.72–7.67 (m, 1H), 7.60 (dd, J = 4.6, 8.5 Hz, 1H), 6.91 (s, 1H), 3.75 (s, 3H), 2.86 (tt, J = 3.5, 11.7 Hz, 1H), 2.62 (s, 3H), 2.04–1.95 (m, 2H), 1.84–1.76 (m, 2H), 1.72 (br d, J = 12.0 Hz, 1H), 1.59 (dq, J = 3.0, 12.3 Hz, 2H), 1.47–1.35 (m, 2H), 1.33–1.22 (m, 1H).

3-Cyclohexyl-7-(3-methoxypyridin-2-yl)-5-(methylsulfonyl)pyrazolo[1,5-a]pyrimidine (27)

To a mixture of 3-cyclohexyl-7-(3-methoxypyridin-2-yl)-5-(methylthio)pyrazolo[1,5-a]pyrimidine (45) (200 mg, 0.56 mmol, 1.0 equiv) in MeOH/H2O (3 mL, 2/1) was added Oxone (285 mg, 1.69 mmol, 3.0 equiv) at 25 °C. The reaction mixture was stirred at 25 °C for 12 h under N2. Then additional Oxone (95 mg, 0.56 mmol, 1.0 equiv) was added to the mixture at 25 °C. The reaction was stirred at 25 °C for 4 h under N2. TLC (Petroleum ether/Ethyl acetate = 3/1, Rf = 0.50) indicated that starting material still remained and one new spot was formed. The mixture was diluted with H2O (2 mL) and extracted with ethyl acetate (3 mL × 3). The combined organic layers were washed with brine (2 mL), dried over Na2SO4, filtered, and concentrated. The crude was purified by MPLC (Petroleum ether/EtOAc = 20–50%) to give the title compound (170 mg, 78%) as a yellow solid. LCMS (ESI): m/z = 387.2 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ = 8.36 (dd, J = 1.2, 4.6 Hz, 1H), 8.33 (s, 1H), 7.80–7.76 (m, 1H), 7.68 (dd, J = 4.6, 8.6 Hz, 1H), 7.55 (s, 1H), 3.78 (s, 3H), 3.48 (s, 3H), 3.01 (s, 1H), 2.05–1.98 (m, 2H), 1.85–1.78 (m, 2H), 1.74 (br d, J = 12.7 Hz, 1H), 1.61 (dq, J = 2.8, 12.4 Hz, 2H), 1.51–1.39 (m, 2H), 1.36–1.26 (m, 1H).

5-(tert-Butylthio)-3-cyclohexyl-7-(3-methoxypyridin-2-yl)pyrazolo[1,5-a]pyrimidine (46)

To a mixture of 3-cyclohexyl-7-(3-methoxypyridin-2-yl)-5-(methylsulfonyl)pyrazolo[1,5-a]pyrimidine (27) (100 mg, 0.26 mmol, 1.0 equiv) in t-BuOH (1.5 mL) was added t-BuSNa (87 mg, 0.78 mmol, 3.0 equiv) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h under N2. LCMS showed the starting material was consumed completely and one main peak with desired mass was detected. The mixture was diluted with H2O (3 mL) and extracted with ethyl acetate (3 mL × 3). The combined organic mixture was washed with brine (2 mL), dried over Na2SO4, filtered and concentrated to give the title compound (120 mg, crude) as brown oil which would be used for the next step directly. LCMS (ESI): m/z = 397.2 [M + H]+.

5-(tert-Butylsulfonyl)-3-cyclohexyl-7-(3-methoxypyridin-2-yl)pyrazolo[1,5-a]pyrimidine (24)

To the mixture of 5-(tert-butylthio)-3-cyclohexyl-7-(3-methoxypyridin-2-yl)pyrazolo[1,5-a]pyrimidine (46) (120 mg, 0.26 mmol, 1.0 equiv) in MeOH/H2O (3 mL) was added Oxone (127 mg, 0.76 mmol, 2.9 equiv) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h under N2. LCMS showed the starting material was consumed completely and 70% with desired mass was detected. The mixture was diluted with H2O (4 mL) and extracted with ethyl acetate (3 mL × 3). The combined organic mixture was washed with brine (2 mL), dried over Na2SO4, filtered, and concentrated to give crude product. The crude product was purified by prep-HPLC (column: Phenomenex luna C 18 100 × 40 mm × 5 μm; mobile phase: [A: H2O (0.04% HCl); B: ACN]; B%: 45.00–75.00%, 8.00 min) to give the title compound (39 mg, 35%) as a yellow solid. LCMS (ESI): m/z = 429.2 [M + H]+;1H NMR (400 MHz, DMSO-d6) δ = 8.36 (dd, J = 1.1, 4.6 Hz, 1H), 8.34 (s, 1H), 7.78 (dd, J = 1.0, 8.6 Hz, 1H), 7.68 (dd, J = 4.6, 8.6 Hz, 1H), 7.50 (s, 1H), 3.79 (s, 3H), 2.99 (tt, J = 3.4, 11.7 Hz, 1H), 2.06–1.98 (m, 2H), 1.84–1.77 (m, 2H), 1.77–1.70 (m, 1H), 1.63 (dq, J = 2.9, 12.4 Hz, 2H), 1.44 (br d, J = 3.3 Hz, 2H), 1.41 (s, 9H), 1.32–1.21 (m, 1H).

Curation of Mpro Sequences in Coronavirus Isolates

Two sequence data sets were assembled for identifying Mpro boundaries in unannotated coronavirus genomes (Figure 1A(i)). The first included RefSeq Mpro protein sequences, identified through having “3C” or “nsp5” in their FASTA headers and totaling between 250 and 350 amino acids in length (Supporting Information, Table S2). These were derived from the NCBI Protein Database and from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (31) platform. The second data set comprised isolate sequences in the NCBI Nucleotide database annotated as being in the Coronaviridae family (Taxon ID 11118), excluding SARS-CoV-2 sequence isolates other than their RefSeq genomes (Supporting Information, Table S1). Protein sequences were used as queries in a tBLASTn (32) search against the translated isolate nucleotide sequences to identify the Mpro regions and amino acid sequences in the viral genomes (Figure 1A(ii)). This data set was filtered to remove identical sequences, sequences less than 250 amino acids in length, and sequences with stop codons or 10 or more ambiguous “X” amino acids. All data were obtained in September 2022.

Identification of Active Site Residues in Isolate Mpro Sequences

SARS-CoV-2 Mpro active site residues were identified through enumerating residues within 5 Å of ligands present in selected solved structures (PDB accessions 5r84, (34) 7mb4, (21) 7vu6 (33)) followed by manual refinement (Supporting Information, Figure S1). Coronavirus Mpro solved structures in the PDB were compiled and structurally aligned to the SARS-CoV-2 Mpro structure using the PyMOL “align” algorithm, annotating equivalent active site residues in these structures through the resulting 3D-superposition. Next, the genome isolate Mpro sequences identified in the previous step were aligned to the structure sequences using BLASTp, (32) and for each isolate, the structure with the lowest e-value was selected as its reference structure. The alignment between each isolate Mpro sequence and its reference structure was used to infer the equivalent active site residues for that sequence, resulting in an array of noncontiguous sequences residue sequences (Figure 1A(iii–iv)).

Clustering Mpro Active Site Residues

An all-by-all distance matrix was constructed by counting the number of identical amino acids across each pair of active sites. This distance matrix was used as input to a standard agglomerative, bottom-up, average linkage hierarchical procedure to cluster the active site residues. Final clusters were generated by identifying subtrees rooted below the empirically chosen horizontal line of eight amino acid differences, and active site leaves of each subtree were grouped as members of a cluster.

Protein Expression and Purification

Genes encoding Mpro homologues were codon optimized and synthesized into a PET-based expression plasmid encoding an N-terminal His-SUMO tag. SUMO was chosen for two reasons: first, activity of Mpro requires an authentic N-terminus and SUMO cleavage leaves no “scar”, unlike other proteases; second, the protein, when fused to SUMO, is inactive, leading to less cytotoxicity in the expression host. We note that we originally tried to use TEV protease, however the cleavage sequence was too similar to the native Mpro cleavage sequence, and these tags were often self-cleaved. The synthesis was outsourced to Twist Biosciences.
Chemically competent Hi-Control BL21(DE3) Escherichia coli cells were transformed with expression plasmids using standard techniques, and a single colony was used to start an overnight culture in Lysogeny Broth (LB) media (68) (with 50 μg/mL kanamycin). This culture was used to inoculate a 1 L culture in Terrific Broth (TB) (69) supplemented with 50 mM sodium phosphate pH 7.0 and 50 μg/mL kanamycin. These cells were grown in Fernbach flasks at 37 °C while shaking at 200 rpm until the OD600 reached between 1–1.5, at which point the temperature was reduced to 19 °C and the cells were induced with 0.5 mM Isopropyl ß-d-1-thiogalactopyranoside (IPTG). Cells were allowed to grow overnight before harvesting by centrifugation (6000g for 30 min) and either stored at −70 °C or immediately processed (storage did not affect the yield or quality of the protein; data not shown).
Cell pellets were resuspended in lysis buffer (50 mM Tris pH 8.0, 400 mM NaCl, 1 mM TCEP) and lysed by 3–5 passes through a cell homogenizer. The lysate was clarified by centrifugation (45,000g for 30 min) and then was flowed through a 5 mL Ni-Sepharose Excel column (pre-equilibrated with 5 column volumes (CVs) of lysis buffer) on an AKTA FPLC. The material was flowed using a sample pump with a flow rate of 5 mL/min.
Following column loading, the column was washed with lysis buffer until the A280 stabilized. The material was then slowly eluted with a linear gradient of elution buffer (50 mM Tris pH 8.0, 400 mM NaCl, 1 mM TCEP, 500 mM imidazole) over 40 column volumes.
Fractions containing Mpro protein were determined by SDS-PAGE analysis and were pooled. ESI-LCMS analysis was used to confirm the correct molecular weight in all cases (occasionally, a mass corresponding to the loss of the N-terminal methionine was observed; this resolved after cleavage of the N-terminal tag).
100 μg of ULP1 was added to the pooled fractions from the IMAC purification to cleave the His-SUMO tag. Cleavage proceeded at room temperature overnight while dialyzing into 3 L of lysis buffer using a 3500 MWCO dialysis cassette. In most cases, substantial precipitate was observed at this step. However, the protein which remained soluble did not further precipitate throughout the rest of the purification or in downstream applications, and by all observations was very stable.
The protein was then flowed over 5 mL of Ni-NTA resin pre-equilibrated with lysis buffer to remove additional contaminants, the His-SUMO tag, and the ULP1. The flow-through was collected and concentrated to approximately 5 mL prior to purification via SEC. A Superdex 75 16/60 column was pre-equilibrated with fresh SEC Buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and the protein was flowed through the column at 1 mL/min while collecting 1.0 mL fractions. Fractions were analyzed by SDS-PAGE and those containing pure Mpro were pooled, concentrated, aliquoted, and stored at −70 °C. Typical yields observed were between 40 and 80 mg of final protein per L of bacterial culture.

X-ray Crystallography

Crystals of SARS-CoV-2 Mpro (1-306) were grown by hanging-drop vapor diffusion at 18 °C. One μL of protein solution (12.4 mg/mL in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and 1 μL reservoir solution (0.1 M MIB pH 7.0, 20% PEG 1500) were mixed above a reservoir of 300 μL. Crystals grew overnight. They were supplemented with 25% glycerol and flash frozen in liquid nitrogen. Crystals of HCoV-OC43 Mpro (1-298) was grown by hanging-drop vapor diffusion at 18 °C. One μL of protein solution (10 mg/mL in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and 1 μL reservoir solution (0.1 M HEPES pH 7.5, 10% polyethylene glycol 8000, 8% ethylene glycol) were mixed above a reservoir of 300 μL. Crystals grew overnight. They were supplemented with 25% glycerol and flash frozen in liquid nitrogen. X-ray diffraction data was collected at Advanced Light Source (beamline 2.0.1.). Data integration and scaling were performed using XDS from AutoProc. The structure was solved by molecular replacement with Phaser using SARS-CoV-2 Mpro as a search model. Subsequence structure refinement was performed using PHENIX suite, and manual model building with Coot. Structural figures were prepared with PyMOL. Data collection and refinement statistics are summarized with Table 5.
Table 5. Crystallographic Statistics (Values in Parentheses are for the Highest Resolution Shell)
 HCoV-OC43 Mpro + 21SARS-CoV-2 Mpro + 14SARS-CoV-2 Mpro + 1
PDB code9C7W9C8Q9C8O
data collection   
resolution range95.21–2.08 (2.19–2.08)48.37–2.03 (2.14–2.03)48.53–2.00 (2.00–2.10)
space groupP 21 21 21C1 21 1P1 21 1
mol. in the ASU412
unit cell [a, b, c (Å)]67.25 129.80 140.07 90 90 9044.57 53.53 115.0344.78 53.87 114.05
90.00 101.12 90.0090.00 101.31 90.00
total reflections403,83397,369116,464
unique reflections73,41133,71334,629
multiplicity (high shell)5.5 (3.8)2.9 (2.4)3.3 4 (3.4)
completeness (%)98.7 (92.3)97.4 (93.3)94.9 (96.9)
mean I/σ (I)7.0 (1.6)9.2 (4.4)8.4 (2.3)
Wilson B-factor39.1623.9831.86
R-merge (high shell)0.111 (0.702)0.045 (0.128)0.060 (0.0374)
R-meas (high shell)0.135 (0.902)0.062 (0.177)0.082 (0.495)
R-pim (high shell)0.076 (0.560)0.036 (0.121)0.044 (0.267)
CC1/2 (high shell)0.983 (0.503)0.992 (0.541)0.998 (0.872)
refinement   
reflections used in refinement72,70062,25434,617
reflections used for R-free358232861698
R-work0.21420.20670.2611
R-free0.24590.25630.2911
CC(work)0.9390.9640.941
CC(free)0.9270.9370.920
number of non-hydrogen atoms979423724567
protein atoms907023504567
solvent662509371
RMS (bonds)0.0080.0080.037
RMS (angles)1.001.000.73
Ramachandran favored (%)1165 (98%)303 (98%)592 (98%)
Ramachandran allowed (%)28 (2%)5 (2%)13 (2%)
Ramachandran outliers (%)1 (0%)0 (0%)1 (0%)
Rotamer outliers (%)9 (1%)3 (1%)2 (0%)
average B-factor39.0923.1733.71

Coronavirus Main Protease Enzymatic Reactions and Compound Testing

Primary screening for SARS-CoV-2 main protease inhibitors was performed using a Rhodamine-110 fluorescent short peptide substrate cleavage assay. Briefly, compounds and controls were dry spotted into 1536-well plates, followed by 50 nM of SARS-CoV-2 Mpro in a volume of 2.5 μL, and incubated for 20 min at room temperature. Following incubation, 2.5 μL of 25 μM Rhodamine-110 peptide substrate was dispensed, and reactions were allowed to incubate at room temperature for 90 min. Fluorescence intensity was then quantified using a PHERAstar plate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
The RFMS assay was run in clear 384-deep well small volume polypropylene plates (Greiner Bio-One, Monroe, NC; cat# 784201). All solutions were prepared with ultrapure Milli-Q purified water (EMD Millipore, Burlington, MA). Reaction buffer consisted of HEPES, pH 7.3 (VWR International, Radnor, PA; cat# J848), sodium chloride (Quality Biological, Gaithersburg, MD; cat# 351-036-101), EDTA (Quality Biological; cat# 351-027-101), and Pluronic F-127 (EMD Millipore; cat# 540025). Buffer was filtered through a 0.2 μm pore size bottle top filter (ThermoFisher, Waltham, MA; cat# 567-0020).
Evaluation of compound mediated inhibition of coronavirus Mpro enzyme activity was determined in an RFMS assay using an Agilent RapidFire 365 autosampler (Agilent, Santa Clara, CA) coupled to an Agilent 6400 Triple Quad LC/MS mass spectrometer. Mpro enzyme was diluted in assay buffer (50 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM EDTA, 0.01% Pluronic F-127); final assay concentrations are listed in the Supporting Information (Table S5) for specific Mpro proteins. Five μL of 2X Mpro enzyme dilution was added into the assay ready plates dry-spotted with compounds (using 8 or 10 point, 3-fold serial dilutions with a top concentration of 50 μM dissolved in DMSO) via an Echo 555 liquid handler (Labcyte, San Jose, CA), and incubated at room temperature for 15 min prior to addition of substrate. To initiate the reaction, 2X substrate (final assay concentrations listed in Table S5) was added to the compound plate and incubated at room temperature (incubation times are listed in the Supporting Information, Table S5). Conditions were kept similar for each Mpro enzyme reaction with enzyme, substrate concentrations and reaction times adjusted to produce 10–20% substrate turnover in the absence of compound. After incubation, the reaction was quenched with 35 μL of stop solution (5% acetic acid, 0.2 nM 13C,15N-AVLQ, diluted in H2O) and then submitted to the RFMS.
Samples on the RapidFire were loaded onto a C18 SPE cartridge (Agilent) in H2O with 0.1% formic acid at a flow rate of 1.5 mL/min. Samples were then eluted with 75:20:5 acetonitrile: H2O: isopropyl alcohol with 0.1% formic acid at a flow rate of 1.0 mL/min.
Multiple reaction monitoring (MRM) transitions corresponding to the substrate and the related products, along with 13C,15N-AVLQ (for signal normalization), were monitored using MassHunter Workstation LC/MS Data Acquisition (Agilent), and peaks integrated using Agilent RapidFire Integrator. Corresponding peptides (Vivitide, Gardner, MA) monitored for each Mpro enzyme are listed in the Supporting Information (Table S6).
Determination of the Km value for the peptide substrate for each Mpro was conducted as described above for compound testing using RFMS using a dilution series of peptide substrate concentration across the indicated incubation time for each enzyme. The experimental values were graphed as velocity as a function of peptide substrate concentration. These values were fitted to a Michaelis–Menten plot using Prism 3.0 (Graphpad, San Diego, CA; Supporting Information, Figure S4).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01404.

  • Machine-readable datasheet file with full cluster composition including isolate accessions, cluster assignment, and annotation (XLSX)

  • Molecular formula strings (CSV)

  • Additional details regarding coronaviridae isolate curation and clustering, lab strain panel composition, and biochemical assay parameters, as well as experimental procedures and 1H NMR for all mentioned compounds (PDF)

Accession Codes

The X-ray diffraction structures of protein–ligand complexes have been deposited in the Protein Data Bank with the accession codes PDB ID 9C7W, 9C8Q, and 9C80 (for HCoV-OC43 Mpro + compound 21, for SARS-CoV-2 Mpro + compound 14, and for SARS-CoV-2 Mpro + compound 1 respectively).

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Dustin Dovala - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States;  Orcidhttps://orcid.org/0000-0002-2278-5027;  Email: [email protected]
    • Stephanie Moquin - Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States;  Email: [email protected]
    • Vanessa M. Marx - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States;  Email: [email protected]
  • Authors
    • David T. Barkan - Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
    • Keira Garland - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Lei Zhang - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Richard T. Eastman - Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States;  Orcidhttps://orcid.org/0000-0003-3580-380X
    • Matthew Hesse - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Mark Knapp - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Elizabeth Ornelas - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Jenny Tang - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Wilian Augusto Cortopassi - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Yu Wang - Discovery Sciences, Novartis Biomedical Research, La Jolla, California 92121, United States
    • Frederick King - Discovery Sciences, Novartis Biomedical Research, La Jolla, California 92121, United States
    • Weiping Jia - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Zachary Nguyen - Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
    • Andreas O. Frank - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States;  Orcidhttps://orcid.org/0000-0002-2330-3974
    • Ryan Chan - Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Eric Fang - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Daniel Fuller - Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
    • Scott Busby - Discovery Sciences, Novartis Biomedical Research, Cambridge, Massachusetts 02139, United States
    • Heidi Carias - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Kristine Donahue - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States;  Orcidhttps://orcid.org/0000-0003-3248-7573
    • Laura Tandeske - Discovery Sciences, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Thierry T. Diagana - Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Nadine Jarrousse - Global Health, Novartis Biomedical Research, Emeryville, California 94608, United States
    • Heinz Moser - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States;  Orcidhttps://orcid.org/0000-0002-8013-2139
    • Christopher Sarko - Global Discovery Chemistry, Novartis Biomedical Research, Emeryville, California 94608, United States
  • Author Contributions

    D.D., S.M., and V.M.M. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Funding

    Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number U19AI171413. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We acknowledge the synthetic contributions of our colleagues at WuXi AppTech. We thank Viktor Hornak, Patrick Rudewicz, Manjunatha Ujjini, and Gu Feng (Novartis) for helpful discussions, as well as Vineet Menachery and Xuping Xie (University of Texas at Galveston, Medical Branch). We also thank Vincent Leonard (Novartis) for inspiring the original clustering concept. We are grateful to the Global Health Alliance Management and Partnering, Legal, and Finance team (Thomas Krucker, Elianna Amin, Daniel Raymond, Marcus Hall, Tracey Heinrich, and Jean Claude Poilevey) for their operational and administrative support.

Abbreviations

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BLAST

basic local alignment search tool

BV-BRC

bacterial and viral bioinformatics resource center

CCR2

CC chemokine receptor 2

CCL2

CC chemokine ligand 2

CCR5

CC chemokine receptor 5

COVID-19

coronavirus disease 2019

CoV

coronavirus

CV

column volume

DMF-DMA

N,N-dimethylformamide dimethyl acetal

EDCI

N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide

FA

formic acid

FPLC

fast protein liquid chromatography

GSH

glutathione

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HCoV

human coronavirus

IBV

infectious bronchitis virus

IMAC

immobilized metal affinity chromatography

IPTG

isopropyl ß-d-1-thiogalactopyranoside

LB

lysogeny broth

LM

liver microsomal

LRCoV

Lucheng rat coronavirus

MDCK

Madin-Darby canine kidney

MERS-CoV

middle eastern respiratory syndrome coronavirus

MIB

malonic acid:imidazole:boric acid

MPLC

medium pressure liquid chromatography

Mpro

main protease

MRM

multiple reaction monitoring

MWCO

molecular weight cutoff

NCBI

National Center for Biotechnology Information

nMS

native mass spectrometry

PAINS

pan-assay interference compounds

PEDV

porcine epidemic diarrhea virus

RFMS

RapidFire mass spectrometry

RP

reverse phase

SARS-CoV

severe acute respiratory syndrome coronavirus

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

SDS-PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SNAr

nucleophilic aromatic substitution

SEC

size exclusion chromatography

SPE

solid phase extraction

SPR

surface plasmon resonance

SUMO

small ubiquitin-like modifier

TB

terrific broth

TCEP

tris(2-carboxyethyl)phosphine

TEV

tobacco etch virus

WSCoV

Wencheng shrew coronavirus

References

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    2
    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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  3. 3
    Zaki, A. M.; van Boheemen, S.; Bestebroer, T. M.; Osterhaus, A. D. M. E.; Fouchier, R. A. M. Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. N. Engl. J. Med. 2012, 367 (19), 1814– 1820,  DOI: 10.1056/NEJMoa1211721
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    Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia
    Zaki, Ali Moh; van Boheemen, Sander; Bestebroer, Theo M.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.
    New England Journal of Medicine (2012), 367 (19), 1814-1820CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    A previously unknown coronavirus was isolated from the sputum of a 60-yr-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clin. data, virus isolation, and mol. identification are presented. The clin. picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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    Brüssow, H.; Brüssow, L. Clinical Evidence That the Pandemic from 1889 to 1891 Commonly Called the Russian Flu Might Have Been an Earlier Coronavirus Pandemic. Microbiol. Biotechnol. 2021, 14, 1860– 1870,  DOI: 10.1111/1751-7915.13889
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    Geoghegan, J. L.; Duchêne, S.; Holmes, E. C. Comparative Analysis Estimates the Relative Frequencies of Co-Divergence and Cross-Species Transmission within Viral Families. PLoS Pathog. 2017, 13 (2), e1006215  DOI: 10.1371/journal.ppat.1006215
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    Daszak, P.; Cunningham, A. A.; Hyatt, A. D. Anthropogenic Environmental Change and the Emergence of Infectious Diseases in Wildlife. Acta Trop. 2001, 78 (2), 103– 116,  DOI: 10.1016/S0001-706X(00)00179-0
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    Chomel, B. B.; Belotto, A.; Meslin, F. X. Wildlife, Exotic Pets, and Emerging Zoonoses. Emerging Infect. Dis. 2007, 13 (1), 6– 11,  DOI: 10.3201/eid1301.060480
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    Wacharapluesadee, S.; Sintunawa, C.; Kaewpom, T.; Khongnomnan, K.; Olival, K. J.; Epstein, J. H.; Rodpan, A.; Sangsri, P.; Intarut, N.; Chindamporn, A.; Suksawa, K.; Hemachudha, T. Group C Betacoronavirus in Bat Guano Fertilizer, Thailand. Emerging Infect. Dis. 2013, 19 (8), 1349– 1351,  DOI: 10.3201/eid1908.130119
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    Joyjinda, Y.; Rodpan, A.; Chartpituck, P.; Suthum, K.; Yaemsakul, S.; Cheun-Arom, T.; Bunprakob, S.; Olival, K. J.; Stokes, M. M.; Hemachudha, T.; Wacharapluesadee, S. First Complete Genome Sequence of Human Coronavirus HKU1 from a Nonill Bat Guano Miner in Thailand. Microbiol. Resour. Announce. 2019, 8 (6), 7– 8,  DOI: 10.1128/MRA.01457-18
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    Smith, K. M.; Anthony, S. J.; Switzer, W. M.; Epstein, J. H.; Seimon, T.; Jia, H.; Sanchez, M. D.; Huynh, T. T.; Galland, G. G.; Shapiro, S. E.; Sleeman, J. M.; McAloose, D.; Stuchin, M.; Amato, G.; Kolokotronis, S. O.; Lipkin, W. I.; Karesh, W. B.; Daszak, P.; Marano, N. Zoonotic Viruses Associated with Illegally Imported Wildlife Products. PLoS One 2012, 7, e29505  DOI: 10.1371/journal.pone.0029505
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    Jones, B. A.; Grace, D.; Kock, R.; Alonso, S.; Rushton, J.; Said, M. Y.; McKeever, D.; Mutua, F.; Young, J.; McDermott, J.; Pfeiffer, D. U. Zoonosis Emergence Linked to Agricultural Intensification and Environmental Change. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (21), 8399– 8404,  DOI: 10.1073/pnas.1208059110
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    11
    Zoonosis emergence linked to agricultural intensification and environmental change
    Jones, Bryony A.; Grace, Delia; Kock, Richard; Alonso, Silvia; Rushton, Jonathan; Said, Mohammed Y.; Mekeever, Declan; Mutua, Florence; Young, Jarrah; Mcdermott, John; Pfeiffer, Dirk Udo
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (21), 8399-8404, S8399/1-S8399/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A systematic review was conducted by a multidisciplinary team to analyze qual. best available scientific evidence on the effect of agricultural intensification and environmental changes on the risk of zoonoses for which there are epidemiol. interactions between wildlife and livestock. The study found several examples in which agricultural intensification and/or environmental change were assocd. with an increased risk of zoonotic disease emergence, driven by the impact of an expanding human population and changing human behavior on the environment. We conclude that the rate of future zoonotic disease emergence or reemergence will be closely linked to the evolution of the agriculture-environment nexus. However, available research inadequately addresses the complexity and interrelatedness of environmental, biol., economic, and social dimensions of zoonotic pathogen emergence, which significantly limits our ability to predict prevent and respond to zoonotic disease emergence.
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  12. 12
    Tomley, F. M.; Shirley, M. W. Livestock Infectious Diseases and Zoonoses. Philos. Trans. R. Soc., B 2009, 364 (1530), 2637– 2642,  DOI: 10.1098/rstb.2009.0133
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    Livestock infectious diseases and zoonoses
    Tomley Fiona M; Shirley Martin W
    Philosophical transactions of the Royal Society of London. Series B, Biological sciences (2009), 364 (1530), 2637-42 ISSN:.
    Infectious diseases of livestock are a major threat to global animal health and welfare and their effective control is crucial for agronomic health, for safeguarding and securing national and international food supplies and for alleviating rural poverty in developing countries. Some devastating livestock diseases are endemic in many parts of the world and threats from old and new pathogens continue to emerge, with changes to global climate, agricultural practices and demography presenting conditions that are especially favourable for the spread of arthropod-borne diseases into new geographical areas. Zoonotic infections that are transmissible either directly or indirectly between animals and humans are on the increase and pose significant additional threats to human health and the current pandemic status of new influenza A (H1N1) is a topical example of the challenge presented by zoonotic viruses. In this article, we provide a brief overview of some of the issues relating to infectious diseases of livestock, which will be discussed in more detail in the papers that follow.
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    Jones, K. E.; Patel, N. G.; Levy, M. A.; Storeygard, A.; Balk, D.; Gittleman, J. L.; Daszak, P. Global Trends in Emerging Infectious Diseases. Nature 2008, 451 (7181), 990– 993,  DOI: 10.1038/nature06536
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    13
    Global trends in emerging infectious diseases
    Jones, Kate E.; Patel, Nikkita G.; Levy, Marc A.; Storeygard, Adam; Balk, Deborah; Gittleman, John L.; Daszak, Peter
    Nature (London, United Kingdom) (2008), 451 (7181), 990-993CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Emerging infectious diseases (EIDs) are a significant burden on global economies and public health. Their emergence is thought to be driven largely by socio-economic, environmental and ecol. factors, but no comparative study has explicitly analyzed these linkages to understand global temporal and spatial patterns of EIDs. Here we analyze a database of 335 EID events' (origins of EIDs) between 1940 and 2004, and demonstrate non-random global patterns. EID events have risen significantly over time after controlling for reporting bias, with their peak incidence (in the 1980s) concomitant with the HIV pandemic. EID events are dominated by zoonoses (60.3% of EIDs): the majority of these (71.8%) originate in wildlife (for example, severe acute respiratory virus, Ebola virus), and are increasing significantly over time. We find that 54.3% of EID events are caused by bacteria or rickettsia, reflecting a large no. of drug-resistant microbes in our database. Our results confirm that EID origins are significantly correlated with socio-economic, environmental and ecol. factors, and provide a basis for identifying regions where new EIDs are most likely to originate (emerging disease hotspots'). They also reveal a substantial risk of wildlife zoonotic and vector-borne EIDs originating at lower latitudes where reporting effort is low. We conclude that global resources to counter disease emergence are poorly allocated, with the majority of the scientific and surveillance effort focused on countries from where the next important EID is least likely to originate.
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  14. 14
    Li, X.; Zhang, L.; Chen, S.; Ouyang, H.; Ren, L. Possible Targets of Pan-Coronavirus Antiviral Strategies for Emerging or Re-Emerging Coronaviruses. Microorganisms 2021, 9 (7), 1479  DOI: 10.3390/microorganisms9071479
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    Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.; Hilgenfeld, R. Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved a-Ketoamide Inhibitors. Science 2020, 368 (6489), 409– 412,  DOI: 10.1126/science.abb3405
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    15
    Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors
    Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf
    Science (Washington, DC, United States) (2020), 368 (6489), 409-412CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compd. in plasma. On the basis of the unliganded structure, we developed the lead compd. into a potent inhibitor of the SARS-CoV-2 Mpro. The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
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    Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; Duan, Y.; Yu, J.; Wang, L.; Yang, K.; Liu, F.; Jiang, R.; Yang, X.; You, T.; Liu, X.; Yang, X.; Bai, F.; Liu, H.; Liu, X.; Guddat, L. W.; Xu, W.; Xiao, G.; Qin, C.; Shi, Z.; Jiang, H.; Rao, Z.; Yang, H. Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors. Nature 2020, 582 (7811), 289– 293,  DOI: 10.1038/s41586-020-2223-y
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    16
    Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors
    Jin, Zhenming; Du, Xiaoyu; Xu, Yechun; Deng, Yongqiang; Liu, Meiqin; Zhao, Yao; Zhang, Bing; Li, Xiaofeng; Zhang, Leike; Peng, Chao; Duan, Yinkai; Yu, Jing; Wang, Lin; Yang, Kailin; Liu, Fengjiang; Jiang, Rendi; Yang, Xinglou; You, Tian; Liu, Xiaoce; Yang, Xiuna; Bai, Fang; Liu, Hong; Liu, Xiang; Guddat, Luke W.; Xu, Wenqing; Xiao, Gengfu; Qin, Chengfeng; Shi, Zhengli; Jiang, Hualiang; Rao, Zihe; Yang, Haitao
    Nature (London, United Kingdom) (2020), 582 (7811), 289-293CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Abstr.: A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the etiol. agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19). Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here, we describe the results of a program that aimed to rapidly discover lead compds. for clin. use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This program focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then detd. the crystal structure of Mpro of SARS-CoV-2 in complex with this compd. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compds.-including approved drugs, drug candidates in clin. trials and other pharmacol. active compds.-as inhibitors of Mpro. Six of these compds. inhibited Mpro, showing half-maximal inhibitory concn. values that ranged from 0.67 to 21.4μM. One of these compds. (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clin. potential in response to new infectious diseases for which no specific drugs or vaccines are available.
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    Trauner, D.; Fischer, C.; Veprek, N.; Peitsinis, Z.; Rühmann, P.; Yang, C.; Spradlin, J.; Dovala, D.; Nomura, D.; Zhang, Y. De Novo Design of SARS-CoV-2 Main Protease Inhibitors. Synlett 2022, 33, 458,  DOI: 10.1055/a-1582-0243
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    Biering, S. B.; Van Dis, E.; Wehri, E.; Yamashiro, L. H.; Nguyenla, X.; Dugast-Darzacq, C.; Graham, T. G. W.; Stroumza, J. R.; Golovkine, G. R.; Roberts, A. W.; Fines, D. M.; Spradlin, J. N.; Ward, C. C.; Bajaj, T.; Dovala, D.; Schulze-Gamen, U.; Bajaj, R.; Fox, D. M.; Ott, M.; Murthy, N.; Nomura, D. K.; Schaletzky, J.; Stanley, S. A. Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2. ACS Infect. Dis. 2021, 7 (8), 2337– 2351,  DOI: 10.1021/acsinfecdis.1c00017
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    18
    Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2
    Biering, Scott B.; Van Dis, Erik; Wehri, Eddie; Yamashiro, Livia H.; Nguyenla, Xammy; Dugast-Darzacq, Claire; Graham, Thomas G. W.; Stroumza, Julien R.; Golovkine, Guillaume R.; Roberts, Allison W.; Fines, Daniel M.; Spradlin, Jessica N.; Ward, Carl C.; Bajaj, Teena; Dovala, Dustin; Schulze-Gamen, Ursula; Bajaj, Ruchika; Fox, Douglas M.; Ott, Melanie; Murthy, Niren; Nomura, Daniel K.; Schaletzky, Julia; Stanley, Sarah A.
    ACS Infectious Diseases (2021), 7 (8), 2337-2351CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in >168 million cases and 3.4 million deaths to date, while the no. of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clin. compds. holds the potential for rapid identification of drugs effective against SARS-CoV-2. We utilized a library of FDA-approved and well-studied preclin. and clin. compds. to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compds. that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compds. with anti-inflammatory activity, and compds. targeting host pathways such as kinases and proteases crit. for SARS-CoV-2 replication. We identified 7 compds. not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compd. screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.
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    Owen, D. R.; Allerton, C. M. N.; Anderson, A. S.; Aschenbrenner, L.; Avery, M.; Berritt, S.; Boras, B.; Cardin, R. D.; Carlo, A.; Coffman, K. J.; Dantonio, A.; Di, L.; Eng, H.; Ferre, R.; Gajiwala, K. S.; Gibson, S. A.; Greasley, S. E.; Hurst, B. L.; Kadar, E. P.; Kalgutkar, A. S.; Lee, J. C.; Lee, J.; Liu, W.; Mason, S. W.; Noell, S.; Novak, J. J.; Obach, R. S.; Ogilvie, K.; Patel, N. C.; Pettersson, M.; Rai, D. K.; Reese, M. R.; Sammons, M. F.; Sathish, J. G.; Singh, R. S. P.; Steppan, C. M.; Stewart, A. E.; Tuttle, J. B.; Updyke, L.; Verhoest, P. R.; Wei, L.; Yang, Q.; Zhu, Y. An Oral SARS-CoV-2 Mpro Inhibitor Clinical Candidate for the Treatment of COVID-19. Science 2021, 374 (6575), 1586– 1593,  DOI: 10.1126/science.abl4784
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    An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19
    Owen, Dafydd R.; Allerton, Charlotte M. N.; Anderson, Annaliesa S.; Aschenbrenner, Lisa; Avery, Melissa; Berritt, Simon; Boras, Britton; Cardin, Rhonda D.; Carlo, Anthony; Coffman, Karen J.; Dantonio, Alyssa; Di, Li; Eng, Heather; Ferre, RoseAnn; Gajiwala, Ketan S.; Gibson, Scott A.; Greasley, Samantha E.; Hurst, Brett L.; Kadar, Eugene P.; Kalgutkar, Amit S.; Lee, Jack C.; Lee, Jisun; Liu, Wei; Mason, Stephen W.; Noell, Stephen; Novak, Jonathan J.; Obach, R. Scott; Ogilvie, Kevin; Patel, Nandini C.; Pettersson, Martin; Rai, Devendra K.; Reese, Matthew R.; Sammons, Matthew F.; Sathish, Jean G.; Singh, Ravi Shankar P.; Steppan, Claire M.; Stewart, Al E.; Tuttle, Jamison B.; Updyke, Lawrence; Verhoest, Patrick R.; Wei, Liuqing; Yang, Qingyi; Zhu, Yuao
    Science (Washington, DC, United States) (2021), 374 (6575), 1586-1593CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    The worldwide outbreak of COVID-19 caused by SARS-CoV-2 has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. We report the discovery and characterization of PF-07321332 (I), an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concns. exceeding the in vitro antiviral cell potency in a phase 1 clin. trial in healthy human participants.
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    Flynn, J. M.; Samant, N.; Schneider-Nachum, G.; Barkan, D. T.; Yilmaz, N. K.; Schiffer, C. A.; Moquin, S. A.; Dovala, D.; Bolon, D. N. A. Comprehensive Fitness Landscape of SARS-CoV-2 Mpro Reveals Insights into Viral Resistance Mechanisms. eLife 2022, 11, e77433  DOI: 10.7554/eLife.77433
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    20
    Comprehensive fitness landscape of SARS-CoV- 2 Mpro reveals insights into viral resistance mechanisms
    Flynn, Julia M.; Samant, Neha; Schneider-Nachum, Gily; Barkan, David T.; Yilmaz, Nese Kurt; Schiffer, Celia A.; Moquin, Stephanie A.; Dovala, Dustin; Bolon, Daniel N. A.
    eLife (2022), 11 (), e77433CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    With the continual evolution of new strains of severe acute respiratory syndrome coronavirus- 2 (SARS-CoV- 2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV- 2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three sep. high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophys. properties crit. to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addn., we found a network of crit. amino acids that phys. bridge the two active sites of the Mpro dimer. The clin. variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.
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  21. 21
    Shaqra, A. M.; Zvornicanin, S. N.; Huang, Q. Y. J.; Lockbaum, G. J.; Knapp, M.; Tandeske, L.; Bakan, D. T.; Flynn, J.; Bolon, D. N. A.; Moquin, S.; Dovala, D.; Kurt Yilmaz, N.; Schiffer, C. A. Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance. Nat. Commun. 2022, 13 (1), 3556  DOI: 10.1038/s41467-022-31210-w
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    Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance
    Shaqra, Ala M.; Zvornicanin, Sarah N.; Huang, Qiu Yu J.; Lockbaum, Gordon J.; Knapp, Mark; Tandeske, Laura; Bakan, David T.; Flynn, Julia; Bolon, Daniel N. A.; Moquin, Stephanie; Dovala, Dustin; Kurt Yilmaz, Nese; Schiffer, Celia A.
    Nature Communications (2022), 13 (1), 3556CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, addnl. treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clin. use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resoln. cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the crit. recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
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    Flynn, J. M.; Huang, Q. Y. J.; Zvornicanin, S. N.; Schneider-Nachum, G.; Shaqra, A. M.; Yilmaz, N. K.; Moquin, S. A.; Dovala, D.; Schiffer, C. A.; Bolon, D. N. A. Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease. ACS Infect. Dis. 2023, 9 (7), 1372– 1386,  DOI: 10.1021/acsinfecdis.3c00125
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    Hoffman, R. L.; Kania, R. S.; Brothers, M. A.; Davies, J. F.; Ferre, R. A.; Gajiwala, K. S.; He, M.; Hogan, R. J.; Kozminski, K.; Li, L. Y.; Lockner, J. W.; Lou, J.; Marra, M. T.; Mitchell, L. J.; Murray, B. W.; Nieman, J. A.; Noell, S.; Planken, S. P.; Rowe, T.; Ryan, K.; Smith, G. J.; Solowiej, J. E.; Steppan, C. M.; Taggart, B. Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19. J. Med. Chem. 2020, 63 (21), 12725– 12747,  DOI: 10.1021/acs.jmedchem.0c01063
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    Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19
    Hoffman, Robert L.; Kania, Robert S.; Brothers, Mary A.; Davies, Jay F.; Ferre, Rose A.; Gajiwala, Ketan S.; He, Mingying; Hogan, Robert J.; Kozminski, Kirk; Li, Lilian Y.; Lockner, Jonathan W.; Lou, Jihong; Marra, Michelle T.; Mitchell Jr., Lennert J.; Murray, Brion W.; Nieman, James A.; Noell, Stephen; Planken, Simon P.; Rowe, Thomas; Ryan, Kevin; Smith III, George J.; Solowiej, James E.; Steppan, Claire M.; Taggart, Barbara
    Journal of Medicinal Chemistry (2020), 63 (21), 12725-12747CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is crit. for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallog. led to the identification of 3 and 4. Preclin. expts. reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an i.v. treatment for COVID-19.
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    Moon, P.; Zammit, C. M.; Shao, Q.; Dovala, D.; Boike, L.; Henning, N. J.; Knapp, M.; Spradlin, J. N.; Ward, C. C.; Wolleb, H.; Fuller, D.; Blake, G.; Murphy, J. P.; Wang, F.; Lu, Y.; Moquin, S. A.; Tandeske, L.; Hesse, M. J.; McKenna, J. M.; Tallarico, J. A.; Schirle, M.; Toste, F. D.; Nomura, D. K. Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**. ChemBioChem 2023, 24 (11), e202300116  DOI: 10.1002/cbic.202300116
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    Flynn, J. M.; Zvornicanin, S. N.; Tsepal, T.; Shaqra, A. M.; Kurt Yilmaz, N.; Jia, W.; Moquin, S.; Dovala, D.; Schiffer, C. A.; Bolon, D. N. A. Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance. ACS Infect. Dis. 2024, 10, 1174,  DOI: 10.1021/acsinfecdis.3c00560
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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. C. The Complete Sequence (22 Kilobases) of Murine Coronavirus Gene 1 Encoding the Putative Proteases and RNA Polymerase. Virology 1991, 180 (2), 567– 582,  DOI: 10.1016/0042-6822(91)90071-I
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    Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307– 317,  DOI: 10.1038/nrd3410
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    The resurgence of covalent drugs
    Singh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, Adrian
    Nature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
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    Resnick, S. J.; Iketani, S.; Hong, S. J.; Zask, A.; Liu, H.; Kim, S.; Melore, S.; Lin, F.-Y.; Nair, M. S.; Huang, Y.; Lee, S.; Tay, N. E. S.; Rovis, T.; Yang, H. W.; Xing, L.; Stockwell, B. R.; Ho, D. D.; Chavez, A. Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity. J. Virol. 2021, 95 (14), e0237420  DOI: 10.1128/JVI.02374-20
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    Sayers, E. W.; Bolton, E. E.; Brister, J. R.; Canese, K.; Chan, J.; Comeau, D. C.; Connor, R.; Funk, K.; Kelly, C.; Kim, S.; Madej, T.; Marchler-Bauer, A.; Lanczycki, C.; Lathrop, S.; Lu, Z.; Thibaud-Nissen, F.; Murphy, T.; Phan, L.; Skripchenko, Y.; Tse, T.; Wang, J.; Williams, R.; Trawick, B. W.; Pruitt, K. D.; Sherry, S. T. Database Resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2022, 50 (D1), D20– D26,  DOI: 10.1093/nar/gkab1112
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    Database resources of the national center for biotechnology information
    Sayers, Eric W.; Bolton, Evan E.; Brister, J. Rodney; Canese, Kathi; Chan, Jessica; Comeau, Donald C.; Connor, Ryan; Funk, Kathryn; Kelly, Chris; Kim, Sunghwan; Madej, Tom; Marchler-Bauer, Aron; Lanczycki, Christopher; Lathrop, Stacy; Lu, Zhiyong; Thibaud-Nissen, Francoise; Murphy, Terence; Phan, Lon; Skripchenko, Yuri; Tse, Tony; Wang, Jiyao; Williams, Rebecca; Trawick, Barton W.; Pruitt, Kim D.; Sherry, Stephen T.
    Nucleic Acids Research (2022), 50 (D1), D20-D26CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A review. The National Center for Biotechnol. Information (NCBI) produces a variety of online information resources for biol., including the GenBank nucleic acid sequence database and the PubMed database of citations and abstrs. published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for the most of these databases. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, RefSeq, SRA, Virus, dbSNP, dbVar, ClinicalTrials.gov, MMDB, iCn3D and PubChem.
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    Olson, R. D.; Assaf, R.; Brettin, T.; Conrad, N.; Cucinell, C.; Davis, J. J.; Dempsey, D. M.; Dickerman, A.; Dietrich, E. M.; Kenyon, R. W.; Kuscuoglu, M.; Lefkowitz, E. J.; Lu, J.; Machi, D.; Macken, C.; Mao, C.; Niewiadomska, A.; Nguyen, M.; Olsen, G. J.; Overbeek, J. C.; Parrello, B.; Parrello, V.; Porter, J. S.; Pusch, G. D.; Shukla, M.; Singh, I.; Stewart, L.; Tan, G.; Thomas, C.; VanOeffelen, M.; Vonstein, V.; Wallace, Z. S.; Warren, A. S.; Wattam, A. R.; Xia, F.; Yoo, H.; Zhang, Y.; Zmasek, C. M.; Scheuermann, R. H.; Stevens, R. L. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): A Resource Combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023, 51 (1 D), D678– D689,  DOI: 10.1093/nar/gkac1003
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    Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T. L. BLAST+: Architecture and Applications. BMC Bioinf. 2009, 10, 421  DOI: 10.1186/1471-2105-10-421
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    BLAST+: architecture and applications
    Camacho Christiam; Coulouris George; Avagyan Vahram; Ma Ning; Papadopoulos Jason; Bealer Kevin; Madden Thomas L
    BMC bioinformatics (2009), 10 (), 421 ISSN:.
    BACKGROUND: Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. RESULTS: We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. CONCLUSION: The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.
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    Unoh, Y.; Uehara, S.; Nakahara, K.; Nobori, H.; Yamatsu, Y.; Yamamoto, S.; Maruyama, Y.; Taoda, Y.; Kasamatsu, K.; Suto, T.; Kouki, K.; Nakahashi, A.; Kawashima, S.; Sanaki, T.; Toba, S.; Uemura, K.; Mizutare, T.; Ando, S.; Sasaki, M.; Orba, Y.; Sawa, H.; Sato, A.; Sato, T.; Kato, T.; Tachibana, Y. Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J. Med. Chem. 2022, 65, 6499,  DOI: 10.1021/acs.jmedchem.2c00117
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    Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19
    Unoh, Yuto; Uehara, Shota; Nakahara, Kenji; Nobori, Haruaki; Yamatsu, Yukiko; Yamamoto, Shiho; Maruyama, Yuki; Taoda, Yoshiyuki; Kasamatsu, Koji; Suto, Takahiro; Kouki, Kensuke; Nakahashi, Atsufumi; Kawashima, Sho; Sanaki, Takao; Toba, Shinsuke; Uemura, Kentaro; Mizutare, Tohru; Ando, Shigeru; Sasaki, Michihito; Orba, Yasuko; Sawa, Hirofumi; Sato, Akihiko; Sato, Takafumi; Kato, Teruhisa; Tachibana, Yuki
    Journal of Medicinal Chemistry (2022), 65 (9), 6499-6512CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. We describe the discovery of S-217622, the 1st oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clin. candidate. S-217622 was discovered via virtual screening followed by biol. screening of an inhouse compd. library, and optimization of the hit compd. using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.
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    Douangamath, A.; Fearon, D.; Gehrtz, P.; Krojer, T.; Lukacik, P.; Owen, C. D.; Resnick, E.; Strain-Damerell, C.; Aimon, A.; Ábrányi-Balogh, P.; Brandão-Neto, J.; Carbery, A.; Davison, G.; Dias, A.; Downes, T. D.; Dunnett, L.; Fairhead, M.; Firth, J. D.; Jones, S. P.; Keeley, A.; Keserü, G. M.; Klein, H. F.; Martin, M. P.; Noble, M. E. M.; O’Brien, P.; Powell, A.; Reddi, R. N.; Skyner, R.; Snee, M.; Waring, M. J.; Wild, C.; London, N.; von Delft, F.; Walsh, M. A. Crystallographic and Electrophilic Fragment Screening of the SARS-CoV-2 Main Protease. Nat. Commun. 2020, 11 (1), 5047  DOI: 10.1038/s41467-020-18709-w
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    Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease
    Douangamath, Alice; Fearon, Daren; Gehrtz, Paul; Krojer, Tobias; Lukacik, Petra; Owen, C. David; Resnick, Efrat; Strain-Damerell, Claire; Aimon, Anthony; Abranyi-Balogh, Peter; Brandao-Neto, Jose; Carbery, Anna; Davison, Gemma; Dias, Alexandre; Downes, Thomas D.; Dunnett, Louise; Fairhead, Michael; Firth, James D.; Jones, S. Paul; Keeley, Aaron; Keseru, Gyorgy M.; Klein, Hanna F.; Martin, Mathew P.; Noble, Martin E. M.; O'Brien, Peter; Powell, Ailsa; Reddi, Rambabu N.; Skyner, Rachael; Snee, Matthew; Waring, Michael J.; Wild, Conor; London, Nir; von Delft, Frank; Walsh, Martin A.
    Nature Communications (2020), 11 (1), 5047CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    A review. Abstr.: COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Addnl., no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallog. screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
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    Tomar, S.; Johnston, M. L.; John, S. E. S.; Osswald, H. L.; Nyalapatla, P. R.; Paul, L. N.; Ghosh, A. K.; Denison, M. R.; Mesecar, A. D. Ligand-Induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus Nsp5 Protease (3CLpro): Implications for Nsp5 Regulation and the Development of Antivirals. J. Biol. Chem. 2015, 290 (32), 19403– 19422,  DOI: 10.1074/jbc.M115.651463
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    Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro)
    Tomar, Sakshi; Johnston, Melanie L.; St. John, Sarah E.; Osswald, Heather L.; Nyalapatla, Prasanth R.; Paul, Lake N.; Ghosh, Arun K.; Denison, Mark R.; Mesecar, Andrew D.
    Journal of Biological Chemistry (2015), 290 (32), 19403-19422CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CLpro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CLpro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CLpro is less efficient at processing a peptide substrate due because MERS-CoV 3CLpro forms a weakly assocd. dimer, while HKU4, HKU5, and SARS-CoV 3CLpro enzymes form tightly assocd. dimers. Anal. ultracentrifugation studies confirm that MERS-CoV 3CLpro is a weakly assocd. dimer (Kd ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CLpro were synthesized and utilized in anal. ultracentrifugation expts. and demonstrate that MERS-CoV 3CLpro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compd. concn. as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, detd. to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CLpro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CLpro dimerization. Activation of MERS-CoV 3CLpro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CLpro inhibitors as antiviral agents.
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    Beach, J. R. A Filtrable Virus, the Cause of Infectious Laryngotracheitis of Chickens. J. Exp. Med. 1931, 54 (6), 809– 816,  DOI: 10.1084/jem.54.6.809
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    Xue, X.; Yu, H.; Yang, H.; Xue, F.; Wu, Z.; Shen, W.; Li, J.; Zhou, Z.; Ding, Y.; Zhao, Q.; Zhang, X. C.; Liao, M.; Bartlam, M.; Rao, Z. Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design. J. Virol. 2008, 82 (5), 2515– 2527,  DOI: 10.1128/JVI.02114-07
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    Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design
    Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe
    Journal of Virology (2008), 82 (5), 2515-2527CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (Mpro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) Mpro and a severe acute respiratory syndrome CoV (SARS-CoV) Mpro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually detd. to elucidate the structural flexibility and substrate binding of Mpro. A monomeric form of IBV Mpro was identified for the first time in CoV Mpro structures. A comparison of these two structures to other available Mpro structures provides new insights for the design of substrate-based inhibitors targeting CoV Mpros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystd. with IBV Mpro and was found to demonstrate in vitro inactivation of IBV Mpro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-assocd. diseases. The structure-based optimization of N3 has yielded two more efficacious lead compds., N27 and H16, with potent inhibition against SARS-CoV Mpro.
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  • Abstract

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    Figure 1

    Figure 1. Mpro clustering procedure and results. (A) Active site curation flowchart; Mpro sequences are extracted from the NCBI Coronavirus Nucleotide database and active site residues are inferred through transitive structure alignment using SARS-CoV-2 as a reference. Dashed lines indicate sequence or structure alignments. Blue spheres are active site pocket residues. (B) Number of sequences following each step of the curation process. (C) Clustering results; trees rooted below the red-dashed line (distance cutoff of 8) represent the final clusters.

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    Figure 2

    Figure 2. Cocrystal structure of 1 covalently bound to the catalytic C145 of SARS-CoV-2 Mpro (PDB ID 9C8O).

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    Figure 3

    Figure 3. Aromatization of the core leads to potency gains after replacement of the carboxylic acid moiety with a sulfone.

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    Figure 4

    Figure 4. Co-structure of 14 covalently bound to the catalytic C145 of SARS-CoV-2 Mpro (PDB ID 9C8Q).

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    Figure 5

    Figure 5. Cocrystal structure of 21 (green) bound to HCoV-OC43 Mpro (rainbow). Compound’s pyrimidine moiety covalently interacts with OC43 at residue C142, which is N142 in wt SARS-CoV-2 Mpro (salmon). (PDB ID 9C7W).

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    Scheme 1

    Scheme 1. Synthesis of Compound 1a
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    aReagents and conditions: (a) 2-cyanoacetic acid (1.0 equiv), EDCI·HCl (1.2 equiv), CH2Cl2, 25 °C, 94%; (b) DMF-DMA (2.0 equiv), toluene, 80 °C; (c) NH2NH2·H2O (2.0 equiv), EtOH, 25–80 °C, 22% over two steps; (d) (E)-4-(2-fluorophenyl)-2-oxobut-3-enoic acid (1.0 equiv), HOAc, 25–80 °C, 33%.

    Scheme 2

    Scheme 2. Synthesis of Compound 13a
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    aReagents and conditions: (a) diethyl malonate (1.5 equiv), NaOEt (2.5 equiv), EtOH, 25–80 °C, 88%; (b) POCl3, 25–80 °C, 71%; (c) 3-methyl-2-(tributylstannyl)pyridine (0.8 equiv), Pd(PPh3)2Cl2 (0.1 equiv), CuI (0.1), dioxane, 130 °C, 8%; (d) NaSMe (1.1 equiv), THF/H2O = 1:1, 20 °C, 5%; (e) m-CPBA (3.5 equiv), CH2Cl2, 20 °C, 34%.

    Scheme 3

    Scheme 3. Synthesis of 21a
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    aReagents and conditions: (a) CS2 (1.0 equiv), i-PrI (2.2 equiv), NaH (2.5 equiv), THF, 25 °C, 20%; (b) 4-(4,4-difluorocyclohexyl)-1H-pyrazol-5-amine (1.0 equiv), piperidine (0.1 equiv), HOAc/H2O = 3/1, 25–110 °C, 30%; (c) m-CPBA (2.0 + 0.5 + 0.5 equiv), CH2Cl2, 0–25 °C, 36%.

    Scheme 4

    Scheme 4. Synthesis of 24a
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    aReagents and conditions: (a) CS2 (1.0 equiv), MeI (2.2 equiv), NaH (2.5 equiv), THF, 25 °C, 75%; (b) 4-cyclohexyl-1H-pyrazol-5-amine (1.0 equiv), piperidine (0.1 equiv), HOAc/H2O = 3/1, 25–110 °C, 19%; (c) Oxone (3.0 eq +1.0 equiv), MeOH/H2O = 2/1, 25 °C, 78%; (d) t-BuSNa (3.0 equiv), t-BuOH, 25 °C; (e) Oxone (2.9 equiv), MeOH/H2O = 2/1, 25 °C, 35% over two steps.

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      Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 Jan. 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 Dec. 2019. Epidemiol. investigations have suggested that the outbreak was assocd. with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 Dec. 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic anal. of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.
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      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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      Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia
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      A previously unknown coronavirus was isolated from the sputum of a 60-yr-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clin. data, virus isolation, and mol. identification are presented. The clin. picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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      Zoonosis emergence linked to agricultural intensification and environmental change
      Jones, Bryony A.; Grace, Delia; Kock, Richard; Alonso, Silvia; Rushton, Jonathan; Said, Mohammed Y.; Mekeever, Declan; Mutua, Florence; Young, Jarrah; Mcdermott, John; Pfeiffer, Dirk Udo
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      A systematic review was conducted by a multidisciplinary team to analyze qual. best available scientific evidence on the effect of agricultural intensification and environmental changes on the risk of zoonoses for which there are epidemiol. interactions between wildlife and livestock. The study found several examples in which agricultural intensification and/or environmental change were assocd. with an increased risk of zoonotic disease emergence, driven by the impact of an expanding human population and changing human behavior on the environment. We conclude that the rate of future zoonotic disease emergence or reemergence will be closely linked to the evolution of the agriculture-environment nexus. However, available research inadequately addresses the complexity and interrelatedness of environmental, biol., economic, and social dimensions of zoonotic pathogen emergence, which significantly limits our ability to predict prevent and respond to zoonotic disease emergence.
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      Tomley, F. M.; Shirley, M. W. Livestock Infectious Diseases and Zoonoses. Philos. Trans. R. Soc., B 2009, 364 (1530), 2637– 2642,  DOI: 10.1098/rstb.2009.0133
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      Livestock infectious diseases and zoonoses
      Tomley Fiona M; Shirley Martin W
      Philosophical transactions of the Royal Society of London. Series B, Biological sciences (2009), 364 (1530), 2637-42 ISSN:.
      Infectious diseases of livestock are a major threat to global animal health and welfare and their effective control is crucial for agronomic health, for safeguarding and securing national and international food supplies and for alleviating rural poverty in developing countries. Some devastating livestock diseases are endemic in many parts of the world and threats from old and new pathogens continue to emerge, with changes to global climate, agricultural practices and demography presenting conditions that are especially favourable for the spread of arthropod-borne diseases into new geographical areas. Zoonotic infections that are transmissible either directly or indirectly between animals and humans are on the increase and pose significant additional threats to human health and the current pandemic status of new influenza A (H1N1) is a topical example of the challenge presented by zoonotic viruses. In this article, we provide a brief overview of some of the issues relating to infectious diseases of livestock, which will be discussed in more detail in the papers that follow.
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      Jones, K. E.; Patel, N. G.; Levy, M. A.; Storeygard, A.; Balk, D.; Gittleman, J. L.; Daszak, P. Global Trends in Emerging Infectious Diseases. Nature 2008, 451 (7181), 990– 993,  DOI: 10.1038/nature06536
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      Global trends in emerging infectious diseases
      Jones, Kate E.; Patel, Nikkita G.; Levy, Marc A.; Storeygard, Adam; Balk, Deborah; Gittleman, John L.; Daszak, Peter
      Nature (London, United Kingdom) (2008), 451 (7181), 990-993CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Emerging infectious diseases (EIDs) are a significant burden on global economies and public health. Their emergence is thought to be driven largely by socio-economic, environmental and ecol. factors, but no comparative study has explicitly analyzed these linkages to understand global temporal and spatial patterns of EIDs. Here we analyze a database of 335 EID events' (origins of EIDs) between 1940 and 2004, and demonstrate non-random global patterns. EID events have risen significantly over time after controlling for reporting bias, with their peak incidence (in the 1980s) concomitant with the HIV pandemic. EID events are dominated by zoonoses (60.3% of EIDs): the majority of these (71.8%) originate in wildlife (for example, severe acute respiratory virus, Ebola virus), and are increasing significantly over time. We find that 54.3% of EID events are caused by bacteria or rickettsia, reflecting a large no. of drug-resistant microbes in our database. Our results confirm that EID origins are significantly correlated with socio-economic, environmental and ecol. factors, and provide a basis for identifying regions where new EIDs are most likely to originate (emerging disease hotspots'). They also reveal a substantial risk of wildlife zoonotic and vector-borne EIDs originating at lower latitudes where reporting effort is low. We conclude that global resources to counter disease emergence are poorly allocated, with the majority of the scientific and surveillance effort focused on countries from where the next important EID is least likely to originate.
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      Li, X.; Zhang, L.; Chen, S.; Ouyang, H.; Ren, L. Possible Targets of Pan-Coronavirus Antiviral Strategies for Emerging or Re-Emerging Coronaviruses. Microorganisms 2021, 9 (7), 1479  DOI: 10.3390/microorganisms9071479
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    15. 15
      Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.; Hilgenfeld, R. Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved a-Ketoamide Inhibitors. Science 2020, 368 (6489), 409– 412,  DOI: 10.1126/science.abb3405
      15
      Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors
      Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf
      Science (Washington, DC, United States) (2020), 368 (6489), 409-412CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compd. in plasma. On the basis of the unliganded structure, we developed the lead compd. into a potent inhibitor of the SARS-CoV-2 Mpro. The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
      >> More from SciFinder ®
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    16. 16
      Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; Duan, Y.; Yu, J.; Wang, L.; Yang, K.; Liu, F.; Jiang, R.; Yang, X.; You, T.; Liu, X.; Yang, X.; Bai, F.; Liu, H.; Liu, X.; Guddat, L. W.; Xu, W.; Xiao, G.; Qin, C.; Shi, Z.; Jiang, H.; Rao, Z.; Yang, H. Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors. Nature 2020, 582 (7811), 289– 293,  DOI: 10.1038/s41586-020-2223-y
      16
      Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors
      Jin, Zhenming; Du, Xiaoyu; Xu, Yechun; Deng, Yongqiang; Liu, Meiqin; Zhao, Yao; Zhang, Bing; Li, Xiaofeng; Zhang, Leike; Peng, Chao; Duan, Yinkai; Yu, Jing; Wang, Lin; Yang, Kailin; Liu, Fengjiang; Jiang, Rendi; Yang, Xinglou; You, Tian; Liu, Xiaoce; Yang, Xiuna; Bai, Fang; Liu, Hong; Liu, Xiang; Guddat, Luke W.; Xu, Wenqing; Xiao, Gengfu; Qin, Chengfeng; Shi, Zhengli; Jiang, Hualiang; Rao, Zihe; Yang, Haitao
      Nature (London, United Kingdom) (2020), 582 (7811), 289-293CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Abstr.: A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the etiol. agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19). Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here, we describe the results of a program that aimed to rapidly discover lead compds. for clin. use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This program focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then detd. the crystal structure of Mpro of SARS-CoV-2 in complex with this compd. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compds.-including approved drugs, drug candidates in clin. trials and other pharmacol. active compds.-as inhibitors of Mpro. Six of these compds. inhibited Mpro, showing half-maximal inhibitory concn. values that ranged from 0.67 to 21.4μM. One of these compds. (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clin. potential in response to new infectious diseases for which no specific drugs or vaccines are available.
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    17. 17
      Trauner, D.; Fischer, C.; Veprek, N.; Peitsinis, Z.; Rühmann, P.; Yang, C.; Spradlin, J.; Dovala, D.; Nomura, D.; Zhang, Y. De Novo Design of SARS-CoV-2 Main Protease Inhibitors. Synlett 2022, 33, 458,  DOI: 10.1055/a-1582-0243
      There is no corresponding record for this reference.
    18. 18
      Biering, S. B.; Van Dis, E.; Wehri, E.; Yamashiro, L. H.; Nguyenla, X.; Dugast-Darzacq, C.; Graham, T. G. W.; Stroumza, J. R.; Golovkine, G. R.; Roberts, A. W.; Fines, D. M.; Spradlin, J. N.; Ward, C. C.; Bajaj, T.; Dovala, D.; Schulze-Gamen, U.; Bajaj, R.; Fox, D. M.; Ott, M.; Murthy, N.; Nomura, D. K.; Schaletzky, J.; Stanley, S. A. Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2. ACS Infect. Dis. 2021, 7 (8), 2337– 2351,  DOI: 10.1021/acsinfecdis.1c00017
      18
      Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2
      Biering, Scott B.; Van Dis, Erik; Wehri, Eddie; Yamashiro, Livia H.; Nguyenla, Xammy; Dugast-Darzacq, Claire; Graham, Thomas G. W.; Stroumza, Julien R.; Golovkine, Guillaume R.; Roberts, Allison W.; Fines, Daniel M.; Spradlin, Jessica N.; Ward, Carl C.; Bajaj, Teena; Dovala, Dustin; Schulze-Gamen, Ursula; Bajaj, Ruchika; Fox, Douglas M.; Ott, Melanie; Murthy, Niren; Nomura, Daniel K.; Schaletzky, Julia; Stanley, Sarah A.
      ACS Infectious Diseases (2021), 7 (8), 2337-2351CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in >168 million cases and 3.4 million deaths to date, while the no. of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clin. compds. holds the potential for rapid identification of drugs effective against SARS-CoV-2. We utilized a library of FDA-approved and well-studied preclin. and clin. compds. to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compds. that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compds. with anti-inflammatory activity, and compds. targeting host pathways such as kinases and proteases crit. for SARS-CoV-2 replication. We identified 7 compds. not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compd. screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.
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      Owen, D. R.; Allerton, C. M. N.; Anderson, A. S.; Aschenbrenner, L.; Avery, M.; Berritt, S.; Boras, B.; Cardin, R. D.; Carlo, A.; Coffman, K. J.; Dantonio, A.; Di, L.; Eng, H.; Ferre, R.; Gajiwala, K. S.; Gibson, S. A.; Greasley, S. E.; Hurst, B. L.; Kadar, E. P.; Kalgutkar, A. S.; Lee, J. C.; Lee, J.; Liu, W.; Mason, S. W.; Noell, S.; Novak, J. J.; Obach, R. S.; Ogilvie, K.; Patel, N. C.; Pettersson, M.; Rai, D. K.; Reese, M. R.; Sammons, M. F.; Sathish, J. G.; Singh, R. S. P.; Steppan, C. M.; Stewart, A. E.; Tuttle, J. B.; Updyke, L.; Verhoest, P. R.; Wei, L.; Yang, Q.; Zhu, Y. An Oral SARS-CoV-2 Mpro Inhibitor Clinical Candidate for the Treatment of COVID-19. Science 2021, 374 (6575), 1586– 1593,  DOI: 10.1126/science.abl4784
      19
      An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19
      Owen, Dafydd R.; Allerton, Charlotte M. N.; Anderson, Annaliesa S.; Aschenbrenner, Lisa; Avery, Melissa; Berritt, Simon; Boras, Britton; Cardin, Rhonda D.; Carlo, Anthony; Coffman, Karen J.; Dantonio, Alyssa; Di, Li; Eng, Heather; Ferre, RoseAnn; Gajiwala, Ketan S.; Gibson, Scott A.; Greasley, Samantha E.; Hurst, Brett L.; Kadar, Eugene P.; Kalgutkar, Amit S.; Lee, Jack C.; Lee, Jisun; Liu, Wei; Mason, Stephen W.; Noell, Stephen; Novak, Jonathan J.; Obach, R. Scott; Ogilvie, Kevin; Patel, Nandini C.; Pettersson, Martin; Rai, Devendra K.; Reese, Matthew R.; Sammons, Matthew F.; Sathish, Jean G.; Singh, Ravi Shankar P.; Steppan, Claire M.; Stewart, Al E.; Tuttle, Jamison B.; Updyke, Lawrence; Verhoest, Patrick R.; Wei, Liuqing; Yang, Qingyi; Zhu, Yuao
      Science (Washington, DC, United States) (2021), 374 (6575), 1586-1593CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The worldwide outbreak of COVID-19 caused by SARS-CoV-2 has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. We report the discovery and characterization of PF-07321332 (I), an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concns. exceeding the in vitro antiviral cell potency in a phase 1 clin. trial in healthy human participants.
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    20. 20
      Flynn, J. M.; Samant, N.; Schneider-Nachum, G.; Barkan, D. T.; Yilmaz, N. K.; Schiffer, C. A.; Moquin, S. A.; Dovala, D.; Bolon, D. N. A. Comprehensive Fitness Landscape of SARS-CoV-2 Mpro Reveals Insights into Viral Resistance Mechanisms. eLife 2022, 11, e77433  DOI: 10.7554/eLife.77433
      20
      Comprehensive fitness landscape of SARS-CoV- 2 Mpro reveals insights into viral resistance mechanisms
      Flynn, Julia M.; Samant, Neha; Schneider-Nachum, Gily; Barkan, David T.; Yilmaz, Nese Kurt; Schiffer, Celia A.; Moquin, Stephanie A.; Dovala, Dustin; Bolon, Daniel N. A.
      eLife (2022), 11 (), e77433CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      With the continual evolution of new strains of severe acute respiratory syndrome coronavirus- 2 (SARS-CoV- 2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV- 2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three sep. high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophys. properties crit. to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addn., we found a network of crit. amino acids that phys. bridge the two active sites of the Mpro dimer. The clin. variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.
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    21. 21
      Shaqra, A. M.; Zvornicanin, S. N.; Huang, Q. Y. J.; Lockbaum, G. J.; Knapp, M.; Tandeske, L.; Bakan, D. T.; Flynn, J.; Bolon, D. N. A.; Moquin, S.; Dovala, D.; Kurt Yilmaz, N.; Schiffer, C. A. Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance. Nat. Commun. 2022, 13 (1), 3556  DOI: 10.1038/s41467-022-31210-w
      21
      Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance
      Shaqra, Ala M.; Zvornicanin, Sarah N.; Huang, Qiu Yu J.; Lockbaum, Gordon J.; Knapp, Mark; Tandeske, Laura; Bakan, David T.; Flynn, Julia; Bolon, Daniel N. A.; Moquin, Stephanie; Dovala, Dustin; Kurt Yilmaz, Nese; Schiffer, Celia A.
      Nature Communications (2022), 13 (1), 3556CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, addnl. treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clin. use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resoln. cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the crit. recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
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    22. 22
      Flynn, J. M.; Huang, Q. Y. J.; Zvornicanin, S. N.; Schneider-Nachum, G.; Shaqra, A. M.; Yilmaz, N. K.; Moquin, S. A.; Dovala, D.; Schiffer, C. A.; Bolon, D. N. A. Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease. ACS Infect. Dis. 2023, 9 (7), 1372– 1386,  DOI: 10.1021/acsinfecdis.3c00125
      There is no corresponding record for this reference.
    23. 23
      Hoffman, R. L.; Kania, R. S.; Brothers, M. A.; Davies, J. F.; Ferre, R. A.; Gajiwala, K. S.; He, M.; Hogan, R. J.; Kozminski, K.; Li, L. Y.; Lockner, J. W.; Lou, J.; Marra, M. T.; Mitchell, L. J.; Murray, B. W.; Nieman, J. A.; Noell, S.; Planken, S. P.; Rowe, T.; Ryan, K.; Smith, G. J.; Solowiej, J. E.; Steppan, C. M.; Taggart, B. Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19. J. Med. Chem. 2020, 63 (21), 12725– 12747,  DOI: 10.1021/acs.jmedchem.0c01063
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      Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19
      Hoffman, Robert L.; Kania, Robert S.; Brothers, Mary A.; Davies, Jay F.; Ferre, Rose A.; Gajiwala, Ketan S.; He, Mingying; Hogan, Robert J.; Kozminski, Kirk; Li, Lilian Y.; Lockner, Jonathan W.; Lou, Jihong; Marra, Michelle T.; Mitchell Jr., Lennert J.; Murray, Brion W.; Nieman, James A.; Noell, Stephen; Planken, Simon P.; Rowe, Thomas; Ryan, Kevin; Smith III, George J.; Solowiej, James E.; Steppan, Claire M.; Taggart, Barbara
      Journal of Medicinal Chemistry (2020), 63 (21), 12725-12747CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is crit. for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallog. led to the identification of 3 and 4. Preclin. expts. reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an i.v. treatment for COVID-19.
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    24. 24
      Moon, P.; Zammit, C. M.; Shao, Q.; Dovala, D.; Boike, L.; Henning, N. J.; Knapp, M.; Spradlin, J. N.; Ward, C. C.; Wolleb, H.; Fuller, D.; Blake, G.; Murphy, J. P.; Wang, F.; Lu, Y.; Moquin, S. A.; Tandeske, L.; Hesse, M. J.; McKenna, J. M.; Tallarico, J. A.; Schirle, M.; Toste, F. D.; Nomura, D. K. Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**. ChemBioChem 2023, 24 (11), e202300116  DOI: 10.1002/cbic.202300116
      There is no corresponding record for this reference.
    25. 25
      Flynn, J. M.; Zvornicanin, S. N.; Tsepal, T.; Shaqra, A. M.; Kurt Yilmaz, N.; Jia, W.; Moquin, S.; Dovala, D.; Schiffer, C. A.; Bolon, D. N. A. Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance. ACS Infect. Dis. 2024, 10, 1174,  DOI: 10.1021/acsinfecdis.3c00560
      There is no corresponding record for this reference.
    26. 26
      Weiss, S. R.; Hughes, S. A.; Bonilla, P. J.; Turner, J. D.; Leibowitz, J. L.; Denison, M. R. Coronavirus Polyprotein Processing. Arch. Virol., Suppl. 1994, 9, 349– 358,  DOI: 10.1007/978-3-7091-9326-6_35
      There is no corresponding record for this reference.
    27. 27
      Lee, H. J.; Shieh, C. K.; Gorbalenya, A. E.; Koonin, E. V.; La Monica, N.; Tuler, J.; Bagdzhadzhyan, A.; Lai, M. M. C. The Complete Sequence (22 Kilobases) of Murine Coronavirus Gene 1 Encoding the Putative Proteases and RNA Polymerase. Virology 1991, 180 (2), 567– 582,  DOI: 10.1016/0042-6822(91)90071-I
      There is no corresponding record for this reference.
    28. 28
      Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307– 317,  DOI: 10.1038/nrd3410
      28
      The resurgence of covalent drugs
      Singh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, Adrian
      Nature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
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    29. 29
      Resnick, S. J.; Iketani, S.; Hong, S. J.; Zask, A.; Liu, H.; Kim, S.; Melore, S.; Lin, F.-Y.; Nair, M. S.; Huang, Y.; Lee, S.; Tay, N. E. S.; Rovis, T.; Yang, H. W.; Xing, L.; Stockwell, B. R.; Ho, D. D.; Chavez, A. Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity. J. Virol. 2021, 95 (14), e0237420  DOI: 10.1128/JVI.02374-20
      There is no corresponding record for this reference.
    30. 30
      Sayers, E. W.; Bolton, E. E.; Brister, J. R.; Canese, K.; Chan, J.; Comeau, D. C.; Connor, R.; Funk, K.; Kelly, C.; Kim, S.; Madej, T.; Marchler-Bauer, A.; Lanczycki, C.; Lathrop, S.; Lu, Z.; Thibaud-Nissen, F.; Murphy, T.; Phan, L.; Skripchenko, Y.; Tse, T.; Wang, J.; Williams, R.; Trawick, B. W.; Pruitt, K. D.; Sherry, S. T. Database Resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2022, 50 (D1), D20– D26,  DOI: 10.1093/nar/gkab1112
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      Database resources of the national center for biotechnology information
      Sayers, Eric W.; Bolton, Evan E.; Brister, J. Rodney; Canese, Kathi; Chan, Jessica; Comeau, Donald C.; Connor, Ryan; Funk, Kathryn; Kelly, Chris; Kim, Sunghwan; Madej, Tom; Marchler-Bauer, Aron; Lanczycki, Christopher; Lathrop, Stacy; Lu, Zhiyong; Thibaud-Nissen, Francoise; Murphy, Terence; Phan, Lon; Skripchenko, Yuri; Tse, Tony; Wang, Jiyao; Williams, Rebecca; Trawick, Barton W.; Pruitt, Kim D.; Sherry, Stephen T.
      Nucleic Acids Research (2022), 50 (D1), D20-D26CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. The National Center for Biotechnol. Information (NCBI) produces a variety of online information resources for biol., including the GenBank nucleic acid sequence database and the PubMed database of citations and abstrs. published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for the most of these databases. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, RefSeq, SRA, Virus, dbSNP, dbVar, ClinicalTrials.gov, MMDB, iCn3D and PubChem.
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    31. 31
      Olson, R. D.; Assaf, R.; Brettin, T.; Conrad, N.; Cucinell, C.; Davis, J. J.; Dempsey, D. M.; Dickerman, A.; Dietrich, E. M.; Kenyon, R. W.; Kuscuoglu, M.; Lefkowitz, E. J.; Lu, J.; Machi, D.; Macken, C.; Mao, C.; Niewiadomska, A.; Nguyen, M.; Olsen, G. J.; Overbeek, J. C.; Parrello, B.; Parrello, V.; Porter, J. S.; Pusch, G. D.; Shukla, M.; Singh, I.; Stewart, L.; Tan, G.; Thomas, C.; VanOeffelen, M.; Vonstein, V.; Wallace, Z. S.; Warren, A. S.; Wattam, A. R.; Xia, F.; Yoo, H.; Zhang, Y.; Zmasek, C. M.; Scheuermann, R. H.; Stevens, R. L. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): A Resource Combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023, 51 (1 D), D678– D689,  DOI: 10.1093/nar/gkac1003
      There is no corresponding record for this reference.
    32. 32
      Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T. L. BLAST+: Architecture and Applications. BMC Bioinf. 2009, 10, 421  DOI: 10.1186/1471-2105-10-421
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      BLAST+: architecture and applications
      Camacho Christiam; Coulouris George; Avagyan Vahram; Ma Ning; Papadopoulos Jason; Bealer Kevin; Madden Thomas L
      BMC bioinformatics (2009), 10 (), 421 ISSN:.
      BACKGROUND: Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. RESULTS: We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. CONCLUSION: The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c%252FhsVegsA%253D%253D&md5=ed9555ffde3a3634e9486de11af75fd8
    33. 33
      Unoh, Y.; Uehara, S.; Nakahara, K.; Nobori, H.; Yamatsu, Y.; Yamamoto, S.; Maruyama, Y.; Taoda, Y.; Kasamatsu, K.; Suto, T.; Kouki, K.; Nakahashi, A.; Kawashima, S.; Sanaki, T.; Toba, S.; Uemura, K.; Mizutare, T.; Ando, S.; Sasaki, M.; Orba, Y.; Sawa, H.; Sato, A.; Sato, T.; Kato, T.; Tachibana, Y. Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J. Med. Chem. 2022, 65, 6499,  DOI: 10.1021/acs.jmedchem.2c00117
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      Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19
      Unoh, Yuto; Uehara, Shota; Nakahara, Kenji; Nobori, Haruaki; Yamatsu, Yukiko; Yamamoto, Shiho; Maruyama, Yuki; Taoda, Yoshiyuki; Kasamatsu, Koji; Suto, Takahiro; Kouki, Kensuke; Nakahashi, Atsufumi; Kawashima, Sho; Sanaki, Takao; Toba, Shinsuke; Uemura, Kentaro; Mizutare, Tohru; Ando, Shigeru; Sasaki, Michihito; Orba, Yasuko; Sawa, Hirofumi; Sato, Akihiko; Sato, Takafumi; Kato, Teruhisa; Tachibana, Yuki
      Journal of Medicinal Chemistry (2022), 65 (9), 6499-6512CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. We describe the discovery of S-217622, the 1st oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clin. candidate. S-217622 was discovered via virtual screening followed by biol. screening of an inhouse compd. library, and optimization of the hit compd. using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.
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    34. 34
      Douangamath, A.; Fearon, D.; Gehrtz, P.; Krojer, T.; Lukacik, P.; Owen, C. D.; Resnick, E.; Strain-Damerell, C.; Aimon, A.; Ábrányi-Balogh, P.; Brandão-Neto, J.; Carbery, A.; Davison, G.; Dias, A.; Downes, T. D.; Dunnett, L.; Fairhead, M.; Firth, J. D.; Jones, S. P.; Keeley, A.; Keserü, G. M.; Klein, H. F.; Martin, M. P.; Noble, M. E. M.; O’Brien, P.; Powell, A.; Reddi, R. N.; Skyner, R.; Snee, M.; Waring, M. J.; Wild, C.; London, N.; von Delft, F.; Walsh, M. A. Crystallographic and Electrophilic Fragment Screening of the SARS-CoV-2 Main Protease. Nat. Commun. 2020, 11 (1), 5047  DOI: 10.1038/s41467-020-18709-w
      34
      Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease
      Douangamath, Alice; Fearon, Daren; Gehrtz, Paul; Krojer, Tobias; Lukacik, Petra; Owen, C. David; Resnick, Efrat; Strain-Damerell, Claire; Aimon, Anthony; Abranyi-Balogh, Peter; Brandao-Neto, Jose; Carbery, Anna; Davison, Gemma; Dias, Alexandre; Downes, Thomas D.; Dunnett, Louise; Fairhead, Michael; Firth, James D.; Jones, S. Paul; Keeley, Aaron; Keseru, Gyorgy M.; Klein, Hanna F.; Martin, Mathew P.; Noble, Martin E. M.; O'Brien, Peter; Powell, Ailsa; Reddi, Rambabu N.; Skyner, Rachael; Snee, Matthew; Waring, Michael J.; Wild, Conor; London, Nir; von Delft, Frank; Walsh, Martin A.
      Nature Communications (2020), 11 (1), 5047CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      A review. Abstr.: COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Addnl., no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallog. screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVeitbbN&md5=a45f2b463cdc866075014aa3496fd253
    35. 35
      Tomar, S.; Johnston, M. L.; John, S. E. S.; Osswald, H. L.; Nyalapatla, P. R.; Paul, L. N.; Ghosh, A. K.; Denison, M. R.; Mesecar, A. D. Ligand-Induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus Nsp5 Protease (3CLpro): Implications for Nsp5 Regulation and the Development of Antivirals. J. Biol. Chem. 2015, 290 (32), 19403– 19422,  DOI: 10.1074/jbc.M115.651463
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      Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro)
      Tomar, Sakshi; Johnston, Melanie L.; St. John, Sarah E.; Osswald, Heather L.; Nyalapatla, Prasanth R.; Paul, Lake N.; Ghosh, Arun K.; Denison, Mark R.; Mesecar, Andrew D.
      Journal of Biological Chemistry (2015), 290 (32), 19403-19422CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CLpro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CLpro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CLpro is less efficient at processing a peptide substrate due because MERS-CoV 3CLpro forms a weakly assocd. dimer, while HKU4, HKU5, and SARS-CoV 3CLpro enzymes form tightly assocd. dimers. Anal. ultracentrifugation studies confirm that MERS-CoV 3CLpro is a weakly assocd. dimer (Kd ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CLpro were synthesized and utilized in anal. ultracentrifugation expts. and demonstrate that MERS-CoV 3CLpro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compd. concn. as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, detd. to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CLpro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CLpro dimerization. Activation of MERS-CoV 3CLpro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CLpro inhibitors as antiviral agents.
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    36. 36
      Beach, J. R. A Filtrable Virus, the Cause of Infectious Laryngotracheitis of Chickens. J. Exp. Med. 1931, 54 (6), 809– 816,  DOI: 10.1084/jem.54.6.809
      There is no corresponding record for this reference.
    37. 37
      Xue, X.; Yu, H.; Yang, H.; Xue, F.; Wu, Z.; Shen, W.; Li, J.; Zhou, Z.; Ding, Y.; Zhao, Q.; Zhang, X. C.; Liao, M.; Bartlam, M.; Rao, Z. Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design. J. Virol. 2008, 82 (5), 2515– 2527,  DOI: 10.1128/JVI.02114-07
      37
      Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design
      Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe
      Journal of Virology (2008), 82 (5), 2515-2527CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (Mpro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) Mpro and a severe acute respiratory syndrome CoV (SARS-CoV) Mpro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually detd. to elucidate the structural flexibility and substrate binding of Mpro. A monomeric form of IBV Mpro was identified for the first time in CoV Mpro structures. A comparison of these two structures to other available Mpro structures provides new insights for the design of substrate-based inhibitors targeting CoV Mpros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystd. with IBV Mpro and was found to demonstrate in vitro inactivation of IBV Mpro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-assocd. diseases. The structure-based optimization of N3 has yielded two more efficacious lead compds., N27 and H16, with potent inhibition against SARS-CoV Mpro.
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    38. 38
      Chasey, D.; Cartwright, S. F. Virus-like Particles Associated with Porcine Epidemic Diarrhoea. Res. Vet. Sci. 1978, 25 (2), 255– 256,  DOI: 10.1016/S0034-5288(18)32994-1
      There is no corresponding record for this reference.
    39. 39
      St John, S. E.; Anson, B. J.; Mesecar, A. D. X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus. Sci. Rep. 2016, 6 (1), 25961  DOI: 10.1038/srep25961
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    40. 40
      McIntosh, K.; Dees, J. H.; Becker, W. B.; Kapikian, A. Z.; Chanock, R. M. Recovery in Tracheal Organ Cultures of Novel Viruses from Patients with Respiratory Disease. Proc. Natl. Acad. Sci. U.S.A. 1967, 57 (4), 933– 940,  DOI: 10.1073/pnas.57.4.933
      40
      Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease
      McIntosh K; Dees J H; Becker W B; Kapikian A Z; Chanock R M
      Proceedings of the National Academy of Sciences of the United States of America (1967), 57 (4), 933-40 ISSN:0027-8424.
      There is no expanded citation for this reference.
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    41. 41
      Woo, P. C. Y.; Lau, S. K. P.; Chu, C.; Chan, K.; Tsoi, H.; Huang, Y.; Wong, B. H. L.; Poon, R. W. S.; Cai, J. J.; Luk, W.; Poon, L. L. M.; Wong, S. S. Y.; Guan, Y.; Peiris, J. S. M.; Yuen, K. Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia. J. Virol. 2005, 79 (2), 884– 895,  DOI: 10.1128/JVI.79.2.884-895.2005
      41
      Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia
      Woo, Patrick C. Y.; Lau, Susanna K. P.; Chu, Chung-ming; Chan, Kwok-hung; Tsoi, Hoi-wah; Huang, Yi; Wong, Beatrice H. L.; Poon, Rosana W. S.; Cai, James J.; Luk, Wei-kwang; Poon, Leo L. M.; Wong, Samson S. Y.; Guan, Yi; Peiris, J. S. Malik; Yuen, Kwok-yung
      Journal of Virology (2005), 79 (2), 884-895CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      Despite extensive lab. investigations in patients with respiratory tract infections, no microbiol. cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here the authors report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-yr-old man with pneumonia who had just returned from Shenzhen, China. Quant. reverse transcription-PCR showed that the amt. of CoV-HKU1 RNA was 8.5 to 9.6×106 copies per mL in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with IgM titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, resp. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic anal. reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, neg. for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an addnl. specimen, with a viral load of 1.13×106 copies per mL, from a 35-yr-old woman with pneumonia. The data support the existence of a novel group 2 coronavirus assocd. with pneumonia in humans.
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    42. 42
      Zhao, Q.; Li, S.; Xue, F.; Zou, Y.; Chen, C.; Bartlam, M.; Rao, Z. Structure of the Main Protease from a Global Infectious Human Coronavirus, HCoV-HKU1. J. Virol. 2008, 82 (17), 8647– 8655,  DOI: 10.1128/JVI.00298-08
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    43. 43
      Chang, H. W.; Egberink, H. F.; Halpin, R.; Spiro, D. J.; Rottie, P. J. M. Spike Protein Fusion Peptide and Feline Coronavirus Virulence. Emerging Infect. Dis. 2012, 18 (7), 1089– 1095,  DOI: 10.3201/eid1807.120143
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    44. 44
      Fu, L.; Ye, F.; Feng, Y.; Yu, F.; Wang, Q.; Wu, Y.; Zhao, C.; Sun, H.; Huang, B.; Niu, P.; Song, H.; Shi, Y.; Li, X.; Tan, W.; Qi, J.; Gao, G. F. Both Boceprevir and GC376 Efficaciously Inhibit SARS-CoV-2 by Targeting Its Main Protease. Nat. Commun. 2020, 11 (1), 4417  DOI: 10.1038/s41467-020-18233-x
      44
      Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease
      Fu, Lifeng; Ye, Fei; Feng, Yong; Yu, Feng; Wang, Qisheng; Wu, Yan; Zhao, Cheng; Sun, Huan; Huang, Baoying; Niu, Peihua; Song, Hao; Shi, Yi; Li, Xuebing; Tan, Wenjie; Qi, Jianxun; Gao, George Fu
      Nature Communications (2020), 11 (1), 4417CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clin. approved anti-HCV drug, Boceprevir, and a pre-clin. inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analog that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural anal. reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide crit. information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.
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    45. 45
      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 (23), 13190– 13195,  DOI: 10.1073/pnas.1835675100
      45
      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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    46. 46
      Woo, P. C. Y.; Lau, S. K. P.; Li, K. S. M.; Poon, R. W. S.; Wong, B. H. L.; Tsoi, H. wah.; Yip, B. C. K.; Huang, Y.; Chan, K. hung.; Yuen, K. yung. Molecular Diversity of Coronaviruses in Bats. Virology 2006, 351 (1), 180– 187,  DOI: 10.1016/j.virol.2006.02.041
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      Woo, P. C. Y.; Lau, S. K. P.; Lam, C. S. F.; Lau, C. C. Y.; Tsang, A. K. L.; Lau, J. H. N.; Bai, R.; Teng, J. L. L.; Tsang, C. C. C.; Wang, M.; Zheng, B.-J.; Chan, K.-H.; Yuen, K.-Y. Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavi. J. Virol. 2012, 86 (7), 3995– 4008,  DOI: 10.1128/JVI.06540-11
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      Zvornicanin, S. N.; Shaqra, A. M.; Huang, Q. J.; Ornelas, E.; Moghe, M.; Knapp, M.; Moquin, S.; Dovala, D.; Schiffer, C. A.; Kurt Yilmaz, N. Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses. Viruses 2023, 15 (3), 781  DOI: 10.3390/v15030781
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      Hamre, D.; Procknow, J. J. A New Virus Isolated from the Human Respiratory Tract. Proc. Soc. Exp Biol. Med. 1966, 121 (1), 190– 193,  DOI: 10.3181/00379727-121-30734
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      Anand, K.; Anand, K.; Ziebuhr, J.; Wadhwani, P. Coronavirus main proteinase (3CL pro) Structure: Basis for Design of Anti-SARS Drugs. Science 2014, 1763 (2003), 1763– 1768,  DOI: 10.1126/science.1085658
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      van der Hoek, L.; Pyrc, K.; Jebbink, M. F.; Vermeulen-Oost, W.; Berkhout, R. J. M.; Wolthers, K. C.; Wertheim-van Dillen, P. M. E.; Kaandorp, J.; Spaargaren, J.; Berkhout, B. Identification of a New Human Coronavirus. Nat. Med. 2004, 10 (4), 368– 373,  DOI: 10.1038/nm1024
      51
      Identification of a new human coronavirus
      van der Hoek, Lia; Pyrc, Krzysztof; Jebbink, Maarten F.; Vermeulen-Oost, Wilma; Berkhout, Ron J. M.; Wolthers, Katja C.; Wertheim-van Dillen, Pauline M. E.; Kaandorp, Jos; Spaargaren, Joke; Berkhout, Ben
      Nature Medicine (New York, NY, United States) (2004), 10 (4), 368-373CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)
      Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-assocd. coronavirus (SARS-CoV). Here we report the identification of a 4th human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-mo-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clin. specimens from individuals suffering from respiratory illness identified 7 addnl. HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.
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    52. 52
      Wang, F.; Chen, C.; Tan, W.; Yang, K.; Yang, H. Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design. Sci. Rep. 2016, 6 (March), 22677  DOI: 10.1038/srep22677
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      Woo, P. C. Y.; Wang, M.; Lau, S. K. P.; Xu, H.; Poon, R. W. S.; Guo, R.; Wong, B. H. L.; Gao, K.; Tsoi, H.; Huang, Y.; Li, K. S. M.; Lam, C. S. F.; Chan, K.; Zheng, B.; Yuen, K. Comparative Analysis of Twelve Genomes of Three Novel Group 2c and Group 2d Coronaviruses Reveals Unique Group and Subgroup Features. J. Virol. 2007, 81 (4), 1574– 1585,  DOI: 10.1128/JVI.02182-06
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      Lin, X. D.; Wang, W.; Hao, Z. Y.; Wang, Z. X.; Guo, W. P.; Guan, X. Q.; Wang, M. R.; Wang, H. W.; Zhou, R. H.; Li, M. H.; Tang, G. P.; Wu, J.; Holmes, E. C.; Zhang, Y. Z. Extensive Diversity of Coronaviruses in Bats from China. Virology 2017, 507 (February), 1– 10,  DOI: 10.1016/j.virol.2017.03.019
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      Wang, W.; Lin, X.-D.; Liao, Y.; Guan, X.-Q.; Guo, W.-P.; Xing, J.-G.; Holmes, E. C.; Zhang, Y.-Z. Discovery of a Highly Divergent Coronavirus in the Asian House Shrew from China Illuminates the Origin of the Alphacoronaviruses. J. Virol. 2017, 91 (17), e00764-17  DOI: 10.1128/JVI.00764-17
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      Mihindukulasuriya, K. A.; Wu, G.; St Leger, J.; Nordhausen, R. W.; Wang, D. Identification of a Novel Coronavirus from a Beluga Whale by Using a Panviral Microarray. J. Virol. 2008, 82 (10), 5084– 5088,  DOI: 10.1128/jvi.02722-07
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      Shi, M.; Lin, X.-D.; Chen, X.; Tian, J.-H.; Chen, L.-J.; Li, K.; Wang, W.; Eden, J.-S.; Shen, J.-J.; Liu, L.; Holmes, E. C.; Zhang, Y.-Z. The Evolutionary History of Vertebrate RNA Viruses. Nature 2018, 556 (7700), 197– 202,  DOI: 10.1038/s41586-018-0012-7
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      Mordecai, G. J.; Miller, K. M.; Di Cicco, E.; Schulze, A. D.; Kaukinen, K. H.; Ming, T. J.; Li, S.; Tabata, A.; Teffer, A.; Patterson, D. A.; Ferguson, H. W.; Suttle, C. A. Endangered Wild Salmon Infected by Newly Discovered Viruses. eLife 2019, 8, e47615  DOI: 10.7554/eLife.47615
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      Miller, A. K.; Mifsud, J. C. O.; Costa, V. A.; Grimwood, R. M.; Kitson, J.; Baker, C.; Brosnahan, C. L.; Pande, A.; Holmes, E. C.; Gemmell, N. J.; Geoghegan, J. L. Slippery When Wet: Cross-Species Transmission of Divergent Coronaviruses in Bony and Jawless Fish and the Evolutionary History of the Coronaviridae. Virus Evol. 2021, 7 (2), veab050  DOI: 10.1093/ve/veab050
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      Wu, Z.; Lu, L.; Du, J.; Yang, L.; Ren, X.; Liu, B.; Jiang, J.; Yang, J.; Dong, J.; Sun, L.; Zhu, Y.; Li, Y.; Zheng, D.; Zhang, C.; Su, H.; Zheng, Y.; Zhou, H.; Zhu, G.; Li, H.; Chmura, A.; Yang, F.; Daszak, P.; Wang, J.; Liu, Q.; Jin, Q. Comparative Analysis of Rodent and Small Mammal Viromes to Better Understand the Wildlife Origin of Emerging Infectious Diseases 06 Biological Sciences 0604 Genetics 11 Medical and Health Sciences 1108 Medical Microbiology. Microbiome 2018, 6 (1), 178  DOI: 10.1186/s40168-018-0554-9
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      Zhu, F.; Duong, V.; Lim, X. F.; Hul, V.; Chawla, T.; Keatts, L.; Goldstein, T.; Hassanin, A.; Tu, V. T.; Buchy, P.; Sessions, O. M.; Wang, L. F.; Dussart, P.; Anderson, D. E. Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats. Viruses 2022, 14 (2), 176  DOI: 10.3390/v14020176
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      Johnson, T. W.; Gallego, R. A.; Edwards, M. P. Lipophilic Efficiency as an Important Metric in Drug Design. J. Med. Chem. 2018, 61 (15), 6401– 6420,  DOI: 10.1021/acs.jmedchem.8b00077
      62
      Lipophilic Efficiency as an Important Metric in Drug Design
      Johnson, Ted W.; Gallego, Rebecca A.; Edwards, Martin P.
      Journal of Medicinal Chemistry (2018), 61 (15), 6401-6420CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Lipophilic efficiency (LipE) is an important metric that has been increasingly applied in drug discovery medicinal chem. lead optimization programs. In this perspective, using literature drug discovery examples, we discuss the concept of rigorously applying LipE to guide medicinal chem. lead optimization toward drug candidates with potential for superior in vivo efficacy and safety, esp. when guided by physiochem. property-based optimization (PPBO). Also highlighted are examples of small structural modifications such as addn. of single atoms, small functional groups, and cyclizations that produce large increases in LipE. Understanding the factors that may contribute to LipE changes through anal. of ligand-protein crystal structures and using structure-based drug design (SBDD) to increase LipE by design is also discussed. Herein we advocate for use of LipE anal. coupled with PPBO and SBDD as an efficient mechanism for drug design.
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      Sakhno, Y. I.; Murlykina, M. V.; Zbruyev, O. I.; Kozyryev, A. V.; Shishkina, S. V.; Sysoiev, D.; Musatov, V. I.; Desenko, S. M.; Chebanov, V. A. Ultrasonic-Assisted Unusual Four-Component Synthesis of 7-Azolylamino-4,5,6,7-Tetrahydroazolo[1,5-a]Pyrimidines. Beilstein J. Org. Chem. 2020, 16, 281– 289,  DOI: 10.3762/bjoc.16.27
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      Chebanov, V. A.; Sakhno, Y. I.; Desenko, S. M.; Chernenko, V. N.; Musatov, V. I.; Shishkina, S. V.; Shishkin, O. V.; Kappe, C. O. Cyclocondensation Reactions of 5-Aminopyrazoles, Pyruvic Acids and Aldehydes. Multicomponent Approaches to Pyrazolopyridines and Related Products. Tetrahedron 2007, 63 (5), 1229– 1242,  DOI: 10.1016/j.tet.2006.11.048
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      Patel, A. S.; Kapuriya, N. P.; Naliapara, Y. T. A Concise [3 + 3] Heteroaromatization Synthetic Strategy Afford Dicarboxamide Functionalized Novel Pyrazolo[1,5-a]Pyrimidines. J. Heterocycl. Chem. 2017, 54 (5), 2635– 2643,  DOI: 10.1002/jhet.2860
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      Thomas, A.; Chakraborty, M.; Ila, H.; Junjappa, H. Cyclocondensation of Oxoketene Dithioacetals with 3-Aminopyrazoles: A Facile Highly Regioselective General Route to Substituted and Fused Pyrazolo]Pyrimidines. Tetrahedron 1990, 46 (2), 577– 586,  DOI: 10.1016/S0040-4020(01)85438-7
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      Bertani, G. Studies on Lysogenesis. I. The Mode of Phage Liberation by Lysogenic Escherichia Coli. J. Bacteriol. 1951, 62, 293– 300,  DOI: 10.1128/jb.62.3.293-300.1951
      68
      Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli
      BERTANI G
      Journal of bacteriology (1951), 62 (3), 293-300 ISSN:0021-9193.
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      Tartof, K. D. Improved Media for Growing Plasmid and Cosmid Clones. Bethesda Res. Lab. Focus. 1987, 9, 12
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01404.

    • Machine-readable datasheet file with full cluster composition including isolate accessions, cluster assignment, and annotation (XLSX)

    • Molecular formula strings (CSV)

    • Additional details regarding coronaviridae isolate curation and clustering, lab strain panel composition, and biochemical assay parameters, as well as experimental procedures and 1H NMR for all mentioned compounds (PDF)

    Accession Codes

    The X-ray diffraction structures of protein–ligand complexes have been deposited in the Protein Data Bank with the accession codes PDB ID 9C7W, 9C8Q, and 9C80 (for HCoV-OC43 Mpro + compound 21, for SARS-CoV-2 Mpro + compound 14, and for SARS-CoV-2 Mpro + compound 1 respectively).


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