Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19
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Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19
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  • Richard T. Eastman
    Richard T. Eastman
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
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    Orcidhttp://orcid.org/0000-0003-3580-380X
  • Jacob S. Roth
    Jacob S. Roth
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
    Albert Einstein College of Medicine, New York, New York 10461, United States
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  • Kyle R. Brimacombe
    Kyle R. Brimacombe
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
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  • Anton Simeonov
    Anton Simeonov
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
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  • Min Shen
    Min Shen
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
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  • Samarjit Patnaik
    Samarjit Patnaik
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
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  • Matthew D. Hall*
    Matthew D. Hall
    National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
    *Tel: 301-480-9928. E-mail: [email protected]
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    Orcidhttp://orcid.org/0000-0002-5073-442X
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Cite this: ACS Cent. Sci. 2020, 6, 5, 672–683
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https://doi.org/10.1021/acscentsci.0c00489
Published May 4, 2020

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Abstract

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The global pandemic of SARS-CoV-2, the causative viral pathogen of COVID-19, has driven the biomedical community to action—to uncover and develop antiviral interventions. One potential therapeutic approach currently being evaluated in numerous clinical trials is the agent remdesivir, which has endured a long and winding developmental path. Remdesivir is a nucleotide analogue prodrug that perturbs viral replication, originally evaluated in clinical trials to thwart the Ebola outbreak in 2014. Subsequent evaluation by numerous virology laboratories demonstrated the ability of remdesivir to inhibit coronavirus replication, including SARS-CoV-2. Here, we provide an overview of remdesivir’s discovery, mechanism of action, and the current studies exploring its clinical effectiveness.

This article not subject to U.S. Copyright. Published 2020 by the American Chemical Society

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Synopsis

We review clinical development of remdesivir, a prodrug with a demonstrated ability to inhibit SARS-CoV-2 replication, which supports its clinical evaluation for COVID-19 treatment.

Introduction

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Coronaviruses are a family of enveloped viruses with a positive-sense, single-stranded RNA genome that infects animal species and humans. Among coronavirus members are those responsible for the common cold, severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome-related coronavirus (MERS), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the causative pathogen of the disease COVID-19). (1)
Coronaviruses primarily cause respiratory and intestinal infections in animals and humans. (2) Discovered in the 1960s, they were originally thought to be only responsible for mild disease, with strains such as HCoV 229E and HCoV OC43 responsible for the common cold. (3) That changed in 2003 with the SARS pandemic and in 2012 with the outbreak of MERS, both zoonotic infections that resulted in mortality rates greater than 10% and 35%, respectively. (4) Both coronaviruses likely emerged from native bat populations, which maintain a broad diversity of coronaviruses, and were transmitted through an intermediate host to humans. Loss of natural habitat and increased exposure to new hosts are likely responsible for the increased frequency of zoonotic infections originating from bats. (5,6) Evidence also supports that the novel coronavirus which emerged in the Wuhan region of China in late 2019 also originated from bats. (7) This novel coronavirus, SARS-CoV-2, resulted in an outbreak of pathogenic viral pneumonia in Wuhan, Hubei Province, China, as reported to the World Health Organization (WHO) in December 2019. Subsequent spread has led to a global pandemic (officially declared by the WHO on March 11, 2020 (8)).
COVID-19 disease appears to be a spectrum of clinical presentations ranging from asymptomatic to severe respiratory failure. Common symptomology at the onset of illness are fever, cough, and general myalgia, with less common symptoms including sputum production, headache, and diarrhea. (9−11) An initial case analysis from China through mid-February 2020 found 14% of cases were associated with severe disease (dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio < 300, and/or lung infiltrates > 50% within 24–48 h), and 5% of cases were critical (i.e., respiratory failure, septic shock, and/or multiple organ dysfunction or failure). (12) A more extensive meta-analysis found a slightly higher severe disease percentage (20.3%). (13) The disease case fatality rate (CFR) varies depending on region, population demographics, and heath care capabilities; for instance, in Italy an overall CFR of 7.2% is estimated, in part driven by the higher proportion of individuals of advanced age compared to China. (14) On the basis of global data, the CFR from COVID-19 based on confirmed cases is estimated to be ∼6.9%. (15) Disease progression to acute respiratory distress syndrome typically occurs in older patients (over 63), often with underlying medical conditions such as hypertension or diabetes; (16) elevated risk of mortality was associated with advanced age, sepsis, blood clotting deficiencies. (17,18) In individuals less than 60 years of age, an increased body to mass index (over 30) was associated with increased disease severity and progression to acute respiratory distress syndrome. (19) Other symptoms, including neurologic symptoms and coagulopathies, have also been reported in a portion of infected individuals. (20−24)
Similar to other coronaviruses, SARS-CoV-2 primarily infects the respiratory and gastrointestinal tract, with a cell tropism of nasal epithelial cells, pneumocytes, and alveolar macrophages in the lung and enterocytes in the bowel. (25−27) Although not limited to only these specific cell types, evidence does support that cell binding via the viral S protein to the host receptor angiotensin-converting enzyme 2 (ACE2) is required for infection (Figure 1). (28,29) Following entry of the virus into the host cell, the virus complex is then translocated to the endosome, where endosomal acid proteases cleave the S protein mediating membrane fusion. (28) The viral genome is released and translated into the viral replicase polyproteins PP1a and PP1ab, which are cleaved into functional proteins by viral proteases. Subgenomic templates for mRNA synthesis and translation of the viral structural proteins occur through discontinuous transcription. (2) Viral genome replication is mediated by the viral replication complex, which includes an RNA-dependent RNA polymerase (RdRp), helicase, exonucleaseN, and other accessory proteins. Subsequent assembly of viral nucleocapsids from the packaged viral genomes and translated viral structural proteins occurs at the endoplasmic reticulum-Golgi intermediate compartment, (30) with infectious virions then released from the cell through exocytosis.

Figure 1

Figure 1. Life cycle of SARS-CoV-2 in host cells. SARS-CoV-2 primarily infects the respiratory tract (nasal epithelial cells, pneumocytes, and alveolar macrophages) and the gastrointestinal tract (enterocytes). The virus enters though direct interaction between the viral S protein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). Following entry, the viral genome is released and translated into the viral replicase polyproteins PP1a and PP1ab, which are cleaved into functional proteins by viral proteases. (2) Viral genome replication is mediated by the viral replication complex, including the RNA-dependent RNA polymerase (RdRp). Viral nucleocapsids are assembled from the packaged viral genomes and translated viral structural proteins and released through exocytosis. Potential targets and postulated mechanism of action for antiviral interventions are shown: blocking virus/host cell interaction through the use of antibodies/nanobodies (and convalescent plasma therapy) or recombinant ACE2 protein; use of hydroxychloroquine (based on in vitro data) to inhibit endosome maturation; use of protease inhibitors to inhibit viral/endosome membrane fusion or viral polypeptide maturation; nucleoside/nucleotide analogues to inhibit viral genome replication.

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As a new disease, SARS-CoV-2 does not have any clinically proven therapeutics. Furthermore, a significant amount of preclinical research was reported in the search for therapeutic treatments for the related viruses SARS and MERS. As the SARS and MERS coronavirus outbreaks did not persist, no therapeutic or vaccine development programs were completed. The consequence is that drug repositioning and repurposing has received a significant amount of attention, (31) and approved agents including hydroxychloroquine, azithromycin, ritonavir, ruxolitinib, and camostat have entered clinical trials to address the current SARS-CoV-2 pandemic (Figure 1). (32−34) Although some candidates do have pre-existing data to support activity against coronaviruses, other repurposing candidates for potential use against SARS-CoV-2 are based on their ability to inhibit SARS-CoV-2 viral replication in vitro. (29,35−37) These include hydroxychloroquine, a known autophagy inhibitor that suppresses lysosomal function, (38) and the serine protease inhibitor camostat. (29) The ability of these compounds to act as prophylactic agents, treat disease, or even modulate viral replication in vivo has not been demonstrated, although clinical evaluation of several of these potential therapeutics is ongoing.
One of the first clinical candidates that has received attention is remdesivir, a pre-existing drug candidate developed by Gilead Sciences as part of an antiviral development effort, with initial results against Ebola virus (EBOV) reported in 2015. (39) It was recently authorized for compassionate use and has now entered controlled clinical trials. Like all other therapeutic approaches for patients with COVID-19, remdesivir was not developed specifically to treat COVID-19, and here we review its discovery and mode of action.

Development of Remdesivir

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Remdesivir (GS-5734) was developed by Gilead Sciences and emerged from a collaboration between Gilead, the U.S. Centers for Disease Control and Prevention (CDC) and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). They sought to identify therapeutic agents for treating RNA-based viruses that maintained global pandemic potential, such as those that indeed emerged following the initiation of the program, including EBOV and the Coronaviridae family viruses exemplified by Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS).
As a starting point for discovery, a library of ∼1000 small molecules focused around nucleoside analogues was compiled, based on prior knowledge of effective antiviral compounds targeting RNA viruses. Nucleosides are poorly cell-permeable (and therefore can have a low hit rate in cell-based screens such as antiviral screens), so modified nucleosides such as monophosphate, ester, and phosphoramidate prodrugs composed a significant portion of the library. Such prodrugs are typically more permeable and metabolized to liberate the nucleoside or phosphorylated nucleoside within cells. (40−42) While the data from the original full screen does not appear to have been disclosed, a 1′-CN modified adenosine C-nucleoside hit (GS-441524), along with a prodrug form of the monophosphate of GS-441524 (GS-5734, later renamed as remdesivir), was found to be highly potent. (43) GS-441524 and its S-acyl-2-thioethyl monophosphate prodrug had previously been reported in 2012 as potent leads from a series of 10-substituted 4-aza-7,9-dideazaadenosine C-nucleosides, with broad activity against a panel of RNA viruses: yellow fever virus (YFV), Dengue virus type 2 (DENV-2), influenza A, parainfluenza 3, and SARS. (44) The primary assay used was the cytoprotection effect (CPE) assay, in which live virus is incubated with a target cell line and the antiviral activity is inferred by the ability of a test agent to rescue cell death, measured using a standard cell viability reagent. (45) In a 2012 study, GS-5734 showed CPE activity against SARS strain Toronto 2 (IC50 = 2.2 μM) without causing cytotoxicity toward the host Vero African green monkey kidney epithelial cells used in the CPE assay (note that different target cells were utilized in viral CPE assays).
When the Ebola outbreak occurred in 2014, the assembled library was utilized to identify and prioritize compounds with efficacy against EBOV. The study by Madelain et al. found that GS-5734 reduced EBOV replication in HeLa cells with an IC50 ≈ 100 nM, and it retained potency in in vivo nonhuman primate EBOV infection models, while GS-441524 was inactive. (46,47) In addition to demonstrating activity against EBOV, Warren et al. showed that remdesivir also had antiviral activity against several other viruses, including the coronavirus MERS, with an IC50 of 340 nM in vitro.
With the demonstration that GS-5734 (remdesivir) possessed broad activity against RNA viruses, multiple groups assessed antiviral activity both in vitro and in vivo, (45,48,49) validating its activity against coronaviruses. Antiviral activity was confirmed against SARS, MERS zoonotic coronaviruses, (49) as well as the circulating human coronaviruses HCoV-OC43 and HCoV-229E, causative agents of the common cold. (50) Furthermore, de Wit et al. demonstrated that remdesivir had both prophylactic and therapeutic activity against MERS in a nonhuman primate in vivo model. (51)
The pharmacokinetics of remdesivir have been summarized in compassionate use documentation published by the European Medicines Agency (EMA, 2020). Remdesivir is administered via an intravenous injection (IV) with a loading dose on day 1 (200 mg in adults, adjusted for body weight in pediatric patients) followed by a daily maintenance dose (100 mg in adults) for up to 10 days. In nonhuman primates, daily administration of 10 mg/kg of remdesivir yielded a short plasma half-life of the prodrug (t1/2= 0.39 h), but sustained intracellular levels of the triphosphate form. (45)
In vitro and preclinical in vivo animal models supported the effectiveness of remdesivir against SARS-CoV-2 and related coronaviruses. These include a recent in vitro study of remdesivir assessing antiviral activity against SARS-CoV-2 (previously known as 2019-nCov, strain nCoV-2019BetaCoV/Wuhan/WIV04/2019) using qRT-PCR quantification of viral copy number in infected Vero E6 cells. This study demonstrated an IC50 of 770 nM and an IC90 equal to 1,760 nM (with cytotoxic concentration >100 mM). (52) In addition, works by Sheahan et al. and de Wit et al. demonstrated in vivo efficacy of remdesivir at inhibiting viral replication and reducing viral related pathology against related coronaviruses. (51,53) These findings, along with the safety profile of remdesivir in the clinical trial assessment against EBOV, (54) support the evaluation of remdesivir as a potential therapeutic drug for repurposing against the SARS-CoV-2 pandemic.
Driven by the EBOV outbreak in 2014 and based on in vitro and animal model in vivo efficacy against EBOV, (45) Gilead Sciences initiated clinical evaluation of remdesivir for EBOV. Gilead pursued FDA evaluation under the FDA’s Animal Rule, permitting the reliance on efficacy findings from animal studies for drugs in which it is not feasible or ethical to conduct human trials. As such, remdesivir was included in a randomized, controlled trial of Ebola virus therapeutics in patients within the Democratic Republic of the Congo (NCT02818582); however, midstudy primary analyses found remdesivir inferior to the antibody based therapeutics MAb114 and REGN-EB3, with respect to mortality, and the remdesivir intervention arm was terminated. (54) Mulangu et al. reported one serious adverse event related to remdesivir, an instance of hypotension, along with elevated creatinine and aspartate aminotransferase plasma levels (a suggestive marker for impaired kidney or liver function, respectively) in remdesivir-treated patients compared to either antibody based therapeutic arms. Although remdesivir was inferior against EBOV based on efficacy compared to antibody therapy, the study arm did provide an initial insight into the safety profile in patients.

Repurposing or repositioning an effective small-molecule therapeutic promises the fastest therapeutic means to stem the tide of the pandemic.

Remdesivir Mode of Action

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Antiviral chemotherapeutic interventions often target specific viral enzymes or attack a weak point of viral replication within the host, such as targeting the divergent RNA-dependent RNA polymerase (RdRp; Figure 2). Nucleoside analogues represent a class of antiviral agents that has proven efficacious against several viruses, including hepatitis B and C as well as HIV. Generally, these fall into three general classes: mutagenic nucleosides, obligate chain terminators, or delayed chain terminators. (55) Ribavirin, a mutagenic nucleoside, targets the viral reliance on an RdRp to catalyze the replication of the RNA genome from the original RNA template. (56,57) In a seminal paper, Crotty et al. demonstrated that the RNA virus poliovirus exists on the edge of viability, due to the proportion of virus particles with deleterious mutations. Furthermore, treatment with concentrations of ribavirin that caused a 9.7-fold increase in mutations was sufficient to induce “error catastrophe,” in effect lethally mutating the poliovirus, reducing infectivity by 99.3%. (58) Obligate chain terminators, such as azidothymidine (AZT), lack the reactive 3′-hydroxyl group, which directly prevents additional DNA synthesis after incorporation. (59) Lastly, delayed chain terminators, which include remdesivir, block transcription despite still possessing the 3′-hydroxyl and thus can still form a phosphodiester bond with the next incorporated nucleotide. However, evidence suggests that the 1′CN substituent of remdesivir sterically clashes with RdRp (residue S861) upon further chain elongation (remdesivir + three additional nucleotides), distorting the positioning of the RNA and hampering translocation to the remdesivir + fourth position (Figure 2). (60)

Figure 2

Figure 2. SARS-CoV-2 genome and RNA-dependent RNA polymerase structure. (a) Representation of the SARS-CoV-2 RNA genome. As SARS-CoV-2 is a positive-sense RNA virus, the genome serves as a direct template for protein translation. Replication of the viral genome requires a functional viral replication complex, including an RNA-dependent RNA polymerase (RdRp). (b) Domain organization of the SARS-CoV-2 RdRp (encoded by nsp12) domains bound to cofactors nsp7 and dimers of nsp8, that serve as essential cofactors that increase polymerase activity. The rendering was based on the cryo-EM structure at a resolution of 2.9-Å, published by Gao et al, 2020 (PDB: 6M71). The nsp12 RdRp domain is shown in green, nsp7 in purple, nsp8 in cyan, nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain in yellow, interface in blue, and a newly identified β-hairpin domain is shown in red. (61) Highlighted is RdRp residue S861, which is predicted to sterically interact with the 1′CN substituent of remdesivir inducing delayed chain termination. (60)

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Remdesivir (GS-5734), a prodrug, is metabolized within cells into an alanine metabolite (GS-704277), further processed into the monophosphate derivative and ultimately into the active nucleoside triphosphate derivative (Figure 3). Nucleotide analogues are not highly cell permeable, and once in the cell they require di- and then triphosphorylation to produce the nucleoside triphosphate (NTP) that can be utilized by the viral RNA-dependent polymerases for genome replication. As such, NTPs can then be misintegrated into viral RNA by the viral RNA-dependent RNA polymerase (RdRP; Figure 2). To address this, an approach to antiviral drug design can employ the utilization of phosphoramidate prodrugs (ProTides, inferred as prodrugs of nucleotides). (62−65) Protides are composed of a nucleoside monophosphate capped with an aryl group and an amino acid ester (a phosphoramidate). Following diffusion into the cell, the prodrug is presumed to metabolize in a sequence of hydrolytic steps that starts with esterase-mediated ester hydrolysis to a carboxylate that cyclizes internally to the phosphonate ejecting the phenoxide; the resultant unstable cyclic anhydride is hydrolyzed open by water to the alanine metabolite GS-704277 whose P–N bond is hydrolyzed by a phosphoramidase-type enzyme (Figure 3). (66−69) This final step liberates the nucleoside monophosphate, which is highly polar, and does not diffuse back across the cell membrane (essentially trapping it within the cell). Subsequent phosphorylation by host cell kinases convert the compound into the NTP analogue that can be used as a substrate by the viral RdRp enzyme. (70) While the nucleoside analogue core of remdesivir, GS-441524, can diffuse into cells, the initial phosphorylation step for nucleosides is rate-limiting (slow), which is believed to account for the reduced antiviral activity of GS-441524 compared to remdesivir. (49,71) This approach has been successfully applied to a number of FDA-approved antiviral drugs including the Gilead products sofosbuvir (for treating HCV) and tenofovir alafenamide (first approved for treating HIV). (64)

Figure 3

Figure 3. Remdesivir and its intracellular conversion. (a) Chemical structures of GS-441524 that compose the nucleoside analogue core (blue) of remdesivir (GS-5734). (b) Intracellular processing of the prodrug remdesivir (GS-5734), the aryloxy phosphoramidate (purple) prodrug of GS-441524 monophosphate. Upon diffusion of remdesivir into the cell, it is metabolized into the nucleoside monophosphate form via a sequence of steps that are presumably initiated by esterase-mediated hydrolysis of the amino acid ester that liberates a carboxylate that cyclizes on to the phosphorus displacing the phenoxide. The unstable cyclic anhydride is hydrolyzed by water to the alanine metabolite GS-704277 whose P–N bond is hydrolyzed by phosphoramidase-type enzymes to liberate the nucleoside monophosphate or nucleotide analog. The artificial nucleoside monophosphate is routed to further phosphorylation events (hijacking the endogenous phosphorylation pathway) yielding the active nucleoside triphosphate analogue form that is utilized by the viral RNA-dependent RNA polymerase (RdRp). Utilization of the GS-441524 nucleoside triphosphate analogue by RdRp inhibits viral replication through inducing delayed chain termination.

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Among the candidate therapies, remdesivir has demonstrated efficacy in both in vitro and in vivo models against coronaviruses

Remdesivir’s antiviral activity, sterically interacting with the viral RdRp to induce delayed chain termination, has been demonstrated in vitro against multiple coronaviruses (SARS, MERS, contemporary human CoV and bat-CoVs). (72) Remdesivir was also shown to perturb pan-CoV RdRp function by inhibiting viral replication of SARS, MERS, and the model β-coronavirus murine hepatitis virus (MHV), even in settings with intact exonuclease proofreading activity. (45) Biochemical data from recombinant respiratory syncytial virus (RSV) RdRp suggested the primary mechanism of action was through delayed chain termination. (73−75) Importantly, remdesivir inhibits viral replication (demonstrated with both Ebola and RSV) in cell-based assays with IC50 values of approximately 100 nM, whereas human RNA Polymerase (RNAP) II and human mitochondrial RNAP are not inhibited in the presence of compound, (75) providing approximately 500-fold selectivity. This selectivity is achieved, at least in part, due to the nucleoside analogues being poor substrates for the human polymerases. (76) Interestingly, in vitro assays demonstrate that the triphosphate form of the inhibitor was incorporated at increased rates compared to natural nucleotide pools, (77) likely adding to strong antiviral potency of remdesivir through premature RNA synthesis termination.

Clinical Studies for COVID-19

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With the COVID-19 outbreak increasing in size and a lack of alternative therapeutics, two clinical trials using remdesivir were designed and initiated in China. On February 5, 2020, a phase 3 randomized, quadruple-blind, placebo-controlled clinical trial was registered at Capital Medical University, with the goal to determine safety and efficacy of remdesivir in patients with mild to moderate SARS-CoV-2 infection (NCT04252664, since suspended). (78) A day later, a second trial (NCT04257656, since terminated) was registered at the same location, focused on patients with advanced COVID-19 respiratory disease. (79) Both trials had planned to track the primary outcome as time to clinical improvement, up to 28 days: normalization of fever, oxygen saturation, and respiratory rate, and alleviation of cough which is sustained for 72 h. Both trials delivered remdesivir as a 200 mg loading dose on the first day, with 9 subsequent days of maintenance dosing at 100 mg; this regime is identical to that utilized in the previous NCT03719586 Ebola trial, which appears to be the model for all subsequent trials involving remdesivir (discussed below; Figure 4 and Table 1, registered trials of remdesivir).
Table 1. Registered Remdesivir Trials for SARS-CoV-2/COVID-19a
study identifierstudy titlestart datestatussponsorinterventionsphasestudy designexpected completion datelocation (s)
NCT04280705bAdaptive COVID-19 Treatment Trial (ACTT)February 21, 2020recruitingNational Institute of Allergy and Infectious Diseases (NIAID)remdesivir; remdesivir placebo3clinical trial, randomized parallel assignmentdouble (participant, investigator)treatmentApril 1, 2023multiple US; multiple Korea; Tokyo, Japan; Singapore
ISRCTN83971151c, NCT04330690Public Health Emergency SOLIDARITY Trial of Treatments for COVID- 19 Infection in Hospitalized PatientsMarch 1, 2020AvailableWorld Health Organizationremdesivir; lopinavir/ritonavir; lopinavir/ritonavir, interferon β-1a; hydroxychloroquine; standard of care3clinical trial, randomizednone (open label)treatmentMarch 25, 2021multiple sites - countries of recruitment: Argentina, Brazil, Canada, Germany, Indonesia, Iran, Norway, Peru, Qatar, South Africa, Spain, Switzerland, Thailand
NCT04292899bStudy to Evaluate the Safety and Antiviral Activity of Remdesivir (GS-5734) in Participants With Severe Coronavirus Disease (COVID-19)March 6, 2020recruitingGilead Sciencesremdesivir; Standard of Care3clinical trial, randomized parallel assignmentnone (open label)treatmentMay 1, 2020multiple US; multiple Hong Kong; Multiple Italy; multiple Korea; multiple Singapore; multiple Spain; multiple Taiwan
NCT04292730b studyStudy to Evaluate the Safety and Antiviral Activity of Remdesivir (GS-5734) in Participants With Moderate Coronavirus Disease (COVID-19) Compared to Standard of Care TreatmentMarch 15, 2020recruitingGilead Sciencesremdesivir; standard of care3clinical trial, randomized parallel assignmentnone (open label)treatmentMay 1, 2020multiple US; multiple Hong Kong; multiple Italy; multiple Korea; multiple Singapore; multiple Spain; multiple Taiwan
NCT04314817bAdverse Events Related to Treatments Used Against Coronavirus Disease 2019March 17, 2020recruitingGroupe Hospitalier Pitie-Salpetriere, CMC Ambroise Paréany drug used to treat COVID-19 observational model, case-only  January 1, 2023AP-HP Assistance Publique Hopitaux de Paris, Paris, France
NCT04315948bTrial of Treatments for COVID-19 in Hospitalized AdultsMarch 22, 2020recruitingInstitut National de la Santé Et de la Recherche Médicale, Franceremdesivir; lopinavir/ritonavir; interferon β-1a; hydroxychloroquine; standard of care3clinical trial, randomized parallel assignmentnone (open label)treatmentMarch 1, 2023multiple France
NCT04321616b, 2020-000982-18dThe Efficacy of Different Antiviral Drugs in (Severe Acute Respiratory Syndrome-Corona Virus-2) SARS-CoV-2March 26, 2020not yet recruitingOslo University Hospitalremdesivir; hydroxychloroquine; standard of care2, 3clinical trial, randomized parallel assignmentnone (open label)treatmentNovember 1, 2020 
2020-001052-18dA Multicenter, Adaptive, Randomized Blinded Controlled Trial of the Safety and Efficacy of Investigational Therapeutics for the Treatment of COVID-19 in Hospitalised Adults availableRegents of the University of Minnesotaremdesivir clinical trial, randomized parallel assignmentdouble (participant, investigator)treatment multiple sites - countries of recruitment: Denmark, Germany, Italy, Portugal, Spain, United Kingdom
NCT04302766bExpanded Access Remdesivir (RDV; GS-5734) availableU.S. Army Medical Research and Development Commandremdesivir expanded access    
NCT04323761bExpanded Access Treatment Protocol: Remdesivir (RDV; GS-5734) for the Treatment of SARS-CoV-2 (CoV) Infection availableGilead Sciencesremdesivir expanded access    
a

Registered remdesivir clinical studies (as of 4/15/2020) for SARS-CoV-2/COVID-19.

b

Clinicaltrials.gov registered.

c

ISRCTN registered (www.isrctn.com).

d

Clinicaltrialsregister.eu registered.

Figure 4

Figure 4. Remdesivir global clinical trials. Shown are the locations of the clinical study sites for the ongoing clinical studies of remdesivir for SARS-CoV-2/COVID-19. Number of sites participating for each respective study, if no specific information was given, shown are the countries participating (e.g., ISRCTN83971151). Listed are the number of sites participating for each respective study, if no detailed information was provided; shown are the number of countries participating. NCT04302766 is an expanded access trial with no specific sites listed in the registration. Figure created with R, (91) utilizing the packages rnaturalearth, (92) sf, (93) and ggplot2. (94)

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Contemporaneous to the development of the Chinese trials, the first cases of COVID-19 were emerging in the USA. On January 20, 2020, a patient reported to urgent care in Snohomish County, Washington with subjective fever and a 4-day history of cough, later to be confirmed as the first positive case of COVID-19 in the USA. (80) On the seventh day of hospitalization and after worsening clinical status, the patient was given IV remdesivir under compassionate use access (Gilead Sciences), with no adverse events observed on infusion. (80) The patient’s clinical condition improved the next day, though concurrent treatment with acetaminophen, ibuprofen, guaifenesin, vancomycin, cefepime, and supplemental oxygen confound the direct interpretation of remdesivir’s impact.
Subsequently, 12 patients were confirmed to be infected with SARS-CoV-2 between January 20, 2020, and February 5, 2020. (81) Of these 12 patients, seven were hospitalized and three received remdesivir (compassionate use access; Gilead Sciences) upon worsening clinical disease. Treatment was continued for 4–10 days with 200 mg IV on the first day and 100 mg each following day. Following the initial dose, all patients experienced “transient gastrointestinal symptoms, including nausea, vomiting, gastroparesis, or rectal bleeding,” although treatment was continued until improvement in respiratory symptoms, with all 12 patients reporting symptom resolution by February 22, 2020. (81) The small sample size and lack of controlled randomization preclude analysis of clinical efficacy or safety.
The National Institute of Allergies and Infectious Diseases (NIAID), NIH initiated the Adaptive COVID-19 Treatment Trial (ACTT), a double-blind, randomized, placebo-controlled phase 3 trial to evaluate the safety and efficacy of remdesivir compared with a remdesivir placebo-control (NCT04280705). (82) NIAID developed this study in part based on the existing Chinese clinical trials in addition to consulting with the WHO. (83) This study is currently recruiting patients, tracking the primary outcome of patient status severity on an eight-point ordinal scale, with multiple secondary outcomes of interest. A total of 75 clinical sites are anticipated to participate in the study, with distribution across the United States, and an estimated primary completion date of April 2023.
Subsequently, Gilead Sciences initiated two clinical trials that began in mid-March, comparing remdesivir to standard of care in patients with moderate or severe coronavirus disease (COVID-19) in an open-label, randomized trial, NCT04292899. (84) This trial will explore the safety and efficacy of remdesivir in combination with standard of care to compare study arms of 5- or 10-day remdesivir dosing on the primary outcome of fever and oxygen saturation. NCT04292730 maintains three study arms to compare remdesivir provided over 5 or 10 days, to standard of care alone, with the primary outcome being the proportion of patients discharged by the 14th day. (85)
To determine the most effective treatments for COVID-19 and ensure sufficient power to observe definitive results, the WHO announced the SOLIDARITY clinical trial, a four-arm trial comparing remdesivir, lopinavir/ritonavir, lopinavir/ritonavir with interferon-β1a, and chloroquine or hydroxychloroquine (ISRCTN83971151). With the goal of reducing trial design time and start-up, the WHO seeks to rapidly facilitate comparison of treatments on a worldwide scale. Data will be analyzed on an interim basis by an independent group of experts, the Global Data and Safety Monitoring Committee, (86) enabling the modification of study design if particular treatments show early promise. As of March 27, 2020, over 70 countries had committed to participating.
In a trial sponsored by the Oslo University Hospital, the WHO NOR (Norwegian)-COVID 19 study is a multicenter, adaptive, randomized, open label study to evaluate the safety and efficacy of hydroxychloroquine, remdesivir, and current standard of care (NCT04321616, 2020-001052-18). (87) The comparative arms of the study are daily remdesivir, hydroxychloroquine loading dose of 800 mg × 2 followed by 400 mg × 2 daily for a total of 10 days, or the standard of care. Primary outcome is all cause in-hospital mortality, with secondary measures of duration of mechanical ventilation, ICU duration, 28-day mortality, viral clearance, readmittance, occurrence of coinfections, and organ dysfunction. Inclusion criteria include confirmed SARS-CoV-2 infection by PCR, 18 years of age, and admittance to the hospital ward or ICU. Importantly, exclusion criteria include prolonged QT interval (>450 ms) due to the known toxicity issues associated with hydroxychloroquine.
An observational study sponsored by the Groupe Hospitalier Pitie-Salpetriere, with collaborator CMC Ambroise Paré, was initiated to investigate adverse events in COVID-19 treatment (NCT04314817). (88) The study will consider events as classified by the international classification of disease ICD-10, and track lopinavir/ritonavir, chloroquine, azithromycin, remdesivir, and interferon-β1a, potentially expanding the scope in the future prior to the primary completion date in January 2021.
The DisCoVeRy trial is an adaptive, open-label, randomized interventional trial that includes five treatment modalities (NCT04315948): standard of care alone or standard of care plus the following: remdesivir, hydroxychloroquine, lopinavir and ritonavir, or lopinavir, ritonavir, and interferon-β1a. (89) The remdesivir dose regime is identical to existing trials, with maintenance dosing continuing up to 10 days. Lopinavir and ritonavir tablets are to be administered every 12 h for 14 days (400 mg of lopinavir /100 mg of ritonavir). In combination with the lopinavir/ritonavir schedule, interferon-β1a will be administered subcutaneously at a dose of 44 μg, for three doses in 6 days (day 1, day 3, day 6). Hydroxychloroquine will be given 400 mg, twice on the first day, followed by 400 mg once daily for 9 days. Initially, the study will include five French hospitals (Paris – Hôpital Bichat-AP-HP, Lille, Nantes, Strasbourg, Lyon) with potential expansion to other participating sites. (90) The primary outcome is the reported disease severity on a seven-point ordinal scale, assessed on the 15th day, with secondary outcomes tracking various physiological and clinical metrics.

Expanded Access

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With the overwhelming influx of compassionate use requests, on March 23, 2020, Gilead Sciences suspended compassionate use access to remdesivir for all cases save children and pregnant women, shifting their focus to support mounting clinical trials and establish a system of expanded access, wherein hospitals or physicians can request emergency use of remdesivir for multiple patients at one time. (95) In an open letter to the public on March 28, 2020, Gilead CEO reported that they had provided over 1000 doses of remdesivir through compassionate use requests. (96) To date, the FDA has granted expanded access treatment protocols for remdesivir, sponsored by the U.S. Army Medical Research and Development Command (NCT04302766) (95) and Gilead Sciences (NCT04323761). (97) The primary objective of these studies is the provision of expanded access to remdesivir for the treatment of SARS-CoV2 infections. Gilead Sciences has acknowledged that production of remdesivir is an involved process, and this is being scaled up to meet demand.

Other Nucleoside Candidates

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Remdesivir is certainly not the only nucleoside analogue that is being investigated for use against SARS-CoV-2, but it is the most clinically advanced. A recent publication by Sheahan et al. describes the ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC, EIDD-1931), that has in vitro activity against SARS-CoV-2 and in vivo against the related SARS virus. (98) Although in preclinical development, EIDD-1931 is orally bioavailable, a significant advantage compared to remdesivir, and has increased potency against viruses containing mutations in RdRp that conferred increased resistance to remdesivir, supporting the potential for a combination therapy to address the risk of SARS-CoV-2 becoming clinically drug resistant. Other clinically approved nucleoside/nucleotide analogues, such as the hepatitis C drug sofosbuvir and HIV drugs alovudine and zidovudine, have also been shown to be active against the SARS RdRp in in vitro biochemical assays and might have the potential to be repurposed against COVID-19. (99) For a general review of nucleoside and nucleotide analogues for cancer and viral diseases, including approved drugs and clinical candidates, please reference Jordheim et al. (100)

While remdesivir represents one compound whose consideration may yet play a role in mitigating the morbidity, mortality, and strain on global healthcare systems caused by COVID-19, ongoing clinical trials will provide much-needed clarity surrounding the repurposing of approved drugs and experimental agents against SARS-CoV-2.

Conclusions

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As the COVID-19 pandemic races across the globe, the scientific community, from academic and government laboratories to small biotechnology companies and multinational pharmaceutical corporations, has mobilized to develop and evaluate potential therapeutics and vaccines. (101−104) Repurposing or repositioning an effective small-molecule therapeutic promises to be the fastest therapeutic means to stem the tide of the pandemic. (105,106) Among the candidate therapies, remdesivir has demonstrated efficacy in both in vitro and in vivo models against coronaviruses. Recently, through a compassionate use indication, remdesivir has supportive evidence for yielding some clinical improvement in COVID-19 patients. (107) In addition, an interim analysis of the Adaptive COVID-19 Treatment Trial (NCT04280705) supports improvement in the primary endpoint for patients receiving remdesivir, compared to control, with a 31% faster time to recovery. (108) Based on these initial findings, the U.S. Food and Drug Administration has issued an Emergency Use Authorization for the emergency use of remdesivir for the treatment of hospitalized COVID-19 patients. With no drug having FDA approval for marketing as a treatment for SARS-CoV-2, this is the first FDA authorization of an investigational therapeutic for use in treating SARS-CoV-2. (109) While remdesivir represents one compound whose recent use authorization may, in part, mitigate the morbidity, mortality, and strain on global healthcare systems caused by COVID-19, additional ongoing clinical trials will provide much-needed clarity surrounding the repurposing of approved drugs and experimental agents against SARS-CoV-2.

Author Information

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  • Corresponding Author
  • Authors
    • Richard T. Eastman - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States;  Orcidhttp://orcid.org/0000-0003-3580-380X
    • Jacob S. Roth - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States;  Albert Einstein College of Medicine, New York, New York 10461, United States
    • Kyle R. Brimacombe - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
    • Anton Simeonov - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
    • Min Shen - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
    • Samarjit Patnaik - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by the National Center for Advancing Translational Sciences, Division of Preclinical innovation. We would like to thank Philip Sanderson for critical reading and suggestions in the editing of the manuscript.

References

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    INTRODUCTION: An epidemic of Coronavirus Disease 2019 (COVID-19) began in December 2019 in China leading to a Public Health Emergency of International Concern (PHEIC). Clinical, laboratory, and imaging features have been partially characterized in some observational studies. No systematic reviews on COVID-19 have been published to date. METHODS: We performed a systematic literature review with meta-analysis, using three databases to assess clinical, laboratory, imaging features, and outcomes of COVID-19 confirmed cases. Observational studies and also case reports, were included, and analyzed separately. We performed a random-effects model meta-analysis to calculate pooled prevalences and 95% confidence intervals (95%CI). RESULTS: 660 articles were retrieved for the time frame (1/1/2020-2/23/2020). After screening, 27 articles were selected for full-text assessment, 19 being finally included for qualitative and quantitative analyses. Additionally, 39 case report articles were included and analyzed separately. For 656 patients, fever (88.7%, 95%CI 84.5-92.9%), cough (57.6%, 95%CI 40.8-74.4%) and dyspnea (45.6%, 95%CI 10.9-80.4%) were the most prevalent manifestations. Among the patients, 20.3% (95%CI 10.0-30.6%) required intensive care unit (ICU), 32.8% presented with acute respiratory distress syndrome (ARDS) (95%CI 13.7-51.8), 6.2% (95%CI 3.1-9.3) with shock. Some 13.9% (95%CI 6.2-21.5%) of hospitalized patients had fatal outcomes (case fatality rate, CFR). CONCLUSION: COVID-19 brings a huge burden to healthcare facilities, especially in patients with comorbidities. ICU was required for approximately 20% of polymorbid, COVID-19 infected patients and hospitalization was associated with a CFR of >13%. As this virus spreads globally, countries need to urgently prepare human resources, infrastructure and facilities to treat severe COVID-19.
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    Onder, G.; Rezza, G.; Brusaferro, S. Case-Fatality Rate and Characteristics of Patients Dying in Relation to COVID-19 in Italy. JAMA 2020, DOI: 10.1001/jama.2020.4683 .
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    Dong, E.; Du, H.; Gardner, L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect. Dis. 2020, 20, 533, DOI: 10.1016/S1473-3099(20)30120-1 .
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    An interactive web-based dashboard to track COVID-19 in real time
    Dong, Ensheng; Du, Hongru; Gardner, Lauren
    Lancet Infectious Diseases (2020), 20 (5), 533-534CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)
    The authors describe the development of an online interactive dashboard, hosted by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, Baltimore, MD, USA, to visualize and track reported cases of coronavirus disease 2019 (COVID-19) in real time. The dashboard, first shared publicly on Jan 22, illustrates the location and no. of confirmed COVID-19 cases, deaths, and recoveries for all affected countries. It was developed to provide researchers, public health authorities, and the general public with a user-friendly tool to track the outbreak as it unfolds. All data collected and displayed are made freely available, initially through Google Sheets and now through a GitHub repository, along with the feature layers of the dashboard, which are now included in the Esri Living Atlas.
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    Bornstein, S. R.; Dalan, R.; Hopkins, D.; Mingrone, G.; Boehm, B. O. Endocrine and metabolic link to coronavirus infection. Nat. Rev. Endocrinol. 2020, DOI: 10.1038/s41574-020-0353-9 .
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    Wu, C. Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern Med. 2020, DOI: 10.1001/jamainternmed.2020.0994 .
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    Zhou, F. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 2020, 395, 1054– 1062,  DOI: 10.1016/S0140-6736(20)30566-3
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    Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study
    Zhou, Fei; Yu, Ting; Du, Ronghui; Fan, Guohui; Liu, Ying; Liu, Zhibo; Xiang, Jie; Wang, Yeming; Song, Bin; Gu, Xiaoying; Guan, Lulu; Wei, Yuan; Li, Hui; Wu, Xudong; Xu, Jiuyang; Tu, Shengjin; Zhang, Yi; Chen, Hua; Cao, Bin
    Lancet (2020), 395 (10229), 1054-1062CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
    Since Dec., 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiol. and clin. characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clin. course of illness, including viral shedding, have not been well described. In this retrospective, multicenter cohort study, we included all adult inpatients (≥18 yr old) with lab.-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demog., clin., treatment, and lab. data, including serial samples for viral RNA detection, were extd. from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors assocd. with in-hospital death. One hundred ninety-one patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. Ninety-one (48%) patients had a comorbidity, with hypertension being the most common (58 patients), followed by diabetes (36 patients) and coronary heart disease (15 patients). Multivariable regression showed increasing odds of in-hospital death assocd. with older age, higher Sequential Organ Failure Assessment (SOFA) score, and d-dimer >1μg/L on admission. Median duration of viral shedding was 20·0 days (IQR 17·0-24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest obsd. duration of viral shedding in survivors was 37 days. The potential risk factors of older age, high SOFA score, and d-dimer >1μg/L could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future.
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    Lighter, J. Obesity in patients younger than 60 years is a risk factor for Covid-19 hospital admission. Clin. Infect. Dis. 2020, ciaa415, DOI: 10.1093/cid/ciaa415 .
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    Baig, A. M.; Khaleeq, A.; Ali, U.; Syeda, H. Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host–Virus Interaction, and Proposed Neurotropic Mechanisms. ACS Chem. Neurosci. 2020, 11, 995– 998,  DOI: 10.1021/acschemneuro.0c00122
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    Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host-Virus Interaction, and Proposed Neurotropic Mechanisms
    Baig, Abdul Mannan; Khaleeq, Areeba; Ali, Usman; Syeda, Hira
    ACS Chemical Neuroscience (2020), 11 (7), 995-998CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    A review. The recent outbreak of coronavirus infectious disease 2019 (COVID-19) has gripped the world with apprehension and a scare of an epic proportion related to its potential to spread and infect the humans' globe wide. As we are in the midst of an ongoing near pandemic outbreak of the COVID-19, the scientists are struggling to understand how it resembles and varies with the severe acute respiratory syndrome coronavirus (SARS-CoV) at the genomic and transcriptomic level. In a short time following the outbreaks, it has been shown that like SARS-CoV, the COVID-19 exploits the angiotensin-converting enzyme 2 (ACE2) receptor to gain entry inside the cells. This finding raises the curiosity of investigating the expression of ACE2 in neurol. tissue and the possible contribution of neurol. tissues damages to the morbidity and mortality of COIVD-19. Here, we investigate the d. of the expression levels of ACE2 in the CNS, the host-virus interaction and relate it to the pathogenesis and complications seen in the recent cases of COVID-19 outbreak. Also, we debate the need for a model of staging COVID-19 based on neurol. tissue involvement.
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    Li, Y.-C.; Bai, W.-Z.; Hashikawa, T. The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients. J. Med. Virol. 2020, 1– 4,  DOI: 10.1002/jmv.25824
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    Mao, L. Neurological Manifestations of Hospitalized Patients with COVID-19 in Wuhan, China: a retrospective case series study. MedRxiv 2020, DOI: 10.1101/2020.02.22.20026500.
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    Wu, Y. Nervous system involvement after infection with COVID-19 and other coronaviruses. Brain, Behav., Immun. 2020,  DOI: 10.1016/j.bbi.2020.03.031 .
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    Magro, C. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases. Transl Res. 2020, S1931-5244(20)30070-0. DOI: 10.1016/j.trsl.2020.04.007 .
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    To, K. F. Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases. J. Pathol. 2004, 202 (2), 157– 63,  DOI: 10.1002/path.1510
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    Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases
    To, K. F.; Tong, Joanna H. M.; Chan, Paul K. S.; Au, Florence W. L.; Chim, Stephen S. C.; Chan, K. C. Allen; Cheung, Jo L. K.; Liu, Esther Y. M.; Tse, Gary M. K.; Lo, Anthony W. I.; Lo, Y. M. Dennis; Ng, H. K.
    Journal of Pathology (2004), 202 (2), 157-163CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
    Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be assocd. with a new coronavirus (SARS-CoV). The clin. and epidemiol. aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biol. behavior of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, pos. signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) anal. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathol. was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs.
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    Chu, H. Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis. 2020, ciaa410, DOI: 10.1093/cid/ciaa410 .
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    Sungnak, W. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 2020, DOI: 10.1038/s41591-020-0868-6 .
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    Letko, M.; Marzi, A.; Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol 2020, 5 (4), 562– 569,  DOI: 10.1038/s41564-020-0688-y
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    Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses
    Letko, Michael; Marzi, Andrea; Munster, Vincent
    Nature Microbiology (2020), 5 (4), 562-569CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
    Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geog. regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.
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    Hoffmann, M. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181 (2), 271– 280, e8.  DOI: 10.1016/j.cell.2020.02.052
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    SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
    Hoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, Stefan
    Cell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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    Ruch, T. R.; Machamer, C. E. The coronavirus E protein: assembly and beyond. Viruses 2012, 4 (3), 363– 82,  DOI: 10.3390/v4030363
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    The coronavirus E protein: assembly and beyond
    Ruch, Travis R.; Machamer, Carolyn E.
    Viruses (2012), 4 (), 363-382CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    A review. The coronavirus E protein is a small membrane protein that has an important role in the assembly of virions. Recent studies have indicated that the E protein has functions during infection beyond assembly, including in virus egress and in the host stress response. Addnl., the E protein has ion channel activity, interacts with host proteins, and may have multiple membrane topologies. The goal of this review is to highlight the properties and functions of the E protein, and speculate on how they may be related.
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    Morse, J. S.; Lalonde, T.; Xu, S.; Liu, W. R. Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV. ChemBioChem 2020, 21, 730– 738,  DOI: 10.1002/cbic.202000047
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    Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV
    Morse, Jared S.; Lalonde, Tyler; Xu, Shiqing; Liu, Wenshe Ray
    ChemBioChem (2020), 21 (5), 730-738CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examn. of the genome sequence shows strong homol. with its better-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA-dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homol. to SARS-CoV. Herein, we suggest four potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad-spectrum anti-coronaviral agents for future epidemics.
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    Harrison, C. Coronavirus puts drug repurposing on the fast track. Nat. Biotechnol. 2020, 38 (4), 379– 381,  DOI: 10.1038/d41587-020-00003-1
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    Coronavirus puts drug repurposing on the fast track
    Harrison Charlotte
    Nature biotechnology (2020), 38 (4), 379-381 ISSN:.
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    Koch, S.; Pong, W. First up for COVID-19: nearly 30 clinical readouts before end of April. https://www.biocentury.com/article/304658/nearly-30-trials-for-covid-19-could-start-to-yield-data-in-the-next-couple-of-months. Accessed April 1, 2020.
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    Zhai, P. The epidemiology, diagnosis and treatment of COVID-19. Int. J. Antimicrob. Agents 2020, 105955, DOI: 10.1016/j.ijantimicag.2020.105955 .
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    The epidemiology, diagnosis and treatment of COVID-19
    Zhai Pan; Ding Yanbing; Wu Xia; Long Junke; Zhong Yanjun; Li Yiming
    International journal of antimicrobial agents (2020), (), 105955 ISSN:.
    In December 2019, the outbreak of the novel coronavirus disease (COVID-19) in China spread worldwide, becoming an emergency of major international concern. SARS-CoV-2 infection causes clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus. Human-to-human transmission via droplets, contaminated hands or surfaces has been described, with incubation times of 2-14 days. Early diagnosis, quarantine, and supportive treatments are essential to cure patients. This paper reviews the literature on all available information about the epidemiology, diagnosis, isolation and treatments of COVID-19. Treatments, including antiviral agents, chloroquine and hydroxychloroquine, corticosteroids, antibodies, convalescent plasma transfusion and vaccines, are discussed in this article. In addition, registered trials investigating treatment options for COVID-19 infection are listed.
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    Riva, L. A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals. bioRxiv , 2020, 2020.04.16.044016.
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    Touret, F. In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication. bioRxiv , 2020, 2020.04.03.023846.
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    Ellinger, B. Identification of inhibitors of SARS-CoV-2 in-vitro cellular toxicity in human (Caco-2) cells using a large scale drug repurposing collection. Research Square , 2020, DOI: 10.21203/rs.3.rs-23951/v1 .
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    Zhan, L.; Li, J.; Wei, B. Autophagy therapeutics: preclinical basis and initial clinical studies. Cancer Chemother. Pharmacol. 2018, 82 (6), 923– 934,  DOI: 10.1007/s00280-018-3688-3
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    Autophagy therapeutics: preclinical basis and initial clinical studies
    Zhan, Lei; Li, Jun; Wei, Bing
    Cancer Chemotherapy and Pharmacology (2018), 82 (6), 923-934CODEN: CCPHDZ; ISSN:0344-5704. (Springer)
    A review. Autophagy captures and degrades intracellular components such as proteins and organelles to sustain metab. and homeostasis. Rapidly accumulating attention is being paid to the role of autophagy in the development of cancer, which makes autophagy attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that autophagy dysregulation is causal in many cases of cancer, with autophagy acting as tumor suppressors or tumor promoters, and autophagy inhibitor or promoter has shown promise in preclin. studies. The autophagy-targeted therapeutics using chloroquine/hydroxychloroquine have reached clin. development for treating cancer, but these drugs are actually not efficient probably because of a reduced penetration within the tumor. In this review, we first discuss the discoveries related to dual function of autophagy in cancer. Then, we provide an overview of preclin. studies and clin. trials involved in the development of autophagy therapeutics and finally discuss the future of such therapies.
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    Warren, T. Nucleotide Prodrug GS-5734 Is a Broad-Spectrum Filovirus Inhibitor That Provides Complete Therapeutic Protection Against the Development of Ebola Virus Disease (EVD) in Infected Non-human Primates. Open Forum Infectious Diseases 2015, 2, DOI: 10.1093/ofid/ofv130.02 .
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    De Clercq, E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discovery 2002, 1, 13– 25,  DOI: 10.1038/nrd703
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    Strategies in the design of antiviral drugs
    De Clercq, Erik
    Nature Reviews Drug Discovery (2002), 1 (1), 13-25CODEN: NRDDAG ISSN:. (Nature Publishing Group)
    A review. A decade ago, just five drugs were licensed for the treatment of viral infections. Since then, greater understanding of viral life cycles, prompted in particular by the need to combat human immunodeficiency virus, has resulted in the discovery and validation of several targets for therapeutic intervention. Consequently, the current antiviral repertoire now includes more than 30 drugs. But we still lack effective therapies for several viral infections, and established treatments are not always effective or well tolerated, highlighting the need for further refinement of antiviral drug design and development. Here, I describe the rationale behind current and future drug-based strategies for combating viral infections.
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    Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009, 4, 1779– 1791,  DOI: 10.1002/cmdc.200900289
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    Aryloxy Phosphoramidate Triesters: a Technology for Delivering Monophosphorylated Nucleosides and Sugars into Cells
    Mehellou, Youcef; Balzarini, Jan; McGuigan, Christopher
    ChemMedChem (2009), 4 (11), 1779-1791CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our labs., known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell-membrane penetration. Upon entering the cell, the masking groups are enzymically cleaved to release the phosphorylated biomol. The application of this technol. to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Addnl., the phosphoramidate technol. has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate-based nucleotides progressing to clin. investigations. Furthermore, the phosphoramidate technol. has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technol., mechanism of action and the application of the technol. to various monophosphorylated nucleosides and sugars will be reviewed.
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    Seley-Radtke, K. L.; Yates, M. K. The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold. Antiviral Res. 2018, 154, 66– 86,  DOI: 10.1016/j.antiviral.2018.04.004
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    The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold
    Seley-Radtke, Katherine L.; Yates, Mary K.
    Antiviral Research (2018), 154 (), 66-86CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
    A review. This is the first of two invited articles reviewing the development of nucleoside-analog antiviral drugs, written for a target audience of virologists and other non-chemists, as well as chemists who may not be familiar with the field. Rather than providing a simple chronol. account, we have examd. and attempted to explain the thought processes, advances in synthetic chem. and lessons learned from antiviral testing that led to a few mols. being moved forward to eventual approval for human therapies, while others were discarded. The present paper focuses on early, relatively simplistic changes made to the nucleoside scaffold, beginning with modifications of the nucleoside sugars of Ara-C and other arabinose-derived nucleoside analogs in the 1960's. A future paper will review more recent developments, focusing esp. on more complex modifications, particularly those involving multiple changes to the nucleoside scaffold. We hope that these articles will help virologists and others outside the field of medicinal chem. to understand why certain drugs were successfully developed, while the majority of candidate compds. encountered barriers due to low-yielding synthetic routes, toxicity or other problems that led to their abandonment.
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    Siegel, D. Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. J. Med. Chem. 2017, 60, 1648– 1661,  DOI: 10.1021/acs.jmedchem.6b01594
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    Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses
    Siegel, Dustin; Hui, Hon C.; Doerffler, Edward; Clarke, Michael O.; Chun, Kwon; Zhang, Lijun; Neville, Sean; Carra, Ernest; Lew, Willard; Ross, Bruce; Wang, Queenie; Wolfe, Lydia; Jordan, Robert; Soloveva, Veronica; Knox, John; Perry, Jason; Perron, Michel; Stray, Kirsten M.; Barauskas, Ona; Feng, Joy Y.; Xu, Yili; Lee, Gary; Rheingold, Arnold L.; Ray, Adrian S.; Bannister, Roy; Strickley, Robert; Swaminathan, Swami; Lee, William A.; Bavari, Sina; Cihlar, Tomas; Lo, Michael K.; Warren, Travis K.; Mackman, Richard L.
    Journal of Medicinal Chemistry (2017), 60 (5), 1648-1661CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clin. candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were crit. for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclin. efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016, 531, 381-385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
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    Cho, A. Synthesis and antiviral activity of a series of 1′-substituted 4-aza-7,9-dideazaadenosine C-nucleosides. Bioorg. Med. Chem. Lett. 2012, 22, 2705– 2707,  DOI: 10.1016/j.bmcl.2012.02.105
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    Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosides
    Cho, Aesop; Saunders, Oliver L.; Butler, Thomas; Zhang, Lijun; Xu, Jie; Vela, Jennifer E.; Feng, Joy Y.; Ray, Adrian S.; Kim, Choung U.
    Bioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2705-2707CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside, e.g. I, were prepd. and evaluated for the potential as antiviral agents. These compds. showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concn. of the corresponding nucleoside triphosphate.
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    Green, N.; Ott, R. D.; Isaacs, R. J.; Fang, H. Cell-based Assays to Identify Inhibitors of Viral Disease. Expert Opin. Drug Discovery 2008, 3, 671– 676,  DOI: 10.1517/17460441.3.6.671
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    Cell-based assays to identify inhibitors of viral disease
    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong
    Expert Opinion on Drug Discovery (2008), 3 (6), 671-676CODEN: EODDBX; ISSN:1746-0441. (Informa Healthcare)
    A review. Background: Antagonizing the prodn. of infectious viruses inside cells requires drugs that can cross the cell membrane without harming the host cells. Objective: It is therefore advantageous to establish the intracellular potency of antiviral drug candidates early in the drug discovery pipeline. Methods: To this end, cell-based assays are being developed and used in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. Although numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate non-specific screening hits. Results/conclusions: Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being used to reach the full potential of cell-based high-throughput screening.
  46. 46
    Agostini, M. L.; Andres, E. L.; Sims, A. C.; Graham, R. L.; Sheahan, T. P.; Lu, X.; Smith, E. C.; Case, J. B.; Feng, J. Y.; Jordan, R.; Ray, A. S.; Cihlar, T.; Siegel, D.; Mackman, R. L.; Clarke, M. O.; Baric, R. S.; Denison, M. R. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio 2018, 9 (2), e00221– 18,  DOI: 10.1128/mBio.00221-18
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    Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease
    Agostini, Maria L.; Andres, Erica L.; Sims, Amy C.; Graham, Rachel L.; Sheahan, Timothy P.; Lu, Xiaotao; Smith, Everett Clinton; Case, James Brett; Feng, Joy Y.; Jordan, Robert; Ray, Adrian S.; Cihlar, Tomas; Siege, Dustin; Mackman, Richard L.; Clarke, Michael O.; Baric, Ralph S.; Denison, Mark R.
    mBio (2018), 9 (2), e00221-18/1-e00221-18/15CODEN: MBIOCL; ISSN:2150-7511. (American Society for Microbiology)
    Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analog GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance assocd. with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concn. values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as detd. by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concns. of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.
  47. 47
    Madelain, V.; Baize, S.; Jacquot, F.; Reynard, S.; Fizet, A.; Barron, S.; Solas, C.; Lacarelle, B.; Carbonnelle, C.; Mentre, F.; Raoul, H.; de Lamballerie, X.; Guedj, J. Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies. Nat. Commun. 2018, 9, 4013,  DOI: 10.1038/s41467-018-06215-z
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    Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies
    Madelain Vincent; Mentre France; Guedj Jeremie; Baize Sylvain; Reynard Stephanie; Fizet Alexandra; Jacquot Frederic; Barron Stephane; Carbonnelle Caroline; Raoul Herve; Solas Caroline; Lacarelle Bruno; de Lamballerie Xavier
    Nature communications (2018), 9 (1), 4013 ISSN:.
    Despite several clinical trials implemented, no antiviral drug could demonstrate efficacy against Ebola virus. In non-human primates, early initiation of polymerase inhibitors favipiravir and remdesivir improves survival, but whether they could be effective in patients is unknown. Here we analyze the impact of antiviral therapy by using a mathematical model that integrates virological and immunological data of 44 cynomolgus macaques, left untreated or treated with favipiravir. We estimate that favipiravir has a ~50% efficacy in blocking viral production, which results in reducing virus growth and cytokine storm while IFNα reduces cell susceptibility to infection. Simulating the effect of delayed initiations of treatment, our model predicts survival rates of 60% for favipiravir and 100% for remdesivir when treatment is initiated within 3 and 4 days post infection, respectively. These results improve the understanding of Ebola immuno-pathogenesis and can help optimize antiviral evaluation in future outbreaks.
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    Jordan, R. Broad-spectrum Investigational Agent GS-5734 for the Treatment of Ebola, MERS Coronavirus and Other Pathogenic Viral Infections with High Outbreak Potential. Open Forum Infect Dis 2017, 4, S737,  DOI: 10.1093/ofid/ofx180.008
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    Varga, A.; Lionne, C.; Roy, B. Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase. Curr. Drug Metab. 2016, 17, 237– 252,  DOI: 10.2174/1389200217666151210141903
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    Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase
    Varga, Andrea; Lionne, Corinne; Roy, Beatrice
    Current Drug Metabolism (2016), 17 (3), 237-252CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)
    A review. Background: To date, the most effective way to treat HIV is to use a highly active antiretroviral therapy (HAART) that combines three or more different drugs. The usual regimen consists of two nucleoside reverse transcriptase inhibitors and either a protease inhibitor, a non-nucleoside reverse transcriptase inhibitor, or an integrase strand transfer inhibitor. Due to the emerging resistance against the nucleoside analogs in use, there is a continuous need for the development of such therapeutic mols. with different structural features. Objectives: In this review, we would like to summarize the state of knowledge of the antiretroviral nucleoside analogs intracellular metab. Indeed, these mols. have to be phosphorylated in the cell, a process that is often a bottleneck, to produce their pharmacol. active triphosphorylated forms. These forms can be used by the HIV reverse transcriptase. Because they lack a 3'-hydroxyl group, they block further extension of the viral DNA, and finally lead to early chain termination. Several kinases can act on the phosphorylation of these drugs; most of them have low nucleoside/nucleotide specificity. On the other hand, there are also nucleotidases in the cell, which can reverse the phosphorylation process, thus shifting the equil. from the active triphosphorylated state to the non-active (not-, mono- or di-phosphorylated) states of these analogs. Conclusion: Here, we would like to bring to the attention of the medicinal chemists that they have to take into account the limitation of the intracellular phosphorylation machinery when designing new nucleoside analog drugs.
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    Brown, A. J. Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase. Antiviral Res. 2019, 169, 104541,  DOI: 10.1016/j.antiviral.2019.104541
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    Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase
    Brown, Ariane J.; Won, John J.; Graham, Rachel L.; Dinnon, Kenneth H., III; Sims, Amy C.; Feng, Joy Y.; Cihlar, Tomas; Denison, Mark R.; Baric, Ralph S.; Sheahan, Timothy P.
    Antiviral Research (2019), 169 (), 104541CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
    The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV.
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    de Wit, E. Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 6771– 6776,  DOI: 10.1073/pnas.1922083117
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    Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection
    de Wit, Emmie; Feldmann, Friederike; Cronin, Jacqueline; Jordan, Robert; Okumura, Atsushi; Thomas, Tina; Scott, Dana; Cihlar, Tomas; Feldmann, Heinz
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (12), 6771-6776CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The continued emergence of Middle East Respiratory Syndrome (MERS) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. Remdesivir (GS-5734) effectively inhibited MERS coronavirus (MERS-CoV) replication in vitro, and showed efficacy against Severe Acute Respiratory Syndrome (SARS)-CoV in a mouse model. Here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of MERS-CoV infection, the rhesus macaque. Prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented MERS-CoV-induced clin. disease, strongly inhibited MERS-CoV replication in respiratory tissues, and prevented the formation of lung lesions. Therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clin. benefit, with a redn. in clin. signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. The data presented here support testing of the efficacy of remdesivir treatment in the context of a MERS clin. trial. It may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-nCoV.
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    Wang, M. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020, 30, 269– 271,  DOI: 10.1038/s41422-020-0282-0
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    Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro
    Wang, Manli; Cao, Ruiyuan; Zhang, Leike; Yang, Xinglou; Liu, Jia; Xu, Mingyue; Shi, Zhengli; Hu, Zhihong; Zhong, Wu; Xiao, Gengfu
    Cell Research (2020), 30 (3), 269-271CODEN: CREEB6; ISSN:1001-0602. (Nature Research)
    In Dec. 2019, a novel pneumonia caused by a previously unknown pathogen emerged in Wuhan, a city of 11 million people in central China. The initial cases were linked to exposures in a seafood market in Wuhan. The pathogen was soon identified as a novel coronavirus (2019-nCoV), which is closely related to severe acute respiratory syndrome CoV (SARS-CoV). Currently, there is no specific treatment against the new virus. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. In this study, we evaluated the antiviral efficiency of FAD-approved drugs including ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and two well-known broad-spectrum antiviral drugs remdesivir (GS-5734) and favipiravir (T-705) against a clin. isolate of 2019-nCoV in vitro. Our findings reveal that remdesivir and chloroquine are highly effective in the control of 2019-nCoV infection in vitro. Since these compds. have been used in human patients with a safety track record and shown to be effective against various ailments, we suggest that they should be assessed in human patients suffering from the novel coronavirus disease.
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    Sheahan, T. P.; Sims, A. C.; Leist, S. R.; Schafer, A.; Won, J.; Brown, A. J.; Montgomery, S. A.; Hogg, A.; Babusis, D.; Clarke, M. O.; Spahn, J. E.; Bauer, L.; Sellers, S.; Porter, D.; Feng, J. Y.; Cihlar, T.; Jordan, R.; Denison, M. R.; Baric, R. S. Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV. Nat. Commun. 2020, 11 (1), 222,  DOI: 10.1038/s41467-019-13940-6
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    Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV
    Sheahan, Timothy P.; Sims, Amy C.; Leist, Sarah R.; Schafer, Alexandra; Won, John; Brown, Ariane J.; Montgomery, Stephanie A.; Hogg, Alison; Babusis, Darius; Clarke, Michael O.; Spahn, Jamie E.; Bauer, Laura; Sellers, Scott; Porter, Danielle; Feng, Joy Y.; Cihlar, Tomas; Jordan, Robert; Denison, Mark R.; Baric, Ralph S.
    Nature Communications (2020), 11 (1), 222CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease assocd. with more than 2468 human infections and over 851 deaths in 27 countries since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathol. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathol. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.
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    Mulangu, S. A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N. Engl. J. Med. 2019, 381, 2293– 2303,  DOI: 10.1056/NEJMoa1910993
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    A randomized, controlled trial of ebola virus disease therapeutics
    Mulangu, Sabue; Dodd, Lori E.; Davey, Richard T., Jr.; Mbaya, Olivier Tshiani; Proschan, Michael; Mukadi, Daniel; Manzo, Mariano Lusakibanza; Nzolo, Didier; Oloma, Antoine Tshomba; Ibanda, Augustin; Ali, Rosine; Coulibaly, Sinare; Levine, Adam C.; Grais, Rebecca; Diaz, Janet; Lane, H. Clifford; Muyembe-Tamfum, Jean-Jacques
    New England Journal of Medicine (2019), 381 (24), 2293-2303CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
    Although several exptl. therapeutics for Ebola virus disease (EVD) have been developed, the safety and efficacy of the most promising therapies need to be assessed in the context of a randomized, controlled trial. methods We conducted a trial of four investigational therapies for EVD in the Democratic Republic of Congo, where an outbreak began in August 2018. Patients of any age who had a pos. result for Ebola virus RNA on reverse-transcriptase-polymerase-chain-reaction assay were enrolled. All patients received std. care and were randomly assigned in a 1:1:1:1 ratio to i.v. administration of the triple monoclonal antibody ZMapp (the control group), the antiviral agent remdesivir, the single monoclonal antibody MAb114, or the triple monoclonal antibody REGN-EB3. The REGN-EB3 group was added in a later version of the protocol, so data from these patients were compared with those of patients in the ZMapp group who were enrolled at or after the time the REGN-EB3 group was added (the ZMapp subgroup). The primary end point was death at 28 days. results A total of 681 patients were enrolled from Nov. 20, 2018, to August 9, 2019, at which time the data and safety monitoring board recommended that patients be assigned only to the MAb114 and REGN-EB3 groups for the remainder of the trial; the recommendation was based on the results of an interim anal. that showed superiority of these groups to ZMapp and remdesivir with respect to mortality. At 28 days, death had occurred in 61 of 174 patients (35.1%) in the MAb114 group, as compared with 84 of 169 (49.7%) in the ZMapp group (P = 0.007), and in 52 of 155 (33.5%) in the REGN-EB3 group, as compared with 79 of 154 (51.3%) in the ZMapp subgroup (P = 0.002). A shorter duration of symptoms before admission and lower baseline values for viral load and for serum creatinine and aminotransferase levels each correlated with improved survival. Four serious adverse events were judged to be potentially related to the trial drugs. conclusions Both MAb114 and REGN-EB3 were superior to ZMapp in reducing mortality from EVD. Scientifically and ethically sound clin. research can be conducted during disease outbreaks and can help inform the outbreak response.
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    Deval, J. Antimicrobial strategies: inhibition of viral polymerases by 3′-hydroxyl nucleosides. Drugs 2009, 69 (2), 151– 66,  DOI: 10.2165/00003495-200969020-00002
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    Antimicrobial strategies: inhibition of viral polymerases by 3'-hydroxyl nucleosides
    Deval, Jerome
    Drugs (2009), 69 (2), 151-166CODEN: DRUGAY; ISSN:0012-6667. (Wolters Kluwer Health)
    A review. Over the past 20 years, nucleoside analogs have constituted an arsenal of choice in the fight against HIV, hepatitis B and C viruses, and herpesviruses. Classical antiviral nucleosides such as zidovudine act as obligate chain terminators. Once incorporated as monophosphates into the viral nucleic acid, they immediately block the progression of the polymerase as a result of their lack of a reactive 3'-hydroxyl (3'-OH) group. This review explores beyond the paradigm of obligate chain termination, from a structural and a mechanistic perspective, the strategy of inhibiting viral polymerases (RNA- and DNA-dependant) with nucleoside analogs contg. a 3'-OH group. Depending on their mechanism of action, these mols. typically fall into the following three categories: (i) delayed chain terminators; (ii) pseudo-obligate chain terminators; or (iii) mutagenic nucleosides. Delayed chain terminators (i.e. penciclovir, cidofovir and entecavir) block the polymerase at an internal position within the viral nucleic acid, whereas R7128 and the 4'C substituted nucleosides do not permit subsequent incorporation events. Ribavirin, 5-hydroxydeoxycytidine and KP1461 are not chain terminators. Instead, they inhibit viral replication after mispairing with the template base, resulting in random mutations that are often lethal. Finally, brivudine, clevudine and other L-nucleosides have unique or yet to be defined mechanisms of inhibition.
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    Snell, N. J. Ribavirin--current status of a broad spectrum antiviral agent. Expert Opin. Pharmacother. 2001, 2 (8), 1317– 24,  DOI: 10.1517/14656566.2.8.1317
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    Ribavirin - current status of a broad spectrum antiviral agent
    Snell, Noel J. C.
    Expert Opinion on Pharmacotherapy (2001), 2 (8), 1317-1324CODEN: EOPHF7; ISSN:1465-6566. (Ashley Publications Ltd.)
    A review. Ribavirin is a very broad-spectrum virustatic antiviral agent, first synthesized in 1972. It is characterized by low toxicity apart from reversible anemia, usually mild. Its multiple mechanisms of action mean that viral resistance rarely develops. It can be administered orally, i.v., or via a nebulizer. It has shown varying degrees of clin. efficacy in a variety of human diseases including respiratory tract infections due to respiratory syncytial virus and influenza, measles, herpesvirus infections, HIV infection, Lassa fever, hemorrhagic fever with renal syndrome, and (in combination with IFN-α) chronic hepatitis C infection. It may well prove of value against other emerging exotic infections (e.g., West Nile virus, Nipah virus).
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    Witkowski, J. T. Design, synthesis, and broad spectrum antiviral activity of 1- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides. J. Med. Chem. 1972, 15 (11), 1150– 4,  DOI: 10.1021/jm00281a014
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    Design, synthesis, and broad spectrum antiviral activity of 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides
    Witkowski, J. T.; Robins, Roland K.; Sidwell, Robert W.; Simon, Lionel N.
    Journal of Medicinal Chemistry (1972), 15 (11), 1150-4CODEN: JMCMAR; ISSN:0022-2623.
    1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (I) [36791-04-5] show high antiviral activity in tissue culture against both DNA and RNA viruses, namely adenovirus, herpes virus types 1 and 2, vaccinia virus, myxoma virus, parainfluenza virus, rhinovirus, coxsackie virus, influenza A2 virus, and influenza B virus. I was active at concns. as low as 1-32 μg/ml, depending on the viruses utilized. I was also effective against established infection with DNA and RNA viruses in vivo in lab. animals. However, I was ineffective against various types of viral encephalitis, presumably due to the inability of I to cross the blood-brain barrier. I did not induce interferon in mice, and apparently affected macromol. processes of viral replication. I was synthesized by treatment of the trimethylsilyl deriv. of Me 1,2,4-triazole-3-carboxylate with an acyl-blocked ribofuranosyl bromide in MeCN at room temp., or by acid-catalyzed fusion of Me 1,2,4-triazole-3-carboxylate with a 1,2,3,5-tetra-O-acylribofuranose to yield a mixt. of blocked Me ester nucleosides, which were sepd. by chromatog. on silica gel; the desired isomer was reacted with NH3-MeOH to yield I.
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    Crotty, S.; Cameron, C. E.; Andino, R. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 6895– 6900,  DOI: 10.1073/pnas.111085598
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    RNA virus error catastrophe: direct molecular test by using ribavirin
    Crotty, Shane; Cameron, Craig E.; Andino, Raul
    Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (12), 6895-6900CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. The important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and it has been proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here, a direct demonstration of error catastrophe using ribavirin as the mutagen and poliovirus as a model RNA virus is described. Ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is obsd. after a single round of virus infection in ribavirin concns. sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, a graph of error catastrophe was constructed showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.
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    Molecular targets for AIDS therapy
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    Science (Washington, DC, United States) (1990), 249 (4976), 1533-44CODEN: SCIEAS; ISSN:0036-8075.
    A review with 137 refs. The development of antiretroviral therapy against acquired immunodeficiency syndrome (AIDS) has been an intense research effort since the discovery of the causative agent, human immunodeficiency virus (HIV). A large array of drugs and biol. substances can inhibit HIV replication in vitro. Nucleoside analogs-particularly those belonging to the dideoxynucleoside family-can inhibit reverse transcriptase after anabolic phosphorylation. 3'-Azido-2',3'-dideoxythymidine (AZT) was the first such drug tested in individuals with AIDS, and considerable knowledge of structure-activity relations has emerged for this class of drugs. However, virtually every step in the replication of HIV could serve as a target for a new therapeutic intervention. In the future, non-nucleoside-type drugs will likely become more important in the exptl. therapy of AIDS, and antiretroviral therapy will exert major effects against the morbidity and mortality caused by HIV.
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    Gordon, C. J.; Tchesnokov, E. P.; Woolner, E.; Perry, J. K; Feng, J. Y.; Porter, D. P; Gotte, M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J. Biol. Chem. 2020, jbc.RA120.013679, DOI: 10.1074/jbc.RA120.013679 .
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    Gao, Y. Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target. bioRxiv , 2020, DOI: 10.1101/2020.03.16.993386 .
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    Mehellou, Y.; Rattan, H. S.; Balzarini, J. The ProTide Prodrug Technology: From the Concept to the Clinic. J. Med. Chem. 2018, 61, 2211– 2226,  DOI: 10.1021/acs.jmedchem.7b00734
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    The ProTide Prodrug Technology: From the Concept to the Clinic
    Mehellou, Youcef; Rattan, Hardeep S.; Balzarini, Jan
    Journal of Medicinal Chemistry (2018), 61 (6), 2211-2226CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. The ProTide technol. is a prodrug approach developed for the efficient intracellular delivery of nucleoside analog monophosphates and monophosphonates. In this approach, the hydroxyls of the monophosphate or monophosphonate groups are masked by an arom. group and an amino acid ester moiety, which are enzymically cleaved-off inside cells to release the free nucleoside monophosphate and monophosphonate species. Structurally, this represents the current end-point of an extensive medicinal chem. endeavor that spans almost three decades. It started from the masking of nucleoside monophosphate and monophosphonate groups by simple alkyl groups and evolved into the sophisticated ProTide system as known today. This technol. has been extensively employed in drug discovery, and it has already led to the discovery of two FDA-approved (antiviral) ProTides. In this work, we will review the development of the ProTide technol., its application in drug discovery, and its role in the improvement of drug delivery and efficacy.
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    Slusarczyk, M.; Serpi, M.; Pertusati, F. Phosphoramidates and phosphonamidates (ProTides) with antiviral activity. Antivir Chem. Chemother Jan-Dec 2018, 26, 2040206618775243,  DOI: 10.1177/2040206618775243
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    Curley, D. Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity. Antiviral Res. 1990, 14 (6), 345– 56,  DOI: 10.1016/0166-3542(90)90053-A
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    Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity
    Curley, Duncan; McGuigan, Christopher; Devine, Kevin G.; O'Connor, Timothy J.; Jeffries, Donald J.; Kinchington, Derek
    Antiviral Research (1990), 14 (6), 345-56CODEN: ARSRDR; ISSN:0166-3542.
    A series of phosphoramidate derivs. of the anti-HIV drug AZT has been prepd. as membrane sol. pro-drugs of the bio-active nucleotide forms and evaluated in vitro against HIV-1. Terminal substituted alkylamines have a pronounced anti-HIV effect: this effect declines upon increasing the length of the methylene spacer. The results are consistent with a mechanism of action involving intracellular cleavage of the phosphoramidate bond, and release of the nucleotide, or a deriv. thereof. Full spectroscopic data are included on the products and their phosphorochloridate precursors.
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    McGuigan, C. Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 1993, 36 (8), 1048– 52,  DOI: 10.1021/jm00060a013
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    Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT
    McGuigan, Christopher; Pathirana, Ranjith N.; Balzarini, Jan; De Clercq, Erik
    Journal of Medicinal Chemistry (1993), 36 (8), 1048-52CODEN: JMCMAR; ISSN:0022-2623.
    Novel aryl phosphate derivs., e.g. I (R = H, CH2Ph, R1 = H; R = Me, R1 = Me, Et, Pr, F, OMe), have been prepd. by phosphorochloridate chem. These materials were designed to act as membrane-sol. prodrugs of the bioactive free nucleotides. In vitro evaluation revealed the compds. to have a pronounced, selective anti-HIV activity in CEM cells; the magnitude of the biol. effect varied considerably depending on the nature of the phosphate blocking group. Moreover, several of the compds. retain marked antiviral activity in TK- (thymidine kinase-deficient) mutant CEM cells in which AZT was virtually inactive. These data strongly support the hypothesis that the AZT phosphate derivs. exert their biol. effects via intracellular release of AZT nucleotide forms and suggest that the potential of nucleoside drugs in antiviral chemotherapy may be enhanced by suitable nucleotide delivery strategies.
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    Murakami, E. Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977. J. Biol. Chem. 2010, 285 (45), 34337– 47,  DOI: 10.1074/jbc.M110.161802
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    Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977
    Murakami, Eisuke; Tolstykh, Tatiana; Bao, Haiying; Niu, Congrong; Steuer, Holly M. Micolochick; Bao, Donghui; Chang, Wonsuk; Espiritu, Christine; Bansal, Shalini; Lam, Angela M.; Otto, Michael J.; Sofia, Michael J.; Furman, Phillip A.
    Journal of Biological Chemistry (2010), 285 (45), 34337-34347CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-β-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixt. of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot anal. showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the prodn. of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. SiRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, resp.
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    Saboulard, D. Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine. Mol. Pharmacol. 1999, 56 (4), 693– 704
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    Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine
    Saboulard, Didier; Naesens, Lieve; Cahard, Dominique; Salgado, Antonio; Pathirana, Ranjith; Velazquez, Sonsoles; Mcguigan, Christopher; De Clercq, Erik; Balzarini, Jan
    Molecular Pharmacology (1999), 56 (4), 693-704CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
    The phosphoramidate triester prodrugs of anti-human HIV 2',3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-mono-phosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metab. of triester prodrugs of d4T and AZT using pure enzymes or different biol. media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-contg. intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme prepns. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alk. phosphatase, and phosphodiesterase.
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    Tobias, S. C.; Borch, R. F. Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs. J. Med. Chem. 2001, 44 (25), 4475– 80,  DOI: 10.1021/jm010337r
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    Synthesis and Biological Studies of Novel Nucleoside Phosphoramidate Prodrugs
    Tobias, Sandra C.; Borch, Richard F.
    Journal of Medicinal Chemistry (2001), 44 (25), 4475-4480CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quant. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs, e.g. I, against L1210 mouse leukemia cells show potent inhibition of cell growth (IC50 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compd. I is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC50 value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomps. to the nucleotide in cell culture medium, suggesting that the naphthoquinone analog is not sufficiently stable to function as a nucleotide prodrug.
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    Venkatachalam, T. K. Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine. Eur. J. Med. Chem. 2005, 40 (5), 452– 66,  DOI: 10.1016/j.ejmech.2004.11.015
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    Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine
    Venkatachalam, T. K.; Samuel, P.; Qazi, S.; Uckun, F. M.
    European Journal of Medicinal Chemistry (2005), 40 (5), 452-466CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Ltd.)
    Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivs. of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivs. and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivs. The authors found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivs. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivs. In addn., the authors show that the enzymic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, the authors describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivs. of stavudine.
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    Schneider, B. Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides. J. Biol. Chem. 1998, 273 (19), 11491– 7,  DOI: 10.1074/jbc.273.19.11491
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    Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides
    Schneider, Benoit; Xu, Ying Wu; Sellam, Oliver; Sarfati, Robert; Janin, Joel; Vernon, Michel; Deville-Bonne, Dominique
    Journal of Biological Chemistry (1998), 273 (19), 11491-11497CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addn. according to a monoexponential time course. The pseudo-first order rate const. was detd. for different concns. of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast assocn. of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate consts. and dissocn. equil. consts. detd. for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equil. dissocn. consts. for ADP, GDP, ddADP, and ddGDP were also detd. by fluorescence titrn. of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to natural NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.
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    Munch-Petersen, B. Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides. J. Biol. Chem. 1991, 266 (14), 9032– 8
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    Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides
    Munch-Petersen, Birgitte; Cloos, Lisbeth; Tyrsted, Gerda; Eriksson, Staffan
    Journal of Biological Chemistry (1991), 266 (14), 9032-8CODEN: JBCHA3; ISSN:0021-9258.
    The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogs was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FDUrd), 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 μM as compared to 0.5 μM for dThd and 9 μM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; neg. cooperativity with dThd. Neg. cooperativity was also obsd. with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxythymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 μM for TK1, and 2, 10, and 78 μM for TK2, resp. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogs intended for use in cells with different expression of TK1 and TK2.
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    Sheahan, T. P. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci. Transl. Med. 2017, 9 (396), eaal3653,  DOI: 10.1126/scitranslmed.aal3653
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    Jordan, P. C. Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase. PLoS Pathog. 2018, 14, e1006889,  DOI: 10.1371/journal.ppat.1006889
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    Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase
    Jordan, Paul C.; Liu, Cheng; Raynaud, Pauline; Lo, Michael K.; Spiropoulou, Christina F.; Symons, Julian A.; Beigelman, Leo; Deval, Jerome
    PLoS Pathogens (2018), 14 (2), e1006889/1-e1006889/23CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
    Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addn. to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using neg.-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3'- terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clin. relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resoln. structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented neg.-strand RNA viruses.
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    Tchesnokov, E. P.; Feng, J. Y.; Porter, D. P.; Gotte, M. Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses 2019, 11 (4), 326,  DOI: 10.3390/v11040326
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    Mechanism of inhibition of Ebola virus RNA-dependent RNA polymerase by remdesivir
    Tchesnokov, Egor P.; Feng, Joy Y.; Porter, Danielle P.; Goette, Matthias
    Viruses (2019), 11 (4), 326CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    Remdesivir (GS-5734) is a 1'-cyano-substituted adenosine nucleotide analog prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compd. is currently under clin. development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochem. studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with ATP (ATP). Enzyme kinetics revealed that EBOV RdRp and Respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ∼4 for EBOV RdRp and ∼3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ∼500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ∼6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1'-cyano modification. Compds. with modifications at the 2'-position show different patterns of inhibition. While 2'-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochem. data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analog and its inhibitory effect warrant further investigation.
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    Warren, T. K. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016, 531, 381– 385,  DOI: 10.1038/nature17180
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    Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys
    Warren, Travis K.; Jordan, Robert; Lo, Michael K.; Ray, Adrian S.; Mackman, Richard L.; Soloveva, Veronica; Siegel, Dustin; Perron, Michel; Bannister, Roy; Hui, Hon C.; Larson, Nate; Strickley, Robert; Wells, Jay; Stuthman, Kelly S.; Van Tongeren, Sean A.; Garza, Nicole L.; Donnelly, Ginger; Shurtleff, Amy C.; Retterer, Cary J.; Gharaibeh, Dima; Zamani, Rouzbeh; Kenny, Tara; Eaton, Brett P.; Grimes, Elizabeth; Welch, Lisa S.; Gomba, Laura; Wilhelmsen, Catherine L.; Nichols, Donald K.; Nuss, Jonathan E.; Nagle, Elyse R.; Kugelman, Jeffrey R.; Palacios, Gustavo; Doerffler, Edward; Neville, Sean; Carra, Ernest; Clarke, Michael O.; Zhang, Lijun; Lew, Willard; Ross, Bruce; Wang, Queenie; Chun, Kwon; Wolfe, Lydia; Babusis, Darius; Park, Yeojin; Stray, Kirsten M.; Trancheva, Iva; Feng, Joy Y.; Barauskas, Ona; Xu, Yili; Wong, Pamela; Braun, Molly R.; Flint, Mike; McMullan, Laura K.; Chen, Shan-Shan; Fearns, Rachel; Swaminathan, Swami; Mayers, Douglas L.; Spiropoulou, Christina F.; Lee, William A.; Nichol, Stuart T.; Cihlar, Tomas; Bavari, Sina
    Nature (London, United Kingdom) (2016), 531 (7594), 381-385CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The most recent Ebola virus outbreak in West Africa, which was unprecedented in the no. of cases and fatalities, geog. distribution, and no. of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clin. efficacy. Here we report the discovery of a novel small mol. GS-5734, a monophosphoramidate prodrug of an adenosine analog, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacol. active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. I.v. administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily i.v. administration of 10 mg kg-1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clin. disease signs and pathophysiol. markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-mol. antiviral compd. against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufg., and clin. studies investigating the drug safety and pharmacokinetics are ongoing.
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    Feng, J. Y. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus. Antimicrob. Agents Chemother. 2016, 60 (2), 806– 17,  DOI: 10.1128/AAC.01922-15
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    Role of mitochondrial RNA polymerase in the toxicity of nucleotide inhibitors of hepatitis C virus
    Feng, Joy Y.; Xu, Yili; Barauskas, Ona; Perry, Jason K.; Ahmadyar, Shekeba; Stepan, George; Yu, Helen; Babusis, Darius; Park, Yeojin; McCutcheon, Krista; Perron, Michel; Schultz, Brian E.; Sakowicz, Roman; Ray, Adrian S.
    Antimicrobial Agents and Chemotherapy (2016), 60 (2), 806-817CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)
    Toxicity has emerged during the clin. development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clin. relevant HCV NI were characterized in biochem. and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-Me guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concns., followed by general cytotoxicity. In contrast, NI contg. multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an assocn. between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clin. testing.
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    Gordon, C. J. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J. Biol. Chem. 2020, 295 (15), 4773– 4779,  DOI: 10.1074/jbc.AC120.013056
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    The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus
    Gordon, Calvin J.; Tchesnokov, Egor P.; Feng, Joy Y.; Porter, Danielle P.; Gotte, Matthias
    Journal of Biological Chemistry (2020), 295 (15), 4773-4779CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
    Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at contg. the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compd. with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained with a steady-state approach suggests that it is more efficiently incorporated than ATP and 2 other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The addnl. 3 nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.
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    ClinicalTrials.gov. Severe 2019-nCoV Remdesivir RCT. https://clinicaltrials.gov/ct2/show/NCT04257656. Accessed April 10, 2020.
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    First case of 2019 novel coronavirus in the United States
    Holshue, Michelle L.; DeBolt, Chas; Lindquist, Scott; Lofy, Kathy H.; Wiesman, John; Bruce, Hollianne; Spitters, Christopher; Ericson, Keith; Wilkerson, Sara; Tural, Ahmet; Diaz, George; Cohn, Amanda; Fox, LeAnne; Patel, Anita; Gerber, Susan I.; Kim, Lindsay; Tong, Suxiang; Lu, Xiaoyan; Lindstrom, Steve; Pallansch, Mark A.; Weldon, William C.; Biggs, Holly M.; Uyeki, Timothy M.; Pillai, Satish K.
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    An outbreak of novel coronavirus (2019-nCoV) that began in Wuhan, China, has spread rapidly, with cases now confirmed in multiple countries. We report the first case of 2019-nCoV infection confirmed in the United States and describe the identification, diagnosis, clin. course, and management of the case, including the patient's initial mild symptoms at presentation with progression to pneumonia on day 9 of illness. This case highlights the importance of close coordination between clinicians and public health authorities at the local, state, and federal levels, as well as the need for rapid dissemination of clin. information related to the care of patients with this emerging infection.
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    Kujawski, S. A. First 12 patients with coronavirus disease 2019 (COVID-19) in the United States. MedRxiv , 2020, DOI: 10.1101/2020.03.09.20032896 .
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  • Abstract

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    Figure 1

    Figure 1. Life cycle of SARS-CoV-2 in host cells. SARS-CoV-2 primarily infects the respiratory tract (nasal epithelial cells, pneumocytes, and alveolar macrophages) and the gastrointestinal tract (enterocytes). The virus enters though direct interaction between the viral S protein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). Following entry, the viral genome is released and translated into the viral replicase polyproteins PP1a and PP1ab, which are cleaved into functional proteins by viral proteases. (2) Viral genome replication is mediated by the viral replication complex, including the RNA-dependent RNA polymerase (RdRp). Viral nucleocapsids are assembled from the packaged viral genomes and translated viral structural proteins and released through exocytosis. Potential targets and postulated mechanism of action for antiviral interventions are shown: blocking virus/host cell interaction through the use of antibodies/nanobodies (and convalescent plasma therapy) or recombinant ACE2 protein; use of hydroxychloroquine (based on in vitro data) to inhibit endosome maturation; use of protease inhibitors to inhibit viral/endosome membrane fusion or viral polypeptide maturation; nucleoside/nucleotide analogues to inhibit viral genome replication.

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    Figure 2

    Figure 2. SARS-CoV-2 genome and RNA-dependent RNA polymerase structure. (a) Representation of the SARS-CoV-2 RNA genome. As SARS-CoV-2 is a positive-sense RNA virus, the genome serves as a direct template for protein translation. Replication of the viral genome requires a functional viral replication complex, including an RNA-dependent RNA polymerase (RdRp). (b) Domain organization of the SARS-CoV-2 RdRp (encoded by nsp12) domains bound to cofactors nsp7 and dimers of nsp8, that serve as essential cofactors that increase polymerase activity. The rendering was based on the cryo-EM structure at a resolution of 2.9-Å, published by Gao et al, 2020 (PDB: 6M71). The nsp12 RdRp domain is shown in green, nsp7 in purple, nsp8 in cyan, nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain in yellow, interface in blue, and a newly identified β-hairpin domain is shown in red. (61) Highlighted is RdRp residue S861, which is predicted to sterically interact with the 1′CN substituent of remdesivir inducing delayed chain termination. (60)

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    Figure 3

    Figure 3. Remdesivir and its intracellular conversion. (a) Chemical structures of GS-441524 that compose the nucleoside analogue core (blue) of remdesivir (GS-5734). (b) Intracellular processing of the prodrug remdesivir (GS-5734), the aryloxy phosphoramidate (purple) prodrug of GS-441524 monophosphate. Upon diffusion of remdesivir into the cell, it is metabolized into the nucleoside monophosphate form via a sequence of steps that are presumably initiated by esterase-mediated hydrolysis of the amino acid ester that liberates a carboxylate that cyclizes on to the phosphorus displacing the phenoxide. The unstable cyclic anhydride is hydrolyzed by water to the alanine metabolite GS-704277 whose P–N bond is hydrolyzed by phosphoramidase-type enzymes to liberate the nucleoside monophosphate or nucleotide analog. The artificial nucleoside monophosphate is routed to further phosphorylation events (hijacking the endogenous phosphorylation pathway) yielding the active nucleoside triphosphate analogue form that is utilized by the viral RNA-dependent RNA polymerase (RdRp). Utilization of the GS-441524 nucleoside triphosphate analogue by RdRp inhibits viral replication through inducing delayed chain termination.

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    Figure 4

    Figure 4. Remdesivir global clinical trials. Shown are the locations of the clinical study sites for the ongoing clinical studies of remdesivir for SARS-CoV-2/COVID-19. Number of sites participating for each respective study, if no specific information was given, shown are the countries participating (e.g., ISRCTN83971151). Listed are the number of sites participating for each respective study, if no detailed information was provided; shown are the number of countries participating. NCT04302766 is an expanded access trial with no specific sites listed in the registration. Figure created with R, (91) utilizing the packages rnaturalearth, (92) sf, (93) and ggplot2. (94)

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      1
      The proximal origin of SARS-CoV-2
      Andersen, Kristian G.; Rambaut, Andrew; Lipkin, W. Ian; Holmes, Edward C.; Garry, Robert F.
      Nature Medicine (New York, NY, United States) (2020), 26 (4), 450-452CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)
      There is no expanded citation for this reference.
    2. 2
      Fields, B. N.; Knipe, D. M.; Howley, P. M. Coronavirus. In Fields Virology, 6th ed.; Wolters Kluwer Health/Lippincott Williams and Wilkins, 2013; pp 825– 858.
      There is no corresponding record for this reference.
    3. 3
      Geller, C.; Varbanov, M.; Duval, R. E. Human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies. Viruses 2012, 4, 3044– 3068,  DOI: 10.3390/v4113044
      3
      Human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies
      Geller, Chloe; Varbanov, Mihayl; Duval, Raphael E.
      Viruses (2012), 4 (), 3044-3068CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      A review. The Coronaviridae family, an enveloped RNA virus family and, more particularly, human coronaviruses (HCoV), were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, esp. in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002-2003, the outbreak of severe acute respiratory syndrome (SARS), due to a newly discovered coronavirus, the SARS-assocd. coronavirus (SARS-CoV); led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1), NL63, HKU1 or SARS-CoV) to survive in different environmental conditions (e.g. temp. and humidity), on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biol. fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections), the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance) make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to survive in the environment and the efficacy of well-known antiseptic-disinfectants against them, with particular focus on the development of new methodologies to evaluate the activity of new antiseptic-disinfectants on viruses.
    4. 4
      Song, Z. From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses 2019, 11 (1), 59
      4
      From SARS to MERS, thrusting coronaviruses into the spotlight
      Song, Zhiqi; Xu, Yanfeng; Bao, Linlin; Zhang, Ling; Yu, Pin; Qu, Yajin; Zhu, Hua; Zhao, Wenjie; Han, Yunlin; Qin, Chuan
      Viruses (2019), 11 (1), 59CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      A review. Coronaviruses (CoVs) have formerly been regarded as relatively harmless respiratory pathogens to humans. However, two outbreaks of severe respiratory tract infection, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), as a result of zoonotic CoVs crossing the species barrier, caused high pathogenicity and mortality rates in human populations. This brought CoVs global attention and highlighted the importance of controlling infectious pathogens at international borders. In this review, we focus on our current understanding of the epidemiol., pathogenesis, prevention, and treatment of SARS-CoV and MERS-CoV, as well as provides details on the pivotal structure and function of the spike proteins (S proteins) on the surface of each of these viruses. For building up more suitable animal models, we compare the current animal models recapitulating pathogenesis and summarize the potential role of host receptors contributing to diverse host affinity in various species. We outline the research still needed to fully elucidate the pathogenic mechanism of these viruses, to construct reproducible animal models, and ultimately develop countermeasures to conquer not only SARS-CoV and MERS-CoV, but also these emerging coronaviral diseases.
    5. 5
      Menachery, V. D.; Graham, R. L.; Baric, R. S. Jumping species-a mechanism for coronavirus persistence and survival. Curr. Opin. Virol. 2017, 23, 1– 7,  DOI: 10.1016/j.coviro.2017.01.002
      5
      Jumping species-a mechanism for coronavirus persistence and survival
      Menachery Vineet D; Graham Rachel L; Baric Ralph S
      Current opinion in virology (2017), 23 (), 1-7 ISSN:.
      Zoonotic transmission of novel viruses represents a significant threat to global public health and is fueled by globalization, the loss of natural habitats, and exposure to new hosts. For coronaviruses (CoVs), broad diversity exists within bat populations and uniquely positions them to seed future emergence events. In this review, we explore the host and viral dynamics that shape these CoV populations for survival, amplification, and possible emergence in novel hosts.
    6. 6
      Omrani, A. S.; Al-Tawfiq, J. A.; Memish, Z. A. Middle East respiratory syndrome coronavirus (MERS-CoV): animal to human interaction. Pathog. Global Health 2015, 109 (8), 354– 62,  DOI: 10.1080/20477724.2015.1122852
      6
      Middle East respiratory syndrome coronavirus (MERS-CoV): animal to human interaction
      Omrani Ali S; Al-Tawfiq Jaffar A; Memish Ziad A
      Pathogens and global health (2015), 109 (8), 354-62 ISSN:.
      The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel enzootic betacoronavirus that was first described in September 2012. The clinical spectrum of MERS-CoV infection in humans ranges from an asymptomatic or mild respiratory illness to severe pneumonia and multi-organ failure; overall mortality is around 35.7%. Bats harbour several betacoronaviruses that are closely related to MERS-CoV but more research is needed to establish the relationship between bats and MERS-CoV. The seroprevalence of MERS-CoV antibodies is very high in dromedary camels in Eastern Africa and the Arabian Peninsula. MERS-CoV RNA and viable virus have been isolated from dromedary camels, including some with respiratory symptoms. Furthermore, near-identical strains of MERS-CoV have been isolated from epidemiologically linked humans and camels, confirming inter-transmission, most probably from camels to humans. Though inter-human spread within health care settings is responsible for the majority of reported MERS-CoV cases, the virus is incapable at present of causing sustained human-to-human transmission. Clusters can be readily controlled with implementation of appropriate infection control procedures. Phylogenetic and sequencing data strongly suggest that MERS-CoV originated from bat ancestors after undergoing a recombination event in the spike protein, possibly in dromedary camels in Africa, before its exportation to the Arabian Peninsula along the camel trading routes. MERS-CoV serosurveys are needed to investigate possible unrecognized human infections in Africa. Amongst the important measures to control MERS-CoV spread are strict regulation of camel movement, regular herd screening and isolation of infected camels, use of personal protective equipment by camel handlers and enforcing rules banning all consumption of unpasteurized camel milk and urine.
    7. 7
      Zhou, P. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579 (7798), 270– 273,  DOI: 10.1038/s41586-020-2012-7
      7
      A pneumonia outbreak associated with a new coronavirus of probable bat origin
      Zhou, Peng; Yang, Xing-Lou; Wang, Xian-Guang; Hu, Ben; Zhang, Lei; Zhang, Wei; Si, Hao-Rui; Zhu, Yan; Li, Bei; Huang, Chao-Lin; Chen, Hui-Dong; Chen, Jing; Luo, Yun; Guo, Hua; Jiang, Ren-Di; Liu, Mei-Qin; Chen, Ying; Shen, Xu-Rui; Wang, Xi; Zheng, Xiao-Shuang; Zhao, Kai; Chen, Quan-Jiao; Deng, Fei; Liu, Lin-Lin; Yan, Bing; Zhan, Fa-Xian; Wang, Yan-Yi; Xiao, Geng-Fu; Shi, Zheng-Li
      Nature (London, United Kingdom) (2020), 579 (7798), 270-273CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Abstr.: Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large no. of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 Dec. 2019, had caused 2,794 lab.-confirmed infections including 80 deaths by 26 Jan. 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence anal. of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addn., 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.
    8. 8
      WHO Director-General’s opening remarks at the media briefing on COVID-19. In WHO Newsletter; https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19--13-april-2020. Accessed April 13, 2020.
      There is no corresponding record for this reference.
    9. 9
      Chen, N. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet 2020, 395, 507– 513,  DOI: 10.1016/S0140-6736(20)30211-7
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      Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study
      Chen, Nanshan; Zhou, Min; Dong, Xuan; Qu, Jieming; Gong, Fengyun; Han, Yang; Qiu, Yang; Wang, Jingli; Liu, Ying; Wei, Yuan; Xia, Jia-an; Yu, Ting; Zhang, Xinxin; Zhang, Li
      Lancet (2020), 395 (10223), 507-513CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      In Dec., 2019, a pneumonia assocd. with the 2019 novel coronavirus (2019-nCoV) emerged in Wuhan, China. We aimed to further clarify the epidemiol. and clin. characteristics of 2019-nCoV pneumonia. In this retrospective, single-center study, we included all confirmed cases of 2019-nCoV in Wuhan Jinyintan Hospital from Jan 1 to Jan 20, 2020. Cases were confirmed by real-time RT-PCR and were analyzed for epidemiol., demog., clin., and radiol. features and lab. data. Outcomes were followed up until Jan 25, 2020. Of the 99 patients with 2019-nCoV pneumonia, 49 (49%) had a history of exposure to the Huanan seafood market. The av. age of the patients was 55·5 years (SD 13·1), including 67 men and 32 women. 2019-nCoV was detected in all patients by real-time RT-PCR. 50 (51%) Patients had chronic diseases. Patients had clin. manifestations of fever (82 [83%] patients), cough (81 [82%] patients), shortness of breath (31 [31%] patients), muscle ache (11 [11%] patients), confusion (nine [9%] patients), headache (eight [8%] patients), sore throat (five [5%] patients), rhinorrhoea (four [4%] patients), chest pain (two [2%] patients), diarrhoea (two [2%] patients), and nausea and vomiting (one [1%] patient). According to imaging examn., 74 (75%) patients showed bilateral pneumonia, 14 (14%) patients showed multiple mottling and ground-glass opacity, and one (1%) patient had pneumothorax. 17 (17%) Patients developed acute respiratory distress syndrome and, among them, 11 (11%) patients worsened in a short period of time and died of multiple organ failure. The 2019-nCoV infection was of clustering onset, is more likely to affect older males with comorbidities, and can result in severe and even fatal respiratory diseases such as acute respiratory distress syndrome. In general, characteristics of patients who died were in line with the MuLBSTA score, an early warning model for predicting mortality in viral pneumonia. Further investigation is needed to explore the applicability of the MuLBSTA score in predicting the risk of mortality in 2019-nCoV infection. National Key R&D Program of China.
    10. 10
      Huang, C. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497– 506,  DOI: 10.1016/S0140-6736(20)30183-5
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      Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China
      Huang, Chaolin; Wang, Yeming; Li, Xingwang; Ren, Lili; Zhao, Jianping; Hu, Yi; Zhang, Li; Fan, Guohui; Xu, Jiuyang; Gu, Xiaoying; Cheng, Zhenshun; Yu, Ting; Xia, Jiaan; Wei, Yuan; Wu, Wenjuan; Xie, Xuelei; Yin, Wen; Li, Hui; Liu, Min; Xiao, Yan; Gao, Hong; Guo, Li; Xie, Jungang; Wang, Guangfa; Jiang, Rongmeng; Gao, Zhancheng; Jin, Qi; Wang, Jianwei; Cao, Bin
      Lancet (2020), 395 (10223), 497-506CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiol., clin., lab., and radiol. characteristics and treatment and clin. outcomes of these patients. All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analyzed data on patients with lab.-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiol. and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. By Jan 2, 2020, 41 admitted hospital patients had been identified as having lab.-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0-58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum prodn. (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0-13·0]). 26 (63%) Of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was assocd. with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiol., duration of human transmission, and clin. spectrum of disease need fulfilment by future studies. Ministry of Science and Technol., Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technol. Commission.
    11. 11
      Li, Q. Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N. Engl. J. Med. 2020, 382, 1199– 1207,  DOI: 10.1056/NEJMoa2001316
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      Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia
      Li, Qun; Guan, Xuhua; Wu, Peng; Wang, Xiaoye; Zhou, Lei; Tong, Yeqing; Ren, Ruiqi; Leung, Kathy S. M.; Lau, Eric H. Y.; Wong, Jessica Y.; Xing, Xuesen; Xiang, Nijuan; Wu, Yang; Li, Chao; Chen, Qi; Li, Dan; Liu, Tian; Zhao, Jing; Liu, Man; Tu, Wenxiao; Chen, Chuding; Jin, Lianmei; Yang, Rui; Wang, Qi; Zhou, Suhua; Wang, Rui; Liu, Hui; Luo, Yinbo; Liu, Yuan; Shao, Ge; Li, Huan; Tao, Zhongfa; Yang, Yang; Deng, Zhiqiang; Liu, Boxi; Ma, Zhitao; Zhang, Yanping; Shi, Guoqing; Lam, Tommy T. Y.; Wu, Joseph T.; Gao, George F.; Cowling, Benjamin J.; Yang, Bo; Leung, Gabriel M.; Feng, Zijian
      New England Journal of Medicine (2020), 382 (13), 1199-1207CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      The initial cases of novel coronavirus (2019-nCoV)-infected pneumonia (NCIP) occurred in Wuhan, Hubei Province, China, in Dec. 2019 and Jan. 2020. We analyzed data on the 1st 425 confirmed cases in Wuhan to det. the epidemiol. characteristics of NCIP. We collected information on demog. characteristics, exposure history, and illness timelines of lab.-confirmed cases of NCIP that had been reported by Jan. 22, 2020. We described characteristics of the cases and estd. the key epidemiol. time-delay distributions. In the early period of exponential growth, we estd. the epidemic doubling time and the basic reproductive no. Among the 1st 425 patients with confirmed NCIP, the median age was 59 yr and 56% were male. The majority of cases (55%) with onset before Jan. 1, 2020, were linked to the Huanan Seafood Wholesale Market, as compared with 8.6% of the subsequent cases. The mean incubation period was 5.2 days, with the 95th percentile of the distribution at 12.5 days. In its early stages, the epidemic doubled in size every 7.4 days. With a mean serial interval of 7.5 days, the basic reproductive no. was estd. to be 2.2. On the basis of this information, there is evidence that human-to-human transmission has occurred among close contacts since the middle of Dec. 2019. Considerable efforts to reduce transmission will be required to control outbreaks if similar dynamics apply elsewhere. Measures to prevent or reduce transmission should be implemented in populations at risk.
    12. 12
      Wu, Z.; McGoogan, J. M. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention. JAMA 2020, 323 (13), 1239– 1242,  DOI: 10.1001/jama.2020.2648
      12
      Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China summary of a report of 72,314 cases from the Chinese center for disease control and prevention
      Wu, Zunyou; McGoogan, Jennifer M.
      JAMA, the Journal of the American Medical Association (2020), 323 (13), 1239-1242CODEN: JAMAAP; ISSN:1538-3598. (American Medical Association)
      The Chinese Center for Disease Control and Prevention recently published the largest case series to date of coronavirus disease 2019 (COVID-19) in mainland China (72,314 cases, updated through Feb. 11, 2020). This Viewpoint summarizes key findings from this report and discusses emerging understanding of and lessons from the COVID-19 epidemic.
    13. 13
      Rodriguez-Morales, A. J. Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis. Travel Med. Infect Dis 2020, 101623,  DOI: 10.1016/j.tmaid.2020.101623
      13
      Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis
      Rodriguez-Morales Alfonso J; Cardona-Ospina Jaime A; Gutierrez-Ocampo Estefania; Villamizar-Pena Rhuvi; Holguin-Rivera Yeimer; Lagos-Grisales Guillermo J; Ramirez-Vallejo Eduardo; Escalera-Antezana Juan Pablo; Alvarado-Arnez Lucia Elena; Bonilla-Aldana D Katterine; Franco-Paredes Carlos; Henao-Martinez Andres F; Paniz-Mondolfi Alberto; Suarez Jose A; Zambrano Lysien I; Villamil-Gomez Wilmer E; Balbin-Ramon Graciela J; Rabaan Ali A; Harapan Harapan; Dhama Kuldeep; Nishiura Hiroshi; Kataoka Hiromitsu; Ahmad Tauseef; Sah Ranjit
      Travel medicine and infectious disease (2020), 34 (), 101623 ISSN:.
      INTRODUCTION: An epidemic of Coronavirus Disease 2019 (COVID-19) began in December 2019 in China leading to a Public Health Emergency of International Concern (PHEIC). Clinical, laboratory, and imaging features have been partially characterized in some observational studies. No systematic reviews on COVID-19 have been published to date. METHODS: We performed a systematic literature review with meta-analysis, using three databases to assess clinical, laboratory, imaging features, and outcomes of COVID-19 confirmed cases. Observational studies and also case reports, were included, and analyzed separately. We performed a random-effects model meta-analysis to calculate pooled prevalences and 95% confidence intervals (95%CI). RESULTS: 660 articles were retrieved for the time frame (1/1/2020-2/23/2020). After screening, 27 articles were selected for full-text assessment, 19 being finally included for qualitative and quantitative analyses. Additionally, 39 case report articles were included and analyzed separately. For 656 patients, fever (88.7%, 95%CI 84.5-92.9%), cough (57.6%, 95%CI 40.8-74.4%) and dyspnea (45.6%, 95%CI 10.9-80.4%) were the most prevalent manifestations. Among the patients, 20.3% (95%CI 10.0-30.6%) required intensive care unit (ICU), 32.8% presented with acute respiratory distress syndrome (ARDS) (95%CI 13.7-51.8), 6.2% (95%CI 3.1-9.3) with shock. Some 13.9% (95%CI 6.2-21.5%) of hospitalized patients had fatal outcomes (case fatality rate, CFR). CONCLUSION: COVID-19 brings a huge burden to healthcare facilities, especially in patients with comorbidities. ICU was required for approximately 20% of polymorbid, COVID-19 infected patients and hospitalization was associated with a CFR of >13%. As this virus spreads globally, countries need to urgently prepare human resources, infrastructure and facilities to treat severe COVID-19.
    14. 14
      Onder, G.; Rezza, G.; Brusaferro, S. Case-Fatality Rate and Characteristics of Patients Dying in Relation to COVID-19 in Italy. JAMA 2020, DOI: 10.1001/jama.2020.4683 .
      There is no corresponding record for this reference.
    15. 15
      Dong, E.; Du, H.; Gardner, L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect. Dis. 2020, 20, 533, DOI: 10.1016/S1473-3099(20)30120-1 .
      15
      An interactive web-based dashboard to track COVID-19 in real time
      Dong, Ensheng; Du, Hongru; Gardner, Lauren
      Lancet Infectious Diseases (2020), 20 (5), 533-534CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)
      The authors describe the development of an online interactive dashboard, hosted by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, Baltimore, MD, USA, to visualize and track reported cases of coronavirus disease 2019 (COVID-19) in real time. The dashboard, first shared publicly on Jan 22, illustrates the location and no. of confirmed COVID-19 cases, deaths, and recoveries for all affected countries. It was developed to provide researchers, public health authorities, and the general public with a user-friendly tool to track the outbreak as it unfolds. All data collected and displayed are made freely available, initially through Google Sheets and now through a GitHub repository, along with the feature layers of the dashboard, which are now included in the Esri Living Atlas.
    16. 16
      Bornstein, S. R.; Dalan, R.; Hopkins, D.; Mingrone, G.; Boehm, B. O. Endocrine and metabolic link to coronavirus infection. Nat. Rev. Endocrinol. 2020, DOI: 10.1038/s41574-020-0353-9 .
      There is no corresponding record for this reference.
    17. 17
      Wu, C. Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern Med. 2020, DOI: 10.1001/jamainternmed.2020.0994 .
      There is no corresponding record for this reference.
    18. 18
      Zhou, F. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 2020, 395, 1054– 1062,  DOI: 10.1016/S0140-6736(20)30566-3
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      Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study
      Zhou, Fei; Yu, Ting; Du, Ronghui; Fan, Guohui; Liu, Ying; Liu, Zhibo; Xiang, Jie; Wang, Yeming; Song, Bin; Gu, Xiaoying; Guan, Lulu; Wei, Yuan; Li, Hui; Wu, Xudong; Xu, Jiuyang; Tu, Shengjin; Zhang, Yi; Chen, Hua; Cao, Bin
      Lancet (2020), 395 (10229), 1054-1062CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      Since Dec., 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiol. and clin. characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clin. course of illness, including viral shedding, have not been well described. In this retrospective, multicenter cohort study, we included all adult inpatients (≥18 yr old) with lab.-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demog., clin., treatment, and lab. data, including serial samples for viral RNA detection, were extd. from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors assocd. with in-hospital death. One hundred ninety-one patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. Ninety-one (48%) patients had a comorbidity, with hypertension being the most common (58 patients), followed by diabetes (36 patients) and coronary heart disease (15 patients). Multivariable regression showed increasing odds of in-hospital death assocd. with older age, higher Sequential Organ Failure Assessment (SOFA) score, and d-dimer >1μg/L on admission. Median duration of viral shedding was 20·0 days (IQR 17·0-24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest obsd. duration of viral shedding in survivors was 37 days. The potential risk factors of older age, high SOFA score, and d-dimer >1μg/L could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future.
    19. 19
      Lighter, J. Obesity in patients younger than 60 years is a risk factor for Covid-19 hospital admission. Clin. Infect. Dis. 2020, ciaa415, DOI: 10.1093/cid/ciaa415 .
      There is no corresponding record for this reference.
    20. 20
      Baig, A. M.; Khaleeq, A.; Ali, U.; Syeda, H. Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host–Virus Interaction, and Proposed Neurotropic Mechanisms. ACS Chem. Neurosci. 2020, 11, 995– 998,  DOI: 10.1021/acschemneuro.0c00122
      20
      Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host-Virus Interaction, and Proposed Neurotropic Mechanisms
      Baig, Abdul Mannan; Khaleeq, Areeba; Ali, Usman; Syeda, Hira
      ACS Chemical Neuroscience (2020), 11 (7), 995-998CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      A review. The recent outbreak of coronavirus infectious disease 2019 (COVID-19) has gripped the world with apprehension and a scare of an epic proportion related to its potential to spread and infect the humans' globe wide. As we are in the midst of an ongoing near pandemic outbreak of the COVID-19, the scientists are struggling to understand how it resembles and varies with the severe acute respiratory syndrome coronavirus (SARS-CoV) at the genomic and transcriptomic level. In a short time following the outbreaks, it has been shown that like SARS-CoV, the COVID-19 exploits the angiotensin-converting enzyme 2 (ACE2) receptor to gain entry inside the cells. This finding raises the curiosity of investigating the expression of ACE2 in neurol. tissue and the possible contribution of neurol. tissues damages to the morbidity and mortality of COIVD-19. Here, we investigate the d. of the expression levels of ACE2 in the CNS, the host-virus interaction and relate it to the pathogenesis and complications seen in the recent cases of COVID-19 outbreak. Also, we debate the need for a model of staging COVID-19 based on neurol. tissue involvement.
    21. 21
      Li, Y.-C.; Bai, W.-Z.; Hashikawa, T. The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients. J. Med. Virol. 2020, 1– 4,  DOI: 10.1002/jmv.25824
      There is no corresponding record for this reference.
    22. 22
      Mao, L. Neurological Manifestations of Hospitalized Patients with COVID-19 in Wuhan, China: a retrospective case series study. MedRxiv 2020, DOI: 10.1101/2020.02.22.20026500.
      There is no corresponding record for this reference.
    23. 23
      Wu, Y. Nervous system involvement after infection with COVID-19 and other coronaviruses. Brain, Behav., Immun. 2020,  DOI: 10.1016/j.bbi.2020.03.031 .
      There is no corresponding record for this reference.
    24. 24
      Magro, C. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases. Transl Res. 2020, S1931-5244(20)30070-0. DOI: 10.1016/j.trsl.2020.04.007 .
      There is no corresponding record for this reference.
    25. 25
      To, K. F. Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases. J. Pathol. 2004, 202 (2), 157– 63,  DOI: 10.1002/path.1510
      25
      Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases
      To, K. F.; Tong, Joanna H. M.; Chan, Paul K. S.; Au, Florence W. L.; Chim, Stephen S. C.; Chan, K. C. Allen; Cheung, Jo L. K.; Liu, Esther Y. M.; Tse, Gary M. K.; Lo, Anthony W. I.; Lo, Y. M. Dennis; Ng, H. K.
      Journal of Pathology (2004), 202 (2), 157-163CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
      Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be assocd. with a new coronavirus (SARS-CoV). The clin. and epidemiol. aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biol. behavior of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, pos. signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) anal. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathol. was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs.
    26. 26
      Chu, H. Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis. 2020, ciaa410, DOI: 10.1093/cid/ciaa410 .
      There is no corresponding record for this reference.
    27. 27
      Sungnak, W. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 2020, DOI: 10.1038/s41591-020-0868-6 .
      There is no corresponding record for this reference.
    28. 28
      Letko, M.; Marzi, A.; Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol 2020, 5 (4), 562– 569,  DOI: 10.1038/s41564-020-0688-y
      28
      Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses
      Letko, Michael; Marzi, Andrea; Munster, Vincent
      Nature Microbiology (2020), 5 (4), 562-569CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
      Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geog. regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.
    29. 29
      Hoffmann, M. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181 (2), 271– 280, e8.  DOI: 10.1016/j.cell.2020.02.052
      29
      SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
      Hoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, Stefan
      Cell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
    30. 30
      Ruch, T. R.; Machamer, C. E. The coronavirus E protein: assembly and beyond. Viruses 2012, 4 (3), 363– 82,  DOI: 10.3390/v4030363
      30
      The coronavirus E protein: assembly and beyond
      Ruch, Travis R.; Machamer, Carolyn E.
      Viruses (2012), 4 (), 363-382CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      A review. The coronavirus E protein is a small membrane protein that has an important role in the assembly of virions. Recent studies have indicated that the E protein has functions during infection beyond assembly, including in virus egress and in the host stress response. Addnl., the E protein has ion channel activity, interacts with host proteins, and may have multiple membrane topologies. The goal of this review is to highlight the properties and functions of the E protein, and speculate on how they may be related.
    31. 31
      Morse, J. S.; Lalonde, T.; Xu, S.; Liu, W. R. Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV. ChemBioChem 2020, 21, 730– 738,  DOI: 10.1002/cbic.202000047
      31
      Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV
      Morse, Jared S.; Lalonde, Tyler; Xu, Shiqing; Liu, Wenshe Ray
      ChemBioChem (2020), 21 (5), 730-738CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examn. of the genome sequence shows strong homol. with its better-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA-dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homol. to SARS-CoV. Herein, we suggest four potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad-spectrum anti-coronaviral agents for future epidemics.
    32. 32
      Harrison, C. Coronavirus puts drug repurposing on the fast track. Nat. Biotechnol. 2020, 38 (4), 379– 381,  DOI: 10.1038/d41587-020-00003-1
      32
      Coronavirus puts drug repurposing on the fast track
      Harrison Charlotte
      Nature biotechnology (2020), 38 (4), 379-381 ISSN:.
      There is no expanded citation for this reference.
    33. 33
      Koch, S.; Pong, W. First up for COVID-19: nearly 30 clinical readouts before end of April. https://www.biocentury.com/article/304658/nearly-30-trials-for-covid-19-could-start-to-yield-data-in-the-next-couple-of-months. Accessed April 1, 2020.
      There is no corresponding record for this reference.
    34. 34
      Zhai, P. The epidemiology, diagnosis and treatment of COVID-19. Int. J. Antimicrob. Agents 2020, 105955, DOI: 10.1016/j.ijantimicag.2020.105955 .
      34
      The epidemiology, diagnosis and treatment of COVID-19
      Zhai Pan; Ding Yanbing; Wu Xia; Long Junke; Zhong Yanjun; Li Yiming
      International journal of antimicrobial agents (2020), (), 105955 ISSN:.
      In December 2019, the outbreak of the novel coronavirus disease (COVID-19) in China spread worldwide, becoming an emergency of major international concern. SARS-CoV-2 infection causes clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus. Human-to-human transmission via droplets, contaminated hands or surfaces has been described, with incubation times of 2-14 days. Early diagnosis, quarantine, and supportive treatments are essential to cure patients. This paper reviews the literature on all available information about the epidemiology, diagnosis, isolation and treatments of COVID-19. Treatments, including antiviral agents, chloroquine and hydroxychloroquine, corticosteroids, antibodies, convalescent plasma transfusion and vaccines, are discussed in this article. In addition, registered trials investigating treatment options for COVID-19 infection are listed.
    35. 35
      Riva, L. A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals. bioRxiv , 2020, 2020.04.16.044016.
      There is no corresponding record for this reference.
    36. 36
      Touret, F. In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication. bioRxiv , 2020, 2020.04.03.023846.
      There is no corresponding record for this reference.
    37. 37
      Ellinger, B. Identification of inhibitors of SARS-CoV-2 in-vitro cellular toxicity in human (Caco-2) cells using a large scale drug repurposing collection. Research Square , 2020, DOI: 10.21203/rs.3.rs-23951/v1 .
      There is no corresponding record for this reference.
    38. 38
      Zhan, L.; Li, J.; Wei, B. Autophagy therapeutics: preclinical basis and initial clinical studies. Cancer Chemother. Pharmacol. 2018, 82 (6), 923– 934,  DOI: 10.1007/s00280-018-3688-3
      38
      Autophagy therapeutics: preclinical basis and initial clinical studies
      Zhan, Lei; Li, Jun; Wei, Bing
      Cancer Chemotherapy and Pharmacology (2018), 82 (6), 923-934CODEN: CCPHDZ; ISSN:0344-5704. (Springer)
      A review. Autophagy captures and degrades intracellular components such as proteins and organelles to sustain metab. and homeostasis. Rapidly accumulating attention is being paid to the role of autophagy in the development of cancer, which makes autophagy attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that autophagy dysregulation is causal in many cases of cancer, with autophagy acting as tumor suppressors or tumor promoters, and autophagy inhibitor or promoter has shown promise in preclin. studies. The autophagy-targeted therapeutics using chloroquine/hydroxychloroquine have reached clin. development for treating cancer, but these drugs are actually not efficient probably because of a reduced penetration within the tumor. In this review, we first discuss the discoveries related to dual function of autophagy in cancer. Then, we provide an overview of preclin. studies and clin. trials involved in the development of autophagy therapeutics and finally discuss the future of such therapies.
    39. 39
      Warren, T. Nucleotide Prodrug GS-5734 Is a Broad-Spectrum Filovirus Inhibitor That Provides Complete Therapeutic Protection Against the Development of Ebola Virus Disease (EVD) in Infected Non-human Primates. Open Forum Infectious Diseases 2015, 2, DOI: 10.1093/ofid/ofv130.02 .
      There is no corresponding record for this reference.
    40. 40
      De Clercq, E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discovery 2002, 1, 13– 25,  DOI: 10.1038/nrd703
      40
      Strategies in the design of antiviral drugs
      De Clercq, Erik
      Nature Reviews Drug Discovery (2002), 1 (1), 13-25CODEN: NRDDAG ISSN:. (Nature Publishing Group)
      A review. A decade ago, just five drugs were licensed for the treatment of viral infections. Since then, greater understanding of viral life cycles, prompted in particular by the need to combat human immunodeficiency virus, has resulted in the discovery and validation of several targets for therapeutic intervention. Consequently, the current antiviral repertoire now includes more than 30 drugs. But we still lack effective therapies for several viral infections, and established treatments are not always effective or well tolerated, highlighting the need for further refinement of antiviral drug design and development. Here, I describe the rationale behind current and future drug-based strategies for combating viral infections.
    41. 41
      Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009, 4, 1779– 1791,  DOI: 10.1002/cmdc.200900289
      41
      Aryloxy Phosphoramidate Triesters: a Technology for Delivering Monophosphorylated Nucleosides and Sugars into Cells
      Mehellou, Youcef; Balzarini, Jan; McGuigan, Christopher
      ChemMedChem (2009), 4 (11), 1779-1791CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our labs., known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell-membrane penetration. Upon entering the cell, the masking groups are enzymically cleaved to release the phosphorylated biomol. The application of this technol. to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Addnl., the phosphoramidate technol. has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate-based nucleotides progressing to clin. investigations. Furthermore, the phosphoramidate technol. has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technol., mechanism of action and the application of the technol. to various monophosphorylated nucleosides and sugars will be reviewed.
    42. 42
      Seley-Radtke, K. L.; Yates, M. K. The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold. Antiviral Res. 2018, 154, 66– 86,  DOI: 10.1016/j.antiviral.2018.04.004
      42
      The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold
      Seley-Radtke, Katherine L.; Yates, Mary K.
      Antiviral Research (2018), 154 (), 66-86CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
      A review. This is the first of two invited articles reviewing the development of nucleoside-analog antiviral drugs, written for a target audience of virologists and other non-chemists, as well as chemists who may not be familiar with the field. Rather than providing a simple chronol. account, we have examd. and attempted to explain the thought processes, advances in synthetic chem. and lessons learned from antiviral testing that led to a few mols. being moved forward to eventual approval for human therapies, while others were discarded. The present paper focuses on early, relatively simplistic changes made to the nucleoside scaffold, beginning with modifications of the nucleoside sugars of Ara-C and other arabinose-derived nucleoside analogs in the 1960's. A future paper will review more recent developments, focusing esp. on more complex modifications, particularly those involving multiple changes to the nucleoside scaffold. We hope that these articles will help virologists and others outside the field of medicinal chem. to understand why certain drugs were successfully developed, while the majority of candidate compds. encountered barriers due to low-yielding synthetic routes, toxicity or other problems that led to their abandonment.
    43. 43
      Siegel, D. Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. J. Med. Chem. 2017, 60, 1648– 1661,  DOI: 10.1021/acs.jmedchem.6b01594
      43
      Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses
      Siegel, Dustin; Hui, Hon C.; Doerffler, Edward; Clarke, Michael O.; Chun, Kwon; Zhang, Lijun; Neville, Sean; Carra, Ernest; Lew, Willard; Ross, Bruce; Wang, Queenie; Wolfe, Lydia; Jordan, Robert; Soloveva, Veronica; Knox, John; Perry, Jason; Perron, Michel; Stray, Kirsten M.; Barauskas, Ona; Feng, Joy Y.; Xu, Yili; Lee, Gary; Rheingold, Arnold L.; Ray, Adrian S.; Bannister, Roy; Strickley, Robert; Swaminathan, Swami; Lee, William A.; Bavari, Sina; Cihlar, Tomas; Lo, Michael K.; Warren, Travis K.; Mackman, Richard L.
      Journal of Medicinal Chemistry (2017), 60 (5), 1648-1661CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clin. candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were crit. for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclin. efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016, 531, 381-385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
    44. 44
      Cho, A. Synthesis and antiviral activity of a series of 1′-substituted 4-aza-7,9-dideazaadenosine C-nucleosides. Bioorg. Med. Chem. Lett. 2012, 22, 2705– 2707,  DOI: 10.1016/j.bmcl.2012.02.105
      44
      Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosides
      Cho, Aesop; Saunders, Oliver L.; Butler, Thomas; Zhang, Lijun; Xu, Jie; Vela, Jennifer E.; Feng, Joy Y.; Ray, Adrian S.; Kim, Choung U.
      Bioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2705-2707CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside, e.g. I, were prepd. and evaluated for the potential as antiviral agents. These compds. showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concn. of the corresponding nucleoside triphosphate.
    45. 45
      Green, N.; Ott, R. D.; Isaacs, R. J.; Fang, H. Cell-based Assays to Identify Inhibitors of Viral Disease. Expert Opin. Drug Discovery 2008, 3, 671– 676,  DOI: 10.1517/17460441.3.6.671
      45
      Cell-based assays to identify inhibitors of viral disease
      Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong
      Expert Opinion on Drug Discovery (2008), 3 (6), 671-676CODEN: EODDBX; ISSN:1746-0441. (Informa Healthcare)
      A review. Background: Antagonizing the prodn. of infectious viruses inside cells requires drugs that can cross the cell membrane without harming the host cells. Objective: It is therefore advantageous to establish the intracellular potency of antiviral drug candidates early in the drug discovery pipeline. Methods: To this end, cell-based assays are being developed and used in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. Although numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate non-specific screening hits. Results/conclusions: Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being used to reach the full potential of cell-based high-throughput screening.
    46. 46
      Agostini, M. L.; Andres, E. L.; Sims, A. C.; Graham, R. L.; Sheahan, T. P.; Lu, X.; Smith, E. C.; Case, J. B.; Feng, J. Y.; Jordan, R.; Ray, A. S.; Cihlar, T.; Siegel, D.; Mackman, R. L.; Clarke, M. O.; Baric, R. S.; Denison, M. R. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio 2018, 9 (2), e00221– 18,  DOI: 10.1128/mBio.00221-18
      46
      Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease
      Agostini, Maria L.; Andres, Erica L.; Sims, Amy C.; Graham, Rachel L.; Sheahan, Timothy P.; Lu, Xiaotao; Smith, Everett Clinton; Case, James Brett; Feng, Joy Y.; Jordan, Robert; Ray, Adrian S.; Cihlar, Tomas; Siege, Dustin; Mackman, Richard L.; Clarke, Michael O.; Baric, Ralph S.; Denison, Mark R.
      mBio (2018), 9 (2), e00221-18/1-e00221-18/15CODEN: MBIOCL; ISSN:2150-7511. (American Society for Microbiology)
      Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analog GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance assocd. with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concn. values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as detd. by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concns. of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.
    47. 47
      Madelain, V.; Baize, S.; Jacquot, F.; Reynard, S.; Fizet, A.; Barron, S.; Solas, C.; Lacarelle, B.; Carbonnelle, C.; Mentre, F.; Raoul, H.; de Lamballerie, X.; Guedj, J. Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies. Nat. Commun. 2018, 9, 4013,  DOI: 10.1038/s41467-018-06215-z
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      Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies
      Madelain Vincent; Mentre France; Guedj Jeremie; Baize Sylvain; Reynard Stephanie; Fizet Alexandra; Jacquot Frederic; Barron Stephane; Carbonnelle Caroline; Raoul Herve; Solas Caroline; Lacarelle Bruno; de Lamballerie Xavier
      Nature communications (2018), 9 (1), 4013 ISSN:.
      Despite several clinical trials implemented, no antiviral drug could demonstrate efficacy against Ebola virus. In non-human primates, early initiation of polymerase inhibitors favipiravir and remdesivir improves survival, but whether they could be effective in patients is unknown. Here we analyze the impact of antiviral therapy by using a mathematical model that integrates virological and immunological data of 44 cynomolgus macaques, left untreated or treated with favipiravir. We estimate that favipiravir has a ~50% efficacy in blocking viral production, which results in reducing virus growth and cytokine storm while IFNα reduces cell susceptibility to infection. Simulating the effect of delayed initiations of treatment, our model predicts survival rates of 60% for favipiravir and 100% for remdesivir when treatment is initiated within 3 and 4 days post infection, respectively. These results improve the understanding of Ebola immuno-pathogenesis and can help optimize antiviral evaluation in future outbreaks.
    48. 48
      Jordan, R. Broad-spectrum Investigational Agent GS-5734 for the Treatment of Ebola, MERS Coronavirus and Other Pathogenic Viral Infections with High Outbreak Potential. Open Forum Infect Dis 2017, 4, S737,  DOI: 10.1093/ofid/ofx180.008
      There is no corresponding record for this reference.
    49. 49
      Varga, A.; Lionne, C.; Roy, B. Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase. Curr. Drug Metab. 2016, 17, 237– 252,  DOI: 10.2174/1389200217666151210141903
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      Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase
      Varga, Andrea; Lionne, Corinne; Roy, Beatrice
      Current Drug Metabolism (2016), 17 (3), 237-252CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)
      A review. Background: To date, the most effective way to treat HIV is to use a highly active antiretroviral therapy (HAART) that combines three or more different drugs. The usual regimen consists of two nucleoside reverse transcriptase inhibitors and either a protease inhibitor, a non-nucleoside reverse transcriptase inhibitor, or an integrase strand transfer inhibitor. Due to the emerging resistance against the nucleoside analogs in use, there is a continuous need for the development of such therapeutic mols. with different structural features. Objectives: In this review, we would like to summarize the state of knowledge of the antiretroviral nucleoside analogs intracellular metab. Indeed, these mols. have to be phosphorylated in the cell, a process that is often a bottleneck, to produce their pharmacol. active triphosphorylated forms. These forms can be used by the HIV reverse transcriptase. Because they lack a 3'-hydroxyl group, they block further extension of the viral DNA, and finally lead to early chain termination. Several kinases can act on the phosphorylation of these drugs; most of them have low nucleoside/nucleotide specificity. On the other hand, there are also nucleotidases in the cell, which can reverse the phosphorylation process, thus shifting the equil. from the active triphosphorylated state to the non-active (not-, mono- or di-phosphorylated) states of these analogs. Conclusion: Here, we would like to bring to the attention of the medicinal chemists that they have to take into account the limitation of the intracellular phosphorylation machinery when designing new nucleoside analog drugs.
    50. 50
      Brown, A. J. Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase. Antiviral Res. 2019, 169, 104541,  DOI: 10.1016/j.antiviral.2019.104541
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      Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase
      Brown, Ariane J.; Won, John J.; Graham, Rachel L.; Dinnon, Kenneth H., III; Sims, Amy C.; Feng, Joy Y.; Cihlar, Tomas; Denison, Mark R.; Baric, Ralph S.; Sheahan, Timothy P.
      Antiviral Research (2019), 169 (), 104541CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
      The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV.
    51. 51
      de Wit, E. Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 6771– 6776,  DOI: 10.1073/pnas.1922083117
      51
      Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection
      de Wit, Emmie; Feldmann, Friederike; Cronin, Jacqueline; Jordan, Robert; Okumura, Atsushi; Thomas, Tina; Scott, Dana; Cihlar, Tomas; Feldmann, Heinz
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (12), 6771-6776CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The continued emergence of Middle East Respiratory Syndrome (MERS) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. Remdesivir (GS-5734) effectively inhibited MERS coronavirus (MERS-CoV) replication in vitro, and showed efficacy against Severe Acute Respiratory Syndrome (SARS)-CoV in a mouse model. Here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of MERS-CoV infection, the rhesus macaque. Prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented MERS-CoV-induced clin. disease, strongly inhibited MERS-CoV replication in respiratory tissues, and prevented the formation of lung lesions. Therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clin. benefit, with a redn. in clin. signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. The data presented here support testing of the efficacy of remdesivir treatment in the context of a MERS clin. trial. It may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-nCoV.
    52. 52
      Wang, M. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020, 30, 269– 271,  DOI: 10.1038/s41422-020-0282-0
      52
      Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro
      Wang, Manli; Cao, Ruiyuan; Zhang, Leike; Yang, Xinglou; Liu, Jia; Xu, Mingyue; Shi, Zhengli; Hu, Zhihong; Zhong, Wu; Xiao, Gengfu
      Cell Research (2020), 30 (3), 269-271CODEN: CREEB6; ISSN:1001-0602. (Nature Research)
      In Dec. 2019, a novel pneumonia caused by a previously unknown pathogen emerged in Wuhan, a city of 11 million people in central China. The initial cases were linked to exposures in a seafood market in Wuhan. The pathogen was soon identified as a novel coronavirus (2019-nCoV), which is closely related to severe acute respiratory syndrome CoV (SARS-CoV). Currently, there is no specific treatment against the new virus. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. In this study, we evaluated the antiviral efficiency of FAD-approved drugs including ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and two well-known broad-spectrum antiviral drugs remdesivir (GS-5734) and favipiravir (T-705) against a clin. isolate of 2019-nCoV in vitro. Our findings reveal that remdesivir and chloroquine are highly effective in the control of 2019-nCoV infection in vitro. Since these compds. have been used in human patients with a safety track record and shown to be effective against various ailments, we suggest that they should be assessed in human patients suffering from the novel coronavirus disease.
    53. 53
      Sheahan, T. P.; Sims, A. C.; Leist, S. R.; Schafer, A.; Won, J.; Brown, A. J.; Montgomery, S. A.; Hogg, A.; Babusis, D.; Clarke, M. O.; Spahn, J. E.; Bauer, L.; Sellers, S.; Porter, D.; Feng, J. Y.; Cihlar, T.; Jordan, R.; Denison, M. R.; Baric, R. S. Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV. Nat. Commun. 2020, 11 (1), 222,  DOI: 10.1038/s41467-019-13940-6
      53
      Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV
      Sheahan, Timothy P.; Sims, Amy C.; Leist, Sarah R.; Schafer, Alexandra; Won, John; Brown, Ariane J.; Montgomery, Stephanie A.; Hogg, Alison; Babusis, Darius; Clarke, Michael O.; Spahn, Jamie E.; Bauer, Laura; Sellers, Scott; Porter, Danielle; Feng, Joy Y.; Cihlar, Tomas; Jordan, Robert; Denison, Mark R.; Baric, Ralph S.
      Nature Communications (2020), 11 (1), 222CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease assocd. with more than 2468 human infections and over 851 deaths in 27 countries since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathol. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathol. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.
    54. 54
      Mulangu, S. A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N. Engl. J. Med. 2019, 381, 2293– 2303,  DOI: 10.1056/NEJMoa1910993
      54
      A randomized, controlled trial of ebola virus disease therapeutics
      Mulangu, Sabue; Dodd, Lori E.; Davey, Richard T., Jr.; Mbaya, Olivier Tshiani; Proschan, Michael; Mukadi, Daniel; Manzo, Mariano Lusakibanza; Nzolo, Didier; Oloma, Antoine Tshomba; Ibanda, Augustin; Ali, Rosine; Coulibaly, Sinare; Levine, Adam C.; Grais, Rebecca; Diaz, Janet; Lane, H. Clifford; Muyembe-Tamfum, Jean-Jacques
      New England Journal of Medicine (2019), 381 (24), 2293-2303CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      Although several exptl. therapeutics for Ebola virus disease (EVD) have been developed, the safety and efficacy of the most promising therapies need to be assessed in the context of a randomized, controlled trial. methods We conducted a trial of four investigational therapies for EVD in the Democratic Republic of Congo, where an outbreak began in August 2018. Patients of any age who had a pos. result for Ebola virus RNA on reverse-transcriptase-polymerase-chain-reaction assay were enrolled. All patients received std. care and were randomly assigned in a 1:1:1:1 ratio to i.v. administration of the triple monoclonal antibody ZMapp (the control group), the antiviral agent remdesivir, the single monoclonal antibody MAb114, or the triple monoclonal antibody REGN-EB3. The REGN-EB3 group was added in a later version of the protocol, so data from these patients were compared with those of patients in the ZMapp group who were enrolled at or after the time the REGN-EB3 group was added (the ZMapp subgroup). The primary end point was death at 28 days. results A total of 681 patients were enrolled from Nov. 20, 2018, to August 9, 2019, at which time the data and safety monitoring board recommended that patients be assigned only to the MAb114 and REGN-EB3 groups for the remainder of the trial; the recommendation was based on the results of an interim anal. that showed superiority of these groups to ZMapp and remdesivir with respect to mortality. At 28 days, death had occurred in 61 of 174 patients (35.1%) in the MAb114 group, as compared with 84 of 169 (49.7%) in the ZMapp group (P = 0.007), and in 52 of 155 (33.5%) in the REGN-EB3 group, as compared with 79 of 154 (51.3%) in the ZMapp subgroup (P = 0.002). A shorter duration of symptoms before admission and lower baseline values for viral load and for serum creatinine and aminotransferase levels each correlated with improved survival. Four serious adverse events were judged to be potentially related to the trial drugs. conclusions Both MAb114 and REGN-EB3 were superior to ZMapp in reducing mortality from EVD. Scientifically and ethically sound clin. research can be conducted during disease outbreaks and can help inform the outbreak response.
    55. 55
      Deval, J. Antimicrobial strategies: inhibition of viral polymerases by 3′-hydroxyl nucleosides. Drugs 2009, 69 (2), 151– 66,  DOI: 10.2165/00003495-200969020-00002
      55
      Antimicrobial strategies: inhibition of viral polymerases by 3'-hydroxyl nucleosides
      Deval, Jerome
      Drugs (2009), 69 (2), 151-166CODEN: DRUGAY; ISSN:0012-6667. (Wolters Kluwer Health)
      A review. Over the past 20 years, nucleoside analogs have constituted an arsenal of choice in the fight against HIV, hepatitis B and C viruses, and herpesviruses. Classical antiviral nucleosides such as zidovudine act as obligate chain terminators. Once incorporated as monophosphates into the viral nucleic acid, they immediately block the progression of the polymerase as a result of their lack of a reactive 3'-hydroxyl (3'-OH) group. This review explores beyond the paradigm of obligate chain termination, from a structural and a mechanistic perspective, the strategy of inhibiting viral polymerases (RNA- and DNA-dependant) with nucleoside analogs contg. a 3'-OH group. Depending on their mechanism of action, these mols. typically fall into the following three categories: (i) delayed chain terminators; (ii) pseudo-obligate chain terminators; or (iii) mutagenic nucleosides. Delayed chain terminators (i.e. penciclovir, cidofovir and entecavir) block the polymerase at an internal position within the viral nucleic acid, whereas R7128 and the 4'C substituted nucleosides do not permit subsequent incorporation events. Ribavirin, 5-hydroxydeoxycytidine and KP1461 are not chain terminators. Instead, they inhibit viral replication after mispairing with the template base, resulting in random mutations that are often lethal. Finally, brivudine, clevudine and other L-nucleosides have unique or yet to be defined mechanisms of inhibition.
    56. 56
      Snell, N. J. Ribavirin--current status of a broad spectrum antiviral agent. Expert Opin. Pharmacother. 2001, 2 (8), 1317– 24,  DOI: 10.1517/14656566.2.8.1317
      56
      Ribavirin - current status of a broad spectrum antiviral agent
      Snell, Noel J. C.
      Expert Opinion on Pharmacotherapy (2001), 2 (8), 1317-1324CODEN: EOPHF7; ISSN:1465-6566. (Ashley Publications Ltd.)
      A review. Ribavirin is a very broad-spectrum virustatic antiviral agent, first synthesized in 1972. It is characterized by low toxicity apart from reversible anemia, usually mild. Its multiple mechanisms of action mean that viral resistance rarely develops. It can be administered orally, i.v., or via a nebulizer. It has shown varying degrees of clin. efficacy in a variety of human diseases including respiratory tract infections due to respiratory syncytial virus and influenza, measles, herpesvirus infections, HIV infection, Lassa fever, hemorrhagic fever with renal syndrome, and (in combination with IFN-α) chronic hepatitis C infection. It may well prove of value against other emerging exotic infections (e.g., West Nile virus, Nipah virus).
    57. 57
      Witkowski, J. T. Design, synthesis, and broad spectrum antiviral activity of 1- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides. J. Med. Chem. 1972, 15 (11), 1150– 4,  DOI: 10.1021/jm00281a014
      57
      Design, synthesis, and broad spectrum antiviral activity of 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides
      Witkowski, J. T.; Robins, Roland K.; Sidwell, Robert W.; Simon, Lionel N.
      Journal of Medicinal Chemistry (1972), 15 (11), 1150-4CODEN: JMCMAR; ISSN:0022-2623.
      1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (I) [36791-04-5] show high antiviral activity in tissue culture against both DNA and RNA viruses, namely adenovirus, herpes virus types 1 and 2, vaccinia virus, myxoma virus, parainfluenza virus, rhinovirus, coxsackie virus, influenza A2 virus, and influenza B virus. I was active at concns. as low as 1-32 μg/ml, depending on the viruses utilized. I was also effective against established infection with DNA and RNA viruses in vivo in lab. animals. However, I was ineffective against various types of viral encephalitis, presumably due to the inability of I to cross the blood-brain barrier. I did not induce interferon in mice, and apparently affected macromol. processes of viral replication. I was synthesized by treatment of the trimethylsilyl deriv. of Me 1,2,4-triazole-3-carboxylate with an acyl-blocked ribofuranosyl bromide in MeCN at room temp., or by acid-catalyzed fusion of Me 1,2,4-triazole-3-carboxylate with a 1,2,3,5-tetra-O-acylribofuranose to yield a mixt. of blocked Me ester nucleosides, which were sepd. by chromatog. on silica gel; the desired isomer was reacted with NH3-MeOH to yield I.
    58. 58
      Crotty, S.; Cameron, C. E.; Andino, R. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 6895– 6900,  DOI: 10.1073/pnas.111085598
      58
      RNA virus error catastrophe: direct molecular test by using ribavirin
      Crotty, Shane; Cameron, Craig E.; Andino, Raul
      Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (12), 6895-6900CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. The important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and it has been proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here, a direct demonstration of error catastrophe using ribavirin as the mutagen and poliovirus as a model RNA virus is described. Ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is obsd. after a single round of virus infection in ribavirin concns. sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, a graph of error catastrophe was constructed showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.
    59. 59
      Mitsuya, H.; Yarchoan, R.; Broder, S. Molecular targets for AIDS therapy. Science 1990, 249 (4976), 1533– 44,  DOI: 10.1126/science.1699273
      59
      Molecular targets for AIDS therapy
      Mitsuya, Hiroaki; Yarchoan, Robert; Broder, Samuel
      Science (Washington, DC, United States) (1990), 249 (4976), 1533-44CODEN: SCIEAS; ISSN:0036-8075.
      A review with 137 refs. The development of antiretroviral therapy against acquired immunodeficiency syndrome (AIDS) has been an intense research effort since the discovery of the causative agent, human immunodeficiency virus (HIV). A large array of drugs and biol. substances can inhibit HIV replication in vitro. Nucleoside analogs-particularly those belonging to the dideoxynucleoside family-can inhibit reverse transcriptase after anabolic phosphorylation. 3'-Azido-2',3'-dideoxythymidine (AZT) was the first such drug tested in individuals with AIDS, and considerable knowledge of structure-activity relations has emerged for this class of drugs. However, virtually every step in the replication of HIV could serve as a target for a new therapeutic intervention. In the future, non-nucleoside-type drugs will likely become more important in the exptl. therapy of AIDS, and antiretroviral therapy will exert major effects against the morbidity and mortality caused by HIV.
    60. 60
      Gordon, C. J.; Tchesnokov, E. P.; Woolner, E.; Perry, J. K; Feng, J. Y.; Porter, D. P; Gotte, M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J. Biol. Chem. 2020, jbc.RA120.013679, DOI: 10.1074/jbc.RA120.013679 .
      There is no corresponding record for this reference.
    61. 61
      Gao, Y. Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target. bioRxiv , 2020, DOI: 10.1101/2020.03.16.993386 .
      There is no corresponding record for this reference.
    62. 62
      Mehellou, Y.; Rattan, H. S.; Balzarini, J. The ProTide Prodrug Technology: From the Concept to the Clinic. J. Med. Chem. 2018, 61, 2211– 2226,  DOI: 10.1021/acs.jmedchem.7b00734
      62
      The ProTide Prodrug Technology: From the Concept to the Clinic
      Mehellou, Youcef; Rattan, Hardeep S.; Balzarini, Jan
      Journal of Medicinal Chemistry (2018), 61 (6), 2211-2226CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. The ProTide technol. is a prodrug approach developed for the efficient intracellular delivery of nucleoside analog monophosphates and monophosphonates. In this approach, the hydroxyls of the monophosphate or monophosphonate groups are masked by an arom. group and an amino acid ester moiety, which are enzymically cleaved-off inside cells to release the free nucleoside monophosphate and monophosphonate species. Structurally, this represents the current end-point of an extensive medicinal chem. endeavor that spans almost three decades. It started from the masking of nucleoside monophosphate and monophosphonate groups by simple alkyl groups and evolved into the sophisticated ProTide system as known today. This technol. has been extensively employed in drug discovery, and it has already led to the discovery of two FDA-approved (antiviral) ProTides. In this work, we will review the development of the ProTide technol., its application in drug discovery, and its role in the improvement of drug delivery and efficacy.
    63. 63
      Slusarczyk, M.; Serpi, M.; Pertusati, F. Phosphoramidates and phosphonamidates (ProTides) with antiviral activity. Antivir Chem. Chemother Jan-Dec 2018, 26, 2040206618775243,  DOI: 10.1177/2040206618775243
      There is no corresponding record for this reference.
    64. 64
      Curley, D. Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity. Antiviral Res. 1990, 14 (6), 345– 56,  DOI: 10.1016/0166-3542(90)90053-A
      64
      Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity
      Curley, Duncan; McGuigan, Christopher; Devine, Kevin G.; O'Connor, Timothy J.; Jeffries, Donald J.; Kinchington, Derek
      Antiviral Research (1990), 14 (6), 345-56CODEN: ARSRDR; ISSN:0166-3542.
      A series of phosphoramidate derivs. of the anti-HIV drug AZT has been prepd. as membrane sol. pro-drugs of the bio-active nucleotide forms and evaluated in vitro against HIV-1. Terminal substituted alkylamines have a pronounced anti-HIV effect: this effect declines upon increasing the length of the methylene spacer. The results are consistent with a mechanism of action involving intracellular cleavage of the phosphoramidate bond, and release of the nucleotide, or a deriv. thereof. Full spectroscopic data are included on the products and their phosphorochloridate precursors.
    65. 65
      McGuigan, C. Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 1993, 36 (8), 1048– 52,  DOI: 10.1021/jm00060a013
      65
      Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT
      McGuigan, Christopher; Pathirana, Ranjith N.; Balzarini, Jan; De Clercq, Erik
      Journal of Medicinal Chemistry (1993), 36 (8), 1048-52CODEN: JMCMAR; ISSN:0022-2623.
      Novel aryl phosphate derivs., e.g. I (R = H, CH2Ph, R1 = H; R = Me, R1 = Me, Et, Pr, F, OMe), have been prepd. by phosphorochloridate chem. These materials were designed to act as membrane-sol. prodrugs of the bioactive free nucleotides. In vitro evaluation revealed the compds. to have a pronounced, selective anti-HIV activity in CEM cells; the magnitude of the biol. effect varied considerably depending on the nature of the phosphate blocking group. Moreover, several of the compds. retain marked antiviral activity in TK- (thymidine kinase-deficient) mutant CEM cells in which AZT was virtually inactive. These data strongly support the hypothesis that the AZT phosphate derivs. exert their biol. effects via intracellular release of AZT nucleotide forms and suggest that the potential of nucleoside drugs in antiviral chemotherapy may be enhanced by suitable nucleotide delivery strategies.
    66. 66
      Murakami, E. Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977. J. Biol. Chem. 2010, 285 (45), 34337– 47,  DOI: 10.1074/jbc.M110.161802
      66
      Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977
      Murakami, Eisuke; Tolstykh, Tatiana; Bao, Haiying; Niu, Congrong; Steuer, Holly M. Micolochick; Bao, Donghui; Chang, Wonsuk; Espiritu, Christine; Bansal, Shalini; Lam, Angela M.; Otto, Michael J.; Sofia, Michael J.; Furman, Phillip A.
      Journal of Biological Chemistry (2010), 285 (45), 34337-34347CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-β-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixt. of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot anal. showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the prodn. of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. SiRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, resp.
    67. 67
      Saboulard, D. Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine. Mol. Pharmacol. 1999, 56 (4), 693– 704
      67
      Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine
      Saboulard, Didier; Naesens, Lieve; Cahard, Dominique; Salgado, Antonio; Pathirana, Ranjith; Velazquez, Sonsoles; Mcguigan, Christopher; De Clercq, Erik; Balzarini, Jan
      Molecular Pharmacology (1999), 56 (4), 693-704CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
      The phosphoramidate triester prodrugs of anti-human HIV 2',3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-mono-phosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metab. of triester prodrugs of d4T and AZT using pure enzymes or different biol. media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-contg. intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme prepns. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alk. phosphatase, and phosphodiesterase.
    68. 68
      Tobias, S. C.; Borch, R. F. Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs. J. Med. Chem. 2001, 44 (25), 4475– 80,  DOI: 10.1021/jm010337r
      68
      Synthesis and Biological Studies of Novel Nucleoside Phosphoramidate Prodrugs
      Tobias, Sandra C.; Borch, Richard F.
      Journal of Medicinal Chemistry (2001), 44 (25), 4475-4480CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quant. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs, e.g. I, against L1210 mouse leukemia cells show potent inhibition of cell growth (IC50 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compd. I is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC50 value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomps. to the nucleotide in cell culture medium, suggesting that the naphthoquinone analog is not sufficiently stable to function as a nucleotide prodrug.
    69. 69
      Venkatachalam, T. K. Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine. Eur. J. Med. Chem. 2005, 40 (5), 452– 66,  DOI: 10.1016/j.ejmech.2004.11.015
      69
      Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine
      Venkatachalam, T. K.; Samuel, P.; Qazi, S.; Uckun, F. M.
      European Journal of Medicinal Chemistry (2005), 40 (5), 452-466CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Ltd.)
      Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivs. of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivs. and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivs. The authors found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivs. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivs. In addn., the authors show that the enzymic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, the authors describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivs. of stavudine.
    70. 70
      Schneider, B. Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides. J. Biol. Chem. 1998, 273 (19), 11491– 7,  DOI: 10.1074/jbc.273.19.11491
      70
      Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides
      Schneider, Benoit; Xu, Ying Wu; Sellam, Oliver; Sarfati, Robert; Janin, Joel; Vernon, Michel; Deville-Bonne, Dominique
      Journal of Biological Chemistry (1998), 273 (19), 11491-11497CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addn. according to a monoexponential time course. The pseudo-first order rate const. was detd. for different concns. of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast assocn. of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate consts. and dissocn. equil. consts. detd. for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equil. dissocn. consts. for ADP, GDP, ddADP, and ddGDP were also detd. by fluorescence titrn. of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to natural NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.
    71. 71
      Munch-Petersen, B. Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides. J. Biol. Chem. 1991, 266 (14), 9032– 8
      71
      Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides
      Munch-Petersen, Birgitte; Cloos, Lisbeth; Tyrsted, Gerda; Eriksson, Staffan
      Journal of Biological Chemistry (1991), 266 (14), 9032-8CODEN: JBCHA3; ISSN:0021-9258.
      The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogs was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FDUrd), 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 μM as compared to 0.5 μM for dThd and 9 μM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; neg. cooperativity with dThd. Neg. cooperativity was also obsd. with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxythymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 μM for TK1, and 2, 10, and 78 μM for TK2, resp. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogs intended for use in cells with different expression of TK1 and TK2.
    72. 72
      Sheahan, T. P. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci. Transl. Med. 2017, 9 (396), eaal3653,  DOI: 10.1126/scitranslmed.aal3653
      There is no corresponding record for this reference.
    73. 73
      Jordan, P. C. Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase. PLoS Pathog. 2018, 14, e1006889,  DOI: 10.1371/journal.ppat.1006889
      73
      Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase
      Jordan, Paul C.; Liu, Cheng; Raynaud, Pauline; Lo, Michael K.; Spiropoulou, Christina F.; Symons, Julian A.; Beigelman, Leo; Deval, Jerome
      PLoS Pathogens (2018), 14 (2), e1006889/1-e1006889/23CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addn. to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using neg.-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3'- terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clin. relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resoln. structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented neg.-strand RNA viruses.
    74. 74
      Tchesnokov, E. P.; Feng, J. Y.; Porter, D. P.; Gotte, M. Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses 2019, 11 (4), 326,  DOI: 10.3390/v11040326
      74
      Mechanism of inhibition of Ebola virus RNA-dependent RNA polymerase by remdesivir
      Tchesnokov, Egor P.; Feng, Joy Y.; Porter, Danielle P.; Goette, Matthias
      Viruses (2019), 11 (4), 326CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      Remdesivir (GS-5734) is a 1'-cyano-substituted adenosine nucleotide analog prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compd. is currently under clin. development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochem. studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with ATP (ATP). Enzyme kinetics revealed that EBOV RdRp and Respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ∼4 for EBOV RdRp and ∼3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ∼500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ∼6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1'-cyano modification. Compds. with modifications at the 2'-position show different patterns of inhibition. While 2'-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochem. data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analog and its inhibitory effect warrant further investigation.
    75. 75
      Warren, T. K. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016, 531, 381– 385,  DOI: 10.1038/nature17180
      75
      Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys
      Warren, Travis K.; Jordan, Robert; Lo, Michael K.; Ray, Adrian S.; Mackman, Richard L.; Soloveva, Veronica; Siegel, Dustin; Perron, Michel; Bannister, Roy; Hui, Hon C.; Larson, Nate; Strickley, Robert; Wells, Jay; Stuthman, Kelly S.; Van Tongeren, Sean A.; Garza, Nicole L.; Donnelly, Ginger; Shurtleff, Amy C.; Retterer, Cary J.; Gharaibeh, Dima; Zamani, Rouzbeh; Kenny, Tara; Eaton, Brett P.; Grimes, Elizabeth; Welch, Lisa S.; Gomba, Laura; Wilhelmsen, Catherine L.; Nichols, Donald K.; Nuss, Jonathan E.; Nagle, Elyse R.; Kugelman, Jeffrey R.; Palacios, Gustavo; Doerffler, Edward; Neville, Sean; Carra, Ernest; Clarke, Michael O.; Zhang, Lijun; Lew, Willard; Ross, Bruce; Wang, Queenie; Chun, Kwon; Wolfe, Lydia; Babusis, Darius; Park, Yeojin; Stray, Kirsten M.; Trancheva, Iva; Feng, Joy Y.; Barauskas, Ona; Xu, Yili; Wong, Pamela; Braun, Molly R.; Flint, Mike; McMullan, Laura K.; Chen, Shan-Shan; Fearns, Rachel; Swaminathan, Swami; Mayers, Douglas L.; Spiropoulou, Christina F.; Lee, William A.; Nichol, Stuart T.; Cihlar, Tomas; Bavari, Sina
      Nature (London, United Kingdom) (2016), 531 (7594), 381-385CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The most recent Ebola virus outbreak in West Africa, which was unprecedented in the no. of cases and fatalities, geog. distribution, and no. of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clin. efficacy. Here we report the discovery of a novel small mol. GS-5734, a monophosphoramidate prodrug of an adenosine analog, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacol. active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. I.v. administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily i.v. administration of 10 mg kg-1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clin. disease signs and pathophysiol. markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-mol. antiviral compd. against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufg., and clin. studies investigating the drug safety and pharmacokinetics are ongoing.
    76. 76
      Feng, J. Y. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus. Antimicrob. Agents Chemother. 2016, 60 (2), 806– 17,  DOI: 10.1128/AAC.01922-15
      76
      Role of mitochondrial RNA polymerase in the toxicity of nucleotide inhibitors of hepatitis C virus
      Feng, Joy Y.; Xu, Yili; Barauskas, Ona; Perry, Jason K.; Ahmadyar, Shekeba; Stepan, George; Yu, Helen; Babusis, Darius; Park, Yeojin; McCutcheon, Krista; Perron, Michel; Schultz, Brian E.; Sakowicz, Roman; Ray, Adrian S.
      Antimicrobial Agents and Chemotherapy (2016), 60 (2), 806-817CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)
      Toxicity has emerged during the clin. development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clin. relevant HCV NI were characterized in biochem. and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-Me guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concns., followed by general cytotoxicity. In contrast, NI contg. multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an assocn. between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clin. testing.
    77. 77
      Gordon, C. J. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J. Biol. Chem. 2020, 295 (15), 4773– 4779,  DOI: 10.1074/jbc.AC120.013056
      77
      The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus
      Gordon, Calvin J.; Tchesnokov, Egor P.; Feng, Joy Y.; Porter, Danielle P.; Gotte, Matthias
      Journal of Biological Chemistry (2020), 295 (15), 4773-4779CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
      Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at contg. the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compd. with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained with a steady-state approach suggests that it is more efficiently incorporated than ATP and 2 other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The addnl. 3 nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.
    78. 78
      ClinicalTrials.gov. Mild/Moderate 2019-nCoV Remdesivir RCT. https://clinicaltrials.gov/ct2/show/NCT04252664. Accessed April 10, 2020.
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      ClinicalTrials.gov. Severe 2019-nCoV Remdesivir RCT. https://clinicaltrials.gov/ct2/show/NCT04257656. Accessed April 10, 2020.
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      Holshue, M. L. First Case of 2019 Novel Coronavirus in the United States. N. Engl. J. Med. 2020, 382 (10), 929– 936,  DOI: 10.1056/NEJMoa2001191
      80
      First case of 2019 novel coronavirus in the United States
      Holshue, Michelle L.; DeBolt, Chas; Lindquist, Scott; Lofy, Kathy H.; Wiesman, John; Bruce, Hollianne; Spitters, Christopher; Ericson, Keith; Wilkerson, Sara; Tural, Ahmet; Diaz, George; Cohn, Amanda; Fox, LeAnne; Patel, Anita; Gerber, Susan I.; Kim, Lindsay; Tong, Suxiang; Lu, Xiaoyan; Lindstrom, Steve; Pallansch, Mark A.; Weldon, William C.; Biggs, Holly M.; Uyeki, Timothy M.; Pillai, Satish K.
      New England Journal of Medicine (2020), 382 (10), 929-936CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      An outbreak of novel coronavirus (2019-nCoV) that began in Wuhan, China, has spread rapidly, with cases now confirmed in multiple countries. We report the first case of 2019-nCoV infection confirmed in the United States and describe the identification, diagnosis, clin. course, and management of the case, including the patient's initial mild symptoms at presentation with progression to pneumonia on day 9 of illness. This case highlights the importance of close coordination between clinicians and public health authorities at the local, state, and federal levels, as well as the need for rapid dissemination of clin. information related to the care of patients with this emerging infection.
    81. 81
      Kujawski, S. A. First 12 patients with coronavirus disease 2019 (COVID-19) in the United States. MedRxiv , 2020, DOI: 10.1101/2020.03.09.20032896 .
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      ClinicalTrials.gov. Adaptive COVID-19 Treatment Trial (ACTT). https://clinicaltrials.gov/ct2/show/NCT04280705. Accessed April 10, 2020.
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      Routh, J. https://www.nih.gov/news-events/news-releases/nih-clinical-trial-remdesivir-treat-covid-19-begins; NIH, NIAID, Accessed April 6, 2020.
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      BusinessWire, Foster City, CA, 2020. https://www.gilead.com/news-and-press/press-room/press-releases/2020/2/gilead-sciences-initiates-two-phase-3-studies-of-investigational-antiviral-remdesivir-for-the-treatment-of-covid-19).
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    85. 85
      ClinicalTrials.gov. Study to Evaluate the Safety and Antiviral Activity of Remdesivir (GS-5734) in Participants With Moderate Coronavirus Disease (COVID-19) Compared to Standard of Care Treatment (NCT04292730). https://clinicaltrials.gov/ct2/show/NCT04292730. Accessed April 10, 2020.
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      WHO. “Solidarity” clinical trial for COVID-19 treatments. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/global-research-on-novel-coronavirus-2019-ncov/solidarity-clinical-trial-for-covid-19-treatments. Accessed April 7, 2020.
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      ClinicalTrials.gov. The Efficacy of Different Anti-viral Drugs in (Severe Acute Respiratory Syndrome-Corona Virus-2) SARS-CoV-2. https://clinicaltrials.gov/ct2/show/NCT04321616. Accessed April 6, 2020.
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      ClinicalTrials.gov. Adverse Events Related to Treatments Used Against Coronavirus Disease 2019 (CovidTox). https://clinicaltrials.gov/ct2/show/NCT04314817. Accessed April 6, 2020.
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      ClinicalTrials.gov. Trial of Treatments for COVID-19 in Hospitalized Adults (DisCoVeRy), https://clinicaltrials.gov/ct2/show/NCT04315948. Accessed April 6, 2020.
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      INSERM. Launch of a European clinical trial against COVID-19. https://presse.inserm.fr/en/launch-of-a-european-clinical-trial-against-covid-19/38737/. Accessed April 9, 2020.
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      R Core Team. R Foundation for Statistical Computing. http://www.R-project.org/; Vienna, Austria, 2019.
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      ClinicalTrials.gov. Expanded Access Remdesivir (RDV; GS-5734). https://clinicaltrials.gov/ct2/show/NCT04302766. Accessed April 6, 2020.
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      O’Day, D. An Open Letter from our Chairman and CEO. https://www.gilead.com/stories/articles/an-open-letter-from-our-chairman-and-ceo. Accessed April 7, 2020.
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      ClinicalTrials.gov. Expanded Access Treatment Protocol: Remdesivir (RDV; GS-5734) for the Treatment of SARS-CoV2 (CoV) Infection. https://clinicaltrials.gov/ct2/show/NCT04323761. Accessed April 11, 2020.
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      Sheahan, T. P.; Sims, A. C.; Zhou, S.; Graham, R. L.; Pruijssers, A. J.; Agostini, M. L.; Leist, S. R.; Schafer, A.; Dinnon, K. H.; Stevens, L. J.; Chappell, J. D.; Lu, X.; Hughes, T. M.; George, A. S.; Hill, C. S.; Montgomery, S. A.; Brown, A. J.; Bluemling, G. R.; Natchus, M. G.; Saindane, M.; Kolykhalov, A. A.; Painter, G.; Harcourt, J.; Tamin, A.; Thornburg, N. J.; Swanstrom, R.; Denison, M. R.; Baric, R. S. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci. Transl. Med. 2020 Published online April 6, 12, eabb5883, DOI: 10.1126/scitranslmed.abb5883 .
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      Ju, J. Nucleotide analogues as inhibitors of SARS-CoV polymerase. BioRxiv , 2020, DOI: 10.1101/2020.03.12.989186 .
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      Jordheim, L. P. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discovery 2013, 12 (6), 447– 64,  DOI: 10.1038/nrd4010
      100
      Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases
      Jordheim, Lars Petter; Durantel, David; Zoulim, Fabien; Dumontet, Charles
      Nature Reviews Drug Discovery (2013), 12 (6), 447-464CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. Nucleoside analogs have been in clin. use for almost 50 years and have become cornerstones of treatment for patients with cancer or viral infections. The approval of several addnl. drugs over the past decade demonstrates that this family still possesses strong potential. Here, we review new nucleoside analogs and assocd. compds. that are currently in preclin. or clin. development for the treatment of cancer and viral infections, and that aim to provide increased response rates and reduced side effects. We also highlight the different approaches used in the development of these drugs and the potential of personalized therapy.
    101. 101
      Amanat, F.; Krammer, F. SARS-CoV-2 Vaccines: Status Report. Immunity 2020, DOI: 10.1016/j.immuni.2020.03.007 .
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      Chen, W. H.; Strych, U.; Hotez, P. J.; Bottazzi, M. E. The SARS-CoV-2 Vaccine Pipeline: an Overview. Curr. Trop. Med. Rep. 2020,  DOI: 10.1007/s40475-020-00201-6
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      Hodgson, J. The pandemic pipeline. Nat. Biotechnol. 2020, DOI: 10.1038/d41587-020-00005-z .
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      Philippidis, A. Catching Up to Coronavirus: Top 60 Treatments in Development. Genetic Engineering & Biotechnology News. https://www.genengnews.com/virology/coronavirus/catching-up-to-coronavirus-top-60-treatments-in-development/. Accessed April 10, 2020.
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    105. 105
      Allison, M. NCATS launches drug repurposing program. Nat. Biotechnol. 2012, 30, 571– 572,  DOI: 10.1038/nbt0712-571a
      105
      NCATS launches drug repurposing program
      Allison, Malorye
      Nature Biotechnology (2012), 30 (7), 571-572CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
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    106. 106
      Kouznetsova, J.; Sun, W.; Martinez-Romero, C.; Tawa, G.; Shinn, P.; Chen, C. Z; Schimmer, A.; Sanderson, P.; McKew, J. C; Zheng, W.; Garcia-Sastre, A. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerging Microbes Infect. 2014, 3, e84,  DOI: 10.1038/emi.2014.88
      106
      Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs
      Kouznetsova, Jennifer; Sun, Wei; Martinez-Romero, Carles; Tawa, Gregory; Shinn, Paul; Chen, Catherine Z.; Schimmer, Aaron; Sanderson, Philip; McKew, John C.; Zheng, Wei; Garcia-Sastre, Adolfo
      Emerging Microbes & Infections (2014), 3 (12), e84CODEN: EMIMC4; ISSN:2222-1751. (Nature Publishing Group)
      In light of the current outbreak of Ebola virus disease, there is an urgent need to develop effective therapeutics to treat Ebola infection, and drug repurposing screening is a potentially rapid approach for identifying such therapeutics. We developed a biosafety level 2 (BSL-2) 1536-well plate assay to screen for entry inhibitors of Ebola virus-like particles (VLPs) contg. the glycoprotein (GP) and the matrix VP40 protein fused to a β-lactamase reporter protein and applied this assay for a rapid drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. We report here the identification of 53 drugs with activity of blocking Ebola VLP entry into cells. These 53 active compds. can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel antagonists, and anticancer/antibiotics. Several of these compds., including microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in animal model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compds. in drug development for the treatment of Ebola virus infection.
    107. 107
      Grein, J. Compassionate Use of Remdesivir for Patients with Severe Covid-19. N. Engl. J. Med. 2020, DOI: 10.1056/NEJMoa2007016 .
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      NIAID News Release, April 29, 2020. NIH Clinical Trial Shows Remdesivir Accelerates Recovery from Advanced COVID-19. (https://www.niaid.nih.gov/news-events/nih-clinical-trial-shows-remdesivir-accelerates-recovery-advanced-covid-19).
      There is no corresponding record for this reference.
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      FDA News Release, May 1, 2020. Remdesivir EUA Letter of Authorization. (https://www.fda.gov/media/137564/download).
      There is no corresponding record for this reference.