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1.
Biochemistry ; 59(33): 3038-3043, 2020 08 25.
Article in English | MEDLINE | ID: covidwho-713002

ABSTRACT

The COVID-19 pandemic threatens to overwhelm healthcare systems around the world. The only current FDA-approved treatment, which directly targets the virus, is the ProTide prodrug remdesivir. In its activated form, remdesivir prevents viral replication by inhibiting the essential RNA-dependent RNA polymerase. Like other ProTide prodrugs, remdesivir contains a chiral phosphorus center. The initial selection of the (SP)-diastereomer for remdesivir was reportedly due to the difficulty in producing the pure (RP)-diastereomer of the required precursor. However, the two currently known enzymes responsible for the initial activation step of remdesivir are each stereoselective and show differential tissue distribution. Given the ability of the COVID-19 virus to infect a wide array of tissue types, inclusion of the (RP)-diastereomer may be of clinical significance. To help overcome the challenge of obtaining the pure (RP)-diastereomer of remdesivir, we have developed a novel chemoenzymatic strategy that utilizes a stereoselective variant of the phosphotriesterase from Pseudomonas diminuta to enable the facile isolation of the pure (RP)-diastereomer of the chiral precursor for the chemical synthesis of the (RP)-diastereomer of remdesivir.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemical synthesis , Adenosine Monophosphate/chemical synthesis , Alanine/chemical synthesis , Betacoronavirus , Caulobacteraceae/enzymology , Coronavirus Infections , Humans , Molecular Structure , Pandemics , Phosphoric Triester Hydrolases/chemistry , Pneumonia, Viral , RNA Replicase/antagonists & inhibitors , Virus Replication/drug effects
2.
J Biol Chem ; 295(15): 4780-4781, 2020 04 10.
Article in English | MEDLINE | ID: covidwho-686585

ABSTRACT

The nucleotide analogue remdesivir is an investigational drug for the treatment of human coronavirus infection. Remdesivir is a phosphoramidate prodrug and is known to target viral RNA-dependent RNA polymerases. In this issue, Gordon et al. identify that remdesivir acts as a delayed RNA chain terminator for MERS-CoV polymerase complexes.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus/drug effects , Coronavirus/enzymology , RNA Replicase/antagonists & inhibitors , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Coronavirus/physiology , Coronavirus Infections/virology , Exonucleases , Humans , Pandemics , Virus Replication/drug effects
3.
Interdiscip Sci ; 12(3): 335-348, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-649384

ABSTRACT

Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Biological Products/pharmacology , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA Replicase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/chemistry , Betacoronavirus/genetics , Biological Products/chemistry , Catalytic Domain/genetics , Computer Simulation , Coronavirus Infections/epidemiology , Databases, Pharmaceutical , Drug Evaluation, Preclinical , Genome, Viral , Host Microbial Interactions/drug effects , Humans , Ligands , Molecular Docking Simulation , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , RNA Replicase/chemistry , RNA Replicase/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
4.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Article in English | MEDLINE | ID: covidwho-611394

ABSTRACT

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/metabolism , Betacoronavirus/enzymology , Enzyme Inhibitors/metabolism , RNA Replicase/antagonists & inhibitors , RNA Replicase/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Amino Acid Sequence , Antiviral Agents/chemistry , Binding Sites , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Binding , RNA Replicase/chemistry , Thermodynamics , Viral Nonstructural Proteins/chemistry
5.
Antiviral Res ; 180: 104857, 2020 08.
Article in English | MEDLINE | ID: covidwho-602131

ABSTRACT

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Infections/virology , Nucleotides/pharmacology , Pneumonia, Viral/virology , RNA Replicase/antagonists & inhibitors , SARS Virus/enzymology , Severe Acute Respiratory Syndrome/virology , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Betacoronavirus/enzymology , Betacoronavirus/genetics , Cidofovir/chemistry , Cidofovir/pharmacology , Cidofovir/therapeutic use , Coronavirus Infections/drug therapy , Dideoxynucleosides/chemistry , Dideoxynucleosides/pharmacology , Dideoxynucleosides/therapeutic use , Ganciclovir/chemistry , Ganciclovir/pharmacology , Ganciclovir/therapeutic use , Guanine/analogs & derivatives , Guanine/chemistry , Guanine/pharmacology , Guanine/therapeutic use , Nucleotides/chemistry , Nucleotides/therapeutic use , Pandemics , Pneumonia, Viral/drug therapy , Prodrugs/chemistry , Prodrugs/pharmacology , Prodrugs/therapeutic use , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , SARS Virus/genetics , Severe Acute Respiratory Syndrome/drug therapy , Stavudine/chemistry , Stavudine/pharmacology , Stavudine/therapeutic use , Valganciclovir/chemistry , Valganciclovir/pharmacology , Valganciclovir/therapeutic use
7.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Article in English | MEDLINE | ID: covidwho-594910

ABSTRACT

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/metabolism , Betacoronavirus/enzymology , Enzyme Inhibitors/metabolism , RNA Replicase/antagonists & inhibitors , RNA Replicase/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Amino Acid Sequence , Antiviral Agents/chemistry , Binding Sites , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Binding , RNA Replicase/chemistry , Thermodynamics , Viral Nonstructural Proteins/chemistry
8.
J Med Microbiol ; 69(6): 864-873, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-436403

ABSTRACT

Introduction. The emergence of SARS-CoV-2 has taken humanity off guard. Following an outbreak of SARS-CoV in 2002, and MERS-CoV about 10 years later, SARS-CoV-2 is the third coronavirus in less than 20 years to cross the species barrier and start spreading by human-to-human transmission. It is the most infectious of the three, currently causing the COVID-19 pandemic. No treatment has been approved for COVID-19. We previously proposed targets that can serve as binding sites for antiviral drugs for multiple coronaviruses, and here we set out to find current drugs that can be repurposed as COVID-19 therapeutics.Aim. To identify drugs against COVID-19, we performed an in silico virtual screen with the US Food and Drug Administration (FDA)-approved drugs targeting the RNA-dependent RNA polymerase (RdRP), a critical enzyme for coronavirus replication.Methodology. Initially, no RdRP structure of SARS-CoV-2 was available. We performed basic sequence and structural analysis to determine if RdRP from SARS-CoV was a suitable replacement. We performed molecular dynamics simulations to generate multiple starting conformations that were used for the in silico virtual screen. During this work, a structure of RdRP from SARS-CoV-2 became available and was also included in the in silico virtual screen.Results. The virtual screen identified several drugs predicted to bind in the conserved RNA tunnel of RdRP, where many of the proposed targets were located. Among these candidates, quinupristin is particularly interesting because it is expected to bind across the RNA tunnel, blocking access from both sides and suggesting that it has the potential to arrest viral replication by preventing viral RNA synthesis. Quinupristin is an antibiotic that has been in clinical use for two decades and is known to cause relatively minor side effects.Conclusion. Quinupristin represents a potential anti-SARS-CoV-2 therapeutic. At present, we have no evidence that this drug is effective against SARS-CoV-2 but expect that the biomedical community will expeditiously follow up on our in silico findings.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , RNA Replicase/antagonists & inhibitors , Animals , Antiviral Agents/therapeutic use , Betacoronavirus/enzymology , Betacoronavirus/genetics , Betacoronavirus/physiology , Coronavirus Infections/virology , Drug Evaluation, Preclinical/methods , Drug Synergism , Humans , Molecular Conformation , Pandemics , Phylogeny , Pneumonia, Viral/virology , RNA Replicase/drug effects , Rifampin/pharmacology , Sequence Alignment , Sequence Analysis, Protein , Virginiamycin/analogs & derivatives , Virginiamycin/pharmacology , Virus Replication/drug effects
9.
Curr Top Med Chem ; 20(16): 1423-1433, 2020.
Article in English | MEDLINE | ID: covidwho-285738

ABSTRACT

Like other human pathogenic viruses, coronavirus SARS-CoV-2 employs sophisticated macromolecular machines for viral host cell entry, genome replication and protein processing. Such machinery encompasses SARS-CoV-2 envelope spike (S) glycoprotein required for host cell entry by binding to the ACE2 receptor, viral RNA-dependent RNA polymerase (RdRp) and 3-chymotrypsin-like main protease (3Clpro/Mpro). Under the pressure of the accelerating COVID-19 pandemic caused by the outbreak of SARS-CoV-2 in Wuhan, China in December 2019, novel and repurposed drugs were recently designed and identified for targeting the SARS-CoV-2 reproduction machinery, with the aim to limit the spread of SARS-CoV-2 and morbidity and mortality due to the COVID-19 pandemic.


Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Drug Repositioning , Pneumonia, Viral/drug therapy , Virus Internalization , Virus Replication , Coronavirus Infections/virology , Cysteine Endopeptidases , Humans , Pandemics , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/virology , RNA Replicase/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors
10.
Nature ; 581(7808): 252-255, 2020 05.
Article in English | MEDLINE | ID: covidwho-265650

Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/chemistry , Betacoronavirus/immunology , Drug Design , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Viral Vaccines , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Animals , Antiviral Agents/chemistry , Azoles/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , China , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Cryoelectron Microscopy , Crystallization , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Evaluation, Preclinical , Germany , High-Throughput Screening Assays , Humans , Mice , National Institutes of Health (U.S.)/economics , National Institutes of Health (U.S.)/organization & administration , Organoselenium Compounds/pharmacology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protease Inhibitors/pharmacology , RNA Replicase/antagonists & inhibitors , RNA Replicase/chemistry , RNA Replicase/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Synchrotrons , Time Factors , United Kingdom , United States , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Proteins/immunology , Viral Vaccines/chemistry , Viral Vaccines/immunology
11.
J Transl Med ; 18(1): 185, 2020 05 05.
Article in English | MEDLINE | ID: covidwho-175840

ABSTRACT

A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , RNA Replicase/antagonists & inhibitors , RNA Replicase/chemistry , Amino Acid Sequence , Betacoronavirus/isolation & purification , Conserved Sequence , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Drug Repositioning , Humans , Models, Molecular , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , RNA Replicase/metabolism , Sequence Alignment , Virus Replication/drug effects , Virus Shedding/drug effects
12.
Science ; 368(6498): 1499-1504, 2020 06 26.
Article in English | MEDLINE | ID: covidwho-154668

ABSTRACT

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , Betacoronavirus/enzymology , RNA Replicase/antagonists & inhibitors , RNA Replicase/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/metabolism , Alanine/pharmacology , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/physiology , Catalytic Domain , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Models, Molecular , Multiprotein Complexes/chemistry , Protein Conformation , RNA Replicase/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication
13.
J Transl Med ; 18(1): 179, 2020 04 22.
Article in English | MEDLINE | ID: covidwho-102130

ABSTRACT

BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Evolution, Molecular , Genome, Viral/genetics , Mutation , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA Replicase/genetics , Adult , Asia/epidemiology , Coronavirus Infections/drug therapy , Coronavirus Infections/mortality , Drug Resistance, Viral/genetics , Europe/epidemiology , Female , Humans , Male , Middle Aged , Mutation Rate , North America/epidemiology , Oceania/epidemiology , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/mortality , RNA Replicase/antagonists & inhibitors , RNA Replicase/metabolism
14.
J Biol Chem ; 295(20): 6785-6797, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-52576

ABSTRACT

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , RNA Replicase/antagonists & inhibitors , Virus Replication/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Betacoronavirus/physiology , Models, Molecular , Sf9 Cells , Spodoptera
15.
Science ; 368(6492): 779-782, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-47347

ABSTRACT

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified ß-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.


Subject(s)
Betacoronavirus/enzymology , RNA Replicase/chemistry , RNA Replicase/ultrastructure , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/ultrastructure , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/metabolism , Alanine/pharmacology , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Catalytic Domain , Cryoelectron Microscopy , Drug Design , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Conformation, beta-Strand , Protein Domains , RNA Replicase/antagonists & inhibitors , RNA Replicase/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
16.
J Microbiol Immunol Infect ; 53(3): 436-443, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-31183

ABSTRACT

An outbreak related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China in December 2019. An extremely high potential for dissemination resulted in the global coronavirus disease 2019 (COVID-19) pandemic in 2020. Despite the worsening trends of COVID-19, no drugs are validated to have significant efficacy in clinical treatment of COVID-19 patients in large-scale studies. Remdesivir is considered the most promising antiviral agent; it works by inhibiting the activity of RNA-dependent RNA polymerase (RdRp). A large-scale study investigating the clinical efficacy of remdesivir (200 mg on day 1, followed by 100 mg once daily) is on-going. The other excellent anti-influenza RdRp inhibitor favipiravir is also being clinically evaluated for its efficacy in COVID-19 patients. The protease inhibitor lopinavir/ritonavir (LPV/RTV) alone is not shown to provide better antiviral efficacy than standard care. However, the regimen of LPV/RTV plus ribavirin was shown to be effective against SARS-CoV in vitro. Another promising alternative is hydroxychloroquine (200 mg thrice daily) plus azithromycin (500 mg on day 1, followed by 250 mg once daily on day 2-5), which showed excellent clinical efficacy on Chinese COVID-19 patients and anti-SARS-CoV-2 potency in vitro. The roles of teicoplanin (which inhibits the viral genome exposure in cytoplasm) and monoclonal and polyclonal antibodies in the treatment of SARS-CoV-2 are under investigation. Avoiding the prescription of non-steroidal anti-inflammatory drugs, angiotensin converting enzyme inhibitors, or angiotensin II type I receptor blockers is advised for COVID-19 patients.


Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/therapeutic use , Amides/therapeutic use , Azithromycin/therapeutic use , Coronavirus Infections/therapy , Drug Combinations , Humans , Hydroxychloroquine/therapeutic use , Immunization, Passive/methods , Lopinavir/therapeutic use , Pandemics , Pyrazines/therapeutic use , RNA Replicase/antagonists & inhibitors , Ritonavir/therapeutic use , Teicoplanin/therapeutic use
17.
Life Sci ; 253: 117592, 2020 Jul 15.
Article in English | MEDLINE | ID: covidwho-14903

ABSTRACT

AIMS: A new human coronavirus (HCoV), which has been designated SARS-CoV-2, began spreading in December 2019 in Wuhan City, China causing pneumonia called COVID-19. The spread of SARS-CoV-2 has been faster than any other coronaviruses that have succeeded in crossing the animal-human barrier. There is concern that this new virus will spread around the world as did the previous two HCoVs-Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS)-each of which caused approximately 800 deaths in the years 2002 and 2012, respectively. Thus far, 11,268 deaths have been reported from the 258,842 confirmed infections in 168 countries. MAIN METHODS: In this study, the RNA-dependent RNA polymerase (RdRp) of the newly emerged coronavirus is modeled, validated, and then targeted using different anti-polymerase drugs currently on the market that have been approved for use against various viruses. KEY FINDINGS: The results suggest the effectiveness of Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir as potent drugs against SARS-CoV-2 since they tightly bind to its RdRp. In addition, the results suggest guanosine derivative (IDX-184), Setrobuvir, and YAK as top seeds for antiviral treatments with high potential to fight the SARS-CoV-2 strain specifically. SIGNIFICANCE: The availability of FDA-approved anti-RdRp drugs can help treat patients and reduce the danger of the mysterious new viral infection COVID-19. The drugs mentioned above can tightly bind to the RdRp of the SARS-CoV-2 strain and thus may be used to treat the disease. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Molecular Docking Simulation , Nucleic Acid Synthesis Inhibitors/chemistry , RNA Replicase/antagonists & inhibitors , Antiviral Agents/chemistry , Coronavirus Infections/drug therapy , Humans , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleosides/analogs & derivatives
18.
J Med Virol ; 92(6): 693-697, 2020 06.
Article in English | MEDLINE | ID: covidwho-8443

ABSTRACT

An outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and it has rapidly spread to almost all parts of the world. For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an important polymerase that catalyzes the replication of RNA from RNA template and is an attractive therapeutic target. In this study, we screened these chemical structures from traditional Chinese medicinal compounds proven to show antiviral activity in severe acute respiratory syndrome coronavirus (SARS-CoV) and the similar chemical structures through a molecular docking study to target RdRp of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that theaflavin has a lower idock score in the catalytic pocket of RdRp in SARS-CoV-2 (-9.11 kcal/mol), SARS-CoV (-8.03 kcal/mol), and MERS-CoV (-8.26 kcal/mol) from idock. To confirm the result, we discovered that theaflavin has lower binding energy of -8.8 kcal/mol when it docks in the catalytic pocket of SARS-CoV-2 RdRp by using the Blind Docking server. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were found between theaflavin and RdRp. Moreover, one π-cation interaction was formed between theaflavin and Arg553 from the Blind Docking server. Our results suggest that theaflavin could be a potential SARS-CoV-2 RdRp inhibitor for further study.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Biflavonoids/chemistry , Catechin/chemistry , Drugs, Chinese Herbal/chemistry , RNA Replicase/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Betacoronavirus/genetics , Biflavonoids/pharmacology , Catalytic Domain , Catechin/pharmacology , Computational Biology/methods , Drugs, Chinese Herbal/pharmacology , Gene Expression , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/enzymology , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , RNA Replicase/antagonists & inhibitors , RNA Replicase/genetics , RNA Replicase/metabolism , SARS Virus/drug effects , SARS Virus/enzymology , SARS Virus/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Thermodynamics , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics , Viral Proteins/metabolism
19.
Life Sci ; 248: 117477, 2020 May 01.
Article in English | MEDLINE | ID: covidwho-2799

ABSTRACT

AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Coronavirus Infections/drug therapy , Guanosine Monophosphate/analogs & derivatives , Pneumonia, Viral/drug therapy , RNA Replicase/antagonists & inhibitors , Ribavirin/chemistry , Sofosbuvir/chemistry , Viral Proteins/antagonists & inhibitors , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Alphacoronavirus/enzymology , Alphacoronavirus/genetics , Amino Acid Sequence , Antiviral Agents/metabolism , Betacoronavirus/genetics , Catalytic Domain , Computational Biology/methods , Coronavirus Infections/virology , Drug Repositioning/methods , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Humans , Molecular Docking Simulation , Pneumonia, Viral/virology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Replicase/chemistry , RNA Replicase/metabolism , Ribavirin/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sofosbuvir/metabolism , Thermodynamics , Uridine Triphosphate/chemistry , Uridine Triphosphate/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism
20.
J Biol Chem ; 295(15): 4773-4779, 2020 04 10.
Article in English | MEDLINE | ID: covidwho-1988

ABSTRACT

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound 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 here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two 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 additional three 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.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Middle East Respiratory Syndrome Coronavirus/enzymology , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA Replicase/antagonists & inhibitors , Virus Replication/drug effects , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Coronavirus/enzymology , Ebolavirus/enzymology , Gene Expression , Nucleic Acid Synthesis Inhibitors/chemistry , RNA , RNA Replicase/genetics , Sf9 Cells , Viral Nonstructural Proteins/genetics
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