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1.
Proteins ; 89(9): 1216-1225, 2021 09.
Article in English | MEDLINE | ID: covidwho-1293319

ABSTRACT

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.


Subject(s)
Coronavirus 3C Proteases/chemistry , SARS Virus/enzymology , SARS-CoV-2/enzymology , Apoenzymes/antagonists & inhibitors , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Models, Molecular , Protease Inhibitors/pharmacology , Protein Multimerization/drug effects , Protein Structure, Quaternary/drug effects
2.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289982

ABSTRACT

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Animals , Benzoates/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Holoenzymes/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Suramin/pharmacology , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
3.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Article in English | MEDLINE | ID: covidwho-710427

ABSTRACT

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Subject(s)
Methyltransferases/chemistry , RNA Helicases/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Betacoronavirus/genetics , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Holoenzymes/chemistry , Holoenzymes/metabolism , Magnesium/metabolism , Methyltransferases/metabolism , Protein Binding , RNA Helicases/metabolism , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
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