Toward wide-spectrum antivirals against coronaviruses: Molecular characterization of SARS-CoV-2 NSP13 helicase inhibitors.
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ABSTRACT:
SUBMITTER: Perez-Lemus GR
PROVIDER: S-EPMC8741187 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.
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Project description:The SARS-CoV-2 helicase Nsp13 is a promising target for developing anti-COVID drugs. In the present study, we have identified potential natural product inhibitors of SARS-CoV-2 Nsp13 targeting the ATP-binding site using molecular docking and molecular dynamics (MD) simulations. MD simulation of the prepared crystal structure of SARS-CoV-2 Nsp13 was performed to generate an ensemble of structures of helicase Nsp13 capturing the conformational diversity of the ATP-binding site. A natural product library of more than 14,000 phytochemicals from Indian medicinal plants was used to perform virtual screening against the ensemble of Nsp13 structures. Subsequently, a two-stage filter, first based on protein-ligand docking binding energy value and second based on protein residues in the ligand-binding site and non-covalent interactions between the protein residues and the ligand in the best-docked pose, was used to identify 368 phytochemicals as potential inhibitors of SARS-CoV-2 helicase Nsp13. MD simulations of the top inhibitors complexed with protein were performed to confirm stable binding, and to compute MM-PBSA based binding energy. From among the 368 potential phytochemical inhibitors, the top identified potential inhibitors of SARS-CoV-2 helicase Nsp13 namely, Picrasidine M, (+)-Epiexcelsin, Isorhoeadine, Euphorbetin and Picrasidine N, can be taken up initially for experimental studies.
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Project description:The raging COVID-19 pandemic caused by SARS-CoV2 has infected millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV2 helicase (Nsp13), which is critical for viral replication and the most conserved non-structural protein within the coronavirus family. Using homology modeling and molecular dynamics approaches, we generated structural models of the SARS-CoV2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ~970,000 chemical compounds against the ATP binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.
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Project description:On account of its crucial role in the virus life cycle, SARS-COV-2 NSP13 helicase enzyme was exploited as a promising target to identify a novel potential inhibitor using multi-stage structure-based drug discovery approaches. Firstly, a 3D pharmacophore was generated based on the collected data from a protein-ligand interaction fingerprint (PLIF) study using key interactions between co-crystallised fragments and the NSP13 helicase active site. The ZINC database was screened through the generated 3D-pharmacophore retrieving 13 potential hits. All the retrieved hits exceeded the benchmark score of the co-crystallised fragments at the molecular docking step and the best five-hit compounds were selected for further analysis. Finally, a combination between molecular dynamics simulations and MM-PBSA based binding free energy calculations was conducted on the best hit (compound FWM-1) bound to NSP13 helicase enzyme, which identified FWM-1 as a potential potent NSP13 helicase inhibitor with binding free energy equals -328.6 ± 9.2 kcal/mol.
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Project description:The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.
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Project description:There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
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Project description:The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but nonstructural protein 13 (nsp13) stands out as a highly conserved and yet understudied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP-dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound, which presents a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 μs of a Gaussian-accelerated molecular dynamics simulation (at least 6 μs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A Gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key structural states of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inchworm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a power stroke-like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.
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