Computational Study of Helicase from SARS-CoV-2 in RNA-Free and Engaged Form.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic that broke out in 2020 and continues to be the cause of massive global upheaval. Coronaviruses are positive-strand RNA viruses with a genome of ~30 kb. The genome is replicated and transcribed by RNA-dependent RNA polymerase together with accessory factors. One of the latter is the protein helicase (NSP13), which is essential for viral replication. The recently solved helicase structure revealed a tertiary structure composed of five domains. Here, we investigated NSP13 from a structural point of view, comparing its RNA-free form with the RNA-engaged form by using atomistic molecular dynamics (MD) simulations at the microsecond timescale. Structural analyses revealed conformational changes that provide insights into the contribution of the different domains, identifying the residues responsible for domain-domain interactions in both observed forms. The RNA-free system appears to be more flexible than the RNA-engaged form. This result underlies the stabilizing role of the nucleic acid and the functional core role of these domains.

Structural Reshaping of the Zinc-Finger Domain of the SARS-CoV-2 nsp13 Protein Using Bismuth(III) Ions: A Multilevel Computational Study.

The identification of novel therapeutics against the pandemic SARS-CoV-2 infection is an indispensable new address of current scientific research. In the search for anti-SARS-CoV-2 agents as alternatives to the vaccine or immune therapeutics whose efficacy naturally degrades with the occurrence of new variants, the salts of Bi3+ have been found to decrease the activity of the Zn2+-dependent non-structural protein 13 (nsp13) helicase, a key component of the SARS-CoV-2 molecular tool kit. Here, we present a multilevel computational investigation based on the articulation of DFT calculations, classical MD simulations, and MIF analyses, focused on the examination of the effects of Bi3+/Zn2+ exchange on the structure and molecular interaction features of the nsp13 protein. Our calculations confirmed that Bi3+ ions can replace Zn2+ in the zinc-finger metal centers and cause slight but appreciable structural modifications in the zinc-binding domain of nsp13. Nevertheless, by employing an in-house-developed ATOMIF tool, we evidenced that such a Bi3+/Zn2+ exchange may decrease the extension of a specific hydrophobic portion of nsp13, responsible for the interaction with the nsp12 protein. The present study provides for a detailed, atomistic insight into the potential anti-SARS-CoV-2 activity of Bi3+ and, more generally, evidences the hampering of the nsp13-nsp12 interaction as a plausible therapeutic strategy.

Punicalagin as an allosteric NSP13 helicase inhibitor potently suppresses SARS-CoV-2 replication in vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) helicase NSP13 plays a conserved role in the replication of coronaviruses and has been identified as an ideal target for the development of antiviral drugs against SARS-CoV-2. Here, we identify a novel NSP13 helicase inhibitor punicalagin (PUG) through high-throughput screening. Surface plasmon resonance (SPR)-based analysis and molecular docking calculation reveal that PUG directly binds NSP13 on the interface of domains 1A and 2A, with a KD value of 21.6 nM. Further biochemical and structural analyses suggest that PUG inhibits NSP13 on ATP hydrolysis and prevents it binding to DNA substrates. Finally, the antiviral studies show that PUG effectively suppresses the SARS-CoV-2 replication in A549-ACE2 and Vero cells, with EC50 values of 347 nM and 196 nM, respectively. Our work demonstrates the potential application of PUG in the treatment of coronavirus disease 2019 (COVID-19) and identifies an allosteric inhibition mechanism for future drug design targeting the viral helicases.

The SARS-CoV-2 helicase as a target for antiviral therapy: Identification of potential small molecule inhibitors by in silico modelling.

Although vaccines that provide protection against severe illness from coronavirus disease (COVID-19) have been made available, emerging variant strains of severe acute respiratory syndrome 2 coronavirus 2 (SARS-CoV-2) are of concern. A different research direction involves investigation of antiviral therapeutics. In addition to structural proteins, the SARS-CoV-2 non-structural proteins are of interest and this includes the helicase (nsp13). In this study, an initial screen of 300 ligands was performed to identify potential inhibitors of the SARS-CoV-2 nsp13 examining the nucleoside triphosphatase site (NTPase activity) as the target region. The antiviral activity of polyphenols has been previously reported in the literature and as a result, the phenolic compounds and fatty acids from the OliveNet™ library were utilised. Synthetic compounds with antimicrobial and anti-inflammatory properties were also selected. The structures of the SARS-CoV and MERS-CoV helicases, as well as the human RECQ-like DNA helicase, DHX9 helicase, PcrA helicase, hepatitis C NS3 helicase, and mouse Dna2 nuclease-helicase were used for comparison. As expected, sequence and structural homology between the various species was evident. A number of broad-spectrum and well-known inhibitors interacted with the NTPase active site highlighting the need to potentially identify more specific inhibitors for SARS-CoV-2. Acetylcysteine, clavulanic acid and homovanillic acid were identified as potential lead compounds for the SARS-CoV-2 helicase. Molecular dynamics simulations were performed with the leads bound to the SARS-CoV-2 helicase for 200 ns in triplicate, with favourable binding free energies to the NTPase site. Given their availability, further exploration of their potential inhibitory activity could be considered.

Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex.

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

SARS-CoV-2 Nucleocapsid Protein Targets a Conserved Surface Groove of the NTF2-like Domain of G3BP1.

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N - G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N - G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1-25, N1-25) reveals that SARS-CoV-2 N1-25 occupies a conserved surface groove of G3BP1NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N1-25 - G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N - G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection.

The stalk domain of SARS-CoV-2 NSP13 is essential for its helicase activity.

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.

Genomics-guided targeting of stress granule proteins G3BP1/2 to inhibit SARS-CoV-2 propagation.

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.

Architecture of a SARS-CoV-2 mini replication and transcription complex.

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase.

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.

High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses.

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

Repurposing potential of posaconazole and grazoprevir as inhibitors of SARS-CoV-2 helicase.

As the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic engulfs millions worldwide, the quest for vaccines or drugs against the virus continues. The helicase protein of SARS-CoV-2 represents an attractive target for drug discovery since inhibition of helicase activity can suppress viral replication. Using in silico approaches, we have identified drugs that interact with SARS-CoV-2 helicase based on the presence of amino acid arrangements matching binding sites of drugs in previously annotated protein structures. The drugs exhibiting an RMSD of ≤ 3.0 Å were further analyzed using molecular docking, molecular dynamics (MD) simulation, and post-MD analyses. Using these approaches, we found 12 drugs that showed strong interactions with SARS-CoV-2 helicase amino acids. The analyses were performed using the recently available SARS-CoV-2 helicase structure (PDB ID: 5RL6). Based on the MM-GBSA approach, out of the 12 drugs, two drugs, namely posaconazole and grazoprevir, showed the most favorable binding energy, - 54.8 and - 49.1 kcal/mol, respectively. Furthermore, of the amino acids found conserved among all human coronaviruses, 10/11 and 10/12 were targeted by, respectively, grazoprevir and posaconazole. These residues are part of the crucial DEAD-like helicase C and DEXXQc_Upf1-like/ DEAD-like helicase domains. Strong interactions of posaconazole and grazoprevir with conserved amino acids indicate that the drugs can be potent against SARS-CoV-2. Since the amino acids are conserved among the human coronaviruses, the virus is unlikely to develop resistance mutations against these drugs. Since these drugs are already in use, they may be immediately repurposed for SARS-CoV-2 therapy.

Structure-Based Virtual Screening Identifies Multiple Stable Binding Sites at the RecA Domains of SARS-CoV-2 Helicase Enzyme.

With the emergence and global spread of the COVID-19 pandemic, the scientific community worldwide has focused on search for new therapeutic strategies against this disease. One such critical approach is targeting proteins such as helicases that regulate most of the SARS-CoV-2 RNA metabolism. The purpose of the current study was to predict a library of phytochemicals derived from diverse plant families with high binding affinity to SARS-CoV-2 helicase (Nsp13) enzyme. High throughput virtual screening of the Medicinal Plant Database for Drug Design (MPD3) database was performed on SARS-CoV-2 helicase using AutoDock Vina. Nilotinib, with a docking value of -9.6 kcal/mol, was chosen as a reference molecule. A compound (PubChem CID: 110143421, ZINC database ID: ZINC257223845, eMolecules: 43290531) was screened as the best binder (binding energy of -10.2 kcal/mol on average) to the enzyme by using repeated docking runs in the screening process. On inspection, the compound was disclosed to show different binding sites of the triangular pockets collectively formed by Rec1A, Rec2A, and 1B domains and a stalk domain at the base. The molecule is often bound to the ATP binding site (referred to as binding site 2) of the helicase enzyme. The compound was further discovered to fulfill drug-likeness and lead-likeness criteria, have good physicochemical and pharmacokinetics properties, and to be non-toxic. Molecular dynamic simulation analysis of the control/lead compound complexes demonstrated the formation of stable complexes with good intermolecular binding affinity. Lastly, affirmation of the docking simulation studies was accomplished by estimating the binding free energy by MMPB/GBSA technique. Taken together, these findings present further in silco investigation of plant-derived lead compounds to effectively address COVID-19.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

Coronavirus helicases: attractive and unique targets of antiviral drug-development and therapeutic patents.

Introduction: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC50 of patented inhibitors ranges between 0.82 µM and 8.95 µM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.Areas covered: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.Expert opinion: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.

Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM.

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Essential functional molecules associated with SARS-CoV-2 infection: Potential therapeutic targets for COVID-19.

The whole world is still suffering substantially from the coronavirus disease 2019 (COVID-19) outbreak. Several protein-based molecules that are associated with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are essential for its functionality, survival, and pathogenesis have been identified and are considered as potential therapeutic targets. These protein-based molecules are either structural/non-structural components of SARS-CoV-2 or host factors, which play a crucial role in this infection. Developing drug molecules against these essential functional molecules to hinder their regular functioning and associated physiological pathways could be promising for successful clinical management of this novel coronavirus infection. The review aims to highlight the functional molecules that play crucial roles in SARS-CoV-2 pathogenesis. We have emphasized how these potential druggable targets could be beneficial in tackling the COVID-19 crisis.

Discovery of COVID-19 Inhibitors Targeting the SARS-CoV-2 Nsp13 Helicase.

The raging COVID-19 pandemic caused by SARS-CoV-2 has infected tens of 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-CoV-2 helicase (Nsp13), which is critical for viral replication and the most conserved nonstructural protein within the coronavirus family. Using homology modeling that couples published electron-density with molecular dynamics (MD)-based structural refinements, we generated structural models of the SARS-CoV-2 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.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Evaluation of the potency of FDA-approved drugs on wild type and mutant SARS-CoV-2 helicase (Nsp13).

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.