Multi-stage structure-based virtual screening approach towards identification of potential SARS-CoV-2 NSP13 helicase inhibitors.
Journal of enzyme inhibition and medicinal chemistry
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 ) bound to NSP13 helicase enzyme, which identified as a potential potent NSP13 helicase inhibitor with binding free energy equals -328.6 ± 9.2 kcal/mol.
10.1080/14756366.2021.2022659
Discovering potential inhibitors against SARS-CoV-2 by targeting Nsp13 Helicase.
Journal of biomolecular structure & dynamics
The rise in the incidence of COVID-19 as a result of SARS-CoV-2 infection has threatened public health globally. Till now, there have been no proper prophylactics available to fight COVID-19, necessitating the advancement and evolution of effective curative against SARS-CoV-2. This study aimed at the nonstructural protein 13 (nsp13) helicase as a promising target for drug development against COVID-19. A unique collection of nucleoside analogs was screened against the SARS-CoV-2 helicase protein, for which a molecular docking experiment was executed to depict the selected ligand's binding affinity with the SARS-CoV-2 helicase proteins. Simultaneously, molecular dynamic simulations were performed to examine the protein's binding site's conformational stability, flexibility, and interaction with the ligands. Key nucleoside ligands were selected for pharmacokinetic analysis based on their docking scores. Selected ligands (cordycepin and pritelivir) showed excellent pharmacokinetics and were well stabilized at the proteins' binding site throughout the MD simulation. We have also performed binding free energy analysis or the binding characteristics of ligands with Nsp13 by using MM-PBSA and MM-GBSA. Free energy calculation by MM-PBSA and MM-GBSA analysis suggests that pritelivir may work as viable therapeutics for efficient drug advancement against SARS-CoV-2 Nsp13 helicase, potentially arresting the SARS-CoV-2 replication.Communicated by Ramaswamy H. Sarma.
10.1080/07391102.2021.1970024
Natural Compounds Inhibit SARS-CoV-2 nsp13 Unwinding and ATPase Enzyme Activities.
ACS pharmacology & translational science
SARS-CoV-2 infection is still spreading worldwide, and new antiviral therapies are an urgent need to complement the approved vaccine preparations. SARS-CoV-2 nps13 helicase is a validated drug target participating in the viral replication complex and possessing two associated activities: RNA unwinding and 5'-triphosphatase. In the search of SARS-CoV-2 direct antiviral agents, we established biochemical assays for both SARS-CoV-2 nps13-associated enzyme activities and screened both and a small in-house library of natural compounds. Myricetin, quercetin, kaempferol, and flavanone were found to inhibit the SARS-CoV-2 nps13 unwinding activity at nanomolar concentrations, while licoflavone C was shown to block both SARS-CoV-2 nps13 activities at micromolar concentrations. Mode of action studies showed that all compounds are nsp13 noncompetitive inhibitors versus ATP, while computational studies suggested that they can bind both nucleotide and 5'-RNA nsp13 binding sites, with licoflavone C showing a unique pattern of interaction with nsp13 amino acid residues. Overall, we report for the first time natural flavonoids as selective inhibitors of SARS-CoV-2 nps13 helicase with low micromolar activity.
10.1021/acsptsci.1c00253
Toward wide-spectrum antivirals against coronaviruses: Molecular characterization of SARS-CoV-2 NSP13 helicase inhibitors.
Science advances
To date, effective therapeutic treatments that confer strong attenuation against coronaviruses (CoVs) remain elusive. Among potential drug targets, the helicase of CoVs is attractive due to its sequence conservation and indispensability. We rely on atomistic molecular dynamics simulations to explore the structural coordination and dynamics associated with the SARS-CoV-2 Nsp13 apo enzyme, as well as their complexes with natural ligands. A complex communication network is revealed among the five domains of Nsp13, which is differentially activated because of the presence of the ligands, as shown by shear strain analysis, principal components analysis, dynamical cross-correlation matrix analysis, and water transport analysis. The binding free energy and the corresponding mechanism of action are presented for three small molecules that were shown to be efficient inhibitors of the previous SARS-CoV Nsp13 enzyme. Together, our findings provide critical fresh insights for rational design of broad-spectrum antivirals against CoVs.
10.1126/sciadv.abj4526
Diketo acid inhibitors of nsp13 of SARS-CoV-2 block viral replication.
Antiviral research
For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain.
10.1016/j.antiviral.2023.105697
Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.
Zeng Jingkun,Weissmann Florian,Bertolin Agustina P,Posse Viktor,Canal Berta,Ulferts Rachel,Wu Mary,Harvey Ruth,Hussain Saira,Milligan Jennifer C,Roustan Chloe,Borg Annabel,McCoy Laura,Drury Lucy S,Kjaer Svend,McCauley John,Howell Michael,Beale Rupert,Diffley John F X
The Biochemical journal
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.
10.1042/BCJ20210201
A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13.
ACS chemical biology
The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.
10.1021/acschembio.4c00244