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

Abstract

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.

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design

“…Although more attention is directed to targeting the RdRp 22,[25][26][27] or the main protease, [28][29][30][31][32] as these are suggested to be more susceptible for binding an inhibitor, [33][34][35] the helicase is also subject to modelling and docking studies. Crucially, however, the pharmacophore and docking studies of the helicase start from the homologous SARS-CoV crystal structure (Figure 2a) [36][37][38][39][40] or the counterpart in SARS-CoV-2 (Figure 2b), 41 which are both apo, lacking the ATP and the ssRNA from the complex.…”

RNA helicases required for viral propagation in humans

“…Due to the successes with other viral infections such as HIV-1 and HCV, the protease and the core polymerase has been the favored targets for small molecule viral inhibitors. In silico studies utilizing the crystal structures of the SARS1 NSP13 helicase quickly appeared in the literature following the recognition of the remarkable conserved homology of the SARS1 and SARS2 proteins and SARS CoV-2 as a world-wide threat [59] , [60] , [61] , [62] , [63] , [64] , [65] . Importantly, these studies made it clear that NSP13 contains multiple druggable structures and pockets predicted to disrupt the helicase activity outside of the canonical NTP binding site.…”

Investigating Novel Thiazolyl-Indazole Derivatives as Scaffolds for SARS-Cov-2 MPro Inhibitors

“…[31,32] Most recently, thiazole-based inhibitors have been reported for SARS-CoV-2. [33,34] Synthetic indazole-based compounds have exhibited function as anti-inflammatory, antiarrhythmic, antitumor, antifungal, antibacterial, and anti-HIV drugs. [35] Indazole-based drugs are also being considered as SARS-CoV-2 M Pro inhibitors.…”

“…M. [33,34] A machine learning approach to identifying investigational or off-market drug targets for SARS-CoV-2 identified an indazole containing compound as one of the most promising with an estimated binding affinity of -9 kcal/mol. [36] Given the wide-ranging and beneficial pharmacological impacts of thiazole-and indazole-containing compounds, and the community's interest in these drugs as potential SARS-CoV-2 M Pro inhibitors, we…”

“…seek to elucidate the usefulness of thiazolyl-indazole derivatives as potential scaffolds for further pharmaceutical development and exploration. [31][32][33][34][35][36]38] In this work, we explore the potential for compounds I -V to function as reversible, noncovalent inhibitors of M Pro through chemical property prediction, biochemical assays, docking analysis, molecular dynamics simulations, and a detailed comparison with substrate binding. We have identified M Pro amino acids that consistently interact with these compounds and our simulations reveal the importance of π interactions in the mechanism of binding.…”