Application of rational computer-aided drug design approaches to discover novel small molecule inhibitors of conserved viral non-structural proteins of SARS-CoV-2

Abstract

The majority of SARS-CoV-2 therapeutic development work has focussed on targeting the spike protein, viral polymerase and proteases. As the pandemic progressed, many studies reported that these proteins are prone to high levels of mutation and can become drug resistant. Thus, it is necessary to not only target other viral proteins such as the non-structural proteins (NSPs) but to also target the most conserved residues of these proteins. In order to understand the level of conservation among these viruses, we have focussed on the conservation across the coronaviruses and then narrowed our focus to conservation of NSPs across coronaviruses. A synergistic combination of bioinformatics, computer-aided drug-design and in-vitro studies can feed into better understanding of SARS-CoV-2 (SC-2) and therefore help in the development of small molecule inhibitors against the NSPs. As part of our initial anti-viral work, a pharmacophore study on NSP15 found a hit molecule (INS316) that made interactions with Ser293, Lys344 and Leu345 residues (Kandwal & Fayne, 2022) which are highly conserved across SC-2. We have performed multiple sequence alignment studies on different ~1 million sequences of SC-2 NSP sequences to identify the most conserved residues. These residues were then visualized on 3D protein X-ray structures using MOE software. We found that there were known and novel binding pocket residues that were highly conserved in our datasets. Our results indicate that these highly conserved pockets can be targeted for developing promising SC-2 inhibitors. We have also performed mutational analysis and have found different mutational hotspots across the NSPs. Our group was selected to enter two international challenges organized by CACHE to find inhibitors for the RNA binding tunnel of SC-2 NSP13 and the Mac1 domain of SC-2 NSP3. We have used a tiered screening workflow which included the use of volume/shape information of the binding pockets (fastROCS), use of in-house pharmacophore generation software MoPBS/MOE and performed docking in the binding pocket using FRED to rank hits for subsequent clustering and to identify hits that bind to these conserved pockets. These hits were found to make interactions with highly conserved amino acid residues present within the binding pockets of the proteins. According to the experimental validation performed by the CACHE organisation, it was reported that two of our NSP3 hits showed activity in the HTRF and SPR assays. For the two hits, structural analogues were identified and retested for activity. A working protocol of Molecular Dynamics (MD) simulation and Absolute Binding Free Energy (ABFE) workflow was developed for the two NSP3 hits using GROMACS. We have also expanded the project to perform hit-to-lead optimization on the two NSP3 hits with the help of the Notre Dame-DCU Seed Grant in Precision Biomedical Technologies funding. Lastly, sequence and structural conservation analysis was performed on Group-IV viruses, primarily comparing helicases and ADP-binding macro domain proteins with SC-2 NSP13 and NSP3, respectively. We found sequence and structural similarity between the Chikungunya virus and SC-2 for helicase and ADP-binding macro domain proteins. This can thus aid in the development of a pan-antiviral inhibitor.