Molecular dynamics and in silico mutagenesis on the reversible inhibitor-bound SARS-CoV-2 main protease complexes reveal the role of lateral pocket in enhancing the ligand affinity

Abstract

The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and there is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (Mpro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 Mpro that resulted in hundreds of X-ray crystal structures of ligand-bound Mpro complexes in the Protein Data Bank (PDB) describing the interactions of different fragment chemotypes within different sites of the Mpro. In this work, we performed rigorous molecular dynamics (MD) simulation of 62 reversible ligand–Mpro complexes in the PDB to gain mechanistic insights about their interactions at the atomic level. Using a total of over 3 µs long MD trajectories, we characterized different pockets in the apo Mpro structure, and analyzed the dynamic interactions and binding affinity of ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets of Mpro. Our analyses unraveled the role of a lateral pocket in the catalytic site in Mpro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in the lateral pocket towards ligand binding and affinity against Mpro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and Mpro dimerization in the ligand association with the target. Thus, comprehensive molecular-level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 Mpro.

Mechanistic Insights into SARS-CoV-2 Main Protease Inhibition Reveals Hotspot Residues

“…Also, the binding pocket of the enzyme is well defined; the aim of the current study is to identify the crucial residues responsible for complex formation, and thus, employing the monomeric conformation for the investigation is reliable. A similar approach has also been reported in other studies. ,− Therefore, we believe that the protocol used in this study could provide a rough estimate of binding events with its surrounding residues and is reliable. The resulting M pro –GC-376* complex was again subjected to a second round of minimization following the aforementioned steps.…”

“…In light of identifying inhibitors specific to M pro , several attempts have been made in the past by screening chemical databases, mechanistic explorations, and mutational analysis . Nevertheless, the studies conducted by Ma et al have shown that using the fluorescence resonance energy transfer (FRET)-based enzymatic assay, several M pro inhibitors were identified with low- to submicromolar-range binding affinity values (0.030 ± 0.008 to >20 IC 50 in μM).…”

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

“…Outside of these well-studied critical M pro sites, there are additional clusters of mutation-intolerant residues. The R131, D197, N203, D289, and E290 lie at the interface of Domain II and Domain III sandwiched between dimers and make up part of a surface identified by structural modeling as a possible distal drug binding pocket ( Bhat et al, 2021 ; Weng et al, 2021 ; Figure 6b ). Within this cluster, a dynamic salt bridge is formed between R131 located on the loop of Domain II connecting β10–11 of the catalytic pocket, and D289 in the α-helical Domain III that has been reported to contribute to the flexibility and structural plasticity of M pro ( Bhat et al, 2021 ).…”

Supervised Molecular Dynamics (SuMD) Insights into the mechanism of action of SARS-CoV-2 main protease inhibitor PF-07321332

“…Meanwhile, the pyrrolidone moiety is inserted in the S1 pocket, interacting with key residues of the oxyanion loop such as Asn142, Gly143, and Ser144, before undergoing a conformational rearrangement around the 18 ns simulation time mark which allows the carbonyl of the pyrrolidone to establish a hydrogen bond with His163. This interaction has been flagged as a conserved interaction across several deposited structures of non-covalent inhibitors 25 . Moreover, this interaction is conserved across all possible substrate peptide crystal structures, where the interacting group is the sidechain of the Glutamine P1 residue 26 .…”