Detailed Insights into the Inhibitory Mechanism of New Ebselen Derivatives against Main Protease (M(pro)) of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)

[Image: see text] SARS-CoV-2 main protease (M(pro)/3CL(pro)) is a crucial target for therapeutics, which is responsible for viral polyprotein cleavage and plays a vital role in virus replication and survival. Recent studies suggest that 2-phenylbenzisoselenazol-3(2H)-one (ebselen) is a potent covalent inhibitor of M(pro), which affects its enzymatic activity and virus survival. Herein, we synthesized various ebselen derivatives to understand the mechanism of M(pro) inhibition by ebselen. Using ebselen derivatives, we characterized the detailed interaction mechanism with M(pro). We discovered that modification of the parent ebselen inhibitor with an electron-withdrawing group (NO(2)) increases the inhibition efficacy by 2-fold. We also solved the structure of an M(pro) complex with an ebselen derivative showing the mechanism of inhibition by blocking the catalytic Cys145 of M(pro). Using a combination of crystal structures and LC–MS data, we showed that M(pro) hydrolyzes the new ebselen derivative and leaves behind selenium (Se) bound with Cys145 of the catalytic dyad of M(pro). We also described the binding profile of ebselen-based inhibitors using molecular modeling predictions supported by binding and inhibition assays. Furthermore, we have also solved the crystal structure of catalytically inactive mutant H41N-M(pro), which represents the inactive state of the protein where the substrate binding pocket is blocked. The inhibited structure of H41N-M(pro) shows gatekeeper residues in the substrate binding pocket responsible for blocking the substrate binding; mutation of these gatekeeper residues leads to hyperactive M(pro).