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)

Abstract

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 .

The impact of SARS-CoV-2 3CL protease mutations on nirmatrelvir inhibitory efficiency. Computational insights into potential resistance mechanisms

“…7 Due to the fact that none of the known human proteases use this recognition sequence, 3CL pro has attracted considerable attention as a target for the development of inhibitors that can act as antivirals with reduced side effects. This has motivated continuous efforts to look for adequate inhibitors of the protease to be used as potential drugs, using both experimental [8][9][10][11][12][13][14] and computational approaches. [15][16][17][18][19] Many of these compounds are covalent inhibitors that contain an electrophilic site able to react with the catalytic cysteine of 3CL pro , Cys145, mimicking the behavior of the peptidic substrate.…”

New insights in the mechanism of the SARS-CoV-2 Mpro inhibition by benzisoselenazolones and diselenides

“…At the same time, in an attempt to resolve the co-crystal structure with the protein, the authors reported that these compounds were able to transfer a hydrogenselenide unit to Cys145 through a S N Ar-like reaction taking place in the M pro active site 22 . A related study was carried out by Kumar et al, in which it was also proved the selenylation of the very same cysteine also by other benzisoselenazolones 23 . Recently, Rana et al reported a series of ebselen close analogs with anti-M pro activity, with compounds 1d and 1e being the best in class.…”

Discovery of the covalent SARS‐CoV‐2 Mpro inhibitors from antiviral herbs via integrating target‐based high‐throughput screening and chemoproteomic approaches

“…13 Among these, Cys145 and Cys44, located in the catalytic pocket of the enzyme, are particularly important for its function. [14][15][16] They form the catalytic dyad that cleaves the viral polyproteins into smaller functional proteins necessary for viral replication. In addition, Cys156 and Cys300 play important roles in forming enzymatically active dimeric forms of the protein.…”

“…The SARS‐CoV‐2 M pro , also known as 3‐chymotrypsin‐like proteases (3CL pro ), is a canonical cysteine protease that contains 12 cysteine residues per monomer 13 . Among these, Cys145 and Cys44, located in the catalytic pocket of the enzyme, are particularly important for its function 14–16 . They form the catalytic dyad that cleaves the viral polyproteins into smaller functional proteins necessary for viral replication.…”