Evidence for Substrate Binding-Induced Zwitterion Formation in the Catalytic Cys-His Dyad of the SARS-CoV Main Protease

Abstract

The coronavirus main protease (M(pro)) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M(pro) and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M(pro) active site that lead to the Cys(-)/His(+) zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate-enzyme and inhibitor-enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M(pro) employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured pKa values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M(pro) activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M(pro) toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors.

The crystal structure of main protease from mouse hepatitis virus A59 in complex with an inhibitor

“…On the other hand, the regulation of M pro activity was investigated to gain a deeper understanding of the cleavage mechanism. First, it was found that the protease activity of M pro could be related to its homodimerization in some way [15,16]. Then, long-distance communication was identified as a temperature-sensitive defect mutant in V184 or F219 could be readily recovered by a second-site mutation (S133 N or H134Y) [17,18].…”

Rapid Identification of Potential Inhibitors of SARS‐CoV‐2 Main Protease by Deep Docking of 1.3 Billion Compounds

“…Concurrently, CoV infected patients administered with protease inhibitors, lopinavir/ritonavir, have shown improved outcome, [1,13] demonstrating the potential of the main protease (Mpro) as the most promising drug target in CoVs [14,15] Hence, a recently published X-ray crystal structure of the SARS-CoV-2 Mpro provides an excellent ground for structure-based drug discovery efforts. [16] Earlier efforts to target SARS-CoV resulted in identification of several covalent Mpro inhibitors targeting the catalytic dyad of the protein defined by His41 and Cys145 [17] residues. However, covalent inhibitors are often marked by adverse drug responses, off-target side effects, toxicity and lower potency.…”

Structural basis for the development of SARS 3CL protease inhibitors from a peptide mimic to an aza‐decaline scaffold

“…Paasche et al have pointed out that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton‐transfer reaction based on MM/MQ analysis of SARS 3CL pro . Unfortunately, our method of study was incapable of accessing this charge state.…”