Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery

Abstract

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre–steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre–steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

Multiscale Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms

“…The rate constant of the second step is ∼1 s −1 ; this step was attributed to the tighter binding of the inhibitor leading to the increase in the fluorescence intensity. 79 The subsequent reaction step, which can be attributed either to a chemical reaction or to conformational changes following the covalent adduct formation, was observed only for the wild-type enzyme, and it is 5 times slower than the preceding non-covalent binding step. We also note that the reported k cat values for M Pro reactions are predominantly derived from steady-state experiments and refer to either the deacylation step or product release.…”

“…As far as comparisons with the experiment is concerned, unfortunately, the experimental kinetic data (i.e., rate constants of individual steps in reactions of M Pro with substrates and covalent inhibitors) are limited. According to the pre-steady-state kinetics study of M Pro from SARS-CoV-2 and SARS-CoV, the acylation step proceeds efficiently and is unlikely to be responsible for the overall steady-state rate constant k cat . These conclusions are supported by computational studies of the peptide bond hydrolysis in the active site of M Pro . ,, The only pre-steady-state kinetic data for the formation of the covalent M Pro -inhibitor complex are reported in ref for the fluorophore-containing compound PF-00835231.…”

“…For both variants of the protein, the non-covalent binding occurs via two steps. The rate constant of the second step is ∼1 s –1 ; this step was attributed to the tighter binding of the inhibitor leading to the increase in the fluorescence intensity . The subsequent reaction step, which can be attributed either to a chemical reaction or to conformational changes following the covalent adduct formation, was observed only for the wild-type enzyme, and it is 5 times slower than the preceding non-covalent binding step.…”

“…We also note that the reported k cat values for M Pro reactions are predominantly derived from steady-state experiments and refer to either the deacylation step or product release. 79,80 ■…”

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro

“…To quantify the activity of SARS-CoV-2 proteases by means of fluorescence, which is ideal for high-throughput screening, several assays have been developed [ 9 ]. The simplest in vitro test for purified proteases is based on recognition site-bearing short peptides labeled with two fluorophores in such a way that upon cleavage the Forster resonance energy transfer (FRET) between them disappears [ 10 , 11 ]. This assay enables the careful quantification of enzymatic activity.…”