Multiple redox switches of the SARS-CoV-2 main protease in vitro provide new opportunities for drug design

Abstract

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for stopping the current COVID-19 pandemic and preventing future outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

Mechanisms of Cysteine‐Lysine Covalent Linkage—The Role of Reactive Oxygen Species and Competition with Disulfide Bonds**

“…[11] At supraphysiological concentrations of H 2 O 2 , 20 mM, NOS bridges were no longer detected due to over-oxidation. [12] The formation of such a cross-link requires the oxidation of both Cys and Lys residues. Cys residues are broadly known to oxidise through the thiol moiety leading to a wide variety of oxidation products such as the thiyl radical, the disulfide bond or the sulfenic acid under mild oxidation conditions or the sulfinic acid under higher oxidant concentrations.…”

In Solution Identification of the Lysine–Cysteine Redox Switch with a NOS Bridge in Transaldolase by Sulfur K-Edge X-ray Absorption Spectroscopy

“…9 Further studies have identified the NOS switch in crystal structures across a variety of systems and organisms including SARS-CoV-2, potentially playing an important role in regulation, cellular defense, and replication. 10,11 Moreover, computational insights have also been laid out on exploratory reaction mechanisms of the formation of the NOS bridge. 12 Even though the NOS bridge's chemical identity was clearly shown by protein crystallography at sub-ångstrom resolution, its presence in solution has so far never been directly demonstrated.…”

“…This causes a reconfiguration of key catalytic residues evoking an increase in enzymatic activity by several orders of magnitude . Further studies have identified the NOS switch in crystal structures across a variety of systems and organisms including SARS-CoV-2, potentially playing an important role in regulation, cellular defense, and replication. , Moreover, computational insights have also been laid out on exploratory reaction mechanisms of the formation of the NOS bridge …”