Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

Abstract

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (M^pro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent M^pro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in M^pro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger M^pro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of M^pro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.

Fig. 1. Chemical structures, in vitro M^pro inhibition and cell-based antiviral assays of ebselen and five derivatives.

In vitro M^pro inhibitory curves of a ebselen, b MR6-7-2, c MR6-17-1, d MR6-18-4, e MR6-26-2 and f MR6-31-2. Inhibition percentage plots are means of n = 3 measurements obtained over three independent experiments and error bars representing the standard error of the mean. g IC₅₀s of M^pro inhibition and EC₅₀s of viral replication in Vero E6 cells. IC₅₀s and EC₅₀s are means (standard error of log(concentration)).

Fig. 2. Crystallographic structures of ligand-free M^pro and the complexes with ebselen and MR6-31-2.

a Cartoon representation of superimposed structures of ligand-free M^pro (magenta), M^pro-ebselen (cyan) and M^pro-MR6-31-2 (green). The M^pro catalytic site is highlighted in a black box and the global root-mean-square derivations (RMSDs) of the compound-treated structures to ligand-free M^pro are given in blue texts. Close-up views of catalytic site of b ligand-free M^pro, c M^pro-ebselen and d M^pro-MR6-31-2. Electron density (2F_o–F_c) map is shown as grey mesh at 1σ. Anomalous signal of selenium is shown as purple mesh at 3σ. Selenium atom, conserved water and other waters are shown as orange, blue and red spheres, respectively. The close contacts below 2.5 Å and hydrogen bonds are shown as yellow and light blue dashes, respectively. The distances are illustrated by black double-headed arrows.

Fig. 3. Representative chromatograph for salicylanilide standard and its formation in the incubation of M^pro with ebselen.

a The peak at 6.36 min retention time corresponds to salicylanilide standard (85.2 ng/mL). b Salicylanilide was detected in the incubation of M^pro with ebselen. c MS/MS spectrum shows the characteristic fragments derived from salicylanilide. d Time-course of salicylanilide formation. Bar chart represents means of n = 3 measurements obtained over three independent experiments and error bars representing the standard error of the mean.

Fig. 4. Chemical mechanism for selenation of M^pro cysteine 145 by ebselen.

His41 assists a water-mediated attack on intermediate 2 leading to a hydrolysis reaction akin to peptide hydrolysis leading to the generation of the hydrolysis product 4.