Structure‐Activity Studies Reveal Scope for Optimisation of Ebselen‐Type Inhibition of SARS‐CoV‐2 Main Protease

Abstract The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID‐19) and other diseases. We report structure‐activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) main protease (Mpro), employing turnover and protein‐observed mass spectrometry‐based assays. The results reveal scope for optimisation of ebselen/ebselen derivative‐ mediated inhibition of Mpro, particularly with respect to improved selectivity.

Application of General Procedure B was followed using triethylamine (167 µL, 1.

General Information
M pro assays were performed exclusively using freshly purified recombinant M pro solution, prepared as reported. [16] Refrozen M pro samples exhibited reduced activity and were not used.
LCMS (Merck, Supelco) grade solvents were used for MS analyses and to prepare buffers. The peptide substrate and M pro solution were diluted in freshly prepared reaction buffer (defined below) to prepare stock solutions. Compound stock solutions were prepared to 10mM in DMSO and diluted using the reaction buffer.

Fluoroscence Assays
The inhibitory activities of compounds 1-12 against M pro were investigated using a spectroscopic assay, monitoring M pro catalysed hydrolysis of the fluorogenic substrate ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide) as reported. [17] In brief, assays were conducted 10% glycerol, 1% DMSO, 0.01% v/v Triton X-100. The initial rates of reaction (measured after 15 min pre-incubation of the inhibitor with the enzyme) were assessed by monitoring the fluorescence intensity at λex = 485 nm and λem = 520 nm. Following the determination of initial rates of reaction, data were fitted using a four-parameter function: log (inhibitor) vs. response, variable slope in GraphPad Prism 5 to obtain IC50 values.

Solid-Phase Extraction Coupled to MS Assays
The inhibitory activity of compounds 1-12 against M pro was also investigated using a RapidFire (RF) 365 high-throughput sampling robot (Agilent) connected to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight (Q-TOF) mass spectrometer as reported. [16] In brief, processed as reported. [16] Protein Observed Mass Spectrometry The reactions of compounds 1-12 with recombinant M pro were investigated using protein observed mass spectrometry coupled to solid phase extraction purification as reported. [16] In brief, the compounds were dispensed across 384 well plates using an acoustic Echo Dispenser