Discovery and Mechanism Study of SARS-CoV-2 3C-like Protease Inhibitors with a New Reactive Group

Abstract

3CL pro is an attractive target for the treatment of COVID-19. Using the scaffold hopping strategy, we identified a potent inhibitor of 3CL pro (3a) that contains a thiocyanate moiety as a novel warhead that can form a covalent bond with Cys145 of the protein. Tandem mass spectrometry (MS/MS) and X-ray crystallography confirmed the mechanism of covalent formation between 3a and the protein in its catalytic pocket. Moreover, several analogues of compound 3a were designed and synthesized. Among them, compound 3h shows the best inhibition of 3CL pro with an IC 50 of 0.322 μM and a k inact /K i value of 1669.34 M −1 s −1 , and it exhibits good target selectivity for 3CL pro against host proteases. Compound 3c inhibits SARS-CoV-2 in Vero E6 cells (EC 50 = 2.499 μM) with low cytotoxicity (CC 50 > 200 μM). These studies provide ideas and insights to explore and develop new 3CL pro inhibitors in the future.

On the origins of SARS-CoV-2 main protease inhibitors

“…It would be beyond the scope of this review to depict all the series of peptide-derived inhibitors made, especially since 2019 an even larger array of warheads has been incorporated in such compounds. 184 Indeed, a non-exhaustive list used in the last few decades includes: aldehydes (or the corresponding bisulfite adduct), 41,185–202 ketones, 203–211 α-ketoamides, 153,185,212–214 Michael acceptors, 194,215–218 4-iminooxazolidin-2-one, 219 reactive halogens, 220–224 β-lactam of some penicillins, 225,226 phenylsulfide, 227 thiocyanate, 228 epoxide, 24 nitriles 229–231 and, last but not least, alkynes. 232,233 Interestingly, aside from a 2005 patent 234 targeting SARS-CoV-1-M pro , the design of boron-containing inhibitors, which is a fairly classic approach for protease inhibition, 235,236 has not been reported in more recent time.…”

Crystallographic Data Collection Using a Multilayer Monochromator on an Undulator Beamline at the Shanghai Synchrotron Radiation Facility

“…Crystallization was carried out at 18 • C using the hanging-drop vapor diffusion method by combining equal volumes (1:1 µL) of the protein and reservoir solution. Crystals were successfully obtained under the conditions of 25% PEG800, 100 mM MES, and pH 5.5 [23].…”

Progress in Research on Inhibitors Targeting SARS-CoV-2 Main Protease (Mpro)

“…Analysis of the frontier molecular orbitals (FMOs) and the molecular electrostatic potential (MEP) of 6a indicated that the 2,3,5-substituted [1,2,4]-thiadiazole moiety has the potential to serve as a covalent warhead, thereby enhancing its inhibitory activity against M pro . Based on the above, they also designed and synthesized inhibitor 3a using a scaffold hopping strategy with thiocyanate as a covalent warhead (Figure E). At the enzyme level, it showed good activity with an IC 50 of 2.214 μM.…”