SARS-CoV-2 Mpro inhibitors(University of Bonn)

The COVID‐19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96‐ well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (Mpro) of SARS‐CoV‐2. Subsequent direct biological screening identifies novel 1,2,3‐triazole derivatives as robust Mpro inhibitors with high anti‐SARS‐CoV‐2 activity. Notably, C5N17B demonstrates sub‐micromolar Mpro inhibitory potency (IC50 = 0.12 µM) and excellent antiviral activity in Calu‐3 cells determined in an immunofluorescence‐based antiviral assay (EC50 = 0.078 µM, no cytotoxicity: CC50 > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC50 = 1.95 µM) and similar efficacy to ensitrelvir (EC50 = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS‐CoV‐2 variants (Gamma, Delta, and Omicron, EC50 = 0.13 – 0.26 µM) and HCoV‐OC43, indicating its broad‐spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir‐resistant strains with T21I/E166V and L50F/E166V mutations in Mpro (EC50 = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non‐covalent multi‐site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up‐and‐coming drug candidate targeting SARS‐CoV‐2 Mpro for clinical therapy.

Although the coronavirus disease 2019 (COVID-19) crisis has passed, there remains a necessity for continuous efforts toward developing more targeted drugs and preparing for potential future virus attacks. Currently, most of the drugs received authorization for the treatment of COVID-19 have exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness. It is still significant to develop novel, structurally diverse small-molecule antiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease (3CL^pro). Herein, we report a class of alkynylamide-based nonpeptidic 3CL^pro inhibitors that can be prepared conveniently by our previously developed one-pot synthetic method. The structure-activity relationships of alkynylamides as SARS-CoV-2 3CL^pro inhibitors have been carefully investigated and discussed in this study. The two stereoisomers of the resulting molecules exhibit stereoselective interaction with 3CL^pro, and the optimized compound (S,R)-4y inhibits 3CL^pro with high potency (IC₅₀ = 0.43 μM), low cytotoxicity, and acceptable cell permeability. Compound (S,R)-4y presents as a noncovalent inhibitor of 3CL^pro against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. The Lineweaver-Burk plots, binding energy, surface plasmon resonance, and molecular docking studies suggest that (S,R)-4y specifically binds to an allosteric pocket of the SARS-CoV-2 3CL^pro. These findings provide a novel class of nonpeptidic alkynylamide-based allosteric inhibitors with high selectivity against SARS-CoV-2 3CL^pro featured by a simplified one-pot synthesis at room temperature in air.