BRD4354 is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease

Numerous organic molecules are known to inhibit the main protease (M Pro ) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of M Pro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of M Pro . The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against M Pro with a IC 50 concentration, that inhibits activity by 50%, of 0.72 0.04 M after 60 min of incubation. The inactivation follows a two-step process with an initial rapid binding step with a K I of 1.9 0.5 M followed by a second slow inactivation step, k inact,max of 0.040 0.002 min 1 . Native mass spectrometry studies indicate that the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of M Pro . Based on these data, a Michael-addition reaction mechanism between M Pro C145 and BRD4354 was proposed. These results encourage further preclinical testing of BRD4354 and investigation of structural analogs of BRD4354 as COVID-19 therapeutics.Graphical Abstract