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 an IC₅₀ (concentration that inhibits activity by 50%) of 0.72 ± 0.04 μM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a K_I of 1.9 ± 0.5 μM followed by a second slow inactivation step, k_inact,max of 0.040 ± 0.002 min^-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where 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 suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.