Structure-guided design of potent and selective covalent inhibitors of SARS-CoV-1 and SARS-CoV-2 papain-like proteases with antiviral activity.

The papain-like protease (PLpro), one of two essential cysteine proteases in human coronaviruses, is indispensable for viral polyprotein processing and immune evasion through deubiquitination and deISGylation of host proteins. This dual role makes PLpro an attractive antiviral target, either as a monotherapy or in combination with other direct-acting antivirals. Guided by structural insights, we designed and synthesized covalent inhibitors that exploit two key binding elements: Glu167 in the α-cleft (Site I), which mediates recognition of ubiquitin and interferon-stimulated gene (ISG15), and the blocking loop 2 (BL2) hydrophobic groove, which stabilizes inhibitor binding and shapes selectivity. The optimized inhibitors exhibited potent enzymatic inhibition (IC50 = 25-100 nM), strong protein stabilization (ΔTm > 20 °C), and submicromolar antiviral activity in SARS-CoV-2-infected A549-ACE2 cells (EC50 = 130-350 nM), with no measurable cytotoxicity (CC50 > 10 μM). Lead compounds 8 (GL-612), 9 (GL-508), and 10 (GL-637) efficiently inactivated PLpro, with GL-637 achieving a kinact/KI > 18,000 M-1 s-1, and demonstrated selectivity by sparing host deubiquitinases USP7 and USP14 at concentrations up to 30 μM. Docking and mechanistic analyses revealed that cyclic amine-substituted benzamides reinforced electrostatic interactions with Glu167, while the (S)-3-fluoropyrrolidin-1-methyl-phenylthiophene scaffold anchored the BL2 groove, disrupting recognition of ubiquitin and ISG15 substrates. This dual engagement strategy improved covalent efficiency, strengthened binding affinity, and translated into superior antiviral potency compared with benchmark compd. 7 (4). Collectively, these findings establish GL-612, GL-508, and GL-637 as valuable structural templates for developing novel PLpro inhibitors that uniquely target Glu167 and the BL2 groove, providing a strong foundation for broad-spectrum coronavirus therapeutics against current and emerging coronaviruses.