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 deIS-Gylation of host proteins. This dual role makes PLpro an attractive antiviral target, either as a monother-apy 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 ISG15, and the 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), 67 (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, strength-ened binding affinity, and translated into superior antiviral potency compared with benchmark compound 4. Collectively, these findings identify GL-612, GL-508, and GL-637 as first-in-class covalent PLpro in-hibitors that uniquely combine Glu167 engagement with BL2 groove occupancy, providing a strong foundation for broad-spectrum coronavirus therapeutic against current and emerging coronaviruses.