Discovery of SARS-CoV-2 PLpro covalent inhibitors enabled by a novel reversible covalent chloroalkyne warhead

Developing covalent inhibitors of the SARS-CoV-2 papain-like protease (PLpro) represents a promising therapeutic strategy, but translation of this approach into in vivo antiviral efficacy remains limited. A major challenge is the shallow and minimally druggable binding environment surrounding the catalytic cysteine, which reflects the enzyme's stringent requirement for P1 and P2 Gly-Gly recognition. In addition, many previously reported covalent PLpro inhibitors have relied on warheads or linker designs with limited metabolic stability or nonspecific thiol reactivity, restricting their progression toward in vivo efficacy. Here, we report the design and synthesis of a novel class of covalent PLpro inhibitors with low intrinsic GSH reactivity, enabled by a chloroalkyne warhead and an optimized linker strategy. The lead compound, ID3-77 (12), potently inhibits PLpro biochemically, demonstrates strong cellular antiviral activity, reduces viral load in a SARS-CoV-2 infection model, and exhibits minimal glutathione labeling. Jump-dilution experiments demonstrate reversible covalent inhibition, while profiling against the cysteine proteases cathepsin B and calpain demonstrates selectivity over these related proteases. Together, these findings establish chloroalkyne as a promising target-directed reversible covalent warhead for PLpro inhibition and provide in vivo proof-of-concept for this chemistry in antiviral drug discovery, with potential for broader application to other cysteine proteases.