Background
SARS-CoV-2 papain-like protease (PLpro) represents a promising target for novel COVID-19 therapeutics due to its essential roles in viral replication and dysregulation of host innate immunity. PLpro is a domain of SARS-CoV-2 non-structural protein 3 (Nsp3) and is one of two cysteine proteases encoded by the virus. Currently, there are no SARS-CoV-2 PLpro inhibitors in clinical development.
Methods
A novel class of PLpro-binding small molecules containing a diarylmethanol substructure was identified from a DNA-encoded library (DEL) screen. The original PLpro DEL binders and their analogs were evaluated in biochemical assays to assess inhibition of PLpro enzymatic function, and in virological assays to characterize antiviral potencies. Lead molecules were also studied in biophysical assays including X-ray crystallography and 2D-NMR to determine their mechanisms of action for viral intervention.
Results
This chemical series demonstrates activity in biochemical and virological SARS-CoV-2 assays in vitro, with the most potent compound having a biochemical IC50 of 271 nM and antiviral EC50 of 33 nM. Two X-ray co-crystal structures revealed that these compounds engaged PLpro through a novel binding mode distinct from that of GRL0617, a known SARS-CoV/SARS-CoV-2 PLpro inhibitor that binds to the BL2-loop region of monomeric PLpro protein. Specifically, these compounds simultaneously engage the BL2-loop region of one PLpro monomer and a cryptic pocket between the Ubl/Thumb domain of another monomer inducing dimerization. This dimer-inducing mechanism of action was verified to be concentration-dependent in solution using analytical SEC and 1D-NMR suggesting these inhibitors act as molecular glues.
Conclusions
There is a great interest in the discovery of inhibitors for SARS-CoV-2 PLpro to complement currently available SARS-CoV-2 therapeutics. In this study, we report the discovery of a class of small molecule inhibitors that targets SARS-CoV-2 PLpro through the novel mechanism of PLpro dimerization. These compounds are proposed to mediate inhibition through the molecular glue-induced self-association of PLpro. This mechanism is different from those of other PLpro inhibitors which mediate inhibition through a single interaction with monomeric PLpro near the substrate binding pocket of the enzyme. Taken together, this class of inhibitors represents a novel and attractive chemical series for potent SARS-CoV-2 inhibition.
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