Crystallographic fragment screening discovers novel micromolar active inhibitors and druggable hotspots of SARS-CoV-2 PLpro.

The COVID-19 pandemic has highlighted the need to develop broad-spectrum antiviral therapeutics targeting rapidly evolving coronaviruses. This research focuses on SARS-CoV-2 PLpro, a conserved viral protease that plays dual roles in viral polyprotein processing and host immune suppression. Using an integrated fragment-based drug discovery (FBDD) approach that combines high-throughput X-ray crystallography and biochemical assays, we systematically screened a diverse library of 800 fragment compounds. Structural characterization identified 129 validated binders occupying 12 distinct binding sites on PLpro. Remarkably, two fragments demonstrated potent micromolar inhibitory activity. Fr12895 inhibited SARS-CoV-2 PLpro protease activity (IC50 = 8.013 μM), as measured using the fluorogenic substrate RLRGG-AMC, while Fr12338 showed inhibition against PLpro deISGylase activity (IC50 = 4.5 μM), as determined with the substrate Ac-ISG15prox-Rh110MP. This provides a case for directly obtaining micromolar-active compounds through crystallographic fragment screening of a small number of random compounds. Detailed structural analysis revealed these fragments engage key functional regions including the blocking loop 2 (BL2) and ubiquitin/ISG15 binding interface through extensive hydrogen-bond networks and hydrophobic interactions. Our study reveals novel micromolar active inhibitors and druggable sites of SARS-CoV-2 PLpro by crystallographic fragment screening, provides crucial scaffold and a structural roadmap for developing broad-spectrum antivirals against coronaviruses.