Structure-guided identification and characterization of a novel pyrazolo[1,5-a]pyrimidine ligand of SARS-CoV-2 main protease.

The SARS-CoV-2 main protease (Mpro) is an essential viral cysteine protease whose function relies on the structural organization of its catalytic cleft and substrate-recognition subsites. To investigate molecular recognition in this enzyme, we performed crystallographic screening using a structurally diverse collection of drug-like small molecules. This approach identified a previously unreported Mpro-ligand complex, in which a substituted pyrazolo[1,5-a]pyrimidine derivative binds in the S2 pocket of the active site, forming a distinct interaction network. Structure analysis of the co-crystal complex revealed key protein-ligand contacts governing binding specificity. Guided by the obtained structure, a focused series of analogues was synthesized to probe the role of heterocyclic substituents in molecular recognition. Microscale thermophoresis and FRET-based assays confirmed direct binding to recombinant Mpro and supported structure-based interpretation of ligand interaction. Notably, structure-guided optimization yielded a compound with submicromolar affinity (KD = 0.2 μM), representing a substantial improvement over the initial hit. Molecular dynamics simulations further supported the stability of the interactions, contributing to effective ligand recognition beyond static docking poses. Our study expands the structural repertoire of small-molecules binding to Mpro and provides a crystallographic and biophysical basis for understanding ligand recognition in coronavirus main proteases.