Hu, A.; Bailey, J. S.; Spina, S. C.; Rajagopal, G.; Phan, N.; Kimmel, B. R.

Targeted covalent inhibitors are a powerful, yet underexplored, class of therapeutics, and current computational covalent screeners are constrained in early drug discovery due to the need for prior knowledge of the target site and limited throughput. We present CovSite, a blind covalent screening tool that identifies candidate reactive residues across the entire protein surface, utilizing only the protein structure and electrophile SMILES. CovSite applies a pipeline of four orthogonal physicochemical filters (nucleophile identification, solvent accessibility, environment-dependent deprotonation prediction, and semi-quantum-mechanical reactivity ranking) to identify potential small-molecule candidate inhibitors. Validated against 2,062 diverse covalent protein-ligand complexes spanning six nucleophilic residue types, CovSite achieves a 98.5% blind target site hit on a held-out benchmark set of 207 cysteine-targeted complexes while reducing the search space by 97.8%. The target-site hit detection exceeds the 53-62% accuracy of popular covalent screening tools operating under non-blind conditions on the same benchmark set. By extending nucleophilic coverage beyond cysteine to include serine, threonine, lysine, histidine, and tyrosine, and completing a screening of a 200-residue protein in two to three minutes on standard hardware, CovSite serves as a platform technology with the potential to address critical gaps in throughput, generalizability, and accuracy in this field of covalent screening. We demonstrate this capability by using CovSite as a blind, ligand-specific approach that enables iterative, machine-learning-driven covalent inhibitor generation that is impractical with existing tools, establishing a foundation for computationally guided covalent drug discovery for novel and understudied targets.