Novel Inhibitors of Carbonic Anhydrase I and II Identified by High Throughput Virtual Screening and Validated by X‐Ray Crystallography

High-throughput virtual screening (HTVS) enables rapid exploration of large chemical spaces to identify novel modulators of biologically relevant targets. In this study, we applied a large-scale docking campaign to discover new inhibitors of the human Carbonic Anhydrase isoforms I and II. Starting from library of 28,000 compounds, iterative Glide HTVS, SP, and XP docking against hCA I and hCA II yielded 81 top-ranked compounds for experimental evaluation. Among these, five compounds showed micromolar inhibition of hCA II, and one also inhibited hCA I. X-ray crystallographic studies of two carboxylate-based inhibitors (1 and 2) revealed distinct binding modes. Compound 1 directly coordinates the catalytic zinc ion, whereas compound 2 adopts two different binding poses: one anchored to the zinc-bound water molecule and another within a surface-accessible pocket near His64. The latter region, which modulates the proton-shuttling mechanism essential for catalysis, represents a less-explored site for inhibitor development. Overall, these findings validate our HTVS workflow and provide a foundation for the structure-guided optimization of carboxylate scaffolds as carbonic anhydrase inhibitors.