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.