Discovery of Subnanomolar and Highly Selective Carbonic Anhydrase II Inhibitors Featuring L-Shaped Scaffolds via Active-Site-Matched Fragment Growing

Glaucoma is a leading cause of irreversible blindness worldwide, and lowering intraocular pressure (IOP) remains the only proven method to delay progression. While topical carbonic anhydrase (CA) inhibitors are established treatments, more potent and selective agents are needed. We report a series of subnanomolar and highly selective human carbonic anhydrase (hCA) II inhibitors developed through a multistage fragment growth strategy. By incrementally expanding a weakly bound sulfanilamide anchor to precisely complement the enzyme's topology, we achieved a remarkable 15,000-fold increase in potency, resulting in subnanomolar inhibition and exceptional selectivity. High-resolution X-ray crystallography revealed a distinct L-shaped configuration, effectively occupying the entire active site and establishing unique interactions with a peripheral α-helix. In rabbit glaucoma models, these inhibitors demonstrated superior IOP-lowering activity. Furthermore, the inhibitors exhibited exceptional metabolic stability, minimal ocular toxicity, and favorable safety profiles. These results demonstrate multistage fragment growth as a robust strategy for designing next-generation glaucoma therapeutics.