Of all isoforms, human carbonic anhydrase II (PF00194; EC 4.2.1.1), which is mostly found in red cells, kidneys, and the eyes, plays a pivotal role in numerous physiological processes, and its dysregulation has been linked to the wide range of illnesses, such as glaucoma. Finding new inhibitors that target carbonic anhydrase II, therefore has great potential in drug discovery. Using drug repurposing approach, this study focused on the investigation of different drugs as Carbonic anhydrase II inhibitors and their structural studies using X-ray crystallography. For this purpose, 100 different drugs were evaluated for bovine and human carbonic anhydrase II inhibitory activity. Among all, two drugs, i.e. acetohexamide (1) and levosulpiride (54) were found to be active, with IC50 = 437.0 ± 0.2 and 1128 ± 0.75 μM, respectively. Mechanistic studies suggested that both drugs are competitive inhibitors of the human carbonic anhydrase II enzyme. The X-ray crystal structure analysis revealed that acetohexamide (1) interacts via terminal acetyl group with the active site residues of the carbonic anhydrase II enzyme, and showed strong hydrogen bonding with Zn, His94, His119, and Asn67. The sulfonamide group of levosulpiride was involved in strong hydrogen bonding with Zn, His94, His119, and Thr199. From in vivo studies, we found that carbonic anhydrase activity was significantly inhibited by the intraperitoneal administration of levosulpiride for up to 5 h. Our findings provide comprehensive insights for the optimization of the pharmacological profile of these drugs, and provide avenues for the exploration of different derivatives of these drugs with enhanced efficacy and fewer adverse effects.
