Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to fit its S 1 , S 1 ', and S 2 ' subsites. This analysis confirmed that the S 1 subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S 1 ' subsite is hydrophobic whereas the S 2 ' subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H 3 N + CH(CH 2 CH 2 SO 3 - )CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K i of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II and cholecystokinin CCK 8 in the central nervous system.