Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols.

Aminopeptidase A (APA) is a highly selective peptidase, which cleaves the N-terminal Glu or Asp residues of biologically active peptides, and has therefore been proposed to be involved in angiotensin II and CCK8 metabolism. Highly potent and selective APA inhibitors are consequently required to study the physiological regulation of these two peptides. Using, as a model, Glu-thiol (4-amino-5-mercaptopentanoic acid), which was the first efficient APA inhibitor described but is however equipotent on APA (0.14 microM) and aminopeptidase N (APN) (0.12 microM), several beta-amino thiol inhibitors have been synthesized. In these molecules, the length of the side chain was varied and the carboxylate group of Glu-thiol was replaced by other negatively charged groups, such as phosphonate, sulfonate, hydroxamate, and thiol. The inhibitory potency of one of these compounds, 22h (S)-3-amino-4-mercaptobutanesulfonate, was found to be nearly 100-fold better for APA than for APN, with an affinity (0.29 microM) almost equivalent to that of Glu-thiol. Hence, this compound is the first selective APA inhibitor reported, and as such, it should be an interesting probe to explore the physiological involvement of APA in the metabolism of neuropeptides like angiotensin II and CCK8.

Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors.

Aminopeptidase A (APA) and aminopeptidase N (APN) are two metallopeptidases which have been suggested to be involved in the enzymatic cascade of the renin-angiotensin system. APA liberates angotensin III from angiotensin II by releasing the N-terminal aspartate, and APN participates in the inactivation of angiotensin III. As the role of angiotensin III in the regulation of blood pressure in the central nervous system and at the periphery is controversial, it was of interest to develop selective and efficient inhibitors of APA. Starting from Glu-thiol(1), which was the first efficient APA inhibitor described, but however is equipotent on APA (Ki = 0.14 microM) and APN (Ki = 0.12 microM), beta-amino thiols bearing various carboxyalkyl chains have been synthesized and their inhibitory potencies measured on both purified enzymes. Compounds containing a carboxylated aromatic ring inhibited APA and APN with Ki values in the micromolar range but were slightly more active on APA. Conversely, inhibitors containing a cyclohexyl ring were more efficient on APN. Various modifications of the structure of Glu-thiol decreased inhibitory activity on both enzymes but increased the selectivity for APA, and compound 9d ((S)-4-amino-6-mercaptohexanoic acid) was 23 times more potent on APA (Ki = 2.0 microM) than on APN (Ki = 45 microM).