Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15

Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[ 1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. R educing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residu e immediately adjacent to the tyrosine with alpha-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a K-i of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolys in-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was inv estigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubati on with all tissues examined. Further modifications to the JA-2 compound fa iled to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24 .15-preferential and biologically stable inhibitor, therefore providing a v aluable tool for further assessing the biological functions of EP24.15.