Bestatin is a non-competitive inhibitor of porcine M1 family glutamyl aminopeptidase: Insights for selective inhibitor design

Abstract

Glutamyl aminopeptidase (APA) is an M1 family membrane-bound ectoenzyme that is a target for the development of antihypertensive and anticancer agents. Bestatin is a natural product described as a classical inhibitor of metallo-aminopeptidases. Although the IC 50 value of bestatin vs human APA has been reported, the mechanism of inhibition is unknown. In the present contribution, we demonstrated that bestatin is a non-competitive (α>1) inhibitor of porcine APA (pAPA), with a K i value of 31.59 µM (α=3.7). A model of the bestatin-pAPA complex predicted that bestatin binds to pAPA similarly to porcine aminopeptidase N (pAPN). The interaction involved catalytic and chelating residues conserved in the M1 family. Additionally, a salt bridge with R877 and a hydrogen bond interaction with T346, both key residues for APA specificity for N-terminal acidic residues were identified. These residues and E213, which forms a hydrogen bond interaction with bestatin, are not conserved in human and porcine APN. The extension of the in silico analysis to amastatin and bestatin analogs probestin, and phebestin, which are APA inhibitors, indicated that they may interact with the same residues. The results indicate that bestatin analogues currently reported to inhibit APN are dual inhibitors of APA and APN and that some APA residues could be targeted to improve inhibitor selectivity.