The Rational Design of Therapeutic Peptides for Aminopeptidase N using a Substrate-Based Approach

Joshi, Shilvi; Chen, Lang; Winter, Michael B.; Lin, Yi-Lun; Yang, Yang; Shapovalova, Mariya; Smith, Paige M.; Liu, Chang; Li, Fang; LeBeau, Aaron M.

Our investigation into the substrate specificity of APN underscored the utility of the MSP-MS assay over conventional techniques for determining aminopeptidase specificity. A number of limitations exist when using single-amino acid chromogenic or fluorogenic substrates to determine aminopeptidase specificity with P1′ reporter groups, as an example, potentially altering the P1 specificity profile. In addition to reporting on prime-side specificity, our global approach also allowed for the identification of individual synthetic peptides that are bona fide substrates and served as a template for APN inhibitor design. APN has emerged as important pericellular protease target in aggressive cancer. We envision that the substrate specificity preferences identified here will both guide the development of next-generation APN-targeted therapeutics as well as aid in the discovery of its endogenous substrates to further expand our knowledge of its role in biology.