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.

The development of targeted agents for APN has been greatly hindered by two factors -the incomplete understanding of APN substrate specificity and, until recently, the paucity of high-resolution atomic structures. Here, we have presented the most comprehensive examination of the substrate specificity of APN to date and detailed the key structural interactions that dictate this specificity. By understanding the molecular architecture of APN at the atomic level and investing its specificity with our global profiling technique, we were able to rationally design peptide inhibitors of its enzymatic activity. The S1 pocket of APN has hydrophobic walls with an open end resulting from the presence of Met349, Phe467 and Tyr472. As predicted from the S1 architecture, the P1 specificity of APN favored hydrophobic residues. Amino acids with large positively charged side chains (such as arginine and lysine) are also tolerated as substrates because their side chains can form hydrophobic and polar interactions with aforementioned residues in the S1 pocket. In contrast, acidic residues (glutamic acid and aspartic acid) and short-branched residues (such as asparagine and proline) are strongly disfavored in the P1 position due to charge and VDW repulsion, respectively.