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 active site of APN is expansive with openings on three sides allowing for the accommodation of large peptide substrates. Its exclusive mono-aminopeptidase activity is the direct result of hydrogen bonds formed between active site residues Gln208, Glu350 and Glu406 with the N-terminal amine group of the peptide substrate. Di-aminopeptidase activity is not observed because favorable hydrogen bonds cannot be made to anchor the peptide in the proper orientation for bond hydrolysis. The importance of these hydrogen bonds in peptide binding was also evident in our inhibitor study in which the N-termini of HSPW peptides were capped with different groups. By removing one or more potential hydrogen bonds from the N-terminus, the capped peptides were unable to bind, resulting in poor inhibition. In addition to these P1 residues, our global analysis revealed previously uncharacterized prime-side specificity features that were dictated by hydrophobic interactions in addition to hydrogen bonding with main chain groups. These important interactions were evident when the HSPW core sequence of our lead inhibitor was truncated. The removal of Trp5 or both Pro4 and Trp5 abrogated the ability of the peptide to inhibit APN. The critical interactions these two residues make with the active site were observed in our modelling experiment documenting that both residues form VDW interactions with Y472. The end result of our study was a cyclic peptide, cyc-LHSPW that was a potent and specific inhibitor of APN. By inhibiting the enzymatic activity of APN, we found that cyc-LHSPW was therapeutic both in vitro and in vivo in a model of highly aggressive neuroendocrine prostate cancer. With this aggressive form of cancer on the rise, it is highly plausible the future treatments for neuroendocrine prostate cancer could involve targeting APN 30 .