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.

To identify sequences for inhibitor development, a selection of 14-mer peptides from the MSP-MS library that underwent complete hydrolysis during the assay were compared using label-free quantitation of peptide cleavage kinetics (Supplemental Figure 2 ). Five-residue long peptides containing P1-P4′ residues from primary cleavages in the library were synthesized and tested for their ability to prevent the cleavage of a fluorogenic alanine substrate by hAPN. From these initial peptides, only nHSPW was able to appreciably inhibit substrate cleavage with an IC 50 of 6.5 μM, whereas the remaining peptides all had IC 50 values above 500 μM ( Table 1) . Replacement of the P1 residue norleucine of nHSPW with either leucine or alanine did not have a significant impact on the IC 50 , resulting in low μM inhibitors. Complete removal of the P1 norleucine, resulting in the 4-mer peptide HSPW, only decreased the potency of the inhibitor to 43 μM.