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 define the key prime-side interactions accounting for the inhibition of these HSPW-containing sequences, the peptide LHSPW was modeled into our pAPN crystal structure (Fig. 4A ). For modeling purposes, the P1 residue was selected as leucine since we had solved a crystal structure with that residue in the P1 position -we were unable to get a crystal structure of norleucine -and leucine was the second most favored P1 residue in our MSP-MS assay. The first three residues (Leu1-His2-Ser3) were modeled from a poly-alanine structure and last two residues (Pro4-Trp5) were from the structure of pAPN complexed with substance P, which also contains a proline at the 4th position (PDB ID: 4HOM). As predicted from the P1 structural data, the side chain of Leu1 in both peptides points into a hydrophobic pocket surrounded by APN residues M349, Q208, and F467 (Fig. 4B) . The side chain of His2 forms a hydrogen bond with the main chain carbonyl oxygen of pAPN residue A346. Pro4 forms a turn in the peptide, whereas the side chains of Ser3 and Trp5 both form VDW interactions with APN residue Y472 (Fig. 4C) .