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.

In agreement with prior P1 specificity profiling using single-amino acid fluorogenic substrates 5 , hAPN displayed broad specificity at the P1 position. In particular, hAPN exhibited a significant preference for P1 hydrophobic residues (such as norleucine, leucine, tryptophan, and alanine), whereas proline, asparagine, and acidic residues (aspartic acid and glutamic acid) were significantly disfavored. We note that norleucine is used as an isostere for methionine in the MSP-MS library. Inspection of individual peptide cleavage events within the MSP-MS time course supported these overarching P1 specificity preferences with N-terminal cleavages being impaired or blocked by disfavored residues at the P1 (or neo-P1) position ( Fig. 1C and Supplemental Figure 2 ). In shown; "n" is norleucine). Residues with a positive percent difference are considered favorable at a given position; residues with a negative percent difference are considered disfavorable. (B) Heat map representation of hAPN P1-P4′ specificity at the 60 min assay time point calculated using Z-scores at each position. Favored residues are colored blue (Z-score > 0) and disfavored residues are colored red (Z-score < 0). iceLogo representations and heat maps for the 15, 240, and 1200 min assay time points are provided (Supplementary Figure 1 ). (C) Example 14-mer peptides from the MSP-MS library are shown with primary and secondary cleavages indicated with a blue arrow. "X" indicates that no cleavage was detected at the indicated position. A progress curve is provided depicting the total cleavages observed at each assay time point.