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.

Peptide synthesis and inhibitor testing. The peptides used in this study were custom synthesized by AAPPTEC of Louisville, KY using traditional Fmoc solid-phase peptide synthesis 40 . All of the peptides were purified and characterized by AAPPTEC. The peptides used in all of the studies were greater than >95% purity as determined by high-performance liquid chromatography. Inhibition assays were performed in triplicate in a 100 mM Tris pH 7.2, Brij 35 0.0002% w/v assay buffer at 37 °C with 0.1 μM APN and 100 μM H-Ala-AMC (fluorophore). Values were recorded by Infinite pro plate reader every 2 minutes for 30 minutes with laser parameters set for excitation at 355 nm and emission at 460 nm. As the inhibition of APN was competitive, the values for the inhibition constant, K i , were determined from the IC50 using the Cheng-Prusoff equation: K i = IC 50 /(1 + [S]/ Km).