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 proteases responsible for the increased proteolysis associated with aggressive forms of cancer represent candidate therapeutic targets. The over-expression of APN on the surface and neo-vasculature of solid tumors has made it the most studied member of the M1 family of metalloproteases and the most targeted. A number of groups have developed molecules targeting APN ranging from monoclonal antibodies to synthetic peptidomimetic inhibitors of its enzymatic activity [19] [20] [21] [22] [23] [24] . Perhaps the most well-known for APN is the cyclic tumor-homing peptide asparagine-glycine-arginine (cyc-NGR) originally discovered through in vivo peptide phage display 25 . This peptide has been radiolabeled for nuclear imaging and has been used to deliver therapeutic peptides and proteins in vivo to APN-expressing tissues [26] [27] [28] . The NGR motif mimics the structure of extracellular matrix proteins that APN is known to bind but not degrade. There are a number of drawbacks associated with the use of the cyc-NGR peptide, foremost among them is deamidation of the asparagine residue 25 . This spontaneous reaction, enhanced when the asparagine is adjacent to a glycine residue, results in the formation of isoaspartate-glycine-arginine (isoDGR) which is a ligand capable of binding to α V β 3 integrin and several other integrins with lower affinity. This deamidation can lead to decreased specificity and potential off target affects 29 . Additionally, as further demonstrated by our study, the cyclic-NGR peptide alone is not therapeutic, rather it is only therapeutic when conjugated to toxins. Thus, there is a great need for low-molecular weight APN-targeted therapeutics that are stable and specific.