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.

EPKVA >500 APN, we next evaluated the ability of cyc-LHSPW to preferentially bind to cells that express surface APN using flow cytometry (Fig. 5B) . For flow cytometry, we synthesized a fluorophore-containing version of the peptide possessing a (Gly) 4 -FITC group coupled to the C-terminal cysteine (Supplemental Figure 4) . The expression of cell surface-associated APN was examined in the androgen receptor negative neuroendocrine prostate cancer cell lines PC3 and DU145 using a commercially available antibody. The cell line PC3 was found to express surface-associated APN, whereas no detectable expression was found for the DU145 cell line by this method. When incubated with both cells lines, the FITC labeled cyc-LHSPW peptide preferentially bound to PC3 cells with no noticeable labelling of DU145 cells. The peptide was next tested on frozen PC3 and DU145 tumor xenografts sections. Preferential labeling of the PC3 xenograft section was evident, furthering attesting to the specificity of cyc-LHSPW by fluorescence microscopy. The potential therapeutic efficacy of cyc-LHSPW was next tested in vitro using the PC3 and DU145 cell lines. The APN inhibitor demonstrated a specific and pronounced therapeutic benefit resulting in the decreased clonal survival of PC3 cells. No significant biological effect was observed on either cell line after treatment with the hydrolyzable LHSPW peptide or the cyc-nHSPW derivative. Prior to in vivo therapeutic efficacy studies, a toxicity study was performed with the i.v. administration of cyc-LHSPW. We found that a single high dose of 100 mg/ kg was well tolerated by the mice; however, multiple doses at this concentration, as would be needed, resulted in cachexia. It was found that a dose of 40 mg/kg administered three time a week for four weeks (t.i.w. x4) was well tolerated, i.e. no signs of morbidity. Mice bearing established PC3 and DU145 xenografts were treated systemically via tail vein injection with 40 mg/kg of the tumor homing APN peptide cyc-NGR or the cyc-LHSPW peptide t.i.w. x4 (Fig. 6B) . Inhibition of tumor growth in the PC3 xenograft mice treated with cyc-LHSPW was significant (P < 0.05) three weeks into the trial when compared to the saline-and cyc-NGR-treated arms. This therapeutic effect persisted to the end of the study with the cyc-LHSPW-treated mice having a tumor volume of 309.3 ± 92.7 mm 3 at day 35. Little therapeutic effect was observed in the cyc-NGR-treated mice with both the saline-and cyc-NGR-treated arms having tumor volumes >1000 mm 3 at the end of the study. PC3 tumors were removed and stained for the cellular proliferation marker Ki67. Both the saline-and cyc-NGR-treated tumors had profoundly higher populations of K i 67 positive cells compared to the cyc-LHSPW-treated tumors (Fig. 6C) . No therapeutic effect observed in the APN-negative DU145 tumors treated with either cyc-LHSPW or cyc-NGR.