Binding and Internalization of Rationally Designed Novel Peptide Ligands Targeted to Tumor Vascular Endothelial Aminopeptidase N (CD13) Receptor

Authors

Md Zahir Uddin, University of the PacificFollow
Yu Zheng, University of the PacificFollow
Xiaoling Li, University of the PacificFollow
Hyun Joo, University of the PacificFollow
Jerry Tsai, University of the PacificFollow
Lisa Wrischnik, University of the PacificFollow
Bhaskara Jasti, University of the PacificFollow

Poster Number

13

Lead Author Affiliation

Drug Targeting and Delivery

Lead Author Status

Doctoral Student

Introduction/Abstract

The CD13, also known as aminopeptidase N (APN), is a 967-residue type-2 cell surface membrane glycoprotein with a number of characteristics of the malignant phenotype such as tumor cell proliferation, secretion, invasion and angiogenesis. CD13 expression on the tumor vascular endothelial cells in breast, kidney, prostate, ovarian, colon, gastric, pancreatic and thyroid cancers is significantly higher than normal cells. We used a rational designing approach based on the Knob-Socket model to develop a series of novel peptide ligands that specifically bind to CD13 receptor.

Purpose

The objective of this study was to characterize the in vitro binding and uptake of the rationally designed novel CD13 targeting peptide ligands.

Method

Four novel peptide ligands designed rationally using Knob-Socket model, the known CD13 targeting CNGRC (C1-C5) peptide (positive control), and GARAG peptide (negative control) with and without Fluorescein isothiocyanate (FITC) label were synthesized by solid phase synthesis method. The synthesized peptides were characterized by ESI-MS and HPLC for identity and purity. To characterize the CD13 protein expression in HT-1080 and MCF-7 cells, flow cytometry studies (BD FACSCalibur) were performed using FITC-conjugated anti-CD13 antibody WM15. CD13 catalysis inhibition assay was carried out to evaluate the binding of the peptides to CD13. Briefly, the CD13 positive HT-1080 cells were plated in 96-well plate and incubated in 100 μM L-Alanine 7-amido-4-methylcoumarin trifluoroacetate (Sigma) with and without (control) the peptides (50 µM) (n=8). The release of fluorescent product 7-amido-4-methylcoumarin was measured on a BioTek Synergy HT microplate reader with λexc of 360 nm and λem of 460 nm after 90 minutes. Statistical differences among treatment groups were determined using 2-tailed unpaired Student’s t-Test with p < 0.05 considered as level of significance. The binding specificity and uptake of the peptides were evaluated in HT-1080 (CD13 expressing) and MCF-7 (control) cell lines using confocal microscopy. The cells were incubated with FITC conjugated peptides at a concentration of 30 μM at 37° C for 60 minutes. Cells were then stained with Alexa Fluor 594 (plasma membrane dye) and visualized under a Leica DMIRE2 confocal laser scanning microscope at 65X magnification with oil immersion.

Results

The synthesized peptides were obtained with more than 90% purity. Flow cytometry study showed that 90.1% of the HT-1080 cells expressed CD13 while only 2.5% of the MCF-7 cells showed CD13 expression, indicating that they can be used as targeted and control cells respectively. CD13 catalysis inhibition assay showed that the release of fluorescent product was significantly lower for all four novel peptides when compared to CNGRC (C1-C5) peptide. CD13 catalysis inhibition assay data (as % of control) with the respective standard error of the mean (SEM) are shown in Table 1. Confocal microscopy showed that the designed peptides and positive control CNGRC (C1-C5) peptide strongly bound to HT-1080 cells (higher fluorescence intensity on cell surface and inside the cells) when compared to negative control GARAG peptide. The peptides only showed background fluorescence in non-CD13 expressing MCF-7 cells indicating the specificity of the peptides to CD13 receptor. The confocal microscopy images are illustrated in Figure 1.

Significance

In vitro studies demonstrated good binding specificity and cellular uptake of the designed novel peptides. The designed peptides also showed significantly higher inhibition of CD13 enzymatic activity as compared to CNGRC (C1-C5) peptide. The designed peptides have potential to be used for targeting CD13 over-expressed cancers.

Location

DUC Ballroom A&B

Format

Poster Presentation

Poster Session

Afternoon