EDB-FN Targeted Peptide-Drug Conjugates for Use against Prostate Cancer

Abstract

Prostate cancer (PCa) is the most common malignancy in men and is the leading cause of cancer-related male mortality. A disulfide cyclic peptide ligand [CTVRTSADC] 1 has been previously found to target extra domain B of fibronectin (EDB-FN) in the extracellular matrix that can differentiate aggressive PCa from benign prostatic hyperplasia. We synthesized and optimized the stability of ligand 1 by amide cyclization to obtain [KTVRTSADE] 8 using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 8 was found to be stable in phosphate buffered saline (PBS, pH 6.5, 7.0, and 7.5) and under redox conditions, with a half-life longer than 8 h. Confocal microscopy studies demonstrated increased binding of ligand 8 to EDB-FN compared to ligand 1. Therefore, we hypothesized that the EDB-FN targeted peptides (1 and 8) conjugated with an anticancer drug via a hydrolyzable linker would provide selective cytotoxicity to the cancer cells. To test our hypothesis, we selected both the normal prostate cell line, RWPE-1, and the cancerous prostate cell lines, PC3, DU-145, LNCaP, and C4-2, to evaluate the anticancer activity of synthesized peptide-drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 13 containing disulfide linkage showed comparable cytotoxicity versus Dox after 72 h incubation in all the cancer cell lines, whereas it was found to be less cytotoxic on RWPE-1, suggesting that it can act as a Dox prodrug. Doce conjugate 14 was found to be less cytotoxic in all the cell lines as compared to drug alone.

Keywords: Fmoc/tBu; antiproliferative assay; conjugation; docetaxel; doxorubicin; extra domain B; fibronectin; peptide–drug conjugate; prostate cancer; solid-phase synthesis; targeting.

Figure 1

Chemical structures of peptide ligands 1 and 8.

Scheme 1

Synthesis of hydrazine-glutarate-GG-FK-C₆-[CTVRTSADC] 5 and hydrazine-glutarate-GG-VCit-C₆-[CTVRTSADC] 6.

Scheme 2

Selected synthesis of C-GFLG-C₆-[KTVRTSADE] 9 containing lactam cyclization.

Scheme 3

Synthesis of Dox-disulfide-pyridyl 10.

Scheme 4

Synthesis of Doce-βA-SMCC 11.

Scheme 5

Conjugation of Dox, Dox-SH, and Doce-βA-SMCC with the peptide targeting moiety through a hydrolyzable linker

Scheme 6

Conjugation of FAM with the peptide targeting moiety through a hydrolyzable linker.

Figure 2

Conjugate 13 in PBS at different pH. The percent remaining of conjugate 13 was quantified using the area under the curve (AUC) in analytical HPLC.

Figure 3

Stability analysis of conjugate 13 under reducing conditions using dithiothreitol (DTT). The experiment was done in PBS at pH 7.4. The percent remaining was quantified using the AUC, and the trendline equation gives the half-life of 2.6 min with an R² of 0.98. Fitting was done online using MyCurveFit.

Figure 4

Stability of peptide 8 ([KTVRTSADE]) in pH 7.4 PBS with DTT. The percent remaining was calculated from the AUC.

Figure 5

Peptide 1 before (a) and after 5 min (b) incubation with DTT. The higher molecular weight shows a full reduction of peptide 1 to the linear counterpart of (CTVRTSADC) within 5 min.

Figure 6

Stability of conjugate 13 (125 µM) in 25% human serum. Note the trendline gives the half-life of 11.8 min with an R² of 0.978. Each point represents an average of three readings.

Figure 7

Analytical HPLC chromatogram over time for Dox-peptide conjugate 13 in human cathepsin B with absorbance detection at 214 nm (top) and 490 nm (bottom). Dox-peptide 13 retention time = 34 min. Dox alone retention time = 30 min. Note the similar shape of the 5, 30, and 60 min incubations. Also, the peak at ~30.5 min showed strong absorbance at both 214 nm and 490 nm.

Figure 8

Cytotoxicity of peptide 9, conjugate 13 (Dox-s-s-CGFLG-C6-[KTVRTSADE]) and Dox on a PC3 prostate cancer cell line after two hours of incubation at various concentrations.

Figure 9

Cell viability of peptide 9, Dox, Doce, Dox–peptide conjugate 13, and Doce–peptide conjugate 14 at 5 μM on prostate epithelial and PCa cell lines (RWPE-1, PC3, LNCap, Du-145) after 72 h using MTS assay.

Figure 10

Time-dependent (24, 48, 72 h) cell viability studies of drugs, peptide 9, and drug–peptide conjugates 13, 14, and physical mixtures of drug and peptides using MTS on (a) prostate C4-2 cell line, a subline of LNCaP without TGF-β, (b) with TGF-β treatment.

Figure 11

Comparative confocal microscopy of Fluorescent tagged peptide 17 and 18. Control (a) shows no treatment; (b) shows FAM-linked disulfide cyclized targeting moiety in the peptide 17; (c) shows FAM linked amide cyclized targeting moiety in the peptide 18; (d), (e), and (f) are Z-slices of images in (a), (b), and (c); (g) shows the unpaired t-statistic reflects peptide 18 has higher binding to EDB-FN as compared to peptide 17.