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

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 dierentiate 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 buered 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

Synthesis and Antiproliferative Activity of Hybrid Peptides for Ovarian and Prostate Cancer

Ovarian carcinoma is the leading malignancy of the female congenital system with a high mortality rate. In ovarian carcinoma, inappropriate activation of BCL6 has been shown to predict poor prognosis and promote tumor progression whereas inhibition reduces proliferation of cancer. During the malignant transformation of ovarian surface epithelium cells, NRP1 expression was found to be threefold upregulated. We hypothesized that developing a hybrid peptide containing a BCL6 peptide inhibitor with an NRP1 peptide activator would enhance cytotoxic effects on the ovarian cancer cells. Therefore, we designed and synthesized a hybrid peptide conjugate (BNP) with a BCL6 binding motif (GLVATVKEAGRSIHEIPREEL) connected via two beta alanine residues as a linker to the NRP1 sequence (RPARPAR). Two control peptides were synthesized with nonfunctional motifs for BCL6 and NRP1 (B’NP and BN’P respectively). The antiproliferative activities of these hybrid peptides were evaluated using ovarian cancer (SKOV3), prostate cancer (PC3), and human embryonic kidney (HEK293) control cell lines. This study indicates that BN’P (peptide with a BCL6-inhibiting sequence without a NRP1-activating sequence) showed no significant inhibition on cell proliferation of HEK293 but a significant reduction of SKOV3 (73%) and PC3 (63%) at a concentration of 1 µM after 48 h of incubation. The PC3 cytotoxicity was found to be independent of BCL6 inhibition and was not likely resulted from BCL6 inhibition. BNP (peptide with a BCL6-inhibiting and NRP1-activating sequence) did not show any synergistic effect because of the nature of tissue penetration cannot be tested in-vitro. This leaves the value of this hybrid peptide for future in-vivo studies

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

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