Fluorescently tagged prostate-specific antigen targeting peptides as models of tumour activated prodrugs

Abstract

Existing chemotherapeutics for metastatic prostate cancer are limited in use due to a lack of selectivity for cancer cells. On account of a high active expression in cancerous prostate tissue, prostate-specific antigen (PSA) is an ideal Tumour Activated Prodrug (TAP) target. This project investigates the design, synthesis, and in vitro testing of model peptide TAPs (mTAPs) targeted to PSA. The mTAPs feature a fluorescent model of a cytotoxin which enables the localisation of the mTAPs to be visualised before and after PSA activation. A strategy of attaching fluorophores to the peptide was developed using copper(I) catalysed azide-alkyne cycloaddition. The mTAPs incorporate a previously reported PSA cleavable peptide sequence (histidine-serine-serine-lysine-leucine-glutamine, HSSKLQ) modified with a negatively-charged tri- or tetra-glutamic acid cellular uptake inactivating group at the N-terminus of the HSSKLQ sequence. Masking the C-terminal carboxylic acid functionality of the mTAP to form a methyl ester group was found to promote uptake of the cleaved fragment. Fluorescence microscopy and in vitro cytotoxicity assays indicated that although the fluorescently tagged HSSKLQ sequence itself does not readily enter cells, the addition of the blocking sequence further reduces cellular uptake and non-selective cytotoxicity. Cytotoxicity was reactivated upon the introduction of PSA and activation was uninhibited by the presence of the negatively charged group. The results of this study provide proof of principle for a strategy by which cellular uptake of a cytotoxin can be activated in the presence of PSA, hence enabling a tumour-selective method of delivering cytotoxicity to prostate cancer cells.