Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

Background: the enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. the asparagine-glycine-arginine (ngr) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase n (apn),which is an ngr receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. the objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-fc) and an ngr-containing peptide fused with yeast cytosine deaminase (ycd),i.e. cngrc-ycd fusion protein,to target apn-expressing cells within the tumor tissues and to convert 5-fc into 5-fluorouracil (5-fu) to kill tumors. results: both ycd and cngrc-ycd proteins were cloned into the pet28a vector and expressed by an escherichia coli host. both ycd and cngrc-ycd proteins were purified and the yields were approximately 20 mg/l with over 95 % purity. the binding assay demonstrated that the cngrc-ycd fusion protein had specific binding affinity toward purified apn recombinant protein and high-apn-expressing cells,including human endothelial cells (huvecs) and various types of human tumor cell lines,but not low-apn-expressing tumor cell lines. moreover,the enzyme activity and cell viability assay showed that the cngrc-ycd fusion protein could effectively convert 5-fc into 5-fu and resulted in significant cell death in both high-apn-expressing tumor cells and huvecs. conclusions: this study successfully constructs a new targeting enzyme-prodrug system,cngrc-ycd fusion protein/5-fc. systematic experiments demonstrated that the cngrc-ycd protein retained both the apn-binding affinity of ngr and the enzyme activity of ycd to convert 5-fc into 5-fu. the combined treatment of the cngrc-ycd protein with 5-fc resulted in the significantly increased cell death of high-apn-expressing cells as compared to that of low-apn-expressing cells. © 2016 li et al.