Drug Design, Development and Therapy (Nov 2017)
Novel production method of innovative antiangiogenic and antitumor small peptides in Escherichia coli
Abstract
Sarra Setrerrahmane,1 Jian Yu,1 Jingchao Hao,1,2 Heng Zheng,3 Hanmei Xu1,3 1The Engineering Research Center of Peptide Drug Discovery and Development, China Pharmaceutical University, Nanjing, Jiangsu, 2College of Pharmacy & the Provincial Key Laboratory of Natural Drug and Pharmacology, Kunming, Yunnan, 3State Key Laboratory of Natural Medicines, Ministry of Education, China Pharmaceutical University, Nanjing, Jiangsu, People’s Republic of China Background: Developing innovative drugs with potent efficacy, specificity, and high safety remains an ongoing task in antitumor therapy development. In the last few years, peptide drugs have become attractive agents in cancer therapy. HM-3, mainly with antiangiogenic effect, and AP25, with an additional antiproliferative effect, are two peptides designed in our laboratory targeting αvβ3 and α5β1 integrins, respectively. The low molecular weight of the two peptides renders their recombinant expression very difficult, and the complicated structure of AP25 makes its chemical synthesis restricted, which presents a big challenge for its development.Methods: Bifunctional peptides designed by the ligation of HM-3 and AP25, using linkers with different flexibility, were prepared using recombinant DNA technology in Escherichia coli. The fusion peptides were expressed in a modified auto-induction medium based on a mixture of glucose, glycerol, and lactose as carbon substrates and NH4+ as nitrogen source without any amino acid or other elements. Subsequently, the antiangiogenic, antiproliferative, and cell adhesion assays were conducted to evaluate the bioactivity of the two fusion peptides.Results: The peptides were successfully expressed in a soluble form without any induction, which allows the culture to reach higher cell density before protein expression occurs. Human umbilical vein endothelial cell migration assay and chick embryo chorioallantoic membrane assay showed, at low doses, a significantly increased antiangiogenic effect (>75%) of the purified products compared with the single molecules. Meanwhile, MTT assay confirmed their enhanced antitumor activity against gastric cancer cell line MGC-803; however, no significant effect was observed on hepatoma HepG2 cells and no cytotoxicity on normal human lens epithelial cell SRA01/04 and human epithelial esophageal cells.Conclusion: Bifunctional molecules with antiangiogenic and antiproliferative effects were obtained by using this technique, which presents an alternative for small peptide production, instead of the conventional chemical method. The increased molecular weight facilitates the peptide expression with a simultaneous improvement in their stability and biological activity. Keywords: innovative drugs, bifunctional peptides, linkers, auto-induction, antiangiogenic, antitumor
