The genetic modification of enzymes towards an anti-cancer prodrug activation system

• Main Author: Mohmmad Kareem, Shokrea Ali
• Published: Bangor University 2013
• Subjects: 615.5
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.664600

Cancer is the most common cause of death worldwide and therefore attracts significant research attention. Directed enzyme-pro drug therapy offers a selective approach which can kill tumour cells whilst limiting the damage to healthy cells. Nitroreductases have potential applications in enzyme/prodrug targeted anti-cancer therapies due to their ability to convert the nontoxic prodrug CB 1954 into a cytotoxic drug that kills malignant cells. E. coli NfsB nitroreductase/CB 1954 has previously been studied but the low affinity of this enzyme for CB 1954 and its poor catalytic efficiency has thus far limited its clinical potential in targeted anti-cancer therapy. This work seeks to overcome this shortfall by finding alternative nitroreductases and xenobiotic reductases from different bacterial strains with improved activity towards CB1954. The genetic modification of the nitroreductases has been investigated to enable the enzymes to be immobilised onto gold-coated magnetic nanoparticles which could subsequently be used to direct the enzymes to solid tumours with a focused magnetic beam. The targeted enzymes would be able to convert a nontoxic prodrug into a cytotoxic drug at the site of the cancer cells thus limiting damage to healthy cells. This thesis reports the successful isolation and characterisation of two nitroreductases, NfnB and PnrA from E. coli K12 and P. putida JLR11 respectively, and two xenobiotic reductases, XenA and XenB from P. putida KT2440. The enzymes were genetically modified by incorporating a series of cysteine tags at the N-termini of the monomer structure to enable the enzymes to be immobilised onto gold coated magnetic nanoparticles. Both bacterial nitroreductases (NfnB and PnrA) and both xenobiotic reductases (XenA and XenB) showed considerable activity when utilised with the prodrug CB 1954. In addition, nitroreductases NfnB and PnrA and the xenobiotic reductase XenB also demonstrated activity against the prodrug SN23862, with PnrA having the greatest activity out of the three in reducing this substrate. The optimum pH for all enzymes was established as pH 7.0, and the optimum temperatures were found to be 30 QC for NfnB, 30-40 QC for PnrA, and 25 QC for XenB. In the in vitro assays, XenB was found to be the most active in causing cell death in cervical and neuroblastoma cancer cells.