Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: effects of combining beneficial single mutations.

Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia colinfsB nitroreductase (E.C. 1.6.99.7)(NTR), sensitises cells to the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which it converts to a potent DNA-crosslinking agent. However, low catalytic efficiency with this non-natural substrate appears to limit the efficacy of this enzyme prodrug combination for eliminating the target cancer cells. To improve this, we aim to engineer NTR for improved prodrug activation. Previously, a number of single amino acid substitutions at six positions around the active site of the enzyme were found to increase activity, resulting in up to approximately 5-fold enhanced cell sensitisation to CB1954. In this study we have made pairwise combinations among some of the best mutants at each of these 6 sites. A total of 53 double mutants were initially screened in E. coli, then the 7 most promising were inserted into an adenovirus vector and compared in SKOV3 human ovarian carcinoma cells for sensitisation to CB1954 and two alternative prodrugs. The most effective mutants, T41L/N71S and T41L/F70A, were 14-17-fold more potent than WT NTR at sensitising the cancer cells to CB1954. The best mutant for activation of the dinitrobenzamide mustard prodrug SN23862 was T41L/F70A (4.8-fold improvement); and S40A/F124M showed 1.7-fold improvement over WT with the nitrobenzylphosphoramide mustard prodrug LH7. In two tumour xenograft models using SKOV3 or human prostate carcinoma PC3, T41L/N71S NTR demonstrated greater CB1954-dependent anti-tumour activity than WT NTR.