Escherichia coli
nitroreductase (NTR) activates the
prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or
tumors to the
prodrug. This is one of several
enzyme-
prodrug combinations that are under development for cancer gene
therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for
CB1954 could improve its therapeutic potential. From the crystal structure of an
enzyme-
ligand complex, we identified nine
amino acid residues within the active site that could directly influence
prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to
CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to
CB1954 than did the WT
enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian
carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to
CB1954 at the clinically relevant
prodrug concentration of 1 micro M than was the WT
enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/
CB1954 combination in cancer gene
therapy.