Abstract
Cyclophosphamide (CPA) is a chemotherapeutic agent that is primarily activated in the liver by cytochrome P4502B6 (CYP2B6) and then transported to the tumor via blood flow. To prevent deleterious secondary effects, P450-based gene-directed enzyme prodrug therapy (GDEPT) consists of expressing CYP2B6 in tumor cells before CPA treatment. Given the relatively low affinity of CYP2B6 for CPA, the aim of our work was to modify CYP2B6 to increase its catalytic efficiency (Vmax/Km) to metabolize CPA into 4′-OH CPA. A molecular model of CYP2B6 was built, and four residues in close contact with the substrate were subjected to mutagenesis. Canine CYP2B11 exhibiting a particularly low Km to CPA, the amino acids exclusively present in the CYP2B11 substrate recognition sequences were substituted in human CYP2B6. All mutants (n = 26) were expressed in Saccharomyces cerevisiae and their enzymatic constants (Km, Vmax) evaluated using CPA as substrate. Five mutants exhibited a 2- to 3-fold higher catalytic efficiency than wild-type CYP2B6. A double mutant, comprising the two most effective mutations, showed a 4-fold increase in Km/Vmax. Molecular dynamic simulations of several mutants were found to be consistent with the observed modifications in catalytic efficiency. Finally, expression of the CYP2B6 114V/477W double mutant, contrary to wt CYP2B6, allowed switching of a resistant human head and neck cancer cell line (A-253) into a sensitive cell line toward CPA. Thus, we were able to obtain a new efficient CYP2B6 mutant able to metabolize CPA, an important step in the GDEPT strategy for human cancer treatment.
Footnotes
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This work was supported by the Region Bourgogne, the Ligue contre le Cancer and Oncodesign S.A. (Dijon, France).
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ABBREVIATIONS: CPA, cyclophosphamide; P450, cytochrome P450; SRS, substrate recognition sequences; CPI, 4-(4-chlorophenyl)imidazole; MD, molecular dynamics; RED, NADPH cytochrome P450 reductase; MOI, multiplicity of infection; RMSD, root-mean-square deviation; SMAS, solvent molecular accessible surface; wt, wild type.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received October 22, 2007.
- Accepted January 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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