Received for publication October 22, 2007.
Revised January 21, 2008.
Accepted for publication January 22, 2008.

Improvement of Cyclophosphamide Activation by CYP2B6 Mutants: from in Silico to ex Vivo

Abstract

Cyclophosphamide (CPA) is a chemotherapeutic agent primarily activated in the liver by cytochrome P4502B6 (CYP2B6) then transported to the tumor via blood flow. To prevent deleterious secondary effects, CYP-based gene-directed enzyme prodrug therapy (GDEPT) consists in 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 the CYP2B6 in order 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 these mutants (n=26) were expressed in Saccharomyces cerevisiae and their enzymatic constants (Km, Vmax) were evaluated using CPA as substrate. Five mutants exhibited a 2 to 3-fold higher catalytic efficiency than CYP2B6wt. A double mutant, comprising the two most effective mutations showed a 4-fold increase in this Km/Vmax. Molecular dynamic simulations of several mutants were found to be in adequacy with the observed modifications of catalytic efficiency. Finally, expression of the CYP2B6 114V/477W double mutant, contrary to CYP2B6wt, allowed to switch a resistant human head and neck cancer cell line (A-253) into a sensitive one towards CPA. Thus, we were able to obtain a new efficient CYP2B6 mutant to metabolize CPA, an important step in a strategy of GDEPT for human cancer treatment.

Key words: Structure-activity relationships and modeling, Cytochrome P450, Overexpression, Pharmacokinetics, metabolism and activation