Improvement of cyclophosphamide activation by CYP2B6 mutants: from in silico to ex vivo.

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Mol Pharmacol. 2008 Apr;73(4):1122-33. Epub 2008 Jan 22.

Erratum in:
Mol Pharmacol. 2008 Jun;73(6):1866.

Improvement of cyclophosphamide activation by CYP2B6 mutants: from in silico to ex vivo.

Nguyen TA, Tychopoulos M, Bichat F, Zimmermann C, Flinois JP, Diry M, Ahlberg E, Delaforge M, Corcos L, Beaune P, Dansette P, André F, de Waziers I.
INSERM UMR-S775, Facultéde Médecine, 45 rue des Saints Pères, 75270 Paris Cedex 06, France.
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 (V(max)/K(m)) 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 K(m) 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 (K(m), V(max))
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 K(m)/V(max). 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.