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

Commissariat à l'Energie Atomique, Institut de Biologie et de Technologies de Saclay, Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique, SB2SM, Gif sur Yvette, France (T.A.N., M.D., F.A.); Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 775, Paris, France; Université Paris Descartes, Paris, France; Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France. (M.T., C.Z., J.P.F., M.D., E.A., P.B., I.W.); Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 517, Dijon, France (F.B.); Institut National de la Santé et de la Recherche Médicale, U613/EA948, Brest, France (L.C.); Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8601, Paris, France; Université Paris Descartes, Paris, France (P.D.)

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.

Address correspondence to: Isabelle de Waziers, INSERM UMR-S775, Facultéde Médecine, 45 rue des Saints Pères, 75270 Paris Cedex 06, France. E-mail: isabelle.waziers{at}univ-paris5.fr