Improvement of cyclophosphamide activation by CYP2B6 mutants: from in silico to ex vivo.
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.
Nguyen
Thien-An
TA
INSERM UMR-S775, Facultéde Médecine, 45 rue des Saints Pères, 75270 Paris Cedex 06, France.
Tychopoulos
Marina
M
Bichat
Florence
F
Zimmermann
Clothilde
C
Flinois
Jean-Pierre
JP
Diry
Monique
M
Ahlberg
Emelie
E
Delaforge
Marcel
M
Corcos
Laurent
L
Beaune
Philippe
P
Dansette
Patrick
P
André
François
F
de Waziers
Isabelle
I
D000595:Amino Acid Sequence; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods*; D003520:Cyclophosphamide / Q000378:metabolism* / Q000494:pharmacology; D065702:Cytochrome P-450 CYP2B6; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment
D018906:Antineoplastic Agents, Alkylating; D008024:Ligands; D050505:Mutant Proteins; D011355:Prodrugs; D003520:Cyclophosphamide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating