title: Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants.
abstract: Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, Amsterdam 1081 HV, The Netherlands.
authors.forename: Galvin
authors.initials: G
authors.lastname: Vredenburg
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, Amsterdam 1081 HV, The Netherlands.
authors.forename: Shalenie
authors.initials: S
authors.lastname: den Braver-Sewradj
authors.affiliation: BioDetection Systems BV, Science Park 406, Amsterdam 1098 XH, The Netherlands.
authors.forename: Barbara M A
authors.initials: BM
authors.lastname: van Vugt-Lussenburg
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, Amsterdam 1081 HV, The Netherlands.
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, Amsterdam 1081 HV, The Netherlands.
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, Amsterdam 1081 HV, The Netherlands. Electronic address: j.c.vos@vu.nl.
authors.forename: J Chris
authors.initials: JC
authors.lastname: Vos
mesh_terms: D065767:Activation, Metabolic; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism* / Q000494:pharmacology; D001859:Bone Neoplasms / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003520:Cyclophosphamide / Q000378:metabolism* / Q000494:pharmacology; D065702:Cytochrome P-450 CYP2B6 / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D006801:Humans; D006900:Hydroxylation; D007069:Ifosfamide / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D009154:Mutation; D012516:Osteosarcoma / Q000188:drug therapy / Q000473:pathology; D010641:Phenotype
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003520:Cyclophosphamide; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D007069:Ifosfamide
keywords: Cyclophosphamide; Cytochrome P450 BM3; Cytotoxicity; Extracellular bioactivation; Ifosfamide