Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

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Abstract
Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, ∼50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only ∼3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation ≥90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only ∼10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low Km (80-160 μM), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

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