Abstract
The chemotherapeutic prodrug dacarbazine (DTIC) has limited efficacy in human malignancies and exhibits numerous adverse effects that arise from systemic exposure to the cytotoxic metabolite. DTIC is activated by CYP1A1 and CYP1A2 catalyzed N-demethylation. However, structural features of these enzymes that confer DTIC N-demethylation have not been characterized. A validated homology model of CYP1A1 was employed to elucidate structure-activity relationships and to engineer CYP1A1 enzymes with altered DTIC activation. In silico docking demonstrated that DTIC orientates proximally to Ser122, Phe123, Asp313, Ala317, Ile386, Tyr259, and Leu496 of human CYP1A1. The site of metabolism is positioned 5.6 Å from the heme iron at an angle of 105.3°. Binding in the active site is stabilized by H-bonding between Tyr259 and the N2 position of the imidazole ring. Twenty-seven CYP1A1 mutants were generated and expressed in Escherichia coli in yields ranging from 9 to 225 pmol P450/mg. DTIC N-demethylation by the E161K, E256K, and I458V mutants exhibited Michaelis-Menten kinetics, with decreases in Km (183–249 μM) that doubled the catalytic efficiency (p < 0.05) relative to wild-type CYP1A1 (Km, 408 ± 43 μM; Vmax, 28 ± 4 pmol · min−1 · pmol of P450−1). The generation of enzymes with catalytically enhanced DTIC activation highlights the potential use of mutant CYP1A1 proteins in P450-based gene-directed enzyme prodrug therapy for the treatment of metastatic malignant melanoma.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Health and Medical Research Council of Australia and the Faculty of Health Sciences of Flinders University.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072124.
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ABBREVIATIONS:
- DTIC
- dacarbazine [5-(3,3-dimethyl-triazenyl)-imidazole-4-carboxamide]
- P450
- cytochrome P450
- AIC
- 5-aminoimidazole-4-carboxamide
- GDEPT
- gene-directed enzyme prodrug therapy
- CPR
- cytochrome P450 NADPH oxidoreductase
- HPLC
- high-performance liquid chromatography
- RMSD
- root-mean-square deviation.
- Received March 6, 2011.
- Accepted August 3, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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