title: Application of homology modeling to generate CYP1A1 mutants with enhanced activation of the cancer chemotherapeutic prodrug dacarbazine.
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 N(2) 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 K(m) (183-249 μM) that doubled the catalytic efficiency (p < 0.05) relative to wild-type CYP1A1 (K(m), 408 ± 43 μM; V(max), 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.
authors.affiliation: Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Lewis
authors.forename: Peter I
authors.initials: PI
authors.lastname: Mackenzie
authors.forename: John O
authors.initials: JO
authors.lastname: Miners
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001483:Base Sequence; D001711:Biotransformation; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D017931:DNA Primers; D003606:Dacarbazine / Q000493:pharmacokinetics*; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011355:Prodrugs / Q000493:pharmacokinetics*; D051381:Rats
chemical_list: D000970:Antineoplastic Agents; D017931:DNA Primers; D011355:Prodrugs; D003606:Dacarbazine; D019363:Cytochrome P-450 CYP1A1