Author: Benjamin Cavell Lewis
Lewis, Benjamin Cavell, 2011 COMPUTATIONAL MODELING AND STRUCTURE-ACTIVITY RELATIONSHIPS OF CYTOCHROME P450 1A1: AN APPROACH TO CYP1A1 GENE DIRECTED ENZYME PRODRUG THERAPY, Flinders University, School of Medicine
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Given the paucity of data relating to the structure of human CYP1A1, an enzyme of considerable toxicological significance, the initial aim of this thesis was to generate a chemically and structurally valid CYP1A1 homology model. CYP1A1 homology models based on the CYP2C5 X-ray crystal structure and a composite of the CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the crystal coordinates of CYP1A2. The model using the CYP1A2 coordinates gave near ideal stereochemical quality and was favored energetically. Automated in silico docking studies identified active-site residues potentially involved in the orientation and binding of the prototypic CYP1A1 substrate, 7-ethoxyresorufin. The most energetically favorable pose placed the carbon atom adjacent to the ether oxygen of 7-ethoxyresorufin at 4.4Å from the heme iron, at an angle of 106.4° to the plane of the heme. The CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresorufin in a catalytically favorable orientation. The validated 3-dimensional structure of CYP1A1 was subsequently employed to elucidate structure-activity relationships with the anticancer prodrug, dacarbazine (DTIC). In silico docking experiments demonstrated that DTIC orientates in close proximity to S122, F123, D313, A317, I386, Y259, and L496. Docking located the site of metabolism of DTIC at 5.6Å from the heme iron at an angle of 105.3° to the plane of the heme. Binding of DTIC in the active-site was stabilized by H-bonding between Y259 and the N2 position of the imidazole ring. Structural modification of the CYP1A1 enzyme to increase its catalytic efficiency (Vmax/Km) for N-demethylation and the subsequent activation of DTIC was facilitated by the CYP1A1 homology model. Twenty-nine CYP1A1 mutants were generated and expressed in E. coli. DTIC N-demethylation by the CYP1A1 E161K, E256K, and I458V mutants exhibited Michaelis-Menten kinetics, with decreases in Km that doubled the catalytic efficiency relative to wild-type (P < 0.05). The kinetics of DTIC N-demethylation by the CYP1A1*2C and CYP1A1*4 polymorphic variants was additionally characterized. There was an approximate 30% reduction in catalytic efficiency of the CYP1A1*2C and CYP1A1*4 variants relative to wild-type. Thus, patients with malignancies who carry either polymorphism may not respond as well to DTIC treatment compared to those expressing the wild-type enzyme. As a chemotherapeutic agent, DTIC has relatively poor clinical activity in human malignancies and exhibits numerous serious adverse effects, which presumably arise from bioactivation in the liver and other tissues resulting in systemic exposure to the cytotoxic metabolite. Gene directed enzyme prodrug therapy (GDEPT) provides a means to enhance the efficacy and reduce the systemic toxicities associated with conventional chemotherapy. Thus, COS-7 and SK-MEL-28 cells were transfected with cDNA encoding an open reading frame (ORF) comprising the CYP1A1 (wild-type or mutant) coding sequence (CDS), a picornaviral 2A cleavage peptide, followed by the cytochrome P450 oxidoreductase (OxR) CDS. Cell line sensitization assays using the wild-type, E161K, E256K, and I458V CYP1A1-2A-OxR constructs were attempted, but the generation of stable cell lines was not successful. However, data obtained from transiently expressed CYP1A1-2A-OxR motifs in COS-7 and SK-MEL-28 cells revealed the synthesis of both holo-CYP1A1 and OxR proteins. This study elucidated important structural characteristics of human CYP1A1 and how manipulation of protein tertiary structure can enhance enzyme function. Combination of kinetic analyses with in silico docking data has allowed interpretation of the structure-activity relationships of CYP1A1 and DTIC. Moreover, the successful generation of CYP1A1 enzymes with catalytically enhanced DTIC activation highlights their potential use as a strategy for P450-based GDEPT in the treatment of metastatic malignant melanoma.
Keywords: Cytochrome P450 1A1,CYP1A1,Gene Directed Enzyme Prodrug Therapy,dacarbazine
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Prof John Miners