The long term aim of the present proposal is to understand the role of human cytochrome P-450 2D6 in the protection from, or predisposition to neurological disease. CYP2D6 is polymorphically expressed in human liver, lung and brain, with over 17 distinct alleles described in the literature, eight of which encode for non-null variants. CYP2D6 plays a key role in the metabolism of many important psychoactive drugs, such as the activation of codeine to morphine, and is known to detoxify MPTP, a thermal breakdown product of 'street heroin' which causes Parkinsonian symptoms in susceptible individuals. Therefore, it is important to know the intrinsic metabolic capability of individual variants of 2D6 in these critical bioactivation and detoxification pathways. Using computer-derived homology models we should be able to rationalize the effects of specific mutations on the ability to bind substrates and inhibitors and the ability to interact with essential cofactors. With the advent of rapid genotyping, it may be possible to predict the outcome of exposure by individuals possessing minor allelic variants toward important environmental agents.
The specific aims of the present proposal are: 1. Creation of cDNAs corresponding to each of the naturally occuring non-null allelic variants of CYP2D6 followed by expression in and purification from an insect cell culture system. Only those proteins that have the capability to bind heme and CO will be carried forth. 2. Characterization of P450 enzymatic activity of each variant toward important substrates of CYP2D6 including MPTP, fluoxetine, codeine and dextromethorphan and rationalization of allelic variability data with homology models for 2D6. Our hypothesis is that different allelic variants of CYP2D6 will show substrate dependent changes in the metabolic clearance (kcat/Km) of these psychoactive substrates. In addition, we believe that isoforms of 2D6 which affect active site serine and threonine residues will show altered partition ratio of dextromethorphan O-demethylated to N-demethylated metabolites.
