Human cytochromes P450 1A1 and 1A2 play an important role in the metabolic activation of chemical carcinogens and in the metabolism of drugs. In particular, P450 1A1, which is present in lungs, is thought to be linked to lung cancer. This is because of the enzyme's ability to oxidize highly toxic compounds, such as polycyclic aromatic hydrocarbons (PAHs), to their carcinogenic derivatives. P450 1A2, a typical hepatic P450, plays a role in the metabolism of drugs and catalyzes metabolic activation of heterocyclic amines to genotoxic products. In order to design effective chemotherapeutic agents that would decrease the incidence of cancer, it is important to elucidate structure-function relationships of these enzymes. Cytochromes P450 1A1 and 1A2 are very similar in sequence (72 percent identity), but differ in substrate and inhibitor specificities. In general, 1A1 exhibits a preference for polyaromatic hydrocarbons, such as benzo(a)pyrene, whereas 1A2 is associated with heterocyclic amine substrates, such as caffeine. The long-term objective of the proposed research is to elucidate the structural basis for those differences. The hypothesis to be tested is that the overall structure of these enzymes is similar and functional differences are caused by the presence of a discrete number of key amino acid residues that govern enzyme function. Substitution of these residues in one enzyme to mimic the other will interconvert activities. This will be tested by molecular modeling of enzyme structure coupled with experimental approaches, such as site-directed mutagenesis and heterologous expression. The 3-dimensional enzyme models will be used to identify key amino acid residues responsible for unique substrate specificities and inhibitor susceptibilities of P450 1A1 and 1A2. The analysis of enzyme-substrate interactions and molecular dynamics simulations will aid in the explanation of functional differences between the two enzymes. Modeling predictions will be followed by the construction, expression and functional evaluation of appropriate mutant proteins, and experimental results will be used to refine models in an iterative approach. Innovative features of the proposal include extensive utilization of molecular dynamics simulations and binding free energy calculations to predict substrate specificity and inhibitor selectivity. The multidisciplinary nature of the project, which combines two markedly different areas of investigation, such as computational chemistry and biochemistry/molecular biology, should prove highly advantageous in studies of complex biochemical and toxicological issues. The proposed research should yield valuable insight into the P450 1A structure and its relationship to enzyme function, and thus provide a basis for the rational design of anticancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15CA085542-01
Application #
6086495
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Grotzinger, Karen R
Project Start
2000-08-15
Project End
2002-07-31
Budget Start
2000-08-15
Budget End
2002-07-31
Support Year
1
Fiscal Year
2000
Total Cost
$146,000
Indirect Cost
Name
West Virginia University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
191510239
City
Morgantown
State
WV
Country
United States
Zip Code
26506
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