Cholesterol removal from different organs always involves cytochrome P450 enzymes (CYP or P450) that play key roles in maintenance of cholesterol homeostasis. Outside the liver, two P450s are particularly important, CYP46A1 and CYP11A1, which metabolize the majority of cholesterol in the brain and steroidogenic tissues, respectively. The role of CYP11A1 is to convert cholesterol to pregnenolone, the precursor of all steroid hormones. The significance of CYP46A1 is still under investigation. Discovered initially as the principal enzyme responsible for cholesterol elimination from the brain via the production of 24S-hydroxycholesterol, CYP46A1 was then found to control cholesterol turnover in this organ and the flow of metabolites required for memory and learning. Mice lacking CYP46A1 have impaired cognition. Likewise, increased CYP46A1 expression has beneficial effects of the brain function and reduces the formation of amyloid plaques in two mouse models of Alzheimer's disease. Our laboratory has characterized CYP46A1 biochemically and structurally and revealed that this enzyme is conformationally flexible. CYP46A1 was found to have the unusual ability for endobiotic-metabolizing P450 to bind molecules of different size and shape including a number of marketed drugs. We established that in most cases drug binding inhibits the CYP46A1 activity as assessed by studies in the reconstituted system in vitro. However, one pharmaceutical, the anti-HIV agent efavirenz significantly upregulates cholesterol 24S-hydroxylation (6-10-fold) mediated by either purified recombinant CYP46A1 or isolated bovine brain microsomes. Also, we found that some of the drugs that inhibit CYP46A1 inhibit CYP11A1 whose crystal structure we determined as well in complex with the sterol intermediate 22R-hydroxycholesterol. In this competing renewal we propose to expand on our previous findings and 1) elucidate the structural basis of drug binding to cholesterol-catabolizing P450s;2) evaluate the in vivo effects of CYP46A1 inhibitors and activators: and 3) investigate heteroactivation of CYP46A1. In vitro, in vivo and in silico studies will be used to achieve the goals of the project. CYP46A1 and CYP11A1 will be co-crystallized with drugs used clinically. Drugs will also be administered to mice to investigate their effect on cerebral cholesterol turnover and brain function. Furthermore, we will determine how efavirenz binds to CYP46A1 and stimulates cholesterol 24-hydroxylation. The results will significantly enhance our understanding of drug binding to P450 enzymes and lead to more selective and safer drugs on the market. In addition, a new direction in treatment of age- and/or disease-associated dementia and amyloid plaque deposition may be developed if we show pharmacological enhancement of CYP46A1 activity in vivo. Overall, we will further advance our knowledge of CYPs 46A1 and 11A1 and the mechanisms of cholesterol hydroxylation, processes of fundamental biological and medical significance.
This research will likely lead to safer drugs on the market and could be critical for development of new compounds to treat age- and Alzheimer's disease-associated problems with memory and brain degeneration.
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