Structure and Function of Cytochrome P450 17A1

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DESCRIPTION (provided by applicant): Cytochrome P450 17A1 (CYP17A1) is a dual-function monooxygenase with a key role in human steroidogenesis. Since CYP17A1 is essential for androgen and estrogen production, understanding how this enzyme functions has practical value in reproductive biology, hormone-responsive chemotherapy, and the understanding of diseases resulting from CYP17A1 defects. To date, investigations of CYP17A1 have been limited by the absence of experimental structural information of this membrane protein. The first structures now show that inhibitors bind very differently from proposed and provide a new opportunity to evaluate CYP17A1 binding, catalysis, and inhibition at a substantially more detailed level. The objective of this proposal is to understand the mechanisms controlling the multifunctional reactions of CYP17A1 through convergent structural, synthetic, and functional approaches. Our central hypothesis is that steroidal substrates bind in an overall orientation similar to that observed for inhibitors in the new structures, but with tight spatial control of liand position and proton delivery directing substrates toward either hydroxylation or lyase reactions. Specifically we will test this hypothesis by 1) generation of X-ray structures that determine substrate binding orientations and interactions with CYP17A1, 2) functional evaluation of key amino acids in substrate binding and catalysis and of proposed mechanisms for hydroxylase vs. lyase reactions, and 3) testing our understanding of CYP17A1 function via the design, synthesis, and evaluation of novel probe substrates and inhibitors. The expected outcome is a detailed understanding of the structural features that control binding and catalysis of native CYP17A1 substrates for both catalytic reactions. The proposed research generates a substantial knowledgebase to guide the design, development, and improvement of more effective pharmaceutical inhibitors with improved selectivity for CYP17A1 and its lyase activity. These outcomes meet NIH goals by probing an important enzyme in hormone biosynthesis that can potentially be manipulated for the treatment of androgen-sensitive and estrogen-responsive cancers, as well as other steroid-related diseases.