Structure and dynamics of clinically-relevant cytochrome P450 enzymes - Summer undergraduate research experience supplement

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Project Summary/Abstract: Human cytochrome P450 enzymes are dynamic and often promiscuous monooxygenases. Some function in the biosynthesis of critical endogenous compounds and are frequent drug targets. Others are dominant factors in drug metabolism, dictating drug clearance and/or prodrug activation. For both, understanding P450 interactions with substrates, inhibitors, and their catalytic partner proteins provides substantial useful information in drug design. Conformational changes that P450s must undergo to channel ligands to the active site and for a single P450 to accommodate many different small molecule scaffolds only randomly become apparent in comparing the X-ray structures achievable. Despite substantial efforts, some key human P450 enzymes have not yielded to crystallization. Many drug substrates have lower active site affinity and/or multiple orientations not suitable to determining clear X- ray structures. There are no structures of human P450 enzymes with reductase or cytochrome b5 catalytic partners bound. As a result, drug design is limited by available structural information. We are employing solution NMR as a viable orthogonal method to obtain the requisite atomic level structural information needed to understand human P450/ligand and P450/protein interactions. While the size, stability, and the absence of information relating individual NMR resonances to the corresponding amino acid have all thus far prevented the determination of any human P450 structures by NMR, we have the combined expertise and preliminary data to demonstrate that this feat is now technically possible. The Pochapsky lab has developed the expertise to determine solution NMR structures of slightly smaller, soluble bacterial P450 enzymes. The Scott lab developed the capacity to generate human membrane P450 enzymes in the isotopically-labeled forms, with the amounts and with the stability required for NMR experiments. Thus, based on substantial preliminary data, we propose to advance strategies for determining human P450 structures by solution NMR while determining the human steroidogenic CYP17A1. Successful completion of this aim will not only further establish the feasibility of NMR structures for human membrane P450 enzymes, but will do so for an important prostate cancer drug target for which additional structural information is essential to further drug design. The current request is for a supplement supporting summer research for Aliyu Alghali, '22 a Brandeis University Wien Scholar student from Sierra Leone. Aliyu will be trained in the expression and purification of CYP17A1 in isotopically labeled form suitable for multidimensional NMR experiments. If time permits, he will also be trained in the acquisition, processing and analysis of the data.