Application of Solution NMR to Membrane Cytochrome P450 17A1 (CYP17A1) and Structure-based Design of Inhibitors with Improved Selectivity for Steroidogenic CYP17A1 over CYP21A2

Monday, August 13, 2018


Dr. Rahul Yadav

Department of Medicinal Chemistry

University of Michigan

Abstract:

Cytochrome P450 17A1 (CYP17A1) is a 57 kDa heme-containing microsomal membrane protein that operates at an intersection between the production of mineralocorticoids, glucocorticoids, and sex steroids. It catalyzes two essential reactions in humans:  hydroxylation of pregnenolone and progesterone at C17, and subsequent C-C bond cleavage of 17α hydroxypregnenolone (17,20-lyase reaction) to generate the first male sex steroid. Previous studies have suggested conformational changes in CYP17A1 upon binding of various substrates and the functional modulator cytochrome b5. Solution NMR was carried out to further investigate these aspects of CYP17A1 structure. The inhibitor abiraterone was used to saturate triple-labeled (2H, 15N, 13C) CYP17A1 for solution NMR studies. Abiraterone has a significant stabilizing effect on CYP17A1 due to its high affinity (KD < 100 nM). Additionally, abiraterone coordinates to the heme iron to yield the low-spin state. Using conventional NMR experiments for backbone resonance assignment, 300 resonances of 458 identifiable resonances have been assigned to date. However, the paramagnetic effect of the heme iron negatively affects the ability to detect resonances close to the active site. Therefore, an alternative method was developed to express human P450 proteins with metal-substituted protoporphyrin IX complexes including cobalt PPIX and other diamagnetic metals such as gallium, silver, and copper protoporphyrin IX. Using these approaches, we have peeked into the conformational changes in the solution structure of CYP17A1.
CYP17A1 is a target for prostate cancer treatment having FDA approved drug “abiraterone”  that inhibit both the activities of CYP17A1. Abiraterone, although clinically efficacious against prostate cancer, is mired with off-target inhibition of a second steroidogenic enzyme, cytochrome P450 21A2 (CYP21A2). Therefore, reduction in off-target inhibition by abiraterone without compromising inhibition against CYP17A1 is desirable. A series of abiraterone analogs were designed and evaluated for improved selectivity for CYP17A1 over CYP21A2. Structural evidence suggest subtle difference in the orientation of progesterone in the active site of CYP17A1 vs CYP21A2 and modifying B-ring substituents of steroid core may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs in this series significantly increased selectivity by decreasing CYP21A2 inhibition up to 84-fold compared with 6.5-fold inhibition for abiraterone. Crystal structure of these analogs shows new contacts with CYP17A1 active site residues, which conserve the tight binding properties of abiraterone. Overall, Reducing the off-target inhibition of CYP21A2 is expected to reduce the undesirable clinical side effects resulting from disruption of glucocorticoid and mineralocorticoid biosynthesis.


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