The cytochromes P450 (CYPs) are responsible for a dazzling array of transformations in the disposition of xenobiotics and biosyntheses of hormones. Over 70% of drugs are metabolized by CYPs; hence tremendous resources are devoted to identification of substrates and inhibitors in drug development to avoid later attrition, or adverse drug interactions following introduction into the clinic. Among these, CYPs involved in steroid hormone biosyntheses are among the key targets for the pharmacotherapy of endocrine disorders and cancers. The range of catalytic selectivity and the breadth of substrates transformed by CYPs are immense; however the interplay of protein dynamics, ligand binding, and catalysis used to achieve this flexibility remains poorly understood. Within this void, nothing is known about how these are impacted by their physiological context. We have adopted human aromatase (CYP19A1), responsible for estrogen biosynthesis from androgens, as a model to address these longstanding and important questions. Phylogenetic analysis supports that CYP19A1 is among the most primordial human CYPs and it is functionally representative of those steroidogenic enzymes catalyzing sequential transformations. The translational relevance of CYP19A1 cannot be underestimated since it has vital role in maintaining numerous tissues and has demonstrated immense value as a pharmacotherapy target for gynecological disorders, cancers and infertility. There are three Specific Aims to test our hypotheses, we will: 1) Map pathways for 19A1 ligand entry, product egress, and delineate the structural underpinnings of selectivity. The working hypotheses are that i) substrates, intermediate products, and inhibitors give rise to unique equilibrium dynamics and ii) that selectivity is conferred by transient bound states as ligands enter and exit the enzyme through distinct channels. 2) Delineate the impact of membrane complexity on catalysis, ligand binding kinetics and global dynamics in 19A1. The working hypotheses are i) Selectivity is dominated by kinetics involving multiple protein states and conformational transitions, ii) catalytic and inhibitory 19A1 complexes display distinct dynamics, and iii) both protein dynamics and binding kinetics are impacted by membrane properties. 3) Determine the architecture of 19A1-NDs in variable environments by small-angle x-ray and neutron scattering. The working hypothesis is that the insertion depth and orientation of 19A1 are determined by the surface and bulk properties of the bilayer.
These Aims are technically- independent, but convergent approaches that are highly synergistic in combination, and will not only allow novel insight into CYP19A1, but also unveil paradigms that will transform our understanding of CYP function.
The proposed research is relevant to public health because it promises to open the door to novel strategies for drug development to target endocrine disorders and cancers with reduced drug-drug interactions and adverse effects. This research will resolve fundamental questions about the functional dynamics of aromatase, as well as validate new tools and unveil paradigms that will be broadly applicable to advancing our understanding of the entire P450 enzyme family.
