Project Summary and Abstract: Liver receptor homolog-1 (LRH-1; NR5A2) is a phospholipid-sensing nuclear receptor (NR) expressed predominantly in the liver, pancreas, and ovaries that plays an important role in metabolic physiologies and pathophysiologies. Specifically, it has been characterized to regulate bile acid metabolism, cholesterol homeostasis, and steroidogenesis, and in turn is involved in various disease states such as type 2 diabetes, atherosclerosis, nonalcoholic fatty liver disease as well as a multitude of cancers. These diseases are all risk factors for metabolic syndrome that affects nearly a third of the American population. This represents a significant health concern as individuals diagnosed with metabolic syndrome have increased risk for developing cardiovascular diseases as well as hepatocellular, gastric, and colon cancers; cancers that have been associated with overexpression of LRH-1. This suggests LRH-1 to be a promising therapeutic target for such metabolic diseases and cancers. However, a lack of a wholistic understanding of the receptors regulatory mechanism presents a significant limitation to the success of LRH-1 as a therapeutic target. This propels the need to develop effective and specific chemical tools to modulate the receptor’s activity and expand our knowledge of its role in both healthy and disease pathways. Similarly to other NRs, LRH-1’s activity is tightly regulated via a multitude of pathways including ligand binding, DNA binding, cellular localization, post- translational modifications (PTMs), and coregulator interactions. However, unlike many other NRs, LRH-1 binds DNA as a monomer and a specific candidate endogenous ligand has yet to be identified. Rather, LRH-1 has been shown to bind an assortment of phospholipids in order to differentially control downstream signaling pathways. Hence, the interplay of additional complementary or conflicting regulatory pathways are essential for tight control of LRH-1 activity. Changes in these regulatory pathways leads to aberrant receptor activity contributing to disease pathophysiology. While the use of chemical approaches to probe NRs in metabolism has increased over the years, the use of chemoproteomics in this area remains limited. Proteomic analysis of NRs remains problematic as NRs are relatively low abundant and tightly bound to chromatin. As such, there is a need for tools to capture endogenous LRH-1 and LRH-1 transcriptional complexes to better understand LRH-1’s ability to control normal physiology or drive pathological processes. I propose the following specific aims develop novel methodologies and extend the understanding of LRH-1 regulatory mechanisms. In Aim 1, I will establish three LRH-1 specific probes to more efficiently probe and/or capture LRH-1 and LRH-1 transcriptional complexes. These include a biotinylated LRH-1 response element oligo probe, a modified DLPC biotin probe, and SR1848, small molecule inhibitor of LRH-1. In Aim 2, I will probe L...