Edited by Norma AllewellLiver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face --stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.Liver receptor homolog 1 (LRH-1; NR5A2) is a nuclear hormone receptor (NR) 3 that controls expression of a diverse set of genes important both in normal physiology and disease. In addition to a vital role during development (1, 2), LRH-1 regulates many genes related to metabolism, proliferation, and cell survival. In the liver, LRH-1 regulates bile acid biosynthesis (3) and reverse cholesterol transport (4, 5), affecting hepatic and circulating cholesterol levels. Glucose metabolism is also regulated by LRH-1 at several points, including GLUT-4-mediated transport (6) and glucose phosphorylation, the latter of which is essential for proper postprandial glucose sensing, flux through glycolysis and glycogenesis pathways, and de novo lipogenesis (7). LRH-1 is a key mediator of the cell stress response through control of genes involved in the hepatic acute phase response (8) and in the cytoprotective resolution of endoplasmic reticulum stress (9). Additionally, LRH-1 can be aberrantly overexpressed in certain cancers and can promote tumor growth through estrogen receptor and ␤-catenin signaling (10 -16).Considering the breadth and significance of these physiological effects, LRH-1 modulators are highly desired as potential therapeutic agents. Chemical modulators would al...

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, Liver Receptor Homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases. Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly-discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of inflammatory bowel disease, the new agonist increases expression of LRH-1-conrolled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility. glucose metabolism suggests therapeutic potential for LRH-1 agonists in metabolic diseases such as nonalcoholic fatty liver disease, type II diabetes, and cardiovascular disease. Indeed, the phospholipid LRH-1 agonist dilauroylphosphatidylcholine (DLPC; PC 12:0/12:0) improves glucose tolerance, insulin sensitivity, and triglyceride levels in obese mice 6 . These anti-diabetic effects occur in an LRH-1-dependent manner and have been primarily attributed to a reduction of de novo lipogenesis 6 . In addition, targeting LRH-1 in the gut has therapeutic potential for inflammatory bowel disease, where LRH-1 overexpression ameliorates disease-associated inflammation and cell death 10 .While small molecule LRH-1 modulators are highly sought, the large and lipophilic LRH-1 binding pocket has been extremely challenging to target. A promising class of agonists developed by Whitby and colleagues features a bicyclic hexahydropentalene core scaffold [11][12] . The beststudied of this class, named RJW100, was discovered as a part of an extensive synthetic effort to improve acid stability and efficacy of a related compound, GSK8470 12 (Figure 1A). We recently determined the crystal structure of LRH-1 bound to RJW100 and made a surprising discovery: it exhibits a completely different binding mode than GSK8470, such that the bicyclic cores of the two agonists are perpendicular to each other (Figure 1A) 13 . As a result, the two compounds use different mechanisms to activate LRH-1 but exhibit similar activation profiles in luciferase reporter assays 13 . A tendency for ligands in this class to bind unpredictably in the hydrophobic pocket has likely been a confounding factor in agonist design; however, insights from the LRH-1-RJW100 structure have provided new strategies to improve activity.In the LRH-1-RJW100 crystal structure, the ligand hydroxyl group contacts a network of water molecules deep in the ligand binding pocket (Figure 1B). This water network coordinates a

Sphingoid bases are cytotoxic for many cancer cell lines, and are thought to contribute to suppression of intestinal tumorigenesis in vivo by ingested sphingolipids. This study explored the behavior of a sphingoid base analog, (2S,3S,5S)-2-amino-3,5-dihydroxyoctadecane (“Enigmol”), that cannot be phosphorylated by sphingosine kinases and is slowly N-acylated, therefore, is more persistent than natural sphingoid bases. Enigmol had potential anti-cancer activity in a National Cancer Institute (NCI-60) cell line screen, and was confirmed to be more cytotoxic and persistent than naturally occurring sphingoid bases using HT29 cells, a colon cancer cell line. Although the molecular targets of sphingoid bases are not well delineated, Enigmol shared one of the mechanisms that has been found for naturally occurring sphingoid bases: to “normalize” the aberrant accumulation of β-catenin in the nucleus and cytoplasm of colon cancer cells due to defect(s) in the adenomatous polyposis coli (APC)/β-catenin regulatory system. Enigmol also had anti-tumor efficacy when administered orally to Min mice, a mouse model with a truncated APC gene product (C57Bl/6JMin/+ mice), decreasing the number of intestinal tumors by half at 0.025 % of the diet (w/w), with no evidence of host toxicity until higher dosages. Enigmol was also tested against the prostate cancer cell lines DU145 and PC-3 in nude mouse xenografts, and suppressed tumor growth in both. Thus, Enigmol represents a novel category of sphingoid base analog that is orally bioavailable and has the potential to be effective against multiple types of cancer.

Peroxisome proliferator-activated gamma coactivator 1- (PGC1) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1 binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1 is known to bind and activate LRH-1, mechanisms through which PGC1 changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1-PGC1 complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1 with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1-PGC1 complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1 induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1 but promoted allosteric signaling from the helix 6/-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1-PGC1 interaction and may illuminate strategies for selective therapeutic targeting of PGC1-dependent LRH-1 signaling pathways.