Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

Structure-based discovery of antagonists of nuclear receptor LRH-1.

Liver receptor homolog 1 (nuclear receptor LRH-1, NR5A2) is an essential regulator of gene transcription, critical for maintenance of cell pluripotency in early development and imperative for the proper functions of the liver, pancreas, and intestines during the adult life. Although physiological hormones of LRH-1 have not yet been identified, crystallographic and biochemical studies demonstrated that LRH-1 could bind regulatory ligands and suggested phosphatidylinositols as potential hormone candidates for this receptor. No synthetic antagonists of LRH-1 are known to date. Here, we identify the first small molecule antagonists of LRH-1 activity. Our search for LRH-1 modulators was empowered by screening of 5.2 million commercially available compounds via molecular docking followed by verification of the top-ranked molecules using in vitro direct binding and transcriptional assays. Experimental evaluation of the predicted ligands identified two compounds that inhibit the transcriptional activity of LRH-1 and diminish the expression of the receptor's target genes. Among the affected transcriptional targets are co-repressor SHP (small heterodimer partner) as well as cyclin E1 (CCNE1) and G0S2 genes that are known to regulate cell growth and proliferation. Treatments of human pancreatic (AsPC-1), colon (HT29), and breast adenocarcinoma cells T47D and MDA-MB-468 with the LRH-1 antagonists resulted in the receptor-mediated inhibition of cancer cell proliferation. Our data suggest that specific antagonists of LRH-1 could be used as specific molecular probes for elucidating the roles of the receptor in different types of malignancies.

Establishment of a molecular diagnostic system for detecting human papillomavirus in clinical samples in Sri Lanka.

Human papillomavirus (HPV) is associated with variety of clinical conditions that range from innocuous lesions to cancer in both men and women. Consensus primer-mediated PCR assays have enabled screening for a broad spectrum of HPV types. We have established a molecular diagnostic system for detecting HPV DNA in clinical samples from STD clinics in Sri Lanka and compared the efficacy of three different primer sets, MY09/11, GP5+/6+ and CPI/IIG primer sets, to determine which primer set or combination of primers is most efficacious in screening for HPV. Cervical and urethral swabs were obtained from 51 patients who were suspected of having HPV. The presence of HPV DNA in swabs was detected by MY09/11 PCR (33%), GP5+/6+ PCR (72%) and CPI/IIG PCR (57%) primers. HPV DNA was detected in 23% of samples by all three methods, in 43% by any two methods, and in 6% only by GP5+/6+ primer set. GP5+/6+ PCR alone was capable of detecting the most number of HPV positives but, any single PCR method for the detection of HPV may underestimate the true prevalence of HPV in clinical samples. Nested PCR assay with MY09/11 and GP5+/6+ primer sets had higher sensitivity than singleplex PCR but, due to the risk of cross contamination in employing nested PCR, it was concluded that GP5+/6+ PCR is more suitable for HPV DNA detection in epidemiologic and clinical follow-up studies in Sri Lanka.