Abstract
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.
Highlights
Liver receptor homolog 1 (LRH-1, NR5A2) regulates functions of liver, intestines, and pancreas; its aberrant activity is associated with tumorigenesis
Modeling of the hLRH-1 ligand-binding domain (LBD) in a Transcriptionally Inactive Conformation—All available crystal structures of the LRH-1 LBD represent the receptor in its active state
Because different orientations for H12 have been observed for estrogen receptor bound by different antagonists [47,48,49,50] (including those that completely abolished the association between H12 and the rest of the LBD [50]), helix H12 of LRH-1 was omitted from the model
Summary
Liver receptor homolog 1 (LRH-1, NR5A2) regulates functions of liver, intestines, and pancreas; its aberrant activity is associated with tumorigenesis. The established transcriptional targets of LRH-1 paired with -catenin include CCND1 and CCNE1 genes as well as MYC genes known for controlling cell differentiation, growth, and proliferation [6, 7, 9] Because these developmental pathways and associated genes are re-activated during tumorigenesis [11,12,13,14,15,16], an aberrant activity of LRH-1 is linked to different types of malignancies, including breast and endometrial cancers as well as intestinal tumors and cancer of the pancreas [17,18,19,20,21,22,23,24]. Candidate modulators have been identified using screening by molecular docking against a model of the LRH-1 ligand-binding domain (LBD) in an antagonized conformation This computational screening was followed by direct binding, transcription, and cell proliferation studies in vitro. The results described and discussed in this work suggest that specific antagonists of LRH-1 could be developed for studies of the receptor’s biological mechanisms as well as therapeutic treatments
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