NR5A2 (Liver receptor homologue-1; LRH1) belongs to the four-member NR5A, or Ftz-F1, subfamily V of nuclear receptors. Murine LRH1 was originally identified due to its sequence homology to Drosophila Fushi tarazu factor-1. Orthologs were subsequently identified in other species including rat, chicken, horse, zebrafish and human. LRH1, and its closest family member steroidogenic factor-1 (SF-1, NR5A1), bind to identical DNA consensus sequences (response elements; REs) and both are able to bind phospholipids in their ligand binding domains (LBDs). LRH1 was shown to regulate expression of Cyp19 (aromatase), suggesting LRH1 as a target for inverse agonists for the treatment of ER-positive breast cancers. Interestingly, recently it was shown that LRH1 promotes motility and invasiveness in both ER-positive and ER-negative breast cancer cells (MDA-MB-231), with remodeling of the actin cytoskeleton and E-cadherin processing observed with LRH1 over-expression. These findings suggest that inhibition of LRH1 activity should be useful in attenuating both migration and invasion in ER-positive and ER-negative breast cancers. In addition to LRH1’s role in cholesterol metabolism and bile acid homeostasis, the receptor can impact expression of markers of acute phase response (APR) to tissue injury. Thus, we endeavored to identify potent and selective LRH1 inverse agonists which may provide new approaches for the treatment of cancer and to blunt APR. We pursued a Center-based Initiative with transfection studies testing the ability of various chemical scaffolds to inhibit LRH-1-mediated activation of the Cyp19-Aromatase-luciferase and the StAR-luciferase reporter, followed by counterscreening against SF-1 and VP16, as well as studies examining the effect of compounds on LRH-1 modulation of the APR markers haptoglobin and serum amyloid A1 and A4 (SAA1 and SAA4). Combined, the studies led to identification of two novel LRH-1 inverse agonist probe compounds ML179 (PubChem CID 45100448) and ML180 (PubChem CID 3238389) with potent activity in breast cancer cells. In reporter assays ML179 and ML180 had potency IC50 values of 320nM and 3.7 μM and maximum efficacy of 40% and 64% repression, respectively. It is unclear at this stage if maximum repression is more important than potency; thus two probes were declared that differ from each other in terms of potency and efficacy. Further optimization is focused on improving both potency and efficacy. Also, it is likely that the selectivity of these probe compounds versus SF1 is cell context-and promoter-dependent. The mechanism of action of these probes will require much more detailed studies which are currently underway.