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.

Probe Structure & Characteristics

ML 180
CID 3238389
SID 99344023

ML 179
CID 45100448
SID 92092843

Recommendations for scientific use of the probe

• What limitations in current state of the art is the probe addressing? Currently there are no inverse agonists described for LRH1. Whitby and coworkers have reported the identification of cis-bicyclo[3.3.0]-oct-2-enes as synthetic agonists for both SF-1 and LRH1 with one compound in particular having the ability to induce the doubling of mRNA levels of SHP, a downstream target of both receptors, in HepG2 cells [14]. While these synthetic ligands are intriguing, several problems remain. First, the functional activity of these compounds was determined using a FRET-based biochemical coactivator recruitment assay using small peptides representing the LXXLL motifs of the cofactor; for LRH1 a peptide derived from TIF2 (SRC2) was used and for SF-1 a 23 amino acid peptide fragment of DAX-1 was used. The complimentary cellular activity of these compounds was described for only one compound. Second, the most potent and efficacious agonists lacked functional selectivity over SF-1. The paper describes two partial agonists of LRH1 that are devoid of SF-1 activity showing promise for the ability to obtain functional selectivity. Perhaps more important was the discovery of a potent partial agonist of SF-1 that was devoid of LRH1 activity, strongly suggesting the ability to discover potent and selective agonists within the NR5A subfamily. To test the utility of these compounds, we synthesized two of the most potent compounds described in the Whitby manuscript and evaluated these molecules for their ability to modulate LRH1 in a cell-based assay. Prior to testing, the structures of these compounds were confirmed by NMR and their high purity was confirmed by LC-MS. Unfortunately, these compounds, although potent in a biochemical assay, appear to be cytotoxic even at modest concentrations. Therefore, the discovery of potent, cell active LRH1 modulators remains a high priority in elucidating the tissue-specific functions of LRH1 and its role in mammalian physiology.

Overall the goals of the LRH1 modulator program are to identify potent and selective in vivo active LRH1 agonists and inverse agonists. Obviously, we need to better understand the function of this important receptor and its role in diseases like breast cancer if we are going to development new treatments. To do that, first we need to accelerate the identification of chemical probes. Prior to the work described in this Probe Report, there were no potent in vivo active LRH1 agonists and no reports of any inverse agonists. Thus, probes ML180 and ML179 are first in class LRH1 inverse agonists and these probes constitute significant contributions to the field.

Additional mechanism of action (MOA) studies are required to determine if inverse agonists of LRH1 function by modulating the receptors activity directly by either displacing co-activators or by recruiting co-repressors. It is also possible that these compounds modulate the post-translational status of the receptor directly by impacting recruitment of transcriptional machinery by affecting the receptors’ occupancy at promoters of LRH1 target genes. PTMs could also modulate the receptor such that it trans-represses target genes in a DNA binding independent fashion.

• What will the probe be used for? Probes ML180 and ML179 can be used in cell biological studies to elucidate the role of LRH1 in metabolic diseases, and tumorigenesis. Recently, LRH1 has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Simpson and coworkers were the first to report high levels of LRH1 expression in preadipocytes and they correlated the expression of LRH1 with transcriptional activation of the aromatase cytochrome p450 (CYP19) gene [1]. Aromatase expression has been shown to be highly upregulated in breast tumors as well as breast adipose tissue surrounding tumors. The hallmark of upregulation of CYP19 expression is a switch in promoters from the normal adipose-specific promoter I.4 to the gonadal type PII promoter on the CYP19 gene [2–4]. The Cyp19PII promoter is cAMP-dependent and stimulators such as prostaglandin E2, forskolin, and PMA dramatically induce LRH1-dependent Cyp19PII expression up to, in some reports, 500 fold. This activity was further induced in a synergistic fashion by coexpressing LRH1 and the transcription factors GATA3 and GATA4 in the presence of a CYP19PII luciferase reporter plasmid following stimulators of the cAMP pathway [5]. Moreover, this increase in aromatase expression is completely abrogated following overexpression of SHP which is a negative regulator of LRH1 activity [6, 7]. It has been shown that upregulation of aromatase expression in breast adipose tissue surrounding tumors is responsible for the 10–50 fold increase in estrogen produced in these cells [8]. Unfortunately, this provides fertile ground for the growth and spread of estrogen-dependent tumors which comprise nearly 75% of all breast cancer occurrences in post-menopausal women [1, 9]. Aromatase inhibitors are used clinically in breast cancer treatment but unfortunately these compounds reduce estrogen production in other tissues giving rise to significant side effects such as bone loss. Therefore, inhibitors or inverse agonists of LRH1-dependent activation of aromatase expression could represent a strategy for the development of novel breast-specific tumor therapies.

In addition to modulating expression of the aromatase gene in breast tumors and surrounding tissues, LRH1 has been implicated in cell proliferation and intestinal cancer. Murine hepatic and pancreatic cells overexpressing LRH1 more than doubled in growth rate and formed colonies in soft agar [10]. LRH1 was shown to mediate these effects by acting in synergy with β-catenin to transactivate CyclinD1 and CyclinE1. Furthermore, these effects were reversed by introducing LRH1 siRNA or by overexpression of SHP demonstrating the functionally specific role of LRH1 in cell proliferation. These studies were further extended to examine the effects of LRH1 expression on intestinal tumorigenesis as LRH1 is highly expressed in rapidly dividing intestinal crypt cells. Auwerx and coworkers examined the effects of reduced LRH1 expression on intestinal tumorigenesis using two independent mouse models of the disease [10]. APCmin/− mice that were made haploinsufficient for LRH1 showed lower LRH1 expression and dramatically less tumor formation than mice with wild-type levels of LRH1 expression. In addition, mice lacking one LRH1 allele that were treated with AOM, a chemical inducer of colon cancer, had significantly reduced aberrant crypt foci than mice that expressed normal levels of LRH1.

More recently, overexpression of LRH1 in both ER positive and ER negative cell lines has been shown to promote motility and cell invasiveness in both ER-positive as well as ER-negative breast cancer cells (MDA-MB-231), with remodeling of the actin cytoskeleton and E-cadherin processing observed [11]. This observation suggests that inverse agonists of LRH1 would also be useful in treating ER negative breast cancer. We present data showing the ability of ML180 to inhibit the growth of MDA-MB-231 cells as well as other ER negative and ER positive cancer cell lines.

• Who in the research community will use the probe? Probes ML180 and ML179 can be used by academic researchers studying cell biology, molecular biology, and tumor biology. In addition, the two identified probes will be useful for any researcher working in the field of metabolism, cancer biology, APR and drug discovery.

• What is the relevant biology to which the probe can be applied? Most nuclear receptors function as homodimers or as heterodimers with the retinoid X receptor (RXR) to bind their cognate DNA response elements in promoter regions of target genes [12–14]. However, members of the NR5A subfamily such as SF-1 and LRH1 bind DNA with high affinity as monomers through interactions between the Ftz-F1-consensus binding site on target genes and the Ftz-F1 box, which is a 26 amino acid stretch at the C-terminus of the DNA binding domain (DBD) of these receptors [15]. Additionally, most nuclear receptors require binding of ligand to become transcriptionally active. The model of activation for most nuclear receptors suggests that ligand binding induces conformational changes in the LBD of the receptor that leads to recruitment of coactivators and basal transcriptional machinery with subsequent activation of target genes. Interestingly, members of the NR5A subfamily appear to be constitutively active when expressed in cells. Recently a number of laboratories have identified the presence of phospholipids in the ligand binding pockets of both SF-1 and LRH1 and their presence leads to the recruitment of coactivators in vitro [16–18]. Whether phospholipids are in fact endogenous ligands of NR5A subfamily receptors remains undetermined. The possibility that the NR5A family represent ligand-independent transcription factors does exist. However, a recent publication by Whitby and coworkers [19] revealed small molecule agonists for both LRH1 and SF-1. These compounds were characterized in a biochemical assay utilizing purified protein. The most potent LRH1 agonist described lacked functional selectivity over SF-1 although a potent partial agonist of SF-1 lacking LRH1 activity was discovered. Partial agonists of LRH1 (EC₅₀ 1.2 μM, 45% relative efficacy) were also described that lacked affinity for SF-1. More importantly, we synthesized and characterized several examples from the Whitby manuscript and these compounds, although potent in a biochemical assay, were cytotoxic even at modest concentrations (~10 μM).

Perhaps more important is a recent publication resulting from our MLPCN screening center describing a HTS campaign for SF-1 [20]. This screen revealed that chemically tractable small molecule inverse agonists of SF-1 can be discovered directly from an HTS campaign. These compounds were inactive in the RORα counterscreen indicating that they do not inhibit luciferase and they are not potent cytotoxic agents. These studies, combined with the Whitby publication, support the notion that potent and selective small molecule agonist and inverse agonists of LRH1 can be discovered.

Crystal structures for both SF-1 and LRH1 have been solved and these structures have provided some clues to the constitutive activity of these receptors [17, 21, 22]. Most nuclear receptors undergo changes in the conformational dynamics of the ligand binding pocket upon ligand binding resulting in stabilization of the AF2 (activation function 2) surface of the coactivator binding interface of the receptors creating a charge clamp to facilitate recruitment and binding of coactivators [12, 14]. In contrast, the crystal structures of SF-1 and LRH1 show that this stabilization of the coactivator interacting region of the LBD’s may occur as a result of a unique fourth sandwich layer formed by helix 2 (H2) [17, 22]. This region of H2 comes into close proximity with H12 in the absence of ligand to constitutively stabilizing AF2 facilitating coactivator binding. This structural feature of the receptor is also present in the murine receptor structure that was obtained with an empty ligand binding pocket. As mentioned above, the crystal structures of both human and mouse SF-1 as well as human LRH1 revealed the presence of bacterial phospholipids in the ligand binding pockets of these receptors. While the exact role of phospholipids in modulating the activity of these receptors is controversial, these findings further confirm that the NR5A receptors are capable of binding ligands that could potentially modulate their activity in vivo.

Relevance to Human Health: In addition to the role of LRH1 in tumorigenesis discussed above, LRH1 has been shown to play a pivotal role in cholesterol metabolism and bile acid homeostasis in the liver [23]. LRH1 transcriptionally regulates cholesterol 7α-hydroxylase (CYP7A1) [24], the rate-limiting enzyme of the bile acid biosynthetic pathway along with sterol 12 α-hydroxylase (CYP8B1) [25], which is required for cholic acid production. Moreover, LRH1 regulates transcription of a number of key enzymes involved in the uptake of cholesterol from tissues and plasma. Cholesterol ester transfer protein (CETP) is an LRH1 target gene that catalyzes the transfer of cholesterol esters from HDL particles to lipoproteins that can be extracted from serum by the hepatic low density lipoprotein receptor [12, 26]. CETP is currently targeted by several pharmaceutical companies for treatment of hypercholesterolemia, although Pfizer recently withdrew its late stage clinical candidate. It is unclear at this point in time if this is a problem with the specific compound or a problem with the mechanism. Perhaps modulation of the pathway instead of inhibition of one step might provide an approach that is better tolerated. In addition, apolipoprotein A1 is transcriptionally regulated by LRH1 and acts as an acceptor molecule for phospholipids and cholesterol coming from peripheral tissues forming pre-HDL particles. These particles mature and are transferred into hepatocytes by the scavenger receptor class B type I cell surface receptor (SR-BI) which is also transcriptionally regulated by LRH1 [27, 28]. Thus, it is clear that LRH1 modulation could play an important role in cholesterol homeostasis.

LRH1 also activates genes involved in inflammatory responses. Delerive and coworkers, were the first to identify LRH1 as a negative regulator of the hepatic acute phase response [29]. Ectopic expression of LRH1 led to reduced expression of pro-inflammatory genes such as haptoglobin, serum amyloid A and C-reactive protein following stimulation of hepatocytes with proinflammatory cytokines IL-1b and IL-6. In addition, LRH1 activated expression of interleukin-1 receptor antagonist (IL-1RA), a potent anti-inflammatory molecule, following induction by IL-1b and IL-6 as well as intraperitoneal injection of lipopolysaccharide [30]. Moreover, partial deficiencies in LRH1 expression in hepatocytes resulted in decreases in expression of IL-1RA and an exaggerated inflammatory response in vitro and in vivo indicating that LRH1 expression leads to the modulation of anti-inflammatory responses. More recently Venteclef and coworkers reported that the GSK selective synthetic agonist induces sumoylation-dependent recruitment of LRH1 to hepatic acute phase response (APR) promoters and this prevents clearance of the corepressor N-CoR resulting in repression of APR genes [31]. Preliminary studies in our lab using Huh7 cells stimulated with IL1β and IL6 results in significant increase in expression of LRH1, haptoglobin (Hp), serum amyloid A-4 (SAA1), and serum amyloid A-4 (SAA4) as determined by qPCR. Surprisingly and in contrast to the anticipated outcome, treatment of these cells with the inverse agonists ML180 and ML179 represses the expression of three of these genes (Hp, SAA1, SAA4) to the level of control cells (unstimulated) and LRH1 to levels below that in control cells. This finding confirms that our inverse agonists can repress the expression of LRH1 and more importantly, suggests that LRH1 inverse agonists can repress APR genes in similar fashion to LRH1 agonists. Clearly the mechanism of action of inverse agonists in this model is likely different than that of agonists. This finding warrants further mechanistic studies which will be carried out as part of this extended probe development project.

1. Introduction

The goal of this project is to identify modulators (agonists and inverse agonists) of the orphan nuclear receptor LRH1, which has been implicated in cancer by enhancing proliferation and cell cycle progression and metabolic disorders through its regulation of genes involved in cholesterol and bile acid homeostasis. In this specific Probe Report we focus on the discovery and characterization of inverse agonists of LRH1.

NR5A2 or Liver receptor homologue-1 (LRH1) is a member of the NR5A, or Ftz-F1, subfamily V nuclear receptors of which there are four members [12]. Murine LRH1 was originally identified due to its sequence homology to the Drosophila Fushi tarazu factor-1 but orthologs have been subsequently identified in several other species including rat, chicken, horse, zebrafish and human [32–37]. LRH1, and its closest family member steroidogenic factor-1 (SF-1, NR5A1), bind to identical DNA consensus sequences (response elements or REs) and both have the ability to bind phospholipids in their ligand binding domains (LBDs) [16–18]. However, LRH1 and SF-1 are expressed in different tissues and thus are considered likely to have non-overlapping, non-redundant functions. SF-1 expression is confined to steroidogenic tissues and adrenals where it regulates development, differentiation, steroidogenesis and sexual determination [13, 35, 37]. LRH1 is highly expressed in tissues of endodermal origin and its expression is essential for normal liver, intestine, and pancreas function. LRH1 has also been shown to be expressed in the ovary and adipose tissue.

In a very recent report, Chand and colleagues investigated the mechanism of action of LRH1 in invasive breast cancer cells [38]. They found that LRH1 promotes motility and cell invasiveness in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells and similar effects were observed in non-tumorigenic mammary epithelial cells. Interestingly, both remodeling of the actin cytoskeleton and E-cadherin processing were observed when LRH1 was over-expressed. These findings implicate LRH1 in promotion of migration and invasion in breast cancer independent of estrogen sensitivity. Together these findings provided strong evidence that LRH1 plays a significant role in tumor formation both in vitro and in vivo. Therefore, the identification of potent and selective LRH1 inverse agonists may provide new approaches for the treatment of cancer.

2. Materials and Methods

The SRIMSC Center Driven Research Project (CDRP) titled “Functional genomics approaches to small molecule discovery” was recently renewed. This project is focused on High throughput Functional Genomics and our overall goal is to continue to apply this platform towards pathway discovery and selectivity profiling of chemical probes emerging from the MLPCN network. During Years 1 and 2 of this MLPCN Center Driven Research Project we designed and constructed a nuclear receptor (NR) library using resources within the TSRI high-throughput cDNA screening platform. Using a combination of small molecule libraries and these novel GAL4 tagged libraries, we profiled signaling pathways of several orphan nuclear receptors. During this profiling, we discovered activity on RORA, RORG, and LRH1. These orphan NRs have been implicated in a wide range of diseases and syndromes including metabolic and immune disorders, cancer, and neurological dysfunction. One of these discoveries resulted in submission and approval of a MLPCN probe report (AIDs 1901, 1954, 2117, 2139) describing the first synthetic modulators of RORA and RORG. We demonstrated that these compounds can repress glucose production in hepatocytes, and expression of IL17 from TH17 cells, which clearly demonstrated the utility of these chemical probes in models of diabetes and autoimmune disease. A second project within the CDRP was to characterize synthetic inverse agonists of LRH1. The significance of these compounds is described in detail above. As a result, several assays were performed in order to identify novel inverse agonists of LRH1. The specific assays are listed in Table 1.

Table 1

Summary of Performed Assays.

2.2. Probe Chemical Characterization

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Compound	SR Number	MLS	CID	SID	Solubility in PBS (μM)	MichaelAcceptor 100 μM GSH trap	Stability in PBS (t1/2 (hr)
PROBE #1 (ML180)	SR-01000621848-2	MLS003153119	CID 3238389	SID 99344023	8.5	No	> 48 hr
PROBE #2 (ML179)	SR-03000001309-1	MLS003153122	CID 45100448	SID 92092843	3.9	No	> 48 hr

Graphical representations of the stability of the new probe compounds are shown in Figure 1. The new probes have been tested in numerous cell-based assays, demonstrating their cellular permeability, solubility, and stability.

Figure 1

Probe Stability.

LC-MS/MS

All analytical methods were in MRM mode where the parent ion was selected in Q1 of the mass spectrometer. The parent ion was fragmented and a characteristic fragment ion monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods were rapidly set up using Automaton^® (Applied Biosystems), where the compounds were listed with their name and mass in an Excel datasheet. Compounds were submitted in a 96-well plate to the HPLC autosampler and slowly injected without a column present. A narrow range centered on the indicated mass was scanned to detect the parent ion. The software then evaluated a few pre-selected parameters to determine conditions that maximized the signal for the parent ion. The molecule was then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass were determined. Three separate collision energies were evaluated to fragment the parent ion and the largest three ions were selected. Each of these three fragment ions was further optimized and the best fragment was chosen. The software then inserted the optimized masses and parameters into a template method and saved it with a unique name that indicated the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern were saved and reviewable later.

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS/MS analysis was used.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration was 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized—a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of-129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [39, 40].

2.3. Probe Preparation: Synthesis of LRH1 Inverse Agonist Probes and Analogs

Figure 2Synthesis of SR-01000621848 (Probe 1: ML180) (CID 3238389)

The first reaction was performed using a known method [41]. Cyclohexylurea (510 mg, 3.52 mmol) and malonic acid (370 mg, 3.52 mmol) were weighted into a sealed tube. Acetic anhydride (671 μL, 7.03 mmol) was added and the vial was heated 7 min in a microwave at 100°C. The mixture was then evaporated, EtOH was added and the product precipitated after triturating crude product. The solid was filtrated to provide 300 mg of 3-cyclohexyl-6-hydroxypyrimidine-2,4(1H,3H)-dione (41%) as white powder. ¹H NMR (400 MHz, (CD₃)₂SO): 1.09 (qt, J = 12.9, 3.4 Hz, 1H), 1.25 (qt, J = 12.9, 3.4 Hz, 2H), 1.52–1.64 (m, 3H), 1.76 (broad d, J = 13.0 Hz, 2H), 2.14 (qd, J = 12.5, 3.4 Hz, 2H), 3.58 (s, 2H), 4.41 (tt, J = 12.2, 3.5 Hz, 1H), 11.19 (s, 1H).

The chlorination reaction was performed using a known method [42]. 3-Cyclohexyl-6-hydroxypyrimidine-2,4(1H,3H)-dione (272 mg, 1.29 mmol) and tetrabutylammonium chloride (771 mg, 2.72 mmol) were weighed in a round bottom flask. Phosphorus oxychloride (2.55 mL) was added and the mixture was heated 4h at 50°C, then cooled to ambient temperature and poured in cold water. The aqueous phase was extracted three times by CHCl₃. The combined organic extracts were dried over Na₂SO₄, filtrated and evaporated. The crude residue was purified by silica gel column and eluted with hexane-EtOAc (60/40) to obtain 216 mg of 6-chloro-3-cyclohexylpyrimidine-2,4(1H,3H)-dione (73%) as a white powder. ¹H NMR (400 MHz, (CD₃)₂SO): 1.10 (qt, J = 12.9, 3.2 Hz, 1H), 1.26 (qt, J = 12.9, 3.1 Hz, 2H), 1.47–1.65 (m, 3H), 1.76 (broad d, J = 12.9 Hz, 2H), 2.25 (qd, J = 12.4, 3.3 Hz, 2H), 4.41 (tt, J = 12.1, 3.8 Hz, 1H), 5.82 (s, 1H), 12.21 (s, 1H).

6-Chloro-3-cyclohexylpyrimidine-2,4(1H,3H)-dione (50 mg, 0.22 mmol) and 1-(3-chlorophenyl)piperazine (129 mg, 0.66 mmol) were weighed into a sealed tube. Ethanol (300 μL) was added and the vial was heated 10 min in a microwave at 150°C. The mixture was directly applied to a silica gel column and eluted with CH₂Cl₂-MeOH (95/5) to obtain 51 mg of SR-01000621848 (61%, purity 99%) as a reddish powder.

FTIR: 2937, 2828, 1691, 1625, 1592, 1430, 1377, 1234, 1195, 1019, 946, 784, 768, 687, 679 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 1.17 (qt, J = 13.2, 3.4 Hz, 1H), 1.40 (qt, J = 13.2, 3.2 Hz, 2H), 1.62–1.76 (m, 3H), 1.85 (broad d, J = 13.0 Hz, 2H), 2.31 (qd, J = 12.6, 3.1 Hz, 2H), 3.27–3.34 (m, 4H), 3.45–3.53 (m, 4H), 4.78 (bt, J = 12.0 Hz, 1H), 4.99 (bs, 1H), 6.80 (dd, J = 8.3, 2.1 Hz, 1H ), 6.87–6.91(m, 2H), 7.20 (d, J = 8.4 Hz, 1H), 10.33 (s, 1H)

¹³C NMR (100 MHz, (CDCl₃)): 25.6, 26.4, 29.0, 46.0, 48.5, 80.2, 114.4, 116.2, 120.6, 130.3, 135.2, 151.5, 153.3, 164.0.

MS (ES-) m/z = 387 (found for C₂₀H₂₅ClN₄O₂-H⁺).

Data for SR-03000001309 (Probe 2: ML179) (CID 45100448)

SR-03000001309

SR-03000001309 (95% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing in the last step 1-(3-chlorophenyl)piperazine by 1-(3-(trifluoromethyl)phenyl)piperazine.

FTIR: 2935, 2854, 1694, 1626, 1450, 1434, 1389, 1353, 1310, 1232, 1161, 1120, 110, 947, 781, 694 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 1.16 (qt, J = 13.1, 3.3 Hz, 1H), 1.40 (qt, J = 13.1, 3.3 Hz, 2H), 1.60–1.76 (m, 3H), 1.83 (broad d, J = 13.6 Hz, 2H), 2.31 (qd, J = 12.4, 3.3 Hz, 2H), 3.32–3.39 (m, 4H), 3.49–3.56 (m, 4H), 4.79 (bt, J = 12.1 Hz, 1H), 5.01 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.1, 2.3 Hz, 1H ), 7.10–7.14 (m, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 10.50 (s, 1H)

¹³C NMR (100 MHz, (CDCl₃)): 25.5, 26.3, 28.9, 46.1, 48.4, 80.2, 112.5 (q, J = 4.4 Hz), 117.1(q, J = 4.1 Hz), 119.2, 124.1 (q, J = 272.7 Hz), 129.8, 131.7 (q, J = 32.1 Hz), 150.6, 153.4, 164.0.

MS (ES-) m/z = 421 (found for C₂₁H₂₅F₃N₄O₂-H⁺).

Data for SR-1310 (Probe Analog)

SR-03000001310

SR-03000001310 (97% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing in the last step 1-(3-chlorophenyl)piperazine by 1-(3-(trifluoromethyl)phenyl)piperazine.

FTIR: 2937, 2856, 1691, 1622, 1575, 1451, 1432, 1423, 1388, 1373, 1241, 1198, 1019, 955, 787 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 1.12 (qt, J = 13.1, 3.1 Hz, 1H), 1.36 (qt, J = 13.1, 3.1 Hz, 2H), 1.61–1.71 (m, 3H), 1.80 (broad d, J = 13.4 Hz, 2H), 2.30 (qd, J = 12.1, 3.2 Hz, 2H), 3.11–3.20 (m, 4H), 3.48–3.59 (m, 4H), 4.77 (bt, J = 12.2 Hz, 1H), 5.01 (bs, 1H), 6.93 (dd, J = 7.9, 1.6 Hz, 1H ), 7.17 (t, J = 8.0 Hz, 1H), 7.23 (dd, J = 8.1, 1.6 Hz, 1H), 10.29 (s, 1H)

¹³C NMR (100 MHz, (CDCl₃)): 25.5, 26.3, 28.9, 46.5, 50.7, 79.9, 118.5, 125.5, 127.6, 127.7, 134.4, 150.2, 153.2, 153.3, 164.1.

MS (ES-) m/z = 421 (found for C₂₀H₂₄Cl₂N₄O₂-H⁺).

Data for SR-1174 (Probe Analog)

SR-03000001174

SR-03000001174 (97% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing at the last step 1-(3-chlorophenyl)piperazine by 7-chloro-4-piperazin-1-ylquinoline.

FTIR: 2922, 2848, 1694, 1620, 1575, 1425, 1377, 1232, 1197, 1012, 874, 867, 829, 822, 790, 729 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 1.05 (qt, J = 13.1, 3.6 Hz, 1H), 1.25–1.42 (m, 2H), 1.57–1.70 (m, 3H), 1.76 (broad d, J = 13.3 Hz, 2H), 2.14 (qd, J = 12.4, 3.1 Hz, 2H), 3.31–3.37 (m, 4H), 3.60–3.67 (m, 4H), 4.77 (bt, J = 11.9 Hz, 1H), 5.04 (d, J = 1.8 Hz, 1H), 6.86 (d, J = 4.9 Hz, 1H ), 7.47 (dd, J = 9.0, 2.2 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 2.1 Hz, 1H), 8.77 (d, J = 4.9 Hz, 1H), 10.51 (s, 1H)

¹³C NMR (100 MHz, (CDCl₃)): 25.6, 26.3, 28.9, 46.3, 51.6, 53.4, 80.5, 109.1, 121.6, 124.5, 126.8, 129.1, 135.4, 150.1, 151.8, 153.3, 156.0, 163.9.

MS (ES-) m/z = 438 (found for C₂₃H₂₆ClN₅O₂-H⁺).

Data for SR-1409 (Probe Analog)

SR-03000001409

SR-03000001409 (97% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing at the first stage cyclohexylurea by phenylurea and at the last step 1-(3-chlorophenyl)piperazine by 1-(3-(trifluoromethyl)phenyl)piperazine.

FTIR: 3049, 2844, 1726, 1700, 1606, 1495, 1449, 1314, 1232, 1165, 1112, 1099, 1074, 950, 778, 697, 689 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 2.99–3.04 (m, 4H), 3.36–3.43 (m, 4H), 5.08 (s, 1H), 6.95–7.01 (m, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.23–7.27 (m, 2H), 7.38–7.44 (m, 2H), 7.44–7.50 (m, 2H), 10.95 (s, 1H).

¹³C NMR (100 MHz, (CDCl₃)): 46.0, 48.0, 79.4, 112.5 (q, J = 4 Hz), 116.9 (q, J = 4 Hz), 119.2, 124.2 (q, J = 272.0 Hz), 128.5, 128.6, 129.1, 129.7, 131.6 (q, J = 31.7 Hz), 134.8, 150.6, 153.5, 163.5.

MS (ES-) m/z = 415 (found for C₂₁H₁₉F₃N₄O₂-H⁺).

Data for SR-1395 (Probe Analog)

SR-03000001395

SR-03000001395 (97% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing at the first stage cyclohexylurea by ethylurea and at the last step 1-(3-chlorophenyl) piperazine by 1-(3-(trifluoromethyl)phenyl)piperazine.

FTIR: 2972, 1718, 1695, 1604, 1585, 1496, 1445, 1311, 1231, 1151, 1122, 1097, 1075, 956, 809, 787, 767, 700 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 1.23 (t, J = 6.9 Hz, 3H), 3.33–3.38 (m, 4H), 3.50–3.56 (m, 4H), 3.95 (q, J = 7.1 Hz, 2H), 5.03 (bs, 1H), 7.10 (dd, J = 8.4, 2.5 Hz, 1H ), 7.12–7.15 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 10.43 (s, 1H)

¹³C NMR (100 MHz, (CDCl₃)): 13.1, 35.4, 46.2, 48.3, 80.1, 112.8 (q, J = 3.7 Hz), 117.2 (q, J = 3.8 Hz), 119.3, 124.0 (q, J = 272.7 Hz), 129.9, 131.7 (q, J = 31.8 Hz), 150.5, 153.1, 153.3, 163.4.

MS (ES-) m/z = 367 (found for C₁₇H₁₉F₃N₄O₂-H⁺).

Figure 3Synthesis of SR-1393 (Probe Analog)

Data for 6-chloro-3-isobutylpyrimidine-2,4(1H,3H)-dione, ¹H NMR (400 MHz, (CDCl₃)): 0.92 (d, J = 6.8 Hz, 6H), 2.12 (n, J = 6.8 Hz, 1H), 3.74 (d, J = 7.5 Hz, 2H), 5.86 (s, 1H), 9.41 (bs, 1H).

SR-03000001393 (97% last step, purity >98%) obtained as a white powder, was synthesized following the same procedures described for SR-01000621848, replacing at the first stage cyclohexylurea by isobutylurea and at the last step 1-(3-chlorophenyl)piperazine by 1-(3-(trifluoromethyl)phenyl)piperazine.

FTIR: 2959, 1698, 1621, 1494, 1447, 1310, 1229, 1165, 1119, 1098, 1075, 994, 950, 785, 696 cm⁻¹.

¹H NMR (400 MHz, (CDCl₃)): 0.94 (d, J = 6.8 Hz, 6H), 2.15 (n, J = 6.8 Hz, 1H), 3.32–3.39 (m, 4H), 3.50–3.56 (m, 4H), 3.73 (d, J = 7.5 Hz, 2H), 5.02 (bs, 1H), 7.10 (dd, J = 8.4, 2.5 Hz, 1H ), 7.12–7.15 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 10.53 (s, 1H).

¹³C NMR (100 MHz, (CDCl₃)): 20.2, 27.2, 46.2, 47.2, 48.3, 80.0, 112.60 (q, J = 3.8 Hz), 117.1 (q, J = 3.8 Hz), 119.3, 124.1 (q, J = 272.9 Hz), 129.9, 131.8 (q, J = 32.0 Hz), 150.6, 153.3, 153.6, 163.9

MS (ES-) m/z = 395 (found for C₁₉H₂₃F₃N₄O₂-H⁺).

3. Results

3.2. Dose Response Curves for Probes and Aromatase Assay

Figure 4Dose response curves for SR-01000621848 (Probe 1, ML180) and SR-01000001309 (Probe 2, ML179) two LRH-1 modulator probes

293T cells were cotransfected with (A and C) full length LRH-1 and Cyp19 aromatase reporter, or (B and D) full length SF-1 and 5xSFRE reporter and treated with various concentrations of either SR-01000621848 (A and B) or SR-03000001309 (C and D) for 20 hours prior to luciferase activity measurement. Relative change was determined by normalizing to vehicle treatment. Treatment with both probes, SR-01000621848 (Max Rep=52% ; IC₅₀=3.7μM) and SR-03000001309 (Max Rep = 46% ; IC₅₀ = 320nM) showed selective efficacy against LRH-1 over SF-1.

These aromatase dose response data are available in PubChem as AID 488782.

3.3. Scaffold/Moiety Chemical Liabilities

There are no known chemical liabilities associated with the probes ML180 and ML179, or the identified probe analogs.

Discussion of SAR Studies

Starting with the initial selective LRH1 inverse agonist hit, SR-1848 (CID 3238389; ML180), we performed an extensive SAR study via analog purchase and synthesis. Three different sites in SR-1848 have been modified. A range of substituted aryl and heteroaryl groups have been introduced in position R₁ (see Table 4 in Section 3.4). In general, aryl units at the R₁ position possessing meta substituents (e.g., meta-Cl as in SR-1848; a CF₃ group at this position in SR-1309, or the substituted quinoline in SR-1174) result in the greatest LRH1 repression. The optimal substituents at the R₂ position—which give rise to the lowest IC₅₀ values for LRH1 inverse agonism, are alkyl groups like isobutyl in SR-1393 or cyclohexyl in many other analogs (e.g., SR-1309), or ethyl in SR-135. Introduction of a substituent at R₃ leads to a significant reduction or elimination of LRH1 activity; the results obtained thus far indicate that R₃ = H is required.

Figure 5Points of Structural Modification for SAR

Table 2SAR of Probes and Selected Analogs

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Structure	Identification Numbers	LRH1 IC50 (μM)	LRH1 Max Repression
	CID 3238389 (ML180, Probe 1) SR-1848	3.7	64% (10 μM)
	CID 45100448 (ML179, Probe 2) SR-1309	0.281	40% (550 nM)
	CID 45382271 (probe analog) SR-1393	0.061	35% (200 nM)
	CID 45480137 (probe analog) SR-1409	0.65	24% (2 μM)
	CID 45382284 (probe analog) SR-1395	0.378	40% (5 μM)
	CID 45100455 (probe analog) SR-1310	0.285	42% (5 μM)
	CID 44825223 (probe analog) SR-1174	4	75% (10 μM)

We selected SR-1848 (ML180) and SR-1309 (ML179) as the probes because these two compounds give the best results in inhibition of cancer cell growth. While SR-1848 is only 3.7 μM IC₅₀ vs. LRH1, it gives the maximum LRH1 repression of all compounds studied to date (64% at 10 μM). Probe ML179 (SR-1309) is 13-fold more potent (IC₅₀) than ML180 (SR-1848) against LRH1, but demonstrates a lower maximum repression of LRH1 transcription compared to ML180 (40% vs. 64%), albeit at a lower concentration. While SR-1393 (CID 45382271) is the most potent LRH1 inverse agonist discovered to date (IC₅₀ = 61 nM), it is weaker in repressing LRH1 transcription (35% at 200 nM) than SR-1309. In tests of these compounds against various cancer cell lines, SR-1309 proved superior to SR-1393 in the majority of cell lines studied, and therefore SR-1309 was declared a probe rather than SR-1393.

3.4. SAR Tables

Table 3SAR of Probe Compounds

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Compound Information						LRH1 %INH (AID 485348)		LRH1 Dose Response (AID 488782)		SF1 Fold Change (AID 488779)		SF1 Dose Response (AID 488780)		VP16 Fold Change (AID 488775)		QPCR mRNA Fold Change (AID 488769)
Sample ID	Structure	Source	MLS ID	SID	CID	LRH-1 AVG % Inhibition (at 5 μM)	SD	LRH-1 IC50	SD	SF-1 Avg Fold Change (at 5 μM)	SD	SF-1 IC50	SD	VP-16 Avg Fold Change (at 5μM)	SD	Haptoglob in mRNA Avg Fold change (at 10 μM)	SD	SAA1 mRNA Avg Fold change (at 10 μM)	SD	SAA4 mRNA Avg Fold change (at 10 μM)	SD
PROBE #1 SR-01000621848-2		P	MLS003 153119	99344023	3238389	52.33% (Active)	0.055	3.7μM (Active)	0.031	4.67% (Inactive)	0.13	> 10 μM (Inactive)	0.067	1.3% (Inactive)	0.012	0.87 (Active)	0.04	0.91 (Active)	0.00	0.55 (Active)	0.09
PROBE #2 SR-03000001309-1		S	MLS003 153122	92092843	45100448	45.67% (Active)	0.051	320nM (Active)	0.075	−1%	0.01	> 10 μM (Inactive)		−6%	0.049	0.86 (Active)	0.04	0.93 (Active)	0.04	0.55 (Active)	0.10

Analogs with “ND” indicates that their activities for a particular anti-target or mechanism-of-action assay were Not Determined due to lack of LRH1 activity compared to probes. Only the two probes were tested in the LRH1 target gene QPCR assay.

Table 4SAR of R1 Analogs

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Compound Information: R1 Analogs						LRH1 %INH (AID 485348)		LRH1 Dose Response (AID 488782)		SF1 Fold Change (AID 488779)		SF1 Dose Response (AID 488780)		VP16 Fold Change (AID 488775)
Sample ID	Structure	Source	MLS ID	SID	CID	LRH-1 AVG %Inhibition (at 5 μM)	SD	LRH-1 IC50	SD	SF-1 Avg Fold Change (at 5 μM)	SD	SF-1 IC50	SD	VP-16 Avg Fold Change (at 5μM)	SD
SR-03000001170-1		S		89649733	44825215	35.33% (Active)	0.045	921nM (Active)	0.069	9.33% (Inactive)	0.08	9.2μM (Inactive)	0.06	0%	0.000
SR-03000001307-1		P		92092833	1484049	45% (Active)	0.026	538nM (Active)	0.065	−3%	0.06	N/D		−7%	0.058
SR-03000001310-1		S	MLS003153123	92092844	45100455	42% (Active)	0.147	430nM (Active)	0.100	3.3% (Inactive)	0.08	N/D		−5%	0.050
SR-01000140561-3		P		89650166	3244825	32% (Active)	0.087	2.6μM (Active)	0.087	−3%	0.06	N/D		−16%	0.124
SR-03000001171-1		S		89649734	44825216	42% (Active)	0.053	4.8μM (Active)	0.038	0.67% (Inactive)	0.11	N/D		−27%	0.058
SR-03000001174-1		S	MLS003153125	89649737	44825223	34.667% (Active)	0.042	4.0μM (Active)	0.242	11.33% (Inactive)	0.04	N/D		7% (Inactive)	0.026
SR-03000001302-1		S		92092825	45100433	32.33% (Active)	0.045	ND		7% (Inactive)	0.07	ND		17% (Active)	0.044
SR-01000621845-2		P		89650164	3240644	1.33% (Inactive)	0.121	ND		−2%	0.11	ND		−9%	0.017
SR-03000001172-1		S		89649735	44825230	36% (Active)	0.036	20μM (Inactive)	0.017	−7%	0.08	N/D		−8%	0.076
SR-03000001173-1		S		89649736	44825240	30.33% (Active)	0.074	80μM (Inactive)	0.027	−4%	0.05	N/D		−4%	0.066
SR-01000140537-3		P		92092838	3241826	18% (Inactive)	0.156	ND		6%	0.11	ND		−12%	0.072
SR-03000001383-1		S		93375297	45382280	16.33% (Inactive)	0.035	ND		−12%	0.12	ND		0%	0.006
SR-03000001384-1		S		93375298	45382275	38.333% (Active)	0.040	13μM (Inactive)	0.015	−4%	0.13	N/D	N/D	−6%	0.085
SR-03000001385-1		S		93375299	45382282	16% (Inactive)	0.095	ND		5% (Inactive)	0.09	ND		14.3% (Active)	0.021
SR-03000001386-1		S		93375300	45382276	36.67% (Active)	0.091	ND		−112%	0.19	ND		ND
SR-03000001387-1		S		93375301	45382268	20.3% (Inactive)	0.091	ND		−83%	0.44	ND		−12%	0.030
SR-03000001388-1		S		93375302	45382285	16% (Inactive)	0.090	ND		13.33% (Inactive)	0.10	ND		16% (Active)	0.090
SR-03000001389-1		S		93375303	45382283	20% (Inactive)	0.046	ND		−9%	0.07	ND		1.3% (Inactive)	0.012
SR-01000140541-4		P		92092841	3571734	5.33% (Inactive)	0.055	ND		4.33% (Inactive)	0.06	ND		0%	0.100
SR-03000001175-1		S		89649738	44825217	0% (Inactive)	0.121	ND		1% (Inactive)	0.06	ND		−5%	0.084
SR-03000001176-1		S		89649739	44825219	2.67% (Inactive)	0.081	ND		−14%	0.17	ND		−4%	0.105
SR-03000001300-1		S		92092823	45100453	−0.30%	0.173	ND		−13%	0.21	ND		−48%	0.157
SR-03000001301-1		S		92092824	45100462	16.67% (Inactive)	0.055	ND		23.67% (Active)	0.08	ND		−5%	0.031
SR-01000607332-2		S		92092826	3237172	11% (Inactive)	0.046	ND		6% (Inactive)	0.05	ND		16.67% (Active)	0.235
SR-03000001390-1		S		93375304	45382269	3.33% (Inactive)	0.042	ND		−49%	0.01	ND		26% (Active)	0.020
SR-03000001391-1		S		93375305	45382272	11%	0.144	ND		5.33% (Inactive)	0.13	ND		13.3% (Active)	0.050
SR-03000001403-1		S		93577916	45480135	16% (Inactive)	0.050	ND		−19%	0.06	ND		1.3% (Inactive)	0.257
SR-03000001404-1		S		93577917	45480133	17% (Inactive)	0.053	ND		−23%	0.06	ND		−4%	0.035
SR-03000001410-1		S		93577923	45480139	3.333% (Inactive)	0.035	ND		9% (Inactive)	0.04	ND		2.67% (Inactive)	0.032
SR-01000140539-2		P		85786762	1479244	1.667% (Inactive)	0.012	ND		4.67% (Inactive)	0.02	ND		1.33% (Inactive)	0.015
SR-03000001303-1		S		92092827	45100460	13.33% (Inactive)	0.115	ND		8.67% (Inactive)	0.10	ND		26% (Active)	0.131
SR-03000001304-1		S		92092828	45100430	4% (Inactive)	0.139	ND		−8%	0.03	ND		11.33% (Inactive)	0.045
SR-03000001305-1		S		92092829	45100439	0%	0.006	ND		1.67% (Inactive)	0.08	ND		8% (Inactive)	0.072
SR-01000621846-2		P		92092839	3239085	2.33% (Inactive)	0.045	ND		9% (Inactive)	0.05	ND		26.3% (Active)	0.110
SR-01000140511-3		P		92092840	3245522	16% (Inactive)	0.183	ND		19.3% (Inactive)	0.14	ND		−6%	0.053
SR-01000140541-4		P		92092841	3571734	5.33% (Inactive)	0.061	ND		5.3% (Inactive)	0.08	ND		0%	0.100
SR-01000101040-3		P		92092842	3571735	10% (Inactive)	0.020	ND		−5%	0.07	ND		5.67% (Inactive)	0.060
SR-01000140507-3		S		92092831	4141058	20.33% (Inactive)	0.101	ND		23% (Inactive)	0.13	ND		0%	0.006
SR-03000001306-1		S		92092830	45100432	8.67% (Inactive)	0.075	ND		5% (Inactive)	0.17	ND		−6%	0.053
SR-01000101020-3		S		92092832	3242688	5.33% (Inactive)	0.136	ND		0%	0.01	ND		9% (Inactive)	0.052
SR-03000001311-1		S		92092845	45100438	14.33% (Inactive)	0.127	ND		23%	0.08	ND		−11%	0.032

ND indicates a compound was not tested in the indicated assay due to lack of LRH1 activity compared to probe in the 3X%INH assay

Table 5SAR of R2 Analogs

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Compound Information: R2 Analogs (with variable R1)						LRH1 %INH (AID 485348)		LRH1 Dose Response (AID 488782)		SF1 Fold Change (AID 488779)		SF1 Dose Response (AID 488780)		VP16 Fold Change (AID 488775)
Sample ID	Structure	Source	MLS ID	SID	CID	LRH-1 AVG %Inhibition (at 5 μM)	SD	LRH-1 IC50	SD	SF-1 Avg Fold Change (at 5 μM)	SD	SF-1 IC50	SD	VP-16 Avg Fold Change (at 5μM)	SD
SR-03000001393-1		S	MLS003153124	93375307	45382271	33.33% (Active)	0.098	110nM (Active)	0.023	−95%	0.15	15μM (Inactive)	0.02	8.33% (Inactive)	0.015
SR-01000097977-3		P		92092835	5310579	22.67% (Inactive)	0.140	ND		−18%	0.05	ND		−5%	0.064
SR-01000008560-2		P		85786757	7080816	14% (Inactive)	0.122	ND		−2%	0.07	ND		−13%	0.095
SR-01000008566-2		P		85786758	7080823	40.67% (Active)	0.116	1.1μM (Active)	0.023	−2%	0.07	N/D		−1%	0.091
SR-01000101016-2		P		85786759	7080833	45% (Active)	0.176	4.2μM (Active)	0.036	−10%	0.10	N/D		4.67% (Inactive)	0.031
SR-03000001394-1		S		93375308	45382279	36.67% (Active)	0.012	570nM (Active)	0.052	−164%	0.21	5μM (Inactive)	0.03	1% (Inactive)	0.017
SR-03000001395-1		S	MLS003 153121	93375309	45382284	39.33% (Active)	0.049	1μM Active)	0.183	−81%	0.01	N/D		1.3% (Inactive)	0.045
SR-03000001409-1		S	MLS003 153120	93577922	45480137	17.67% (Inactive)	0.040	ND		−3%	0.10	ND		−3%	0.148
SR-03000001392-1		S		93375306	45382277	3% (Inactive)	0.010	ND		1.3% (Inactive)	0.09	ND		6% (Inactive)	0.046
SR-01000101326-2		P		85786761	7080827	2.33% (Inactive)	0.076	ND		10.3% (Inactive)	0.05	ND		−7%	0.040
SR-01000101018-2		P		85786760	6623965	3.67% (Inactive)	0.035	ND		7% (Inactive)	0.03	ND		−7%	0.095
SR-03000001149-1		S		89649715	44825226	0%	0.173	ND		2% (Inactive)	0.02	ND		2.67% (Inactive)	0.031
SR-03000001308-1		P		92092834	4621936	5.667% (Inactive)	0.136	ND		0%	0.08	ND		−7%	0.030
SR-01000101008-3		P		92092836	7080829	9.67% (Inactive)	0.087	ND		26.67% (Active)	0.11	ND		−10%	0.100
SR-01000769836-2		P		92092837	7080831	3.33% (Inactive)	0.049	ND		30% (Active)	0.04	ND		0%	0.006
SR-03000001405-1		S		93577918	45480136	21.3% (Inactive)	0.047	ND		3% (Inactive)	0.06	ND		−1%	0.206
SR-03000001406-1		S		93577919	45480138	29.67% (Active)	0.025	ND		−24%	0.12	ND		20.33% (Active)	0.307
SR-03000001407-1		S		93577920	45480134	2% (Inactive)	0.020	ND		3.67% (Inactive)	0.04	ND		2% (Inactive)	0.062

ND indicates a compound was not tested in the indicated assay due to lack of LRH1 activity compared to probe in the 3X%INH assay

3.5. Cellular Activity

The probe is active in a variety of cell-based assays performed by Dr. Griffin. Importantly, these assays demonstrate that Probe #1 (ML180) and Probe #2 (ML179) are potent in a variety of breast cancer cell lines. These MTT data are available in PubChem as AID 504928.

Figure 6Probes #1 ML180 (1848) and #2 ML179 (SR03-1309) Potency in 4 Different Breast Cancer Cell Lines (Dose Response Curves)

Function Assays: Star Reporter Assays

LRH1 inverse agonist probe compounds (powder samples) were also tested in assays to monitor their effects on the promoter activity of the Steroidogenic acute regulatory protein (Star). These data are available in PubChem as AID 504933. The dose response curves for each probe are below, in Figure 7.

Figure 7

Dose response curves for SR-01000621848 (Probe 1, ML180) and SR-01000001309 (Probe 2, ML179) two LRH-1 modulator probes. 293T cells were cotransfected with full length LRH1 and StAR reporter and were treated with various concentration of SR-01000621848 (more...)

3.6. Profiling Assays

Selectivity of the probe compounds over the nuclear receptor SF-1 has been monitored and is presented in the previous sections. ML180 did not modulate GAL4-VP16. In addition, probe 1 (ML180; 1848) has been tested against a library of all 48 human nuclear receptors [43]. Preliminary analysis suggests that ML180 has little activity on other nuclear receptors (see Figure 8). Note that SR-1848 (ML180) is not active on LRH1 in this assay. We have confirmed that ML180 is active only on full length LRH1 and inactive on the Gal4-LBD-LRH1 construct used in the assay d. These data can also be found in PubChem as AID 504934.

Figure 8

ML180 Nuclear Receptor Promiscuity.

ML180 (MLS003153119 = CID 3238389) was tested in 470 assays in PubChem and was active in 15 AIDs (3.19%). Eleven of these are protein targets with five of the 11 being cytochrome P450’s. With this knowledge we will closely monitor P450 activity of ML180 analogs. Two other targets were RORA (actual hit is in the SF-1 counterscreen to RORA) and the SF-1 assay. This is the origin of the probes as SF-1 is the closest family member to LRH1. However, as we show above, while ML180 is active on GAL4-SF1, it is not active on full length receptor. The remaining four targets are DNA damage-inducible transcript 3, E3 ubiquitin-protein ligase Mdm2, Mdm4, and centromeric isoform d. Activity is these assays could be indirect and LRH1 dependent.

ML180 was also active in; 1) Luminescence Cell-Based Primary HTS to Identify Inhibitors of Beta Cell Apoptosis. [Primary Screening], 2) Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Beta Cell Apoptosis [Confirmatory], 3) uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation [Primary Screening], and 4) HTS to identify inhibitors of zVAD Induced Cell Death in L929 Cells [Primary Screening].

We are confident that ML180 is not an inhibitor of luciferase as we see selectivity over SF-1 in a luminescence-based luciferase reporter assay.

4. Discussion

4.2. Mechanism of Action Studies

Figure 9AProbe MOA (Step 1)

LRH1 is a constitutively active nuclear receptor that binds to identical response elements as SF-1 in promoter regions of LRH1 target genes. The scheme above suggests that LRH1 is associated with NR coactivators such as PGC1a and this interaction drives transactivation of LRH1 target genes. Upon binding to LRH1, inverse agonists such as ML180 alter the conformational dynamics of the receptor causing dissociation of co-activator. It is also likely that inverse agonists enhance LRH1 interaction with NR co-repressors such as SHP.

Figure 9BProbe MOA (Step 2)

Figure 9CProbe MOA (Step 3)

Acute Phase Response (APR)- Preliminary studies in our lab using Huh7 cells stimulated with IL1β and IL6 results in significant increase in expression of LRH1, haptoglobin (Hp), serum amyloid A-4 (SAA1), and serum amyloid A-4 (SAA4) as determined by qPCR (see PubChem AID 488769). Surprisingly and opposite of the anticipated outcome, treatment of these cells with the inverse agonists ML180 and ML179 represses the expression of three of these genes (Hp, SAA1, SAA4) to the level of control cells (unstimulated) and LRH1 to levels below that in control cells. This finding confirms that our inverse agonists can repress the expression of LRH1 and more importantly, suggests that LRH1 inverse agonists can repress APR genes in similar fashion to LRH1 agonists. Clearly the mechanism of action of inverse agonists in this model is likely different than that of agonists. This finding warrants further mechanistic studies which will be carried out as part of this extended probe development project.

Figure 10Hp, SAA1 & SAA4 Expression in the Presence and Absence of Probes

The above QPCR data have been submitted to PubChem as AID 488769.

LRH1 Modulation of Aromatase Expression – preliminary studies in our lab show that treatment of hepG2 cells with SR-1848 reduces the expression of aromatase cytochrome p450 gene (Cyp19). Treatment of these cells with a siRNA for LRH1 also reduces the expression of aromatase. Treatment of cells with siRNA for LRH1 blunts the effects of SR-1848 (probe ML180) on aromatase expression. These results have been submitted to PubChem as AID 488782. Optimization of Cyp19 knockdown is ongoing. The relevance of these studies is found in prior discovery that aromatase is highly upregulated in breast cancers as well as breast tissue surrounding tumors.

Figure 11Reduction of Cyp19 aromatase mRNA expression by ML-180 (SR-1848) is LRH-1 dependent.

HepG2 cells treated with ML-180 (SR1848) demonstrate significant reduction in expression of Cyp19 aromatase. This effect is blunted in cells treated with LRH-1 siRNA that have reduced expression of LRH-1.

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