Potent, Selective and Cell Penetrant Inhibitors of SF-1 by Functional Ultra-High-Throughput Screening

Madoux, Franck; Li, Xin; Chase, Peter; Zastrow, Gina; Cameron, Michael D.; Conkright, Juliana J.; Griffin, Patrick R.; Thacher, Scott M.; Hodder, Peter

doi:10.1124/mol.108.045963

Cited by 46 publications

(33 citation statements)

References 37 publications

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“…The first indication that repressors could be identified for the NR5A family of NRs was revealed by a publication from the Scripps Florida Molecular Libraries Probe Centers Network describing a high-throughput screen campaign for SF-1 (Madoux et al, 2008). In this report, chemically tractable small molecule repressors of SF-1 were discovered in a highthroughput screen campaign where the primary assay was a Gal4-SF-1/UAS (upstream activation sequence) luciferase reporter assay using the constitutively active herpes virus protein 16 fused to Gal4 (Gal4-VP16) and the yeast UAS luciferase reporter as a control for nonspecific transcriptional modulation and cytotoxicity.…”

Section: Resultsmentioning

confidence: 99%

Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

et al. 2014

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The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl) piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1-expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.

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Section: Resultsmentioning

confidence: 99%

Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

et al. 2014

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“…Alternative explanation for the negative results of the HTS screen may be a nature of the compound collection. Even though the NIH compound library has been successfully used in the past for identification of small-molecule ligands for NRs, 34 its diversity may not be deep enough to include structures resembling endogenous NR2E3 ligands present in the photoreceptor cells of the retina. If this is the case, the use of the TR-FRET assay described in this study in another, more diverse and abundant compound collection may yield the desired NR2E3 agonists.…”

Section: Discussionmentioning

confidence: 99%

In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3

Qin

Knapinska²,

Dobri³

et al. 2013

Journal of Ocular Pharmacology and Therapeutics

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Purpose: NR2E3 is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. NR2E3-specific modulators may prolong photoreceptor survival in patients with dry age-related macular degeneration and other forms of retinal degeneration. To definitively establish NR2E3 as a photoreceptor protection target, identification of small-molecule NR2E3 modulators and their testing in animal models of retinal degeneration are required. Development of the high-throughput screen (HTS)-compatible screen for small-molecule NR2E3 modulators is the first step toward this goal. Methods: Purification protocol for isolation of the functionally competent soluble NR2E3 protein after its expression in the insect Sf9 cells was developed. The time-resolved fluorescence energy-transfer (TR-FRET) assay assessing agonist-sensitive interaction between apo-NR2E3 and transcriptional corepressor RetCOR was used for characterization of the previously reported putative NR2E3 agonist, Compound 11a, and to conduct the HTS for novel small-molecule NR2E3 modulators (direct and inverse agonists). A counterscreen TR-FRET assay that measures the affect of test compounds on PPARg interaction with corepressor NCOR was used for assessing the specificity of compounds identified in the HTS. Results: We developed the cell-free TR-FRET assay for small-molecule NR2E3 modulators, which is based on agonist-induced disruption of the interaction between GST-tagged apo-NR2E3 and MBP-tagged fragment of transcriptional corepressor RetCOR. Compound 11a, a putative NR2E3 agonist, did not affect the NR2E3-RetCOR interaction, as was established by its titration in the developed assay. The assay was miniaturized for an ultralow-volume 1,536-well format and automated into 3 simple pipetting steps. Consistent with excellent assay performance, the test runs established a Z¢-score within the 0.6-0.8 range. Analysis of the mid-size National Institutes of Health collection of 315,001 structurally diverse drug-like compounds confirmed excellent assay performance, but did not reveal NR2E3-specific agonists or inverse agonists. Conclusions: A robust and reliable TR-FRET assay for small-molecule NR2E3-specific modulators suitable for the analysis of million compound-strong HTS libraries was developed. A previously described putative NR2E3 agonist, Compound 11a, is unlikely to represent a direct NR2E3 agonist. Application of the developed assay for screening of a more abundant and diverse compound collection be required for identification of synthetic NR2E3 ligands.

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“…5B) even at high compound concentrations. Second, the effect of Nexinhibs on neutrophil metabolic activity was measured in cells after their exposure to increasing concentrations of compounds in a dose-response format, using a luminescence-based ATP detection system, as described previously (35). This assay is based on the analysis of the number of viable cells in culture by the quantification of the ATP present in the lysates, an indicator of metabolically active cells.…”

Section: High-throughput Screening (Hts) For Inhibitors Of Rab27amentioning

confidence: 99%

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation

Johnson¹,

Ramadass²,

He³

et al. 2016

Journal of Biological Chemistry

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Edited by Dennis VoelkerNeutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the upregulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

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Potent, Selective and Cell Penetrant Inhibitors of SF-1 by Functional Ultra-High-Throughput Screening

Cited by 46 publications

References 37 publications

Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation

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