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Human RORa Reporter Assay Kit

SIZES SKU
1 x 96-well format assays IB04011
3 x 32 assays in 96-well format IB04011-32
1 x 384-well format assays IB04012
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human RAR-related Orphan Receptor Alpha (RORa). INDIGO’s ROR Alpha reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the ROR Alpha. In addition to ROR Alpha Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human ROR Alpha. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeNuclear Hormone Receptor
SpeciesHuman
Receptor FormHybrid
Assay ModeInverse Agonist
Kit Components
  • RORa Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • All trans Retinoic Acid, (ref. inverse-agonist; in DMSO)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Shipping RequirementsDry Ice
Storage temperature-80C

Data

Agonist dose-response performance of the Human RORα assay. Agonist response of the Human RORα Reporter Cells is demonstrated using 27-Hydroxy Cholesterol (27OHC; Cayman Chemical), 7-dehydro Cholesterol (Sigma), and CGP 52608 (Sigma). Values of Fold-Activation are plotted against concentration. 27OHC provides greater than a 2-fold increase in RORα activity above the already high endogenous activity level. Z' values confirm the robust performance of the agonist-mode RORα assay. When contemplating concentration ranges for screening test compounds of unknown bioactivity, it is important to note the great disparity in potencies between inverse-agonists and theses agonist reference compounds: ATRA IC50 1 µM.
Inverse-agonist dose-response performance of the Human RORα assay. Inverse-agonist analyses of Human RORα Reporter Cells using All trans-Retinoic Acid (ATRA; provided). Luminescence was quantified using a GloMax-Multi+ luminometer (Promega). Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n ≥ 6). Z’ value was calculated as described by Zhang, et al. (1999). Non-linear regression was performed using GraphPad Prism software.

Target Background

RAR-related orphan receptor alpha (ROR-alpha), also known as NR1F1 is a nuclear receptor encoded by the RORA gene. The protein encoded by this gene is a member of the NR1 subfamily of nuclear hormone receptors. It can bind as a monomer or as a homodimer to hormone response elements upstream of several genes to enhance the expression of those genes. The specific functions of this protein are not known, but it has been shown to interact with NM23-2, a nucleoside diphosphate kinase involved in organogenesis and differentiation, as well as with NM23-1, the product of a tumor metastasis suppressor candidate gene. Four transcript variants encoding different isoforms have been described for this gene.

INDIGO’s RAR-related Orphan Receptor Alpha (RORα) assay includes proprietary mammalian cells engineered to provide high-level expression of Human RORα. Human RAR Orphan Receptor Alpha Inverse Agonist Assay Reporter Cells also incorporate a responsive luciferase reporter gene. Quantifying expressed luciferase activity at the assay endpoint provides a sensitive surrogate measure of changes in RORα activity in treated cells. Endogenous molecular activators maintain RORα in a state of constitutive high-level activity. Therefore, the principle application of this reporter assay system is in the screening of test samples to quantify inverse-agonist or agonist activities that they may exert against human RORα.

Citations

Discovery of a novel RORγ antagonist with skin-restricted exposure for topical treatment of mild to moderate psoriasis

Clinical success of IL-17/IL-23 pathway biologics for the treatment of moderate to severe psoriasis suggests that targeting RORγt, a master regulator for the proliferation and function of Th17 cells, could be an effective alternative. However, oral RORγ antagonists (VTP43742, TAK828) with high systemic exposure showed toxicity in phase I/II clinical trials and terminated development. To alleviate the potential safety concerns, identifying compounds with skin-restricted exposure amenable for topical use is of great interest. Systematic structure activity relationship study and multi-parameter optimization led to the discovery of a novel RORγ antagonist (SHR168442) with desired properties for a topical drug. It suppressed the transcription of IL-17 gene, leading to reduction of IL-17 cytokine secretion. It showed high exposure in skin, but low in plasma. Topical application of SHR168442 in Vaseline exhibited excellent efficacy in the imiquimod-induced and IL-23-induced psoriasis-like skin inflammation mouse models and correlated with the reduction of Th17 pathway cytokines, IL-6, TNFα and IL-17A. This work demonstrated restricted skin exposure of RORγ antagonist may provide a new topical treatment option as targeted therapeutics for mild to moderate psoriasis patients and may be suitable for the treatment of any other inflammatory disorders that are accessible locally.
2021-04-28

Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination

Regeneration of myelin is mediated by oligodendrocyte progenitor cells (OPCs), an abundant stem cell population in the CNS and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the central nervous system (CNS) underlies a number of neurological diseases, including multiple sclerosis (MS) and diverse genetic diseases1–3. Using high throughput chemical screening approaches, we and others have identified small molecules that stimulate oligodendrocyte formation from OPCs and functionally enhance remyelination in vivo4–10. Here we show a broad range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51 (cytochrome P450, family 51), TM7SF2, or EBP (emopamil binding protein), a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to OPCs in purified form while analogous sterols lacking this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism-of-action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.
2018-07-25

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RAR-related Orphan Receptor Alpha (RORa, NR1F1)

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