INDIGO Biosciences, Inc. is a leading provider of nuclear receptor and in vitro toxicology solutions that accelerate scientific decision-making. We supplement the world’s largest portfolio of nuclear receptor kits and services and in vitro toxicology solutions with greater results readability, reproducibility, and faster turnaround times. Our solutions, plus supportive team and reliable science and platforms, aim to reduce the time, cost, and risk associated with the discovery process.


Product Categories

Technical Drawings

The following drawings make good additions to seminars and classes or just use them as a quick learning tool. Use freely, but please acknowledge the NR Resource. Any addition or corrections, please contact the NRR

NR Nomenclature

NR phylogeny

Structure of NRs

DNA Binding domain

Basic gene expression by NRs

Transcriptional regulation by NRs

NR Ligands

Agonist versus antagonist

NRs and metabolism summary

NRs and metabolism Phase 1

NRs and metabolism Phase 2

NRs and metabolism Phase 3

Mechanisms of endocrine disruption

Mechanism of endocrine disruption

Mechanism of metabolic desruption

NRs and cellular metabolites

NRs and dietary bioactives

Hepatotoxicity IBIPlot

NR expression by function

NR expression by GI and Metabolism

Reverse Cholesterol Transport and NRs

Types of NR Ligands

SHRs Summary

SHR Mechanism of Action

SHR Structure

Conformational Change

Types of ligands (static)

AhR Pathway

AR Pathway

CAR Pathway

FXR Pathway

LRH-1 Pathway

LXR Pathway

PPARα Pathway

PPARβ/δ Pathway

RXRα Pathway

PPARɣ Pathway

PXR Pathway

Retinoic Acid Receptor Alpha (RARα)

RORα Receptor

Why INDIGO?

Get what you need from the world’s largest portfolio of nuclear receptors

INDIGO has the largest portfolio of cell-based nuclear receptor assays in the world, helping you identify compounds with the highest selectivity and the lowest potential for unwanted effects and off-target responses. Our broad portfolio of nuclear receptors makes us the preferred source for single receptor or full-panel screenings. Whether for nuclear receptor or in vitro toxicology solutions, our intellectual property, ease of use, and quick turnarounds will get you to the next phase of discovery, faster.

Make confident decisions with clear results about your compounds

To empower confident decision-making throughout the discovery process, INDIGO’s technology generates clear single receptor or full-panel screening results, making for better interpretation and more accurate data. Employing a luminescence-based method and our proprietary CryoMiteTM preservation process, we provide reproducible results lot to lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Be supported in your study with a committed team of experts

INDIGO is a leading provider of nuclear receptor and in vitro toxicology solutions, recognized for our focus on client support. Committed to accelerating the discovery process for you, our team goes the extra mile by offering complimentary study consults, designs, and research, and providing a comprehensive review of the results with you. As objective experts focused on your success, it’s our duty to ensure the results we provide inform assured next steps.

Save time and money with accelerated lab results and decisions

At INDIGO, our cell-based assay kits and services accelerate scientific decision-making by reducing time, cost, and risk associated with the discovery process. We know that time is money during discovery and aim to get you to the next phase faster or prevent unnecessary steps forward. For quick data and decisions, our kits deliver results in just 24 hours. And, beyond saving time, our kits and services also provide the most comprehensive data for your investment.

Screen your compounds using reliable science, platforms, and people

INDIGO provides a superior combination of expertise, kits, services, and support to leading through startup organizations, ranging from pharmaceutical companies and biotechs to university labs and CROs. Lab directors across the globe trust us for single receptor or full-panel screenings at every stage of discovery, leveraging our industry-leading platforms, knowledgeable people, and proprietary science.

Technology

OUR PRODUCT PLATFORM

INDIGO Biosciences’ receptor assay products are cell-based, luciferase reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells typically present greater than 95% cell viability and are ready for immediate use. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments. Test compounds may be screened for agonist or antagonist activities against nuclear receptors expressed within the cytoplasm and nuclear environments of healthy, dividing mammalian cells.

Our reporter assays capitalize on the extremely low background, high sensitivity, and broad linear dynamic range of bio-luminescence reporter gene technology. INDIGO’s reporter cells incorporate the cDNA encoding beetle luciferase, a 62 kD protein originating from the North American firefly (Photinus pyralis). Luciferase catalyzes the mono-oxidation of D-luciferin in a Mg+2-dependent reaction that consumes O2 and ATP as co-substrates, and yields as products oxyluciferin, AMP, PPi, CO2, and photon emission. Luminescence intensity of the reaction is quantified using a luminometer and is reported in terms of Relative Light Units (RLU’s). Thus, quantifying changes in luciferase expression in the test sample-treated reporter cells provides a sensitive surrogate measure of changes in activity.

INDIGO’s luciferase reporter cell systems are engineered to provide optimal assay sensitivity and dynamic range when quantifying receptor activity. Following ligand-activation, the receptor complex acts to induce expression of the luciferase reporter. Upon addition of detection reagent, the intensity of light emission from the luciferase reaction directly correlates to the activation status of the specific receptor. All INDIGO reporter assay kits incorporate a detection reagent specially formulated to provide stable light emission between 5 and 90+ minute after initiating the luciferase reaction, thus allowing users the ability to dispense detection reagent into all assay wells prior to commencing activity measurements. This eliminates the need for a luminometer equipped with injectors and allows plates to be processed in batch, which dramatically reduces the start-to-finish read time of assay plates.

flowchart

PRODUCT FORMATS

INDIGO Biosciences’ luciferase reporter assays are optimally configured for processing in 96- and 384-well format assay plates. Single plate kits (1x 96- and 1x 384-well) are offered as all-inclusive assay systems. Each kit includes luciferase reporter cells, an assay plate, all required reagents (optimized culture media, positive-control agonist, and detection reagent), as well as a detailed Technical Manual.

Bulk Reagent Packs are offered to accommodate the needs of HTS users. These products are scaled for 10x 96- and 10x 384-well plate assays and provide luciferase reporter cells, optimal culture media, positive-control ligand, luciferase detection reagent, and a detailed Technical Manual and Protocol Quick Guide. Bulk Reagent Packs do NOT include assay plates because HTS facilities typically stock large quantities of such plates.

Please note that, regardless of assay format, Luciferase Reporter Cells are single-use reagents. Once thawed, Reporter Cells may not be refrozen or reused. Assay kits provide sufficient extra volumes of all reagents to accommodate the needs of performing mechanical transfers and test compound dilutions. However, extra volumes of reporter cells and Luciferase Detection Reagent should be discarded after each assay set-up.

Five basic configurations of our luciferase reporter assay kits are offered:

  • 3x 32 assays in a 96-well format plate.
    This all-inclusive kit contains reporter cells and sufficient materials to perform three distinct groups of 32 assays. Each group will comprise 32 nuclear receptor assays, and will utilize four 8-well strips affixed to a 96-well frame (provided). If desired, however, reagents may be combined to perform either 64 or all 96 assay reactions at one time.
  • 1x 96-well assay.
    This all-inclusive kit format contains reporter cells and all additional materials needed and is intended for users wishing to dedicate one entire 96-well plate to performing nuclear receptor assays.
  • 1x 384-well assay.
    This all-inclusive kit format is intended for users wishing to dedicate one entire 384-well plate to performing nuclear receptor assays.
  • 960 assay Bulk Reagent Pack for (10x) 96-well plates.
    This kit format is intended to accommodate HTS applications in which a minimum of ten (10) 96-well plates will be dedicated to a single screening run. Assay plates are NOT supplied with this product. Hence, users must provide white, sterile, cell-culture treated 96-well assay plates.
  • 3,840 assay Bulk Reagent Pack for (10x) 384-well plates.
    This kit format is intended to accommodate HTS applications in which a minimum of ten (10) 384-well plates will be dedicated to a single screening run. Assay plates are NOT supplied with this product. Hence, users must provide white, sterile, cell-culture treated 384-well assay plates.

All Bulk Reagent Packs are manufactured on-demand, and require a maximal lead time of 12 business days for delivery. We welcome inquiries pertaining to the custom manufacture of alternative volume Bulk Reagent Packs. Please let us know how we may better help you achieve your research goals!

All Luciferase Reporter Assay System products are shipped on dry ice for overnight delivery anywhere within the United States.

HOW TO USE OUR KITS: A QUICK GUIDE VIDEO

Perform nuclear receptor assays with easy-to-use kits from INDIGO Biosciences. This short video provides a brief overview of what to expect when working with our kits, including unpacking and storing kit contents, setting up and performing the assays, and reviewing data and results.

*Please note, this video is for INDIGO’s non-pre-incubation assays. INDIGO’s pre-incubation assays involve minor but crucial additional Day 1 steps and different dispensed volumes. Always refer to your Technical Manual (included in all kit shipments) for the most up-to-date information and proper technique.

Introduction

Nuclear receptors (NRs) act as sensors for various intracellular molecules including hormones and fatty acid metabolites. The effects of these ligands can be understood by the fact that numerous genes involved in the cellular processes, such as general homeostasis, growth, and defense against microbes, are under the control of NRs. By understanding which diseases, pathways and networks each NR participates, it is easier to anticipate the consequences of regulation of unintended targets by a compound of interest. Although the dominant paradigm in drug discovery is to design maximally selective ligands to act on individual NR targets, many effective drugs act via modulation of multiple proteins rather than single targets.

Advances in systems biology are revealing a phenotypic robustness and a network structure that strongly suggests that exquisitely selective compounds, compared with multitarget drugs, may exhibit lower than desired clinical efficacy. This new appreciation of the role of polypharmacology has significant implications for the two major sources of attrition in drug development, efficacy and toxicity. Thus defining the biological niche of each NR as well as understanding overlapping pathways and functions provides valuable context for evaluating a compound’s liabilities and promise.

In order to characterize the selectivity and potential off-target events, INDIGO offers a comprehensive screen of all our cell-based assays to profile your compound(s).

Nuclear Receptor Profiling

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying those with the highest selectivity and hence lowest potential for off-target and unwanted effects. Due to regulation of metabolic enzymes by several NRs, there is increasing interest in understanding the potential of drug-drug and drug-nutrient interactions of potential drug leads. INDIGO provides a comprehensive list of optimized, robust and selective whole-cell NR assays, ideally suited for examining selectivity as well as potential drug-drug interactions. By relying on INDIGO’s profiling service and our nuclear receptor experts, you can make those critical decisions on your compounds with the highest degree of confidence.

When you perform NR profiling with INDIGO, we provide you with all the data and analysis you need to make those critical decisions on the fate of your compounds.

INDIGO's IBIPlot

INDIGO’s IBIPlot

INDIGO’s Detailed Report Provides:

  •      All the raw data for each receptor and mode
  •      Summary tables and plots of your data for each receptor and mode including non-linear regression analysis, EC50 and peak activity
  •     Detailed statistical analysis and IBIPlotTM to visualize the chemical-by-chemical profile of activity across all receptors in your study (available upon request)
  •      All data is secure and confidential

Benefits of INDIGO’s Profiling Services

  •      Ability to study the largest portfolio of optimized, cell-based Nuclear Receptor assays with accuracy, precision and reproducibility
  •      Unlike biochemical and direct binding interaction systems, INDIGO’s assays can be used to study agonists, antagonists and inverse agonists
  •      Flexible options with the capability of studying our whole library of assays or choose from predefined panels or create your own
  •      Rapid turnaround time with a detailed report evaluating your compounds’ efficacy, potency and selectivity

We are the Nuclear Receptor experts and can assist you in in the critical decisions regarding compound liabilities.

Panels

Defining the system that each NR participates can be approached in several ways such as sequence similarity, potential disease implication or transcriptional networks. The latter is particularly helpful since it encompasses NRs to which there are no known endogenous ligands (orphan receptors) or have few selective pharmacologic agents to evaluate biological consequences. By choosing a select group of receptors to study (a predesigned panel), it is possible to better understand the biological and toxicologic effects of your compounds. INDIGO has pre-designed several NR panels for you to choose. Of course, you can also design your own panel of receptors (see the Disease State chart below) or talk to our experts to assist in developing a plan of study.

Background

Historically, primary hepatocytes have been the preferred in vitro model for assessing drug-induced expression of drug metabolizing enzymes. There are two major limitations to the use of human liver cells or their derivatives. First, there are currently limited sources of fresh human hepatocytes worldwide and, when available, they often suffer from low viability and high batch -to-batch variability. Second, the freezing process used to preserve primary hepatocytes and the transformation process needed to make stable and proliferating cell lines results in changes in cell differentiation, proliferation, and metabolic processes.

Immortalized human heptoma (i.e., HepG2) and hepato-carcinoma cell lines are sometimes used owing to their unlimited proliferative potential. However, a serious limitation to using transformed cell lines is their decreased, or absent, expression of hepatocyte differentiation markers. These include nuclear receptors and other xenobiotic-sensing receptors and, hence, their target genes (e.g., CYPs) whose expression are critical to correctly assessing the potential liability of induced drug-drug interactions.

Using these model systems has led to an inability to predict hepatotoxicity at the preclinical stage. This has been a key reason for xenobiotic-induced liver injury being a major cause of human morbidity and mortality.

upcyte® Hepatocytes

upcyte® technologies has developed a novel technique which allows for the generation of human hepatocyte cultures with the ability to proliferate while maintaining many differentiated functions. upcyte® hepatocytes, which are human donor-derived hepatocytes established by upcyte® technologies GmbH, have the attributes of limited proliferation while maintaining their native high levels of constitutive and inducible xenobiotic metabolizing enzyme activities.

Like primary hepatocytes, confluent cultures of upcyte® hepatocytes express liver-specific proteins, produce urea, and store glycogen. Importantly, the induction profiles of cytochrome p450 (CYP) enzyme activities are similar to those of the primary hepatocytes. Thus, upcyte® hepatocytes combine the characteristics and advantages of primary hepatocytes with the added practical advantage of having access to the same donor cells for use in iterative, large-scale experiments over extended periods – making them ideal for drug discovery.

upcyte markers

Benefits

  1. upcyte® hepatocytes did not form colonies in soft agar and are not immortalized ancorage-independent cells.
  2. Confluent cultures expressed liver-specific proteins, produced urea, and stored glycogen.
  3. CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte® hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses.
  4. Phase 2 activities (UGTs, SULTs, and GSTs) were comparable to activities in freshly isolated hepatocytes.
  5. upcyte® hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells. The cytotoxicity of of aflatoxin B1 was decreased in upcyte® hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. upcyte® hepatocytes differentiated between ten hepatotoxic and eight non-heptatotoxic compounds.
  6. In conclusion, upcyte® hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte® hepatocytes for CYP induction and cytotoxicity screening.

upcyte® Hepatocytes are used by INDIGO Biosciences for these reasons, through commercial license agreement with upcyte® technologies, to provide our customers with the most reliable science and platforms to accelerate scientific decisions.

Products

Product Family Product Number Product Description
In Vitro Toxicology ULC1003-48 in vitro Screening for Drug-Induced Hepatotoxicity, 2x 48 Assays in 96-well format
Gene Expression UGE1003-48 Expression Profiling of Clinically Relevant CYPs,

Nuclear Hormone Receptor Introduction

Figure 1 Phylogeny and nomenclature of NRs

Unlike receptors found on the cell surface, members of the nuclear hormone receptor (NR) superfamily are restricted to metazoan organisms such as nematodes, insects, and vertebrates. These proteins are intracellular transcription factors that directly regulate gene expression in response to lipophilic molecules. They affect a wide variety of functions, including fatty acid metabolism, reproductive development, and detoxification of foreign substances. Many of the NRs act as ligand-inducible transcription factors, responding to endogenous and exogenous chemicals. However, the vast majority of known receptors do not have an identifiable physiologically relevant ligand, and are deemed orphan receptors. To date, over 300 NRs have been cloned, although there are only 48 members of the family in humans.  Early classification of these receptors was based on ligands, DNA binding properties or other functional characterization. A more systematic classification has been proposed, based on sequence similarity, much like that employed for cytochrome P450s. Phylogenic analysis has shown six subfamilies (NR1-6) with various groups and individual genes(1). As discussed below, most NRs have the same basic structure. The most highly conserved region is the C4 zinc finger domain, which as described above, is a DNA binding motif.

Nuclear Hormone Receptor Structure to Function

Nuclear Hormone Receptor Functional Domains

NRs generally follow a standard blueprint. While most NRs have all of these elements, there are a number of exceptions, some of which will be discussed below. The N terminus of the NR, sometimes called the modulator, hypervariable or A/B domain, has transactivation activity, termed activation function 1 (AF-1). This acidic activation domain (AD) is ligand-independent, or constitutively functional. The A/B domain’s sequence and length are highly variable between receptors (i.e. GR versus RXR) and among receptor subtypes (RXRα versus RXRβ). In addition, this region is the most frequent site of alternative splicing and secondary start sites and contains a variety of kinase recognition sequences. For these reasons, it is thought that the variable N- terminal sequences may be responsible for the receptor-, species-, and cell type-specific effects as well as promoter context-dependent properties of NR transactivation.

NRs bind to hormone response elements (HREs) in their target promoters through the DNA binding domain (DBD) or C domain. Composed of two zinc fingers, the DBD is the most conserved region within the NR superfamily. The first zinc finger contains the proximal-or P-box region, an alpha helix that which is responsible for high-affinity recognition of the “core half-site” of the response element. Located within the second zinc finger is the distal or D-box, an α-helix which lies perpendicular to P-box helix, and is a site that mediates receptor dimerization. NRs bind to DNA as heterodimers, homodimers, or monomers, depending on the class of NR. The steroid hormone receptors GRPRERAR and MR (receptors for glucocorticoid, progesterone, estrogen, androgen and mineralocorticoids, respectively) bind to DNA as homodimers and recognize a palindromic response elements. However, thyroid, retinoid, vitamin D and peroxisome proliferator receptors (TR, RAR, VDR and PPAR), as well as most orphan receptors, bind to DNA as a heterodimer with retinoid-x-receptor (RXR). However, the three dimensional structure of the RXR heterodimer complex produces different DNA binding affinities. Response elements may be direct repeats (DRx, AGGTCA-Nx-AGGTCA, where N is any nucleotide and x is any number of residues from 0-10), everted  repeats (ERx, ACTGGA-Nx-AGGTCA) or inverted repeat (IRx, AGGTCA-Nx-ACTGGA).

Immediately adjacent to the DNA binding domain is the D or hinge domain. This particular region has an ill-defined function. The hinge domain contains the carboxy-terminal extension (CTE) of the DBD, which may be involved in recognizing the extended 5’ end of the HRE. The D-domain appears to allow for conformational changes in the protein structure following ligand binding. Also, this region may contain nuclear localization signals and protein-protein interaction sites.

The sequence of the ligand binding domain (LBD) or E/F domain varies substantially between NRs, but they all share a common structure of 11-13 a-helices organized around a hydrophobic binding pocket. Residues within the binding pocket confer specificity, determining whether the LBD will accept steroid hormones, retinoid compounds or the host of xenobiotic ligands that affect receptor function. Ligand-dependent activation requires the presence of activation function 2 (AF-2), located at the extreme C terminus of the NR. LBDs also contain nuclear localization signals, protein interaction with dimerization motifs for heat shock proteins, coregulators and other transcription factors.

Exceptions to the Rule

There are several NRs that do not follow the basic structure function paradigm. For example, the constitutively active receptor (CAR) allows constitutive transcription of the target gene in the absence of ligand. Transcriptional activity is be suppressed by binding to ligands androstenol and androstanol, unlike other NRs examined. Another important exception to the classic model of NR activation comes from receptors that do not require DNA binding. DAX-1 and SHP lack DBDs completely, and transgenic mice bearing a GR that does not bind DNA are viable and fertile even though at least some GR functions are crucial for survival. NRs can also have important biological effects without ligand binding. Most NRs are phosphoproteins, and it is now known that many of these proteins are activated by crosstalk with other signal transduction pathways such as those responding to EGF and TGF-a.

Nuclear Hormone Receptor Basic Mechanism of Action

The mechanism of action of nuclear hormone receptors can take one of two basic forms, that of steroid hormone receptors (SHRs) or that of retinoid/thyroid/Vitamin D receptors . In the absence of ligand, the transcriptionally inactive SHRs MRPRGRAR and ER are sequestered in a large complex comprising the receptor, heat shock protein-90 (HSP90), Hsp70, FKBP52/51 and possibly other proteins. The cellular localization of this inactive complex is somewhat controversial and cytoplasmic or nuclear localization may be observed depending on the cell type and the conditions examined; however, the central dogma is that SHRs are cytosolic in the un-liganded form. One consequence of hormone binding to receptor is a distinct conformational change in receptor structure (discussed below). This conformational change marks the beginning of the signal transduction process. In the case of the GR subfamily (GRARMRPR), hormone binding elicits a dissociation of hsps and the release of a monomeric receptor from the complex. Genetic analysis and in vitro protease digestion experiments indicate that the conformational changes in receptor structure induced by agonists are similar but distinct from those produced by antagonists. However, both conformations appear to be incompatible with hsp binding.

The TR, RAR and VDR receptors do not avidly interact with hsps and are localized predominantly in the nucleus in the absence of ligand. Some unliganded NRs of this class may interact with DNA and act as transcription repressors. This may be the result of interaction with co-repressor proteins. An interesting exception to this observation is the constitutively active receptor (CAR) that is transcriptionally active in the absence of its ligand. Hormone induced conformational changes also occur upon activation of this class of NR, suggesting that alteration of receptor shape by ligands is a key step in the activation pathway.

Evidence suggests that receptors of the GR subfamily (SHRs) cooperatively bind to DNA as homodimers. The TR, RAR, VDRPPAR and most of the orphan receptors form heterodimers with other members of the intracellular receptor superfamily. TR, RAR, PPAR and VDR can utilize RXRs as partners for heterodimer formation. The DNA site of contact depends on certain sequences within the C-domain, namely the proximal (P-box) and distal (D-box) zinc finger motifs (see description of the C-domain above).  The P-box determines the half-site recognized, while the D-box determines the spacing between half-sites. Following activation, the SHRs receptors are capable of interacting with DNA, and both classes of NRs (SHRs and TR/RAR) can now recruit co-activators. The DNA bound NR complex is now a substrate for general transcription apparatus and the initiation of transcription commence.

Nuclear Hormone Receptor Ligands and Activators

Nuclear Hormone Receptor Natural and xenobiotic ligands

Ligands for NRs are as varied as the proteins themselves. Although the structures of these compounds is varied, a few generalized comments can be made. All ligands are lipophilic and can easily transverse the plasma membrane as well as the nuclear membrane, if required. The affinity (Kd) of the ligand-receptor complex is generally in the nM range, but can vary from pM to mM. It should be noted however, that the concentrations of each natural ligand should approach their Kd to be considered a physiologically-relevant ligand. This is of particular importance when considering reclassifying (or adopting) an orphan receptor in the process of reverse endocrinology. Xenobiotic agonists and antagonists share structure features with the natural effector molecules, although the similarities are often cryptic. Some receptors, such as PPARγ, have a large ligand-binding cavity that allows for the association of a variety of endogenous ligands.

Nuclear Hormone Receptor Ligand-induced activation

Structural studies of empty and ligand-bound LBDs have led to the “mousetrap” model of NR activation. The ligand is attracted to the trap, the receptor’s electrostatic potential, and a conformational change takes place, preventing the ligand’s exit. In the same way that the sprung mousetrap is more stable than the primed trap, ligand binding to the NRs ligand-binding domains stabilizes their structures relative to the unliganded receptor. The ligand forms an integral part of the hydrophobic core of the liganded LBD. This structural change is different for ligands that are full agonists versus those that are partial agonists or antagonists. Much attention is focused on the accessibility of the AF-2 domain to accessory proteins.  The AF-2 domain can serve as an activator of transcription when excised from the rest of the protein and linked to a heterologous DNA-binding domain. In this model, binding of the ligand molecule induces a conformational change in the LBD, whereby the AF-2 sequences fold back against the binding pocket, obstructing the opening and causing rearrangements in adjacent helices. In the process, a new surface is revealed that recruits specific transcriptional coactivators. This model may explain why receptor antagonists block transactivation; these compounds do not induce the proper conformational rearrangements in the LBD, interfering with the formation of the transcriptional activation complex.

Reverse Endocrinology

The increasing use of bioinformatics and the spread of genome projects has lead to the discovery of hundreds of proteins which share structural characteristics with NRs. When a NR is discovered without any knowledge of its natural ligand, it was dubbed an orphan nuclear receptor (ONR).  Efforts to understand ONR function and identify their physiological ligands (a process known as reverse endocrinology) have led to the discovery of novel metabolic pathways involving the PPARliver X receptor (LXR), and farnesoid X receptor (FXR), novel classes of ligands (benzoates, terpenoids), and alternative mechanisms for NR receptor regulation and function.

The cloning of the intracellular receptor cDNAs and their reconstruction into ligand responsive transcription units has allowed for the identification of new agonist/antagonists. The “cis-trans” or reporter assay consists of transfecting a receptor expression vector and a vector carrying a receptor-responsive reporter into mammalian cells. Induction of the reporter gene activity (i.e. luciferase, chloramphenicol acetyl transferase, b-galactosidase) reflects the transcriptional activity of the transfected receptor induced by hormone. The identification of receptor antagonists is more complex as it is sometimes difficult to distinguish true receptor antagonism from toxicity. The domain structure of  NRs allows for construction of reporter assays without knowledge of the DNA binding domain of the protein. A common means to assess ligand activation of a receptor is to perform a “finger-swap” whereby the LBD for the novel NR is placed downstream of a heterologous DBD of another, well-characterized protein. Examples of DBD used include the C-domain of GR as well as the transcription factor Gal4. Activation of this chimeric protein by agonists for the novel LBD would regulate a reporter that contains the appropriate DNA binding site (i.e a GRE for proteins containing the C-domain of GR). This type of approach allows for rapid screening of chemicals and may be used to find both xenobiotic and endogenous ligands.

Ligand-independent activation

An emerging concept for intracellular receptors is that they function not only as transducers of nuclear effects of steroids, hormones and nutrients, but also as key points of convergence of multiple signal transduction pathways. The first realization of this “cross-talk” between signaling cascades came from the observation that transcriptional activity of PRERTR and COUP-TF is modulated by the neurotransmitter dopamine. The specific activation of the dopamine D1 receptors results in activation of at least two distinct signal transduction processes in the cell. Mutation of a specific serine site close to the carboxyl terminus of the NR can prevent dopamine activation, with no effect on the ability of the ligand to activate. The two activation pathways may differentially phosphorylate this serine. A similar phenomenon is seen with other signaling pathways, in particular polypeptide growth factors such as insulin-like growth factor (IGF-1), transforming growth factor a (TGFa) and epidermal growth factor (EGF).

Citations

  1. Nuclear Receptors Nomenclature Committee (1999) A unified nomenclature system for the nuclear receptor superfamily, Cell 97, 161-163.
  2. Chawla, A., Repa, J. J., Evans, R. M., and Mangelsdorf, D. J. (2001) Nuclear receptors and lipid physiology: opening the X-files, Science 294, 1866-1870.
  3. Bourguet, W., Germain, P., and Gronemeyer, H. (2000) Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions and pharmacological implications, Trends Pharmacol Sci 21, 381-388.
  4.  Wahli, W., and Martinez, E. (1991) Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression, Faseb J 5, 2243-2249.
  5. Francis, G. A., Fayard, E., Picard, F., and Auwerx, J. (2003) Nuclear receptors and the control of metabolism, Annu Rev Physiol 65, 261-311.
  6. Khan, S. A., and Vanden Heuvel, J. P. (2003) Role of nuclear receptors in the regulation of gene expression by dietary fatty acids (review), J Nutr Biochem 14, 554-567.
  7. Smirnov, A. N. (2002) Nuclear receptors: nomenclature, ligands, mechanisms of their effects on gene expression, Biochemistry (Mosc) 67, 957-977.
  8. Ward, R. D., and Weigel, N. L. (2009) Steroid receptor phosphorylation: Assigning function to site-specific phosphorylation, Biofactors .
  9. Weigel, N. L., and Moore, N. L. (2007) Steroid receptor phosphorylation: a key modulator of multiple receptor functions, Mol Endocrinol 21, 2311-2319.
  10.  Weigel, N. L., and Moore, N. L. (2007) Kinases and protein phosphorylation as regulators of steroid hormone action, Nucl Recept Signal 5, e005.
  11. Weigel, N. L., and Zhang, Y. (1998) Ligand-independent activation of steroid hormone receptors, J Mol Med 76, 469-479.
  12. Weigel, N. L. (1996) Steroid hormone receptors and their regulation by phosphorylation, Biochem J 319 ( Pt 3), 657-667

INDIGO’s effects-based bioassay solutions compliment traditional analytical chemistry approaches for the examination of water quality and can help protect human and environmental health.

Contaminant Detection

Are Traditional Water Quality Methods Enough?

Traditional techniques rely on analyses and data for individual contaminants. Real world exposure generally occurs as mixtures of different chemical compounds. According to the EPA this presents risks posed to humans and the environment from:

  • chemicals without toxicity information
  • the presence and concentrations of other compounds
  • chemicals that work together to increase the toxic potential

To understand the risk posed by complex samples cell-based assay are needed to characterize cumulative effects on humans and other organisms.

Cell-Based Assays: The Next Wave in Evaluating Water Quality

Cell-based reporter assays such as those provided by INDIGO Biosciences screen for total bioactivity for a specific pathway of importance. They can detect the toxicity of unknown chemicals and can account for the cumulative effects simplifying the issues posed by complex mixtures in analytical methods. INDIGO has an extensive list of cellular bioassays that are predictive of cellular toxicity pathways including endocrine disruption, altered xenobiotic metabolism and adaptive stress responses.

INDIGO’s assays can detect the cumulative effect of bioactive compounds in extracted environmental matrices and provide important biological context. They are:

  • All-Inclusive
  • Sensitive
  • Cost Effective
  • Reproducible
  • and Easy to Perform

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Kits

INDIGO has an extensive list of bioassay kits for receptors important in Environmental Monitoring for contaminant detection and quantification in samples which include:

 

Lipid and Energy Metabolism

  • LXRβ (NR1H2)
  • PPARγ (NR1C3)
  • RXRα (NR2B1)
  • TRβ (NR1A2)

Basal metabolism

  • LXRα (NR1H3)
  • MR (NR3C2)
  • RARγ (NR1B3)
  • RXRβ (NR2B2)
  • RXRγ (NR2B3)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their water testing needs. Our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, INDIGO kits provide reproducible results lot-to-lot.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Services

 

INDIGO is a leading provider of cell-based reporter assay solutions, recognized for our focus on providing a superior combination of expertise, service, and support. Organizations across the globe leverage our industry-leading platforms, knowledgeable people, and proprietary science for single receptor or full-panel screenings. Committed to ensuring the results we provide inform assured next steps, our team goes the extra mile by offering complimentary study consultation and design. Service assays include a positive control reference compound and ‘vehicle’ control for every experiment. At the completion of the study a formal study report including helpful graphics, summaries, and insights, as well as all data files are provided to the client. Our scientists are also available to provide a comprehensive review of the results with you once your service study is complete. To receive a quote for your proposed study, contact us to discuss your desired study parameters.

 

Growth Factor Receptors

Growth factor receptors (receptors that bind to a growth factor) are the initial step in a cell’s signaling cascade for cell differentiation and proliferation. The growth / survival signal is initially carried by these receptor ligands which then bind to cell-surface receptor tyrosine kinases (RTKs). Growth factor receptors utilize the JAK/STAT, PI3 kinase, and MAP kinase pathways, as well as transcription factors like signal transducers or SMAD proteins. While growth factors act on different cell types, their signal pathways often overlap, and this shared mechanism has generated a significant amount of interest in cancer research.

Because of their effect on cell growth, research surrounding growth factor receptors typically focuses on their ability to pinpoint cancer treatments. Once growth factors bind to their receptor, a signal transduction pathway occurs within the cell to ensure the cell is functioning properly. In cancer cells, however, this pathway may never turn on or off. In certain cancers, these receptors are often overexpressed, corresponding to uncontrolled proliferation or differentiation. In addition, the expression of mutant forms of growth factor proteins may also lead to cancer. Tyrosine receptors are often a target for cancer therapies for this same reason.

Available Receptors

Epidermal Growth Factor Receptor (EGFR): In many cancer types, mutations affecting EGFR expression or activity could result in cancer progression. Deficient signaling of the EGFR is associated with diseases such as Alzheimer’s, while over-expression is associated with the development of a wide variety of tumors. Interruption of EGFR signaling can prevent the growth of the EGFR-expressing tumors and improve the patient’s condition.

Growth Hormone Receptor: Best known for regulating growth, the growth hormone receptor has other critical biological functions including metabolism regulation and controlling physiological processes related to the cardiovascular, reproductive, gastrointestinal, and renal systems. Growth hormone signaling is also an important regulator of aging and plays a key role in cancer development, as it is involved in multiple biological and physiological actions contributing to cell differentiation and proliferation.

Transforming Growth Factor Beta Receptor: Mutations in the critical TGFbR systems are responsible or a wide spectrum of developmental disorders and adult disease states, including cancer, cardiovascular disease, inflammation, diabetes, fibrosis, obesity, and wound healing. TGFbR signaling not only acts as a tumor suppressor but has been shown both in vitro and in vivo to act as a powerful stimulator of tumor progression.

Vascular Endothelial Growth Factor Receptor 2: VEGF is important in both vasculogenesis (the formation of the circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGFR is primarily localized to, and significantly unregulated on, tumor vasculature – blood and/or lymphatic – supporting the majority of solid-state cancers. Without adequate blood supply solid cancers cannot grow beyond a certain size; cancers that express VEGFR however, are able to grow and metastasize. Overexpression of VEGFR can also cause vascular disease in the retina of the eye and other parts of the body. VEGFR signaling inhibitors have shown clinical efficacy in a range of solid tumor types, including colorectal, lung, and breast cancer.

Kits

Kits are offered in different assay formats to accommodate researchers’ needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications. Assay systems are all inclusive, providing reporter cells, optimized growth media, media for diluting test compounds, a reference agonist or inhibitor, luciferase detection reagent, a cell culture-ready assay plate, and a detailed protocol. All kits are shipped on dry ice.

Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup.

INDIGO’s assay kits provide the convenience of an all-inclusive cell-based assay system. In addition to Reporter Cells, provided are two optimized media for use in recovering the cryopreserved cells and for diluting test samples, the reference agonist or inhibitor of the reporter system, Luciferase Detection Reagents, and a cell culture-ready assay plate.

Assay Kit & Platforms

Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.

Services

Service Assays: Human

Service assays include an reference compound and ‘vehicle’ control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, contact a Customer Service Representative to discuss your desired study parameters.

Research Areas

Bile Acid and Xenobiotics, Cancer, Cardiovascular Disease, Circadian Rhythm, Diabetes, Inflammation, Obesity, Wound Healing

FAQs

Do I need any special equipment to utilize INDIGO’s assay kits?

The equipment and supplies needed to utilize INDIGO’s assay kits are
standard in most labs. Specifically, the materials you will need to
provide include:

 

Day 1

  • Cell culture-rated laminar flow hood
  • 37°C, humidified 5% CO2 incubator for mammalian cell culture
  • 37°C water bath
  • 70% alcohol wipes
  • 8- or 12-channel electronic, repeat-dispensing pipettes and sterile
    tips
  • Disposable media basins, sterile
  • Sterile multi-channel media basins (such as the Heathrow Scientific “Dual-Function Solution Basin”), or deep-well plates, or appropriate similar vessel for generating dilution series of reference compound(s) and test compound(s)
  • Optional: antagonist reference compound
  • Optional: clear 96-well assay plate, sterile, cell culture treated,
    for viewing cells on Day 2

Day 2

  • Plate-reading luminometer

Can INDIGO’s Nuclear Receptor Assay Kits be used for diagnostic purposes?

INDIGO’s kits are intended for research purposes only, and not for diagnostic or therapeutic use in humans.

What is the difference between the 1×96-well and 3×32 assays in a 96-well plate formats?

INDIGO’s 1×96-well assay kits contain materials to perform assays in a single 96-well assay plate. Our 3×32-well assay kits contain materials to perform three distinct groups of assays in a 96-well plate format. Reagents are configured so that each group will comprise 32 assays. If desired, however, reagents may be combined to perform either 64 or 96 assays. Please note, the aliquot of Reporter Cells is provided as a single-use reagent. Once thawed, reporter cells can NOT be refrozen or maintained in extended culture with any hope of retaining downstream assay performance. Therefore, extra volumes of these reagents should be discarded after assay setup.

Do you have a specific value for positive nuclear receptor activations for your assay kits?

2-fold is what we have found to be historically, mathematically significant.

Do you have a specific value for cell viability that provides reliable nuclear receptor data?

A 15% drop in live cells is considered statistically significant.

Do INDIGO kits use Fetal Bovine Serum?

The compound screening medium (CSM) included in nearly all INDIGO kits contains 10% charcoal-stripped fetal bovine serum (FBS). (The exceptions to this are our RORg assays(5%) and TGF-beta (2%).) If your test substance is human serum, we do recommend serum-free medium, which is available at no charge but must be requested at the time of order.

Which wavelength should I use to read my plate?

INDIGO’s assays are luminescence-based, not fluorescence-based. Therefore, one does not designate specific wavelengths (or filters of any kind) when reading in luminescence mode. Total photon emission is quantified.

Is it okay to plate the test compounds into the wells then immediately add the cells after they have been properly reconstituted?

You always want to add the compounds to the cells, rather than adding the cells to the compounds, per the assay protocol.

What concentration of organic solvent carried over into the assay wells is acceptable for INDIGO reporter assays?

The concentration of organic solvent carried over into the assay wells should not exceed 0.1%. We recommend no more than 0.4% DMSO carry-over into the assay wells.

Can compound screening medium (CSM) and cell recovery medium (CRM) be reused after the initial thaw? (3×32-well assay kit-specific)

It is okay to thaw and keep the CSM/CRM refrigerated between uses. However, we recommend it only be stored at 4°C for a week or less, and that for any time longer than that, that it be refrozen.

Can I thaw and refreeze the 4mM Staurosporine provided in your LCMA kits?

Yes, we recommend keeping it at -20°C.

Can I thaw and refreeze LCMA buffer and LCMA substrate?

Yes, you can thaw the LCMA buffer and keep it at 4°C. You can freeze/thaw the LCMA substrate up to three times.

Can Tryptan Blue be used to check viability (QA) before running the assays?

We do not recommend Tryptan Blue for checking cell viability, as it can be inaccurate for recently thawed cells that are recovering from the trauma of thawing.

How are your products shipped?

All of INDIGO’s assay kits are shipped on dry ice.
Upon receipt, individual kit components may be stored at the temperatures indicated on their respective labels. Alternatively, the entire kit may be further stored at -80°C. To ensure maximal viability, Reporter Cells must be maintained at -80°C until immediately prior to use. The date of product expiration is printed on the Product Qualification Insert (PQI) enclosed with each kit.

What kind of data will I receive from my screening study?

Upon completion of your study, you will receive a detailed study report (PDF format) including all assay methods and validation, the raw data and calculations of your study (Excel format), and graphing files of all data (GraphPad Prism format). In addition, you are always welcome to contact INDIGO’s team for assistance in interpretation of your data.

How long does INDIGO retain client compounds once a study has been completed?

INDIGO retains compounds for three months after reporting results, then disposes of or returns the compounds. If you want us to return your compounds or to store them for more than three months, you must contact us to make appropriate arrangements prior to the three-month window expiring. We reserve the right to charge a disposal fee. We may charge a monthly fee for long-term storage. We will store all compounds under locked, temperature-controlled conditions. An internal chain of custody is maintained for each lot of compound received. Additional information relating to product and service agreements can be found in our Terms and Conditions.

How much of my compound should I send for a screening study?

If test samples are provided as concentrated stock solution, we prefer a 1000x concentration stock relative to the highest treatment concentration (e.g., provide > 10mM stocks if the highest assay concentration is 10 uM). This ensures that the concentration of organic solvent carried over into the assay wells is ~0.1%. INDIGO will not perform studies that require greater than 0.4% DMSO carry-over into the assay wells. You will also need to disclose the solvent used to generate the Master Stock (DMSO preferred—please inquire about the use of alternative organic solvents). Please provide approximately 50uL of 1000x concentrated stock solutions. If test samples are provided in powder form, they must be pre-weighed by the client. INDIGO does not perform manual weighing of test materials. Please provide no more than 5 mg of dry material. You will also need to provide the molecular weight (MW; g/mol) of each test compound. If test samples are provided as neat liquids, provide the Molar concentration of the liquid or provide a pre-weighed volume of the liquid sample (e.g., x mg/100ul). You will also need to provide the molecular weight (MW; g/mol) of each test compound. Aqueous solutions may be submitted, but they must be filter-sterilized and devoid of preservatives and chelating agents. Additional charges will be applied if extensive sample handling is required to generate “assay-ready” stocks.

Why do you recommend running the LCMA for antagonist and inverse-agonist modes, but not agonist mode?

While you are certainly welcome to run the LCMA with agonist mode, we do not typically recommend needing to do so. The reason for this is because cytotoxicity would produce a downward curve. In agonist mode, an agonist response creates an upwards curve and if there are any cytotoxic effects they typically occur towards the top of the curve at the highest doses. Therefore, you would see the downward turn at those levels. In antagonist or inverse-agonist mode, the curve is downward to begin with. In this case, the LCMA is recommended to confirm if the downward curve is a true response or due to cytotoxic effects of the treatment compound.

How many replicates do you recommend?

You can design your study to include as many replicates as you would like. If you are looking to utilize your data to publish, at least three replicates are recommended.

Can I choose the exact concentrations I want my compounds to be screened at?

Treatment concentrations are prepared at INDIGO using serial dilutions in fixed increments; the starting concentration and increment of dilution is specified by the customer via our Study Work Order sheet that accompanies all screening studies. For accurate determination of EC50 and/or IC50 values, we recommend at minimum spanning a 5,000-fold concentration range over 8 doses. This strategy requires a 3.33-fold or 4-fold increment of serial dilution. The minimum doses recommended for EC50 and/or IC50 value is 7 test concentrations.

How many dose concentrations do you recommend?

If you are looking for an initial go/no-go response, we recommend 1-3 dose concentrations. For EC50/IC50 values, we recommend at least 7-8, with 7 being the absolute minimum. You are welcome to do more dose concentrations if preferred, and we find that many of our customers opt to do 10 doses for a full EC50/IC50 curve.

Why don’t INDIGO’s ERα and ERβ assays produce the same EC50 values for 17β-estradiol? Likewise, for your TRα and TRβ assays run against triiodothyronine? There are reports that the respective EC50 values should be the same for these related pairs of nuclear receptors.

First, it is important to understand that EC50 (and IC50) values are not constants. There are numerous variables that impact these, and other, assay metrics. Consequently, these values can vary greatly between different assay types and between different related receptors. Regarding this last point: Neither ERα and ERβ, or TRα and TRβ, are simple iso-forms of each other; rather, they are all distinct receptors encoded by separate genes. As an example, the two ER proteins share only 45% sequence identity. If one includes ‘conserved’ substitutions between the two amino acid sequences, the two receptors still display only 60% sequence homology. While the two ERs have evolved to bind the physiological activator 17β-estradiol, it would be quite surprising if these two different nuclear receptors had the same activation profiles within the cellular context. It is also worth noting that the two ERs display strikingly disparate ligand preferences and activation/inhibition profiles for many other bioactive chemicals. INDIGO’s nuclear receptor assays are cell-based trans-activation assays, which rely on a complex orchestration of the many molecular processes that occur in vivo. In brief, these include: the reporter cells take up exogenous drug, which also experiences competitive interactions with serum-based and cellular macromolecules > binding of the drug to the ligand-binding domain of the nuclear receptor, which initiates the shedding of co-repressor molecules and recruitment of co-activator molecules > nuclear-translocation > binding of the activated receptor complex to the genetic response elements of the gene promoter > formation of a complete transcription complex > induced expression of the target gene (in our case, Luciferase) > translation of mRNA. The mechanics of such cell-based trans-activation assays are in
sharp contrast to in vitro ligand-binding assays and/or co-factor recruitment assays. Such assays measure a simple A+B → AB interaction between two purified molecules, typically polypeptides corresponding to the (much more highly conserved) ligand-binding domain sequences of the respective receptors, or to very small polypeptides corresponding to a specific protein-protein interaction sequence of a co-activator protein.
While useful for some applications, these various in vitro assay formats do not represent the complex molecular interactions that occur between ligands and the native receptors within the cellular environment. Therefore, when comparing the data sets from all of these various assay types, one can expect to see widely varying assay metrics, including respective EC50/IC50 values.

Can INDIGO’s nuclear receptor assays be used to test processed extracts derived from waste-water treatment samples, environmental samples, plant samples, etc.?

Yes. Preferably the extracts will be aqueous preparations that are: 1.) homogenous solutions with no particulate material, and 2.) sterile. If either is not true, the sample should be passed through a 0.2um syringe-tip filter. Preferably aqueous preparations will be no less than 4x-concentrated relative to the final treatment concentration in the assay. If the extract is prepared in DMSO, a stock of no less than 250x-concentrated (preferably more concentrated) is required. Residual DMSO carried over into any of INDIGO’s nuclear receptor assays should never exceed 0.4%. Higher DMSO concentrations will degrade assay performance.

Do INDIGO’s RORγ reporter cells express the RORγt isoform of the receptor?

First, it is important to understand the basic molecular biology behind the RORγ and RORγt isoforms. In humans, RORγ and RORγt derive from the same gene. However, expression of the respective transcripts is driven by tissue-specific alternative promoters, resulting in transcripts with variant exon 1 sequences. Consequently, the translated proteins have unique N-terminal amino acid sequences that encode unique DNA binding domains (DBD) for RORγ and RORγt. Importantly, the RORγ and -γt isoforms have common Ligand Binding Domains, as well as all other functional/structural domains. Therefore, it is their differential expression between tissues and cell types, and their variant DBD sequences, that account for their different physiological activities (target gene expression profiles). INDIGO’s RORγ reporter cells express an engineered cDNA that encodes a hybrid receptor. Specifically, the N-terminal amino acid sequence encoding either the RORγ DBD or the RORγt DBD has been replaced with amino acid sequence encoding the yeast GAL4-DBD. Hence, INDIGO’s reporter cells express a Gal4(DBD)-RORγ hybrid receptor—a “generic RORγ”—that is blind to the isoform differences in function and target gene expression profiles that otherwise exist in vivo. The specific utility of INDIGO’s RORγ assay is to quantify and rank interactions that occur between test compounds and the RORγ LBD. These reporter cells will show RORγ inverse-agonist and agonist responses that may be induced through drug interactions with the receptor.

Can the INDIGO’s ‘inverse-agonist’ receptor assays (RORα, RORγ, ERRα, ERRγ, CAR-1, and LRH-1) be set up to suppress the high constitutive activity of the receptor, thereby allowing the assessment of agonist activities?

Yes. When one is looking for agonist responses exclusively it is a common strategy to suppress the constitutive activity of the receptor by co-treating with a fixed concentration of the inverse-agonist reference compound provided with the assay kit. For example, this strategy is frequently used by researchers screening for agonists of RORγ. RORγ reporter cells are co-treated with a fixed concentration of Ursolic Acid (INDIGO’s reference inverse-agonist) and varying concentrations of the user’s test compounds. Compounds that are putative agonists of RORγ will show a significant dose-dependent increase in RORγ activity above the suppressed level.

What is an inverse-agonist nuclear receptor assay? Are these assays run separately in “agonist” and “inverse-agonist” mode?

Several nuclear receptors express high-level constitutive activity that is (seemingly) independent of a ligand interaction. The RORs, the ERRs, CAR-1, and LRH-1 are nuclear receptors with this activity profile, and they are loosely referred to as “inverse-agonist receptors/assays.” In practice, however, when a bio-active compound associates with the receptor’s ligand binding domain, there will be one of two functional outcomes: 1.) There will be a dose-dependent decrease in the receptor’s constitutive activity (an inverse-agonist response), or 2.) there will be a moderate dose-dependent increase in activity above the receptor’s already high constitutive level (an agonist response). INDIGO’s assays are capable of showing both of these alternative response in a single conventional assay setup.

What is the best way to normalize data (e.g., cell number, protein content)?

Normalizing RLU data to cell numbers or protein content is not something we do or recommend. The suspension of reporter cells is a ‘master reagent’ that should be dispensed with high precision into all wells of the assay plate. Any significant downstream variability in cell number or health will be the result of cytotoxicity induced by the test compound treatments. It is certainly important to perform cytotoxicity analyses for antagonist-mode assays (and inverse-agonist mode assays) to identify and weed out false-positive data, and INDIGO’s Live Cell Multiplex (LCM) Assay works very well for that purpose. However, attempting to normalize RLU data from treated cells that are in metabolic crisis to untreated, healthy control cells can result in gross misinterpretations of the data. When performing cell-based trans-activation assays (which rely on the coordinated cell-cycle processes of gene induction, transcription, translation, mRNA and protein turn-over) “healthy cells” and “dying cells” are not equal units, so attempting to normalize assay data to any cell-related endpoint is not a sound method.

Can you tell me what the reference agonist/antagonist is for your receptor assays?

View our list of reference compounds. We do utilize commercially available reference agonists for our assays, and this reference agonist is included in each assay kit. We do use reference antagonists where a reference is known and commercially available. Some of our antagonist assays do not have reference antagonists available.

Do INDIGO’s nuclear receptor assays express the native, full-length receptor?

Reporter cells included in INIDIGO’s steroid hormone nuclear assay kits (ERα, ERβ, AR, PGR, MR, GR) express the native, full-length receptors. INDIGO’s other nuclear receptor assays, however, include reporter cells that express hybrid nuclear receptors. In these cases, the respective receptor’s native N-terminal sequence comprising the DNA Binding Domain (DBD) has been replaced with sequence encoding the yeast Gal4-DBD. All other native NR functional/structural domains (ligand binding domain, hinge region, and various activation domains) are present in these hybrid receptors. These reporter cells also contain the firefly luciferase reporter gene functionally linked to the upstream genetic response element for Gal4. Consequently, once a bioactive compound associates with the ligand binding domain of the hybrid receptor, only the luciferase reporter gene is induced. Ligand-activation of the hybrid receptor will not induce collateral expression of target genes that are otherwise regulated by the native nuclear receptor.

What cell lines/types are used?

INDIGO’s nuclear receptor assays utilize proprietary human and non-human mammalian cells engineered to provide constitutive, high-level expression of the designated receptor. Specific cell type information for each assay is proprietary and available only through consultation with INDIGO’s technical team following a screening service or assay kit purchase.

Are INDIGO assays binding assays, or does INDIGO offer binding assays?

INDIGO’s assays are not binding assays. They are cell-based trans-activation assays, and the principal application is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that the compounds may exert against the nuclear receptors. INDIGO reporter systems utilize firefly luciferase reporter gene technology, and while there is a binding taking place, our assays do not measure it. Instead, the luciferase light response is measured which correlates to the activation status of the receptor (either activation or inhibition). Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in receptor activity. It will tell the scientist the significance (strength) of the interaction by the level of light emitted. In addition, cell-based assays are more sensitive and able to detect smaller levels of activation.

Assays • By Receptor

INDIGO Kits and Services

INDIGO Biosciences’ cell-based, luciferase reporter assay kits feature receptor-specific reporter cells prepared using our unique CryoMite™ process which typically present greater than 95% cell viability and are ready for immediate use. INDIGO’s assay kits are engineered to provide optimal assay sensitivity and dynamic range when quantifying receptor activity. All assays are available as all-inclusive kits and  as contract screening services.

Androgen Receptor (AR; NR3C4)
Aryl Hydrocarbon Receptor (AhR)
Cannabinoid Type 1 Receptor (CB1R, CNR1)
Constitutive Androstane Receptor-1 (CAR-1; NR1I3i1) Constitutive Androstane Receptor-2 (CAR-2; NR1I3i2) Constitutive Androstane Receptor-3 (CAR-3; NR1I3i3)
Epidermal Growth Factor Receptor 1 (EGFR1)
Estrogen Receptor Alpha (ERα; NR3A1) Estrogen Receptor Beta (ERβ; NR3A2)
Estrogen-related Receptor Alpha (ERRα; NR3B1) Estrogen-related Receptor Gamma (ERRγ; NR3B3)
Farnesoid X Receptor (FXR; NR1H4)
Glucocorticoid Receptor (GR; NR3C1)
Growth Hormone Receptor (GHR)
Liver Receptor Homolog-1 (LRH-1; NR5A2)
Liver X Receptor Alpha (LXRα; NR1H3) Liver X Receptor Beta (LXRβ; NR1H2)
Mineralocorticoid Receptor (MR; NR3C2)
Nuclear Factor kappa-light-chain enhancer of activated B cells (NF-kB)
Nuclear Factor (erythroid-derived 2)-like 2 (Nrf2)
Peroxisome Proliferator-Activated Receptor Alpha (PPARα; NR1C1) Peroxisome Proliferator-Activated Receptor Beta/Delta (PPARβ/δ; NR1C2) Peroxisome Proliferator-Activated Receptor Gamma (PPARγ; NR1C3)
Pregnane X Receptor (PXR; NR1I2)
Progesterone Receptor (PGR; NR3C3)
Retinoic Acid Receptor Alpha (RARα; NR1B1) Retinoic Acid Receptor Beta (RARβ; NR1B2) Retinoic Acid Receptor Gamma (RARγ; NR1B3)
RAR-related Orphan Receptor Alpha (RORα; NR1F1) RAR-related Orphan Receptor Gamma (RORγ; NR1F3)
Retinoid X Receptor Alpha (RXRα; NR2B1) Retinoid X Receptor Beta (RXRβ; NR2B2) Retinoid X Receptor Gamma (RXRγ; NR2B3)
TEAD4/YAP (Hippo Pathway)
Thyroid Hormone Receptor Alpha (TRα; NR1A1) Thyroid Hormone Receptor Beta (TRβ; NR1A2)
Transforming Human Growth Factor Beta Receptor I/II (TGFβR)
Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)
Vitamin D Receptor (VDR; NR1I1)

Many labs are interested in cross-species comparisons, especially contrasting human nuclear receptor activity to common laboratory animals. Determining a drug candidate’s cross-activity with human xenobiotic-sensing receptors provides important early indications of that drug’s potential for downstream drug-drug interactions. With animal studies required by the FDA, selecting the animal model that provides the most representative human-surrogate is critical to assessing a potential drug’s likelihood of unwanted effects. Cell-based assay models are crucial to help make this determination prior to entering ADMET studies.

With more than 30 ortholog assays – including rat, mouse, dog, monkey, and zebrafish – available as kits and/or services, and others available for custom development, INDIGO helps researchers screen the right animal, before trial.

Rat Androgen Receptor (rAR; nr3c4) Zebrafish Androgen Receptor (zAR; nr3c4)
Rat Aryl Hydrocarbon Receptor (rAhR) Zebrafish Aryl Hydrocarbon Receptor (zAhR)
Mouse Constitutive Androstane Receptor (mCAR; nr1i1) Rat Constitutive Androstane Receptor (rCAR; nr1i1)
Zebrafish Estrogen Receptor Alpha (zERα; nr3a1)
Mouse Farnesoid X Receptor (mFXR; nr1h4) Rat Farnesoid X Receptor (rFXR; nr1h4)
Cyn Monkey Farnesoid X Receptor (cFXR; nr1h4) Dog Farnesoid X Receptor (dFXR; nr1h4)
Mouse Glucocortioid Receptor (mGR; nr3c1) Rat Glucocorticoid Receptor (rGR; nr3c1)
Zebrafish Glucocortioid Receptor (zGR; nr3c1)
Mouse Liver X Receptor Alpha (mLXRα; nr1h3)
Mouse Liver X Receptor Beta (mLXRβ; nr1h2) Rat Liver X Receptor Beta (rLXRβ; nr1h2)
Mouse Peroxisome Proliferator-Activated Receptor Alpha (mPPARα; nr1c1) Rat Peroxisome Proliferator-Activated Receptor Alpha (rPPARα; nr1c1)
Cyn Monkey Peroxisome Proliferator-Activated Receptor Alpha (cPPARα; nr1c1) Dog Peroxisome Proliferator-Activated Receptor Alpha (dPPARα; nr1c1)
Mouse Peroxisome Proliferator-Activated Receptor Beta/Delta (mPPARβ/δ; nr1c2) Rat Peroxisome Proliferator-Activated Receptor Beta/Delta (rPPARβ/δ; nr1c2)
Cyn Monkey Peroxisome Proliferator-Activated Receptor Beta/Delta (cPPARβ/δ; nr1c2) Dog Peroxisome Proliferator-Activated Receptor Beta/Delta (dPPARβ/δ; nr1c2)
Rodent (Mouse/Rat) Peroxisome Proliferator-Activated Receptor Gamma (mrPPARγ; nr1c3) Cyn Monkey Peroxisome Proliferator-Activated Receptor Gamma (cPPARγ; nr1c3)
Zebrafish Peroxisome Proliferator-Activated Receptor Gamma (zPPARγ; nr1c3)
Mouse Pregnane X Receptor (mPXR; nr1i2) Rat Pregnane X Receptor (rPXR; nr1i2)
Cyn Monkey Pregnane X Receptor (cPXR; nr1i2) Dog Pregnane X Receptor (dPXR; nr1i2)
Rat Progesterone Receptor (rPGR; nr3c3)
Zebrafish Retinoic Acid Receptor Alpha, isoform A (zRARαa; nr1b1)
Mouse RAR-related Orphan Receptor Gamma (mRORγ; nr1f3)
Zebrafish Thyroid Hormone Receptor Beta (zTRβ; nr1a2)

INDIGO’s nuclear receptor reporter assay panels contain all materials needed to perform either 32 or 48 assays for each receptor included. PPAR, RAR, and RXR Panels are available in a 3×32-well format; ER, LXR, and TR Panels are in a 2×48-well format, all designed to fully utilize a single 96-well plate. All reagents are supplied with sufficient extra volume to accommodate the needs of performing separate groups of assays. When screening a smaller number of compounds against all variants of a receptor, INDIGO’s Assay Panel kits allow researchers to save time, cost, and risk by screening against multiple receptors in one easy-to-use kit.

Estrogen Receptor PANEL (ERα; ERβ) Liver X Receptor PANEL (LXRα; LXRβ) Peroxisome Proliferator-Activated Receptor PANEL (PPARα; PPARβ/δ; PPARγ)
Retinoic Acid Receptor PANEL (RARα; RARβ; RARγ) Retinoid X Receptor PANEL (RXRα; RXRβ; RXRγ) Thyroid Hormone Receptor PANEL (TRα; TRβ)

In Vitro Screening for Drug-Induced Hepatotoxicity using upcyte® Hepatocytes

The emergence of liver toxicity is major reason for the termination of clinical drug trials, as well as post-market withdrawal of approved drugs. The assay kit for in vitro screening for drug-induced hepatotoxicity allows researchers to rapidly identify those compounds that induce liver toxicity. The kit utilizes upcyte® hepatocytes, prepared using our proprietary CryoMiteTM process, which yields high viability post-thaw and provides the convenience of immediately dispensing cells into assay plates. This all-inclusive assay kit allows users to bring processes previously available only as contract screening services into their own labs. Learn more.

Human MDR1 / P-Glycoprotein Drug Interaction Assay

Determining if a drug candidate will have incidental interactions with P-Glycoprotein (P-gp, aka MDR-1, or ABCB1) is an important component of the safety assessment process. A drug that is either a substrate or inhibitor of MDR1 transporter activity can significantly alter the rate of absorption, distribution, metabolic conversion, and eventual excretion of co-administered drugs, thereby shifting their therapeutic effects and toxicologic profiles. Because of this, assessing a new drug’s potency as an interactor with P-gp, and thus its potential liability for inducing downstream drug-drug interactions, is mandated by the FDA. Our all-inclusive assay kit for the assessment of MDR1 drug interaction allows users to rapidly assess drug candidates as either inhibitors, substrates, or non-substrates of P-gp, and make critical decisions about potential drug candidates with confidence. Learn more.

Expression Profiling of Clinically Relevant CYPs Utilizing upcyte® Hepatocytes

Assessing drug-induced changes in the expression of Cytochrome P450 (CYP) genes provides a reliable predictive indicator of altered metabolic activity in vivo. It is estimated that CYPs are involved in 70% to 80% of drugs currently on the market, making understanding their metabolic actions crucial to the drug development process. Our gene expression assay kit provides optimized reagents for the culturing and treatment of upcyte® hepatocytes to assess drug-induced changes in the expression of seven clinically relevant CYPs: CYP3A4, CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1. Learn more.

ASSAYS • By Disease State

Nuclear Receptor Disease State Affiliations

DISEASE STATENUCLEAR RECEPTORS
Alzheimer’s DiseaseERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3)
AtherosclerosisLXRα (NR1H3); LXRβ (NR1H2); Nf-kB; PPARα (NR1C1); RORα (NR1F1)
Autoimmune DiseasesAR (NR3C4); AhR; ERα (NR3A1); ERβ (NR3A2); GR (NR3C1); MR (NR3C2); NFAT; PGR (NR3C3); PPARα (NR1C1); PPARγ (NR1C3); RARα (NR1B1); RARβ (NR1B2); RARγ (NR1B3); RORα (NR1F1); RORγ (NR1F3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3); TEAD4/YAP; VDR (NR1I1)
Bile Acid & Xenobiotic MetabolismAhR; CAR (NR1I3); EGFR1; FXR (NR1H4); LRH-1 (NR5A2); PXR (NR1I2); RORγ (NR1F3); VDR (NR1I1)
CancerAR (NR3C4); CB1R;EGFR1; EPOR; ERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3); FGFR1/2; GHR; LRH-1 (NR5A2); Nf-kB; NFAT; Nrf2; PPARδ (NR1C2); PPARγ (NR1C3); RARα (NR1B1); RARβ (NR1B2); RARγ (NR1B3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3);TEAD4/YAP; TGFβR; TPOR; VDR (NR1I1); VEGFR2
Cardiovascular DiseaseCB1R; EPOR; ERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3); FGFR1/β-Klotho; GHR; TEAD4/YAP; TGFβR; VEGFR2
Circadian RhythmERRα (NR3B1); RORα (NR1F1); RORγ (NR1F3); TRα (NR1A1); TRβ (NR1A2); VEGFR2
CNS, Circadian, & Basal MetabolismEPOR; LXRβ (NR1H2); MR (NR3C2); NFAT; RARβ (NR1B2); RORα (NR1F1); RORγ (NR1F3); RXRβ (NR2B2); TRα (NR1A1); TRβ (NR1A2);
DiabetesCB1R; EGFR1; FGFR/β-Klotho; GHR; GR (NR3C1); LXRα (NR1H3); LXRβ (NR1H2); PPARγ (NR1C3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3); TGFβR
Drug-Drug InteractionsAhR; CAR (NR1I3); FXR (NR1H4); LRH-1 (NR5A2); LXRα (NR1H3); Nrf2; PPARα (NR1C1); PXR (NR1I2); VDR (NR1I1)
Drug-Nutrient InteractionAhR; CAR-2 (NR1I3i2); CAR-3 (NR1I3i3); FXR (NR1H4); Nrf2; PXR (NR1I2); VDR (NR1I1)
DyslipidemiaFXR (NR1H4); LRH-1 (NR5A2); LXRα (NR1H3); LXRβ (NR1H2); PPARα (NR1C1); RORα (NR1F1); TRα (NR1A1); TRβ (NR1A2); VDR (NR1I1)
EndocrinologyAR (NR3C4); ERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3); MR (NR3C2); PGR (NR3C3)
Environmental ToxicologyAR (NR3C4); AhR; ERα (NR3A1); ERβ (NR3A2); ERRα (NR3B1); ERRγ (NR3B3); Nrf2; PPARγ (NR1C3); PGR (NR3C3); TRα (NR1A1); TRβ (NR1A2)
FertilityEPOR; ERα (NR3A1); ERRγ (NR3B3)
InflammationCB1R; EGFR1; FGFR1/β-Klotho; GHR; GR (NR3C1); Nf-kB; NFAT; Nrf2; PPARα (NR1C1); PPARδ (NR1C2); PPARγ (NR1C3); RORα (NR1F1); RORγ (NR1F3); TGFβR
Lipid Metabolism & EnergyCB1R; ERRα (NR3B1); FGFR1/β-Klotho; GR (NR3C1); LXRα (NR1H3); PPARα (NR1C1); PPARδ (NR1C2); PPARγ (NR1C3); TRβ (NR1A2)
Metabolic DiseaseFGFR1/β-Klotho; FXR (NR1H4); GHR; GR (NR3C1); LRH-1 (NR5A2); LXRα (NR1H3); LXRβ (NR1H2); PPARα (NR1C1); PPARδ (NR1C2); PPARγ (NR1C3); TRα (NR1A1); VDR (NR1I1)
NAFLD/NASHCAR (NR1I3); FGFR1/β-Klotho; FXR (NR1H4); LXRα (NR1H3); LXRβ (NR1H2); PPARα (NR1C1); PPARδ (NR1C2); PPARγ (NR1C3); PXR (NR1I2); RARα (NR1B1); RARβ (NR1B2); RORγ (NR1F3); TRα (NR1A1); VDR (NR1I1)
ObesityCB1R; ERβ (NR3A2); FGFR1/β-Klotho; GHR; GR (NR3C1); PPARδ (NR1C2); PPARγ (NR1C3); TGFβR; TRα (NR1A1); TRβ (NR1A2)
OsteoporosisAR (NR3C4); ERα (NR3A1); ERβ (NR3A2); ERRα (NR3B1); ERRγ (NR3B3); GHR; PGR (NR3C3); VDR (NR1I1)
PsoriasisRARα (NR1B1); RARβ (NR1B2); RARγ (NR1B3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3); VDR (NR1I1)
Reproduction & DevelopmentAR (NR3C4); CB1R; ERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3); FGFR1/2; GHR; NFAT; PGR (NR3C3); RARα (NR1B1); RARγ (NR1B3); RXRα (NR2B1); RXRγ (NR2B3); TEAD4/YAP
Retinoid PanelRARα (NR1B1); RARβ (NR1B2); RARγ (NR1B3); RORα (NR1F1); RORγ (NR1F3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3)
Steroid ReceptorAR (NR3C4); ERα (NR3A1); ERβ (NR3A2); ERRγ (NR3B3); GR (NR3C1); MR (NR3C2); PGR (NR3C3)
SteroidogenesisLRH-1 (NR5A2)
ToxicologyAhR; CAR (NR1I3); FXR (NR1H4); Nf-kB; Nrf2; PPARα (NR1C1); TRα (NR1A1)
Wound HealingAR (NR3C4); EGFR1; ERα (NR3A1); ERβ (NR3A2); FGFR1/2; PPARδ (NR1C2); PXR (NR1I2); RARα (NR1B1); RARβ (NR1B2); RARγ (NR1B3); RXRα (NR2B1); RXRβ (NR2B2); RXRγ (NR2B3); TGFβR
Xenobiotic MetabolismAhR; CAR (NR1I3); Nrf2;PXR (NR1I2)

Receptors For Anemia and Chronic Kidney Disease Research

Nuclear receptors and other receptors are targets for anemia and chronic kidney disease research due to their involvement in the signaling pathways for metabolism, immune response, and inflammation. Understanding the mechanisms underlying the involvement of various receptors in the pathogenesis of certain anemias and chronic kidney disease, as well as the complications and comorbidities that result from it may offer targets for the development of new treatments.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in anemia and renal disease research include:

  • AR (NR3C4)
  • AhR
  • ERα (NR3A1)
  • ERRα (NR3B1)
  • GR (NR3C1)
  • NF-kB
  • PPARα (NR1C1)
  • TPOR
  • TRα (NR1A1)
  • TRβ (NR1A2)
  • VDR (NR1I1)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their anemia and chronic kidney disease research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Anemia

Anemia is a condition in which the blood has a low number of red blood cells. This resulting in reduced oxygen flow to the body. Anemia is the most common blood condition in the United Stated affecting almost 6% of the population. There are three main causes for anemia including, blood loss, lack of red blood cell production, and high rates of red blood cell apoptosis.

Symptoms may include fatigue, cold hands and feet, pale or yellow skin, shortness of breath, lightheadedness, or a fast heartbeat. Comorbidities of anemia are hypertension, hypothyroidism, Cancer, cardiovascular disease, rheumatologic disease, and chronic kidney disease.

Chronic Kidney Disease

Chronic kidney disease (chronic renal disease) includes a variety of conditions that damage the kidneys and decrease their functioning. As kidney disease progresses, less waste gets filtered out, and it can build to high levels. There is an increased risk for developing chronic kidney disease for those with a family history of kidney disease, as well as those with the comorbidities diabetes, high blood pressure, and cardiovascular disease. More than 37 million people in the United States may have chronic kidney disease.

There typically are no symptoms in the early stages of chronic kidney disease, but as kidney damage progresses, signs and symptoms can include, nausea, vomiting, loss of appetite, fatigue and weakness, sleep problems, more frequent urination, swelling of feet and ankles, persistent itching, chest pain, and shortness of breath. Complications of chronic kidney disease include, fluid retention, cardiovascular disease, anemia, decreased sex drive, damage to the CNS, decreased immune response, pericarditis, and end-stage kidney disease. Chronic kidney disease can be diagnosed using a blood test to measure the glomerular filtration rate or a urine test to check for albumin. Imaging tests, and kidney biopsy can also be performed to understand the type of kidney disease and how much damage has occurred to recommend treatment.

Once damage to the kidney has occurred it is permanent. Treatment of chronic kidney disease largely depends on the underlying cause, but usually consists of measures to help control signs and symptoms and slow the progression of the disease. If the disease progresses to end-stage kidney disease when less than 15% of the kidney is functioning, treatments to maintain health include dialysis and kidney transplantation.

Receptors For Autoimmune Disease and Inflammation Research

Nuclear receptors and other receptors are targets for autoimmune research due to their involvement in the signaling pathways for inflammation and immunity. Autoimmune diseases also cause damage to the endocrine system causing over or underproduction of certain hormones. This makes therapies that are full or partial agonists, or antagonists of nuclear hormone receptors involved in endocrine function important for managing perturbations caused by specific autoimmune diseases.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in autoimmune and inflammation research include:

  • AR (NR3C4)
  • ERα (NR3A1)
  • ERβ (NR3A2)
  • GR (NR3C1)
  • MR (NR3C2)
  • NFAT
  • PGR (NR3C3)
  • PPARα (NR1C1)
  • PPARγ (NR1C3)
  • RARα (NR1B1)
  • RARβ (NR1B2)
  • RARγ (NR1B3)
  • RORα (NR1F1)
  • RORγ (NR1F3)
  • RXRα (NR2B1)
  • RXRβ (NR2B2)
  • RXRγ (NR2B3)
  • TEAD/YAP (Hippo Pathway)
  • VDR (NR1I1)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their autoimmune and inflammation research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results, making for more precise data and allowing better interpretation. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Disease State Background

A healthy immune system defends the body against disease and infection, but if the immune system malfunctions, it can mistakenly attack healthy cells, tissues, and organs. Collectively diseases that result from dysfunction of the immune system, whether from abnormally low activity or over activity, are called autoimmune disease. These attacks can affect any part of the body, weakening bodily function and even turn life-threatening. More than 23 million people in the United States have an autoimmune disease and there are more than 80 known types of autoimmune diseases including type 1 diabetes, rheumatoid arthritis, psoriasis, and multiple sclerosis.

Many autoimmune diseases have similar symptoms. One of the classic signs of an autoimmune disease is the redness, heat, pain, and swelling caused by inflammation. Though inflammation is critical for the body’s normal immune responses, chronic inflammation has been shown to cause tissue damage. For these reasons and others, avenues for autoimmune disease research include the role of inflammation associated with immune system response.

Treatments for autoimmune diseases vary depending on the disease, but mostly consist of ways to reduce symptoms and reduce the immune systems abnormal response. Currently, the cause for autoimmune diseases is unknown but, due to the rise of incidences of autoimmune disease, researchers suspect environmental factors play a role.

Receptors For Cancer Research

Understanding the mechanisms underlying the involvement of receptors in tumorigenesis of various types of cancer may offer targets for the development of new oncology treatments.

Associated Receptors

Specific receptors that have shown to be promising therapeutic targets for drug discovery in oncology research include:

  • AR (NR3C4)
  • AhR
  • EGFR1
  • ERα (NR3A1)
  • ERβ (NR3A2)
  • ERRγ (NR3B3)
  • GHR
  • LRH-1 (NR5A2)
  • NFAT
  • PPARδ/PPARβ (NR1C2)
  • PPARγ (NR1C3)
  • RARα (NR1B1)
  • RARβ (NR1B2)
  • RARγ (NR1B3)
  • RXRα (NR2B1)
  • RXRβ (NR2B2)
  • RXRγ (NR2B3)
  • TEAD/YAP (Hippo Pathway)
  • TGFβR
  • VDR (NR1I1)
  • VEGFR2

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their cancer research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Nuclear Receptors

Nuclear receptors have long been targets in oncology research and the role of dysregulated nuclear receptor mediated signaling pathways in tumorigenesis has been well documented in a variety of cancers. Small molecule drugs that modulate nuclear receptors have proven to be effective therapeutic targets, such as with antagonists of the androgen receptor (AR) in patients with prostate cancer or the estrogen receptor alpha (ERα) in patients with ERα positive breast cancer. Nuclear receptors also play critical roles in the signaling pathways for inflammation and immunity and these pathways have been widely implicated in oncogenesis and may be potent therapeutic avenue for new cancer treatments.

Growth Factor Receptors

Growth factor receptors are important targets in oncology because mutations that lead to constitutive activation of these signal pathways result in the progression of various types of cancers. Overexpression of receptor tyrosine kinases (RTK), which include the epidermal growth factor receptor (EGFR) family and the vascular endothelial growth factor receptor (VEGFR) family, among others, have been found in many tumors. Mutations in serine/threonine-specific protein kinase receptors such as transforming growth factor-beta (TGFβR) have also been linked to growth of many tumors due to defects in the cellular growth inhibition response to TGFβ. As discovery research continues, growth factors and growth factor signaling pathways remain promising targets in the development of oncologic therapeutics.

Receptors For Cardiovascular Disease Research

Nuclear receptors and other receptors are critical regulators of metabolism, inflammation, and regeneration. Understanding the mechanisms underlying the involvement of nuclear receptors, as well as other receptors in the pathogenesis of cardiovascular disease and atherosclerosis may offer targets for the development of new treatments.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in cardiovascular disease and atherosclerosis research include:

  • AhR
  • ERα (NR3A1)
  • ERβ (NR3A2)
  • ERRγ (NR3B3)
  • GHR
  • LXRα (NR1H3)
  • LXRβ (NR1H2)
  • PPARα (NR1C1)
  • RORα (NR1F1)
  • TEAD/YAP (Hippo Pathway)
  • TGFβR
  • VEGFR2

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their cardiovascular disease research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Disease State Background

Cardiovascular disease or heart disease describes a range of conditions that affect the cardiovascular system including blood vessel diseases, arrhythmias, and cardiomyopathy. Causes including diabetes, smoking, stress, obesity, and an existing family history have been associated with cardiovascular disease. A major indicator of developing cardiovascular disease is atherosclerosis. Atherosclerosis happens when plaque builds up in and on the walls of the arteries. This buildup of plaque narrows the arteries, making it hard for blood to flow.

Signs and symptoms vary for different types of cardiovascular disease, but often include fatigue, high blood pressure, chest pain, and shortness of breath. Complications associated with cardiovascular disease can lead to aneurysms, cardiac arrest, heart failure, stroke, and death. In fact, cardiovascular diseases are regularly among the top global causes of death reported by the World Health Organization. Diagnosis of cardiovascular disease typically involves an electrocardiography, echocardiography, or stress testing to screen for the disease.

Initial treatment of cardiovascular disease largely focuses on diet, lifestyle changes, as well as medication. If these treatments are not enough, surgical intervention may be required.

Receptors For Diabetes Research

Nuclear receptors and other receptors are targets for diabetes research due to their involvement in the signaling pathway for carbohydrate metabolism, lipid metabolism, immunity, and inflammation. Understanding the mechanisms underlying the involvement of various receptors in the pathogenesis of diabetes mellitus, as well as the complications that result from the disease may offer targets for the development of new treatments.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in diabetes research include:

  • EGFR
  • GHR
  • GR (NR3C1)
  • LXRα (NR1H3)
  • LXRβ (NR1H2)
  • PPARγ (NR1C3)
  • RXRα (NR2B1)
  • RXRβ (NR2B2)
  • RXRγ (NR2B3)
  • TGFβR

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their diabetes research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Disease State Background

Diabetes mellitus is a progressive metabolic disorder with diverse pathological manifestation in which blood glucose levels are elevated due to the bodies inability to maintain glucose homeostasis. It is estimated that more than 34 million people in the United States have diabetes, with one in five not knowing they have it. There are three main types of diabetes: type 1, type 2, and gestational diabetes. Type 1 diabetes is thought to be caused by an autoimmune response that stops the body from producing insulin. Type 2 diabetes occurs when the body either doesn’t produce enough insulin, or it resists insulin. Of the two types, type 2 is the most prevalent with about 90-95% of the diabetic population being type 2. Gestational diabetes develops during pregnancy and typically ends when the baby is born but increases the risk of developing type 2 later in life.

Signs and symptoms of diabetes include increased thirst, frequent urination, extreme hunger, unexplained weight loss, fatigue, blurred vision, slow-healing sores, and presence of ketones in the urine. Complications associated with diabetes when left untreated can include Alzheimer’s disease, cardiovascular disease, neuropathy, nephropathy, and retinopathy, as well as toe, foot, or leg amputation. There are multiple ways to test and determine if someone has diabetes including an A1C test, Random Plasma Glucose Test, Fasting Plasma Glucose (FPG), Oral Glucose Tolerance Test (OGTT), or keto test.

The exact cause of type 1 and type 2 diabetes is unknown, though both are thought to be caused by a combination of a genetic predisposition and environmental factors. While the exact environmental factors are also unknown, being overweight is strongly linked to the development of type 2. No cure for diabetes currently exists though treatments to manage the disease involve a combination of medications, exercise, diet, lifestyle changes, and insulin injections.

Receptors For Endocrine Research

Nuclear receptors are a critical part of endocrine functioning. Understanding the mechanisms underlying the involvement of nuclear receptors in the pathogenesis of endocrine disorders may offer targets for the development of new treatments, as well as creating better treatments for correcting hormone imbalances caused by endocrine disorders.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in endocrine research include:

  • AR (NR3C4)
  • EPOR
  • ERα (NR3A1)
  • ERβ (NR3A2)
  • ERRγ (NR3B3)
  • MR (NR3C2)
  • PGR (NR3C3)
  • TRα (NR1A1)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their endocrine research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Disease State Background

Endocrinology is the study of the messenger system comprising feedback loops of hormones released by internal glands. Hormones are used to communicate between organs and tissues for physiological regulation of multiple functions including autonomic functions, circadian rhythm, development and reproduction, metabolism, mood, and stress response.

There are many common endocrine disorders that cause disruptions in the endocrine system including diabetes mellitus, acromegaly, Addison’s disease, Cushing’s syndrome, and hyperthyroidism. Treatments for endocrine disorders vary depending on the specific disorder, but often focus on correcting the hormone imbalance.

Another source of study in endocrine research is from chemicals both man-made and natural that are full agonist, partial agonist, or antagonist of the receptors that function in the endocrine system. Perturbation caused by these chemicals, sometimes referred to as Endocrine Disrupting Chemicals (EDCs) can range in their physiological effects from tumorigenesis to birth defects and other developmental disorders. Many of these chemicals are lipophilic and are known to bioaccumulate in tissues. For those interested in endocrine disrupting chemicals, learn more in this white paper from INDIGO Nuclear Receptors & Endocrine / Metabolic Disruption.

Nuclear Receptors for Non-Alcoholic Fatty Liver Disease (NASH/NAFLD) Research

NASH Nuclear Receptors

Understanding the mechanisms underlying the involvement of nuclear receptors in the pathogenesis of Non-Alcoholic Fatty Liver Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH) may offer targets for the development of new treatments for this liver disease. The most common nuclear receptors used in drug and treatment discovery are ligand-activated nuclear receptors, including peroxisome proliferator-activated receptor alpha (PPARα), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), which were first identified as key regulators of the responses against chemical toxicants. Numerous studies using mouse disease models and human samples have revealed critical roles for these receptors and others, such as PPARβ/δ, PPARγ, farnesoid X receptor (FXR), and liver X receptors (LXR), in maintaining nutrient/energy homeostasis in part through modulation of the gut-liver-adipose axis.

NAFLD is associated with altered nuclear receptor function and perturbations along the gut-liver axis. These perturbations include obesity, abnormal hepatic lipid metabolism, increased inflammation, and insulin resistance. Nuclear receptors are essential to understanding the physiology and pathology of liver diseases like NAFLD/NASH, as they are at the crossroads of metabolism, inflammation, and regeneration. Modulation of nuclear receptors has been shown to reduce hepatic steatosis, inflammation, insulin resistance, fibrosis, and obesity, making them attractive – and effective – therapeutic targets.

NASH Nuclear Receptors

Current NASH research indicates drug and treatment discovery relies on Nuclear Receptor activation, specifically:

Our NASH nuclear receptor assay products are cell-based reporter assay systems. They feature engineered nuclear receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against human nuclear receptors expressed within the cytoplasm and nuclear environments of healthy, dividing mammalian cells.

INDIGO Biosciences works closely with clients to provide the appropriate Nuclear Receptors for Non-Alcoholic Fatty Liver Disease Research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results, making for better interpretation and more accurate data. Employing a luminescence-based method and our proprietary CryoMite preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Disease State Background

Non-Alcoholic Fatty Liver Disease (NAFLD) is characterized by liver inflammation and the buildup of extra fat in liver cells not caused by excessive alcohol consumption. The liver naturally contains some fat, however, when the weight of the fat content of the liver is more than 5-10%, the condition is known as fatty liver (steatosis). NAFLD is the most common liver disorder in developed countries with more than 100 million adults and children affected in the United States alone. NAFLD occurs in people of all races and ethnicities, though research shows it to be most common in those of Latin American descent.

Non-Alcoholic Steatohepatitis (NASH) is the most extreme form of NAFLD, and is a major cause of cirrhosis of the liver and liver cancer.

Non-Alcoholic Fatty Liver Disease

The majority of people with NAFLD have no symptoms, though some may experience fatigue, dull upper right quadrant abdominal discomfort, and/or mild jaundice. Diagnosis typically occurs following abnormal liver function tests from routine blood test. Common findings are elevated liver enzymes and a liver ultrasound showing steatosis. A liver biopsy is the only widely accepted test for definitively distinguishing NASH from other forms of liver disease. Current estimates indicate that about 20 percent of people with NAFLD have NASH.

According to P&S Market Research, as of March 2017, more than 50 potential therapeutic candidates were in the pipeline. Certain factors – including unknown etiology, complex patho-physiology, and high treatment costs – make understanding how associated nuclear receptors respond and react key to further development.

Related Articles

Liver Foundation

DDNews Special Focus on Nonalcoholic Steatohepatitis; Date of publication: March 2017; DDNews

Nonalcoholic Steathohepatits Therapeutics Market; Date of publication: 2017; P&S Market Research

Treatment of non-alcoholic fatty liver disease; Date of publication: May 2006; BMJournal

The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease; Date of publication: March 2005; Clinics in Liver Disease; vol 8, issue 3

Receptors For Obesity Research

Nuclear receptors and other receptors are targets for obesity research due to their involvement in the signaling pathway for regulating metabolism, as well as energy and lipid homeostasis. Understanding the mechanisms underlying the involvement of various receptors in the pathogenesis of obesity, as well as the complications and comorbidities that result from it may offer targets for the development of new treatments.

Associated Receptors

Specific receptors that are implicated as promising therapeutic targets for drug discovery in obesity research include:

  • ERβ (NR3A2)
  • GHR
  • GR (NR3C1)
  • PPARδ (NR1C2)
  • PPARγ (NR1C3)
  • TGFβR
  • TRα (NR1A1)
  • TRβ (NR1A2)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their diabetes research. To empower confident decision-making throughout the discovery process, our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, we provide reproducible results lot-to-lot about the efficacy, potency, and selectivity of your compounds, plus comprehensive lab reports that include helpful graphics, summaries, and insights.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Disease State Background

Obesity is defined as abnormal or excessive fat accumulation that presents a risk to health. A body mass index (BMI) over 30 is considered obese, though this is not always accurate as some athletes have high BMI’s due to a high level of muscle mass. Obesity is primarily caused by an imbalance between calories consumed and calories expended which can result from a combination of genetic and environmental factors. Environmental factors leading to obesity can include medical conditions, physical activity, dietary patterns, and medications.

The signs and symptoms of obesity are excessive body fat, shortness of breath, trouble sleeping, skin problems, fatigue, and pain. Long term risks of obesity include developing serious diseases and comorbidities including NAFLD/NASH, hypertension, dyslipidemia, type 2 diabetes mellitus, cardiovascular disease, stroke, gallbladder disease, sleep apnea, and certain types of cancer. Obesity is considered a serious global health problem due to its increasing prevalence and comorbidities

Toxicology Solutions

Product FamilyProduct NumberProduct DescriptionTechnical Manual
In Vitro ToxicologyULC1003-48in vitro Screening for Drug-Induced Hepatotoxicity, 2x 48 Assays in 96-well formatTechnical Manual

Kit

In Vitro Screening for Drug-Induced Hepatotoxicity using upcyte® Hepatocytes

The principal application of this assay is to rapidly screen test compounds to identify those that induce acute liver cell toxicity.

This kit includes two aliquots of cryopreserved Luminescent Reporter Hepatocytes (upcyte®), donor 10-13, isolated from an adult Caucasian female, that have been further modified to constitutively express the luciferase enzyme. The level of luciferase activity expressed in the cells is dependent on the complex coordination of normal cellular processes, including the coupled rates of energy metabolism, transcription, translation, and the turnover of their respective mRNA and protein macromolecules. The hepatocytes are prepared using INDIGO’s proprietary CryoMite process, which yields high cell viability post-thaw, and provides the convenience of immediately dispensing cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. An overnight culture period allows full recovery of the post-thaw hepatocytes and the establishment of a confluent cell monolayer that is ready to receive the user’s test compounds.

In addition to two aliquots of the reporter hepatocytes, the kit provides two cell culture-ready assay plates, optimized Cell Culture Media (CCM) for use in all steps of the assay procedure (cell thawing, seeding, and preparation of the treatment media), luciferase detection reagent, and a reference compound that provides a positive control for hepatotoxicity.

The reagents and materials provided in this assay kit are formatted to allow the user to choose between two alternative assay setups. In one scenario 48 culture wells may be setup at two different times. In the other assay scenario 96 culture wells may be setup at one time.

For more information, view the Technical Manual.

Services

A primary application of INDIGO’s in vitro toxicology platform is to screen chemicals for induction and activity of hepatic drug metabolizing enzymes.

upcyte® hepatocytes exhibit basal CYP1A2, CYP2B6, CYP2C9, and CYP3A4, which are all induced by prototypical inducers. As with primary human hepatocytes, the induction response is donor-dependent; therefore, INDIGO offers a panel of different donors with a range of induction responses.

In Vitro Toxicology

Utilizing this unique platform, INDIGO’s services lab can examine your compound’s potential to cause liver toxicity through metabolic activation or by induction of drug metabolism enzymes. upcyte® hepatocytes contain equivalent activity of several cytochrome P450’s compared to human hepatocytes, thus making examination of metabolism-dependent toxicity biologically relevant. In addition, upcyte® hepatocytes can strengthen and quantify changes in the expression of target genes regulated by PXR, CAR, AhR, LXRs, LRH-1, PPARs, and Nrf2.

Drug-Drug Interactions

Drugs that induce xenobiotic metabolizing enzymes responsible for their own metabolism or that of a co-administered drug are a major source of concern in drug discovery. Human upcyte® hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes and are an important model for studying drug-drug interactions (DDI).

CYP Inhibition

Cytochrome P450 (CYP) enzymes play a major role in the metabolism of the majority of xenobiotics. Cells that perform reliably in the inhibition assays should have reproducible Phase I and II enzyme activities at levels that allow for a good dynamic range for inhibition. upcyte® hepatocytes have donor-dependent basal enzyme activities and represent a reliable tool for xenobiotic inhibition studies.

CYP Induction

CYP Induction

upcyte® hepatocytes exhibit basal CYP1A2, CYP2B6, CYP2C9, and CYP3A4 which are all produced by prototypical inducers. As with primary human hepatocytes, the induction response is donor-dependent; therefore, we offer a panel of different donors with a range of induction responses.

Cytotoxicity

There is an increasing demand to develop more predictive models for liver toxicity due to the high attrition rate of drugs causing liver damage once they enter the market. upcyte hepatocytes v primary human hepatocytesImmortalized hepatic cell lines are often used for cytotoxicity screening since they grow continuously, are easily available, and can be standardized across laboratories. However, cell lines tend to have low or lacking Phase I and II activities and/or transporter functions and this can lead to false negative results when assessing drug toxicity.

Gene Expression

Knowing that your compounds regulate the activity of a specific nuclear receptor using INIDGO’s kits or services is a great first step in characterizing and prioritizing potential new drugs. However, this is just a beginning to fully understanding the biologic or toxicologic effects that may ensue. Coupling our expertise in gene expression and cell biology with the upcyte® platform, we can examine the effects of your compounds on a coordinated and functional response. Whether via examination of gene expression (qPCR, microarray, or Next Gen Sequencing) or addressing biochemical endpoints, we can help you evaluate whether your compounds have effects on hepatic steatosis, mitochondrial toxicity, or other forms of liver damage.

Background

Xenobiotic-induced liver injury is a major cause of human morbidity and mortality. A key reason for this problem is our inability to predict hepatotoxicity at the preclinical stage using currently available model systems. Such models include in vivo animal models and in vitro models based on human-derived liver cells or transformed cell systems. Species differences in xenobiotic disposition and mechanisms of cytotoxicity can make whole animal studies unreliable for extrapolation to human. In addition, whole animal models are costly and of low throughput. Therefore, it is essential to develop in vitro models that are more predictive of hepatotoxicity, particularly those that are based on human or “humanized” component cells.

There are two major limitations to the use of human liver cells or their derivatives. First, there are currently limited sources of fresh human hepatocytes worldwide and, when available, they often suffer from low viability and high batch-to-batch variability. Second, the freezing process used to preserve primary hepatocytes and the transformation process needed to make stable and proliferating cell lines results in changes in cell differentiation, proliferation, and metabolic processes.

INDIGO’s in vitro toxicology platform was developed to meet the increasing demand for more predictive models for liver toxicity due to the fact that hepatotoxicity remains a major reason for drug withdrawal from pharmaceutical development and clinical use. Consequently, the use of hepatocytes for the early identification of drug candidates that induce acute hepatotoxicity provides a powerful predictive tool that can inform drug development decisions.

Application of this platform is to screen chemicals for induction and activity of hepatic drug metabolizing enzymes (DME). which have been linked to Non-Alcoholic Fatty Liver Disease, Steatosis, Cholestasis and other conditions.

The platform now utilizes upcyte® human liver cells to assess chemical and drug-induced toxicity.

Product FamilyProduct NumberProduct DescriptionTechnical Manual
Gene ExpressionUGE1003-48Expression Profiling of Clinically Relevant CYPs,Technical Manual

Kit

Expression Profiling of Clinically Relevant CYPs Utilizing upcyte® Hepatocytes

The reagents and materials provided in the assay kit for the Expression Profiling of Clinically Relevant CYPs are formatted to allow for two alternative cell culture setups. In one scenario 48 culture wells may be set up at two different times. In the other assay scenario 96 culture wells may be set up at one time. The kit includes two aliquots of upcyte® hepatocytes, donor 10-03, isolated from an adult Caucasian female. These hepatocytes are cryopreserved using INDIGO’s proprietary CryoMite™ process, which yields high cell viability post-thaw, and provides the convenience of immediately dispensing cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. During an overnight culture period for cell recovery the hepatocytes will form a confluent monolayer that is ready to receive treatment media containing the user’s test compounds.

The kit provides two aliquots of upcyte® hepatocytes, two cell culture-ready assay plates, optimized Cell Culture Medium (CCM) for use in all steps of the assay procedure (cell thawing, seeding, and preparation of treatment media), and three reference compounds (rifampicin, β-naphthoflavone, and chenodeoxycholic acid (CDCA)) that activate one or more of the primary xenobiotic-sensing receptors: PXR, CAR, AhR, and FXR. Upon activation, these nuclear receptors modulate the expression of the clinically relevant CYP genes. Also included are seven sets of validated qPCR primers for quantifying drug-induced changes in the expression of CYP3A4, CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1, as well as primers for ACTB (β-actin; the internal control used to normalize all CYP gene expression data).

Please note: This kit does not include reagents or protocols for cell lysis, RNA isolation, cDNA preparation, or qPCR assays.

For more information, view the Technical Manual.

Services

Gene Expression Services

Knowing that your compounds regulate the activity of a specific nuclear receptor using INDIGO’s services are a great first step in characterizing and prioritizing potential new drugs. However, this is just a beginning to fully understanding the biologic (or toxicologic) effects that may ensue. Gene expression is a widely used approach for characterizing biological perturbations, defining the molecular mechanisms of diseases and making critical decisions about risks associated with compounds of interest.

qPCR

Quantitative real time PCR (qPCR) is the gold standard for gene expression analysis. Our team of experts in nuclear receptor biology and toxicology will provide the know-how in gene selection, primer design and mRNA accumulation analysis.

Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is possible. If a particular sequence (DNA or RNA) is abundant in the sample, amplification is observed in earlier cycles; if the sequence is scarce, amplification is observed in later cycles.

Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real-time PCR instruments that measure fluorescence while performing temperature changes needed for the PCR cycles. Standard curves were made using serial dilutions from pooled cDNA samples. Real Time PCR was performed using the SYBR Green PCR Mater Mix according to the manufacturer’s protocol and amplified on the ABI Prism 7300 Sequence Detection system.

QPCR Solutions:

  • INDIGO offers predesigned panels of optimized primers for nuclear receptor target genes including PXR, CAR, and PPARs.
  • Experience in gene selection for disease and pathway-specific studies including inflammation-, cancer-, diabetes-, and drug metabolism-related gene expression.
  • We will provide data on mRNA levels relative to a housekeeping gene, statistical analysis and interpretation in our study report.

 

Cell Culture

A variety of cell lines and primary cells are available for study. Cell growth, maintenance, and treatment conditions are determined upon consultation.

Microarray

INDIGO also provides comprehensive DNA microarray solutions, using commercial offerings from Affymetrix, for use in biomarker discovery, gene ontology analysis and predictive toxicology. All array services include quality control of array performance data and statistical, clustering and pathway analysis needs.

Microarray Solutions:

  • INDIGO offers the Affylmetrix microarray platform for comprehensive analysis of gene expression in human, rat and mouse tissues.
  • In addition to providing support for purchase of the appropriate array for your study, we will perform RNA extraction, cDNA labeling, RNA quality control, array hybridization and data collection.
  • Our suite of data analysis includes generating lists of genes significantly regulated under your treatment conditions as well as gene ontology and pathway analysis.

 

Reverse Cholesterol Transport

Atherosclerotic plaques contain macrophages that have ingested large amounts of cholesteryl ester, forming lipid droplets and gaining the appearance of a “foam cell.” During the process of reverse cholesterol transport (RCT), excess peripheral cholesterol is scavenged by tissue macrophages, which process cholesterol and transport it to the liver via HDL for excretion. In macrophage, control of the initial steps of RCT (cholesterol uptake and efflux) is manifested by a variety of NRs, in particular the PPARs and LXRs. As part of INDIGO’s RCT assay, THP-1 cells are converted to foam cells upon treatment with oxidized LDL and are loaded with a fluorescent cholesterol derivative. Subsequently, the effects of clients’ compounds of interest are examined for their ability to induce the transport of fluorescent cholesterol to HDL.

Clinically Relevant CYP Enzymes

Cytochrome p450 (CYP) enzymes are responsible for the Phase I metabolism of most drugs. And, it is noteworthy that the expression of cytochrome p450 (CYP) genes are predominantly regulated by ligand-activated receptors/transcription factors such as pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (CAR, NR1I3), aryl hydrocarbon receptor (AhR), farnesoid x receptor (FXR, NR1H4), glucocorticoid receptor (GR, NR3C1), and to lesser degrees liver X receptors (LXR, NR1C3), vitamin D receptor (VDR, NR1I1), and peroxisome proliferator-activated receptor alpha (PPARα, NR1C1). Consequently, drugs that activate any of these xenobiotic sensing receptors can dramatically change the endogenous levels of CYP expression in the liver, potentially impacting the rate of their own metabolism, as well as the metabolism of all other co-administered drugs. Of particular concern are metabolic outcomes that transform a drug to greater potency, or to an altered bioactivity.

Assessing drug-induced changes in the expression of CYP genes provides a reliable predictive indicator of altered (either heightened or inhibited) metabolic activities leading to drug-drug interactions in vivo. Cytochrome p450 enzymes with the greatest clinical relevance belong to CYP1, 2, and 3 families. It is estimated that they are involved in the metabolism of 70-80% of drugs currently on the market. For example, CYP3A4 is estimated to be involved in ~30% of all prescription drugs.

INDIGO’s assay kit for the Expression Profiling of Clinically Relevant CYPs contains optimized reagents for the culturing and treatment of upcyte® hepatocytes to assess drug-induced changes in seven clinically-relevant CYPs: CYP3A4. CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP 2E1.

Product FamilyProduct NumberProduct DescriptionTechnical Manual
Human P-Glycoprotein / MDR1HPGP-48Human P-Glycoprotein / MDR1 Drug Interaction Assay in 2x 48-well formatTechnical Manual

Kit

The materials provided in the assay kit for MDR1 / Human P-Glycoprotein Drug Interaction are formatted to allow for two independent 48-well assay setups. Each aliquot of HCT-Pgp Cells is provided as a single-use assay reagent and provides sufficient volume to dispense 48 assay wells of HCT-Pgp cells into one 96-well plate. If desired, the two aliquots of HCT-Pgp cells may be thawed, combined to generate a single cell suspension, and then dispensed into all wells of an assay plate.

The kit contains two aliquots of HCT-Pgp cells and two black, sterile, collagen-coated 96-well assay plates. In addition to HCT-Pgp cells and assay plates, this kit includes Cell Recovery Medium (CRM-p) used to perform a rapid thaw of the -80°C HCT-Pgp cells, Compound Screening Medium supplemented with daunorubicin (CSM+DR) used to prepare drug treatment media, Wash Buffer, two aliquots of Lysis Reagent, and the potent reference inhibitor Tariquidar.

HCT-Pgp cells are a proprietary cell line derived from human colorectal adenocarcinoma cells that have been extensively selected for high-level expression of native P-glycoprotein. The primary application of this MDR1 drug interaction assay kit is to rapidly assess drug candidates as either inhibitors, substrates, or non-substrates of P-glycoprotein. This is accomplished by co-treating the cells with varying concentrations of the test drug and a fixed concentration of daunorubicin, a well-characterized fluorescent substrate for P-glycoprotein mediated transport.

For more information, view the Technical Manual.

Background

What is MDR1?

Human P-glycoprotein (P-gp) is also known as MDR1 or ABCB1, the human multidrug resistance protein 1. It is a 170kDa transmembrane glycoprotein that functions as an ATP-dependent efflux transporter present in the human body’s epithelial tissues.

P-gp is comprised of twelve membrane-spanning domains and two cytosolic nucleotide-binding domains. The transmembrane domains comprise several substrate binding pockets capable of interacting with a broad range of both endogenous and foreign small molecule chemotypes. Xenobiotic substrates of P-gp range from pollutants, such as those encountered through unintended exposure to industrial and agricultural chemicals, to small molecule drugs that are intentionally administered for therapeutic benefit.

P-gp is highly expressed in gastrointestinal epithelium, liver, pancreatic and kidney cells, and capillary endothelial cells that establish the blood-brain barrier. Multidrug resistance research has found MDR1 expressed at high levels on the lines of transformed and tumor cells. In combination with the activities of Cytochrome P450 oxidases, the robust efflux activity of P-gp plays a critical role in limiting the absorption and systemic physiological distribution of xenobiotics and facilitating their ultimate elimination from the body. The MDR1 transporter role is important for drug-drug interaction (DDI) and drug safety assessments.

Drug Safety Assessment

Determining if a drug candidate has incidental activity as either a substrate or an inhibitor of the P-gp transporter is a vital component of the drug safety assessment process. A drug that is a P-gp substrate or an inhibitor can profoundly alter the rate of absorption, distribution, metabolic conversion, and eventual excretion (ADME) of co-administered drugs, thereby significantly shifting their respective therapeutic efficacies and toxicologic profiles. Assessing a drug’s potency as an interactor with P-gp, and thus its potential liability for inducing downstream drug-drug interactions, is mandated by the FDA.

The MDR1 multidrug resistance transporter impacts public health and is associated with nosocomial infections and developments with antibiotics and mortality. MDR1 is also often cited as a transporter for drug-drug interaction in product labels and particularly in the use of digoxin. The EMA and FDA recommend at minimum in vitro MDR1 tests for interaction.

Toxicity continues to be a primary cause for compound attrition and long development timelines, causing companies to increasingly integrate safety assessment principles into earlier phases of the drug discovery process. A new discipline has emerged called Discovery Toxicology, which utilizes many in vitro tools to prioritize compounds at the earliest phases of drug discovery.

Background

A typical testing scheme for a small molecule drug begins with large numbers of compounds and high-throughput assays. As the number of viable leads is reduced, incrementally more predictive but lower throughput assays identify those leads with the most drug-like properties and optimal in vitro and in vivo efficacy. Compounds that successfully meet preclinical efficacy, ADME, pharmacokinetics, and safety criteria are nominated as candidates for formal development.

Historically, within pharmaceutical companies the functions of discovery and development, including preclinical safety assessment, were performed by discreet organizations within the company. As toxicity continues to be a primary cause for compound attrition and long development cycle times, companies have increasingly integrated safety assessment principles into earlier phases of the drug discovery process, and a new discipline has emerged called Discovery Toxicology

INDIGO’s approach to Discovery Toxicology is to provide research services to assist in prioritizing drug leads, found either through our Drug Discovery services or products, or through your own effort. We provide a series of in vitro assays in the broad categories of Prospective Screens, Safety Pharmacology, and Retrospective Screens.

Drug Discovery

Modern drug discovery involves the identification of screening hits, medicinal chemistry, and optimization of those hits to increase the affinity, selectivity, efficacy/potency, metabolic stability, and bioavailability. INDIGO’s kits and services are ideal for drug discovery because they provide a convenient way to examine a compound for its ability to regulate nuclear receptor activity in agonist, inverse-agonist, or antagonist mode.

Using our robust cell-based reporter assays and nuclear receptor expertise allows you to better identify and prioritize drug leads. INDIGO’s research lab can aid in the identification of targets of interest and help your research with complimentary study consults and design. The INDIGO team can also aid in reducing the number of viable leads with high-throughput screening of compounds for efficacy and potency. Our scientists aim to get you to the next phase of discovery faster and prevent unnecessary steps backward by providing the most comprehensive data for your investment with fast turnaround times.

Whether you are just beginning the drug discovery process, or have narrowed down viable leads using INDIGO’s services or through your own effort, INDIGO’s cell-based assays allow you to examine a compounds efficacy and potency in cells, in your own lab, with assay data ready in just 24 hours. Our cell-based reporter assays are designed to quantify nuclear receptor activity with clear, reproducible data lot-to-lot. This allows for the prioritization of leads by learning the efficacy of a compound in vitro, as well as to establish the EC50/IC50 values and potential recommended dosing levels. This can be done with human receptors as well as those of orthologs to decide which animal model would be best for in vivo animal trials.

More information on INDIGO’s Assay Kit Platform & Formats

INDIGO’s research services and assay kits provide a convenient way to examine your compound’s ability to regulate nuclear receptor activity in robust, cell-based reporter assays.

View the full portfolio of INDIGO’s cell-based assays

Prospective Screens

At INDIGO, we specialize in mechanistic and molecular toxicology, in particular those events related to Nuclear Receptors or other transcription factors, and offer prospective screens that predict potential adverse effects of your compounds of interest.

Drug-Drug Interactions NRs represent one of the most important families of drug targets, resulting from their roles as transcription factors and therefore regulators of gene expression and physiological functions. Since drug metabolism and transport proteins have several substrates, increases in the expression of these biotransformation systems may affect the activity of many drugs and nutrients, hence, drug-drug and drug-nutrient interactions. In particular, PXR and CAR have been implicated in affecting the expression of cytochrome P450s and other drug metabolism enzymes. INDIGO provides several NR assays that are predictive of potential liabilities due to drug-drug interactions.

Phenotypic Screens The INDIGO laboratory has extensive experience examining events that are downstream of nuclear receptor activation. Examples include quantification of gene expression by real-time PCR, adipogenesis assays, and gene expression microarrays. In addition, we can develop or utilize cell culture systems to examine the biological response of your compounds. Whether you start with your own RNA samples, tissues, or cells or utilize our cell culture service, INDIGO can design primers and quantify mRNA expression. More information on gene expression solutions from INDIGO.

Safety Pharmacology

Reporter Assay Panels Defining the system each receptor participates in can be approached in several ways such as sequence similarity, potential disease implication, or transcriptional networks. The latter is particularly helpful since it encompasses receptors to which there are no known endogenous ligands (orphan receptors) or have few selective pharmacologic agents to evaluate biological consequences. By choosing a select group of receptors to study, it is possible to better understand the biological and toxicological effects of your compounds. INDIGO has pre-designed several panels for you to choose, or you can talk to our experts to design your own panel of receptors.

Nuclear Receptor Profiling A major focus in the current discovery of drugs targeting nuclear receptors is identifying those with the highest selectivity and therefore lowest potential for off-target and unwanted effects. Due to regulation of metabolic enzymes by several nuclear receptors, there is increasing interest in understanding the potential of drug-drug and drug-nutrient interactions of potential drug leads. INDIGO provides a comprehensive list of optimized, robust, and selective whole-cell receptor assays, ideally suited for examining selectivity as well as potential interactions.

More on Nuclear Receptor Profiling and Panels from INDIGO

Retrospective Screens

Retrospective screens are often done in response to a troubling finding in clinical studies such as hepatotoxicity in laboratory animals or unexpected failures in human trials. INDIGO’s large portfolio of ortholog model assays can help determine if the enzyme induction response observed in laboratory animals is concordant with humans. Mouse, rat, dog, monkey, and/or zebrafish models are currently available for many receptors, with more available for custom development.

Learn more about INDIGO’s Ortholog Assays

Environmental Testing Solutions from INDIGO

INDIGO’s effects-based bioassay solutions compliment traditional analytical chemistry approaches for the examination of water quality and can help protect human and environmental health.

Contaminant Detection

Are Traditional Water Quality Methods Enough?

Traditional techniques rely on analyses and data for individual contaminants. Real world exposure generally occurs as mixtures of different chemical compounds. According to the EPA this presents risks posed to humans and the environment from:

  • chemicals without toxicity information
  • the presence and concentrations of other compounds
  • chemicals that work together to increase the toxic potential

To understand the risk posed by complex samples cell-based assay are needed to characterize cumulative effects on humans and other organisms.

Cell-Based Assays: The Next Wave in Evaluating Water Quality

Cell-based reporter assays such as those provided by INDIGO Biosciences screen for total bioactivity for a specific pathway of importance. They can detect the toxicity of unknown chemicals and can account for the cumulative effects simplifying the issues posed by complex mixtures in analytical methods. INDIGO has an extensive list of cellular bioassays that are predictive of cellular toxicity pathways including endocrine disruption, altered xenobiotic metabolism and adaptive stress responses.

INDIGO’s assays can detect the cumulative effect of bioactive compounds in extracted environmental matrices and provide important biological context. They are:

  • All-Inclusive
  • Sensitive
  • Cost Effective
  • Reproducible
  • and Easy to Perform

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Kits

INDIGO has an extensive list of bioassay kits for receptors important in Environmental Monitoring for contaminant detection and quantification in samples which include:

 

Lipid and Energy Metabolism

  • LXRβ (NR1H2)
  • PPARγ (NR1C3)
  • RXRα (NR2B1)
  • TRβ (NR1A2)

Basal metabolism

  • LXRα (NR1H3)
  • MR (NR3C2)
  • RARγ (NR1B3)
  • RXRβ (NR2B2)
  • RXRγ (NR2B3)

Our receptor specific assays are cell-based reporter assay systems. They feature engineered receptor-specific reporter cells prepared using our unique CryoMite™ process. Once thawed, reporter cells are ready for immediate use. Test compounds can be screened for agonist or antagonist activities against receptors.

INDIGO Biosciences works closely with clients to provide the appropriate reporter specific assays for their water testing needs. Our technology generates clear single receptor or full-panel screening results. Employing a luminescence-based method and our proprietary CryoMite™ preservation process, INDIGO kits provide reproducible results lot-to-lot.

Learn more about INDIGO Biosciences’ Assay Kit Platforms & Formats

Services

 

INDIGO is a leading provider of cell-based reporter assay solutions, recognized for our focus on providing a superior combination of expertise, service, and support. Organizations across the globe leverage our industry-leading platforms, knowledgeable people, and proprietary science for single receptor or full-panel screenings. Committed to ensuring the results we provide inform assured next steps, our team goes the extra mile by offering complimentary study consultation and design. Service assays include a positive control reference compound and ‘vehicle’ control for every experiment. At the completion of the study a formal study report including helpful graphics, summaries, and insights, as well as all data files are provided to the client. Our scientists are also available to provide a comprehensive review of the results with you once your service study is complete. To receive a quote for your proposed study, contact us to discuss your desired study parameters.

 

INDIGlo Luciferase Detection Reagent Kit

Product Family Product Number Product Description
LDR LDR-10 10 mL each of 2x-concentrated INDIGlo LDR and Dilution Buffer
LDR-25 25 mL each of 2x-concentrated INDIGlo LDR and Dilution Buffer
LDR-50 50 mL each of 2x-concentrated INDIGlo LDR and Dilution Buffer

INDIGlo Luciferase Detection Reagent

INDIGlo Luciferase Detection Reagent (LDR) is formulated to deliver steady, long-lived luminescence signal from mammalian reporter cells expressing firefly luciferase. For convenience, the reagent is provided ‘ready to use’, without the requirement to first re-constitute powder-form components.

INDIGlo LDR works equally well in quantifying expressed luciferase activity from 96- or 384-well format reporter assays. The resulting light emission is stable over a 2 hr period (Figure 1), thereby making INDIGlo LDR an ideal, economical detection reagent for both low- and high-throughput batch processing of assay plates.

INDIGlo LDR is formulated as a 2x-concentrated reagent, thereby allowing greater versatility in its use. The 2x-concentrated reagent will be used when using 384-well or 1536-well assay plates that are processed using a homogenous assay strategy.

Alternatively, when processing 96-well assay plates either a homogenous or non-homogenous assay strategy may be used. For convenience, this kit provides an optimized Dilution Buffer for use in diluting INDIGlo LDR when performing non-homogenous assays.

Regardless of one’s assay setup preference INDIGlo LDR delivers rapid lysis of the reporter cells, robust luminescence signal, and sensitive quantification of expressed luciferase activities.

INDIGlo LDR potency is stable following repeated freeze and thaw cycles. INDIGlo LDR was frozen at -20°C and thawed either 1, 2 or 3 times, then used to quantify luciferase activity from HEK-293:luc cells cultured in a white 96-well assay plate. No loss of reagent potency is observed between these conditions.

INDIGlo LDR performance from both Homogenous and Non-Homogenous assays. Dose-response analyses of Human AhR (INDIGO #IB06001, 96-well assay) was performed using the reference agonist MeBIO. AhR reporter cells were plated across two white assay plates, MeBIO treatment media were added, and the assay plates incubated for 24 hr. Plates were then processed with luciferase detection reagents in either a.) a homogenous or b.) a non-homogenous assay format. Resulting assay metrics are also shown from using another extended-glo detection reagent (Vendor X).

INDIGlo Luciferase Detection Reagent Components and Storage Conditions

INDIGlo Luciferase Detection Reagent kits provide two reagents in equal volumes:

  • INDIGlo LDR, formulated as a 2x-concentrated reagent
  • Dilution Buffer, for optional use if performing non-homogenous assays

INDIGlo LDR is packaged in amber tubes, sealed under argon, then frozen. Upon receipt, both reagents should be further stored at -20°C. INDIGlo LDR is stable for at least 6 months from the date of its manufacture; the expiration date is printed on the Product Qualification Insert that is enclosed in each kit.

After INDIGlo LDR has been thawed for the first time, it may be subjected to an additional three freeze / thaw cycles without loss of potency. If more than three thaw-cycles are anticipated, it is advised to dispense the first-time thawed reagent into smaller single-use aliquots, which are then stored frozen.

Live Cell Multiplex Assay Kits & Services

Product FamilyProduct NumberProduct DescriptionTechnical Manual
LCMALCM01LCMA 1×96-wells Reagent PackTechnical Manual
LCMALCM05LCMA 5×96-wells Reagent PackTechnical Manual
LCMALCM10LCMA 10×96-wells Reagent PackTechnical Manual

The Live Cell Multiplex (LCM) Assay provides an efficient fluorescence-based method of quantifying the relative number of live cells resident in treated wells of an assay plate. While the LCM Assay may be performed as a stand-alone assay, it has been specifically optimized to be run in multiplex with any of INDIGO’s 96-well, 2×48-well, or 3×32-well Nuclear Receptor Reporter Assay System products.

The LCM assay allows users to validate their primary Nuclear Receptor Assay data by determining if their test compound treatments exert mitogenic, cytostatic or cytotoxic activities on the reporter cells. The occurrence of such adverse non-specific effects will always undermine the accurate assessment of a test compound’s potency and/or efficacy as a modulator of nuclear receptor function.

When screening test compounds for antagonist activities it is particularly important to quantify changes in the relative number of live reporter cells at the assay endpoint. Test compounds that exert cytostatic, cytotoxic, or cytolytic activities invariably generate “false-positive” results in an antagonist screen. In such cases, the observed drop in luciferase activity will be incorrectly attributed to inhibition of the nuclear receptor by the test compound. In reality, however, the treatment compound has pushed the reporter cells into division arrest, apoptosis, necrosis, or lysis.

The LCM assay allows user’s to validate their primary Nuclear Receptor Assay data by determining if their test compound treatments exert mitogenic, cytostatic or cytotoxic activities on the reporter cells. The occurrence of such adverse non-specific effects will always undermine the accurate assessment of a test compound’s potency and/or efficacy as a modulator of nuclear receptor function.

When screening test compounds for antagonist activities it is particularly important to quantify changes in the relative number of live reporter cells at the assay endpoint. Test compounds that exert cytostatic, cytotoxic, or cytolytic activities invariably generate “false-positive” results in an antagonist screen. In such cases, the observed drop in luciferase activity will be incorrectly attributed to inhibition of the nuclear receptor by the test compound. In reality, however, the treatment compound has pushed the reporter cells into division arrest, apoptosis, necrosis, or lysis.

Live Cell Multiplex Format
The fluorescence-based LCM Assay and the luminescence-based INDIGO NR Assay are performed sequentially using the same assay wells. The text in blue corresponds to the LCM Assay portion of the multiplex protocol. Text in black corresponds to the standard protocol used for each of INDIGO’s 96-well format Nuclear Receptor Reporter Assays.

Performance

Live Cell Multiplex performance
RFU = % Live Cells. The LCM Assay provides a direct correlation between % RFU and % Live Cells in an assay well.

Ortholog Assays

Ortholog Kits and Services

Many labs are interested in cross-species comparisons, especially contrasting human nuclear receptor activity to common laboratory animals. Determining a drug candidate’s cross-activity with human xenobiotic-sensing receptors provides important early indications of that drug’s potential for downstream drug-drug interactions. With more than 30 ortholog kits and services available, INDIGO’s assay solutions provide the critical data you need, while lowering the time, cost, and risk associated with drug discovery.

Rat Androgen Receptor (rAR; nr3c4) Zebrafish Androgen Receptor (zAR; nr3c4)
Rat Aryl Hydrocarbon Receptor (rAhR) Zebrafish Aryl Hydrocarbon Receptor (zAhR)
Mouse Constitutive Androstane Receptor (mCAR; nr1i1) Rat Constitutive Androstane Receptor (rCAR; nr1i1)
Zebrafish Estrogen Receptor Alpha (zERα; nr3a1)
Mouse Farnesoid X Receptor (mFXR; nr1h4) Rat Farnesoid X Receptor (rFXR; nr1h4)
Cyn Monkey Farnesoid X Receptor (cFXR; nr1h4) Dog Farnesoid X Receptor (dFXR; nr1h4)
Mouse Glucocortioid Receptor (mGR; nr3c1) Rat Glucocorticoid Receptor (rGR; nr3c1)
Zebrafish Glucocortioid Receptor (zGR; nr3c1)
Mouse Liver X Receptor Alpha (mLXRα; nr1h3)
Mouse Liver X Receptor Beta (mLXRβ; nr1h2) Rat Liver X Receptor Beta (rLXRβ; nr1h2)
Mouse Peroxisome Proliferator-Activated Receptor Alpha (mPPARα; nr1c1) Rat Peroxisome Proliferator-Activated Receptor Alpha (rPPARα; nr1c1)
Cyn Monkey Peroxisome Proliferator-Activated Receptor Alpha (cPPARα; nr1c1) Dog Peroxisome Proliferator-Activated Receptor Alpha (dPPARα; nr1c1)
Mouse Peroxisome Proliferator-Activated Receptor Beta/Delta (mPPARβ/δ; nr1c2) Rat Peroxisome Proliferator-Activated Receptor Beta/Delta (rPPARβ/δ; nr1c2)
Cyn Monkey Peroxisome Proliferator-Activated Receptor Beta/Delta (cPPARβ/δ; nr1c2) Dog Peroxisome Proliferator-Activated Receptor Beta/Delta (dPPARβ/δ; nr1c2)
Rodent (Mouse/Rat) Peroxisome Proliferator-Activated Receptor Gamma (mrPPARγ; nr1c3) Cyn Monkey Peroxisome Proliferator-Activated Receptor Gamma (cPPARγ; nr1c3)
Zebrafish Peroxisome Proliferator-Activated Receptor Gamma (zPPARγ; nr1c3)
Mouse Pregnane X Receptor (mPXR; nr1i2) Rat Pregnane X Receptor (rPXR; nr1i2)
Cyn Monkey Pregnane X Receptor (cPXR; nr1i2) Dog Pregnane X Receptor (dPXR; nr1i2)
Rat Progesterone Receptor (rPGR; nr3c3)
Zebrafish Retinoic Acid Receptor Alpha, isoform A (zRARαa; nr1b1)
Mouse RAR-related Orphan Receptor Gamma (mRORγ; nr1f3)
Zebrafish Thyroid Hormone Receptor Beta (zTRβ; nr1a2)

Pre-clinical studies utilize animals as human surrogates to assess a drug’s pharmacokinetic and toxicologic profiles. These studies are often confounded by the fact that the ligand preferences and response dynamics can vary dramatically between the human receptor and the corresponding ortholog receptors, with even the receptors of closely related species, such as mouse and rat, sometimes exhibiting dramatically different ligand preferences and responses.[1] This creates a challenge when selecting the most human-relevant animal model for pre-clinical studies.

With animal studies required by the FDA, selecting the animal model that provides the most representative human-surrogate is critical to assessing a potential drug’s likelihood of unwanted effects. This is why cell-based assay models are crucial to help make this determination prior to entering ADMET studies.

With more than 30 ortholog assays – including rat, mouse, dog, monkey, and zebrafish – available as kits and/or services, and others available for custom development, INDIGO helps researchers screen the right animal, before trial.

What to know more about choosing the right ortholog model? Find more information on profiling drug activity of human and ortholog xenobiotic-sensing receptors in this scientific poster. or view related articles in the Related Articles tab.

Discovery and development research depends on reliable, high-performing assays designed to meet your needs. INDIGO currently offers numerous ortholog nuclear receptor assays in rat, mouse, dog, non-human primate, and zebrafish, though if you need an assay for a species or nuclear receptor not currently available, our services lab can clone and optimize an assay for your research.

We supplement the world’s largest portfolio of nuclear receptor assay kits and services and in vitro toxicology solutions with greater results readability, reproducibility, and faster turnaround times. Our custom assay kit solutions, plus supportive team and reliable science and platforms aim to reduce time, cost, and risk associated with the discovery process. Contact us today and Request a Quote to discuss your research needs.

Species Differences in Pregnane X Receptor Activation: Examination of common laboratory animal species

INTRODUCTION Nuclear receptors (NRs) are ligand-dependent transcription factors found in many species that regulate the expression of important target genes involved in a spectrum of developmental and physiological processes. In addition to ligand binding, the transcriptional activities of NRs are also modulated through a range of protein-protein interactions with coregulatory proteins, either with coactivator orREAD MORE

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INDIGO Expands Discovery Platform to Include Zebrafish Assays

Expansion Meets Industry Demand for Pre-Clinical Models for Cancer and Environmental Research State College, PA (23 January 2020) – INDIGO Biosciences, the recognized industry leader in nuclear receptor research, has announced the addition of four zebrafish assays to its portfolio. These additions both expand INDIGO’s robust portfolio of in vitro animal model assay systems andREAD MORE

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Identifying qualitative differences in PPARα signaling networks in human and rat hepatocytes and their significance for next generation chemical risk assessment methods

ABSTRACT In this paper, we evaluate the PPARα signaling network in rats, examining transcriptional responses in primary hepatocytes exposed to a PPARα specific ligand, GW7647. These transcriptomic studies were complemented with ChIP-seq studies of PPARα binding and transcription binding motif identification for PPARα responsive genes. We also conducted a limited study of GW7647 dosing the inREAD MORE

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Quinazolinone derivative BNUA‐3 ameliorated [NDEA+2‐AAF]‐ induced liver carcinogenesis in SD rats by modulating AhR‐CYP1B1‐Nrf2‐Keap1 pathway

ABSTRACT Cytochrome P450 1B1, considered as one of the novel chemotherapeutic targets involved in cancer prevention and therapy is also associated with the conversion of procarcinogens into their active metabolites. The aryl hydrocarbon receptor (AhR) is responsible for mediating different biological responses to a wide variety of environmental pollutants and also causes transcriptional activation ofREAD MORE

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Intestinal TGR5 agonism improves hepatic steatosis and insulin sensitivity in Western diet-fed mice

ABSTRACT Takeda G protein-coupled receptor 5 (TGR5) agonists induce systemic release of glucagon-like peptides (GLPs) from intestinal L cells, a potentially therapeutic action against metabolic diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and Type 2 diabetes. Historically, TGR5 agonist use has been hindered by side effects, including inhibition of gallbladder emptying.READ MORE

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Involvement of estrogen receptor α in pro-pruritic and pro-inflammatory responses in a mouse model of allergic dermatitis

ABSTRACT It has been reported that endogenous or exogenous estrogens can affect the immune system, resulting in immune disorders; however, their direct involvement in such conditions remains to be demonstrated. The purpose of this study was to investigate whether estrogen receptors (ER) are directly implicated in pro-pruritic and pro-inflammatory reactions in cutaneous allergy. Initially, enhancement of the pro-inflammatoryREAD MORE

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Bitter melon seed oil increases mitochondrial content in gastrocnemius muscle and improves running endurance in sedentary C57BL/6 J mice

ABSTRACT The α-eleostearic acid (α-ESA) in bitter melon seed oil (BMSO) is efficiently converted by the body into rumenic acid. The objective of this study was to investigate effects of BMSO on skeletal muscle fiber-type switch and endurance capacity in mice, with or without exercise training. In a 3 x 2 factorial design, C57BL/6 JREAD MORE

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Disruption of Nuclear Receptor Signaling Alters Triphenyl Phosphate-Induced Cardiotoxicity in Zebrafish Embryos

ABSTRACT Triphenyl phosphate (TPHP) is an unsubstituted aryl phosphate ester used as a flame retardant and plasticizer within the United States. Using zebrafish as a model, the objectives of this study were to rely on (1) mRNA-sequencing to uncover pathways disrupted following embryonic TPHP exposure and (2) high-content screening to identify nuclear receptor ligands thatREAD MORE

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Sedaxane—Use of Nuclear Receptor Transactivation Assays, Toxicogenomics, and Toxicokinetics as Part of a Mode of Action Framework for Rodent Liver Tumors

ABSTRACT Experimental data demonstrate a mode of action (MOA) for liver tumors in male rats and mice treated with sedaxane that starts with activation of CAR, followed by altered expression of CAR-responsive genes, increased cell proliferation, and eventually clonal expansion of preneoplastic cells, leading to the development of altered foci and tumors. This MOA isREAD MORE

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Perfluorooctane Sulfonate-Induced Hepatic Steatosis in Male Sprague Dawley Rats is not Attenuated by Dietary Choline Supplementation

ABSTRACT Perfluorooctane sulfonate (PFOS) is an environmentally persistent chemical. Dietary 100 ppm PFOS fed to male mice and rats for four weeks caused hepatic steatosis through an unknown mechanism. Choline deficient diets can cause hepatic steatosis. A hepatic choline:PFOS ion complex was hypothesized to cause this effect in mice. This study tested whether dietary choline supplementationREAD MORE

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Discovery and development research depends on reliable, high-performing assays designed to meet your needs. If your R&D requires assays beyond INDIGO’s existing offerings, our experienced team will assist in your research on nuclear receptors, cell-based reporter assays, gene expression, or toxicology screening. We can help create high-throughput NR reporter gene assays, develop ortholog assays, or a variety of other services. We supplement the world’s largest portfolio of nuclear receptor assay kits and services and in vitro toxicology solutions with greater results readability, reproducibility, and faster turnaround times. Our custom assay kit solutions, plus supportive team and reliable science and platforms aim to reduce time, cost, and risk associated with the discovery process. Contact us today and Request a Quote to discuss your research needs.

Examples of Custom Assay Development Services Available

Custom Nuclear Receptor Assay: Interested in a human nuclear receptor that is not in our current portfolio? We can clone and optimize an assay for services use in our laboratories or as assay kits to be utilized in yours.

Ortholog Nuclear Receptor Assay: Many labs are interested in cross-species comparisons, especially contrasting human nuclear receptor activity to common laboratory animals such as rat, mouse, dog, and non-human primate. While INDIGO currently offers numerous NR assays in human, rat, and mouse, along with several in dog and non-human primate, though if you need an assay for a species not currently available, our services lab can clone and optimize an assay for your research.

Selective Receptor Modulation (SRM) Assays: The concept that ligands for members of the NR superfamily should be classified as either “agonists” or as “antagonists” is often an oversimplification. For example, tamoxifen acts in vivo as an estrogen receptor (ER) antagonist in the breast but as an ER agonist in bone and uterus. The reason for these opposite actions is provided by numerous NR-associated proteins such as corepressors / coactivators that interact with a particular ligand receptor complex. Thus a new concept of selective receptor modulators (SRMs) has emerged to account for the different contextual actions of NR ligands, depending on the cellular milieu in which this chemical acts. INDIGO has at its disposal a variety of coactivator and corepressor constructs, as well as LXXLL containing peptides for examination of protein-protein interaction in the mammalian to-hybrid assay.

Nuclear Receptor Assay in a Specific Cell Line: INDIGO’s team has optimized its reporter assays in particular cell line, which vary depending on the receptor. If this cell line is not where you need the information, let INDIGO’s staff know and we can develop and optimize the appropriate conditions.

Nuclear Receptor Assay with a Specific Reporter: Similar to using a specific cell line, if our optimized reporter gene is not what you need, INDIGO will incorporate a different reporter gene in the customized assay.

Please note: Custom assays are developed under a non-exclusive arrangement. Although the constructs developed for the client may be sent for their own use, INDIGO retains the right to add the newly developed assay as part of our services and/or kit portfolio.


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