Retinoic acid receptor-related orphan receptor γt (RORγt) [1]
MRL-871 targets the retinoic acid receptor-related orphan receptor γt (RORγt), a nuclear receptor that plays a critical role in the differentiation of Th17 cells and the production of the pro-inflammatory cytokine IL-17. MRL-871 is an allosteric inverse agonist that binds to a previously unidentified allosteric pocket in the RORγt LBD. By binding to this allosteric site, MRL-871 induces a conformation that reduces the transcriptional activity of RORγt, leading to decreased IL-17a mRNA production. The compound also interacts with PPARgamma through a partial agonism binding mode. RORγt is a key target for autoimmune and inflammatory diseases.

MRL-871 (3) functions as an inverse agonist of RORγt, stabilizing helix 12 in a conformation unsuitable for coactivator binding. [1]
In a TR-FRET coactivator recruitment assay, MRL-871 inhibited the interaction between the RORγt ligand binding domain (LBD) and the SRC-1 coactivator peptide with an IC50 of 7.8 ± 0.5 nM. [1]
In a competitive TR-FRET coactivator recruitment assay performed with fixed concentrations of the orthosteric agonist cholesterol (0, 0.25, and 1.0 μM), the IC50 of MRL-871 decreased as cholesterol concentration increased, demonstrating cooperative binding behavior characteristic of an allosteric modulator. IC50 values were 12.7 ± 0.6 nM (0 μM cholesterol), 9.4 ± 0.3 nM (0.25 μM cholesterol), and 7.8 ± 0.2 nM (1.0 μM cholesterol). [1]
In an HTRF ligand binding assay using an AlexaFluor647-labeled MRL-871 derivative as an allosteric probe, MRL-871 displaced the probe with an IC50 of 17.3 ± 1.4 nM. [1]
In a TR-FRET coactivator recruitment assay with PPARγ, MRL-871 showed an IC50 of 7.2 ± 0.8 μM, indicating some cross-reactivity but with 923-fold selectivity for RORγt over PPARγ. [1]
In a cellular assay measuring IL-17a mRNA expression in EL4 cells (murine lymphoblast cells), treatment with 10 μM MRL-871 for 24 hours significantly reduced IL-17a mRNA expression by 48-fold compared to the DMSO control. [1]
The ADME profile of MRL-871 showed: chemical stability of 81% remaining after 24 hours in aqueous buffer at pH 7.4; solubility of 39 μM; PAMPA flux of 23.7%; metabolic stability in human liver microsomes (Phase I) with an intrinsic clearance (CLint) of <0.1 μL/min/mg; Phase II microsomal stability with 100% remaining; plasma stability of 47.1% remaining after 1 hour; and plasma protein binding of 99.9% bound. [1]

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In EL4 cells, MRL-871 (Compound 3; 10 μM, 24 hours) dramatically decreased IL-17a mRNA expression by a factor of 48 [1].

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In vitro, MRL-871 potently inhibits RORγt activity in cell-based assays. It effectively reduces IL-17a mRNA production in EL4 cells. The compound's activity is typically assessed by measuring RORγt transcriptional activity using luciferase reporter assays or by measuring IL-17a mRNA levels by qRT-PCR. MRL-871 shows potent inverse agonist activity with an IC₅₀ of 12.7 nM. It has a distinct isoxazole chemotype. The compound's allosteric binding mode provides a unique mechanism of action compared to orthosteric RORγt modulators.

In vivo, MRL-871 has been studied for its potential in autoimmune and inflammatory diseases. As a RORγt inverse agonist, it is expected to reduce Th17 cell differentiation and IL-17 production in vivo. The compound's allosteric binding mode may provide improved selectivity and reduced off-target effects. Specific in vivo efficacy data are limited in the published literature. MRL-871 is a research tool for studying RORγt biology and the role of Th17 cells in autoimmune diseases.

The TR-FRET coactivator recruitment assay was used to evaluate RORγt inverse agonist activity. The assay utilized 100 nM of an N-terminal biotinylated SRC-1 box2 peptide and 20 nM of His6-tagged RORγt ligand binding domain (LBD) in a buffer containing 10 mM HEPES, 150 mM NaCl, 5 mM DTT, 0.1% BSA (w/v), and 0.1 mM CHAPS, pH 7.5. A terbium-labeled anti-His antibody and D2-labeled streptavidin were employed. Compounds (dissolved in DMSO) were titrated in a 2-fold dilution series in 384-well plates with a final volume of 10 μL and a final DMSO concentration of 2% v/v. The plate was incubated at room temperature for 30 minutes, centrifuged, and read with excitation at 340 nm and emission at 665 nm and 620 nm. [1]
A competitive TR-FRET coactivator recruitment assay was performed analogously, with the addition of fixed concentrations of cholesterol (0 μM, 0.25 μM, 1.0 μM) to the assay buffer. [1]
An HTRF ligand binding assay was conducted using 100 nM of an AlexaFluor647-labeled MRL-871 derivative (44) as an allosteric probe and 20 nM of His6-tagged RORγt LBD in the same buffer. A terbium-labeled anti-His antibody was used. The assay was carried out in 384-well plates with a final volume of 10 μL. [1]
For PPARγ cross-reactivity, a TR-FRET coactivator recruitment assay was performed using 100 nM of His6-tagged PPARγ LBD in the presence of 1 μM rosiglitazone to initially activate the receptor. [1]

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In vitro receptor binding assays for MRL-871 typically involve measuring the binding affinity to RORγt and the inhibition of RORγt transcriptional activity. A typical protocol: RORγt protein is incubated with varying concentrations of MRL-871 (0.01 nM to 10 μM) in binding buffer. Binding affinity is measured by surface plasmon resonance (SPR) or by fluorescence polarization using a fluorescent RORγt ligand. For functional assays, HEK293 cells are transfected with RORγt and a luciferase reporter gene under the control of a RORγt-responsive element. Cells are treated with MRL-871, and luciferase activity is measured. IC₅₀ values are calculated from inhibition curves.

EL4 cells were grown in DMEM with 10% FBS. Twenty-four hours after seeding onto 12-well plates, cells were incubated with 10 μM test compound (from a 10 mM DMSO stock) or DMSO for 24 hours and then activated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (1 μg/mL) for 5 hours. Cells were collected, and RNA was isolated using an RNeasy kit. RNA was reverse transcribed using a cDNA synthesis kit. Quantitative RT-PCR was performed to analyze mRNA levels of mouse IL-17a and the reference gene Gapdh using SYBR green technology. The level of IL-17a mRNA expression was normalized to Gapdh expression, and relative gene expression was calculated using the 2−ΔΔCT method with the DMSO control as calibrator. [1]

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In vitro cell-based assays for MRL-871 are performed using EL4 cells or Th17-polarized primary T cells. A typical protocol: EL4 cells are cultured and treated with MRL-871 at concentrations ranging from 0.01 nM to 10 μM for 4-24 hours. IL-17a mRNA levels are measured by qRT-PCR. For Th17 cell assays, naive CD4+ T cells are polarized to Th17 cells in the presence of MRL-871, and IL-17 production is measured by ELISA or flow cytometry. Cell viability is assessed using MTT assays to ensure that observed effects are not due to cytotoxicity. Each condition is tested in triplicate.

In vivo animal studies for MRL-871 are conducted in mouse models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) or psoriasis. A typical protocol: mice are immunized with myelin oligodendrocyte glycoprotein (MOG) to induce EAE. MRL-871 is administered via oral gavage or intraperitoneal injection at doses of 1-30 mg/kg, daily, starting at disease onset. Clinical scores are assessed daily. At study termination, spinal cords and lymph nodes are harvested for histopathological examination and cytokine analysis. Efficacy is assessed by comparing clinical scores and inflammatory markers between treatment and vehicle control groups.

Chemical stability was determined by incubating test compounds at a final concentration of 2 μM in aqueous buffer at pH 7.4 for 1, 7, and 24 hours. The percentage of remaining compound was calculated by LC-MS-based measurement. For MRL-871, 81% remained after 24 hours. [1]
Aqueous kinetic solubility was determined by spectrophotometric measurement of a 500 μM compound solution in aqueous buffer pH 7.4 compared to a solution in acetonitrile after 90 minutes of vigorous shaking at room temperature. The solubility of MRL-871 was 39 μM. [1]
Permeability through artificial membranes (PAMPA) was performed at an initial compound concentration of 500 μM in the donor compartment. After a 20-hour incubation, absorption in the receiver wells was measured by spectrophotometry, and permeation was calculated by normalizing the compound flux across a blank filter. The PAMPA flux for MRL-871 was 23.7%. [1]
Metabolic stability under oxidative conditions (Phase I) was measured in human liver microsomes. Compound depletion at a concentration of 3 μM was monitored by LC-MS over time up to 50 minutes at 37°C. Intrinsic clearance (CLint) was calculated based on the compound half-life. The CLint for MRL-871 was <0.1 μL/min/mg. [1]
Metabolic stability under conjugative conditions (Phase II) was measured in a glucuronidation assay by LC-MS-based determination of the percentage of compound remaining after incubation with liver microsomes supplemented with UDPGA for 1 hour at 37°C. For MRL-871, 100% remained. [1]
Plasma stability was measured by LC-MS-based determination of the percentage of compound remaining after incubation at a concentration of 5 μM in 100% plasma for 1 hour at 37°C. For MRL-871, 47.1% remained after 1 hour. [1]
Plasma protein binding was assessed by equilibrium dialysis. Plasma was incubated with the test compound at a concentration of 5 μM for 6 hours at 37°C, followed by LC-MS-based determination of final compound concentrations. For MRL-871, 99.9% was bound to plasma proteins. [1]

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Pharmacokinetic properties of MRL-871 have been characterized in preclinical studies. The compound is soluble in DMSO. Its plasma half-life, volume of distribution, protein binding, and oral bioavailability have been evaluated in preclinical models. The compound is metabolized in the liver, and its metabolites are eliminated via the renal and biliary routes. Specific pharmacokinetic parameters are available from preclinical study reports. The compound should be stored at -20°C for long-term stability.

Toxicological data for MRL-871 are limited to preclinical studies. The compound has not undergone formal toxicology testing for regulatory purposes. Standard laboratory safety precautions should be followed when handling MRL-871: use of personal protective equipment (gloves, safety goggles, lab coat) and handling in a well-ventilated fume hood. The compound should be stored at -20°C for long-term stability. No genotoxicity, carcinogenicity, or reproductive toxicity data are available. Researchers should consult the safety data sheet (SDS) before handling.

[1]. Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists. J Med Chem. 2020 Jan 9;63(1):241-259.

MRL-871 (3) is a potent RORγt inverse agonist that targets an allosteric binding site within the RORγt ligand binding domain, distinct from the orthosteric pocket. This binding site is located between helices 4, 5, 11, and 12, and the ligand directly interacts with the activation function loop between H11 and H12 (AF-2 domain), forcing helix 12 into an unusual conformation that prevents coactivator recruitment. [1]
MRL-871 was originally discovered by Merck and has been used as a tool compound for studying allosteric modulation of RORγt. It was used as the reference compound for the in silico pharmacophore screen in this study. [1]
MRL-871 displays some cross-reactivity with PPARγ, showing an IC50 of 7.2 μM in a PPARγ coactivator recruitment assay, which is 923-fold selective for RORγt. [1]

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Additional information for MRL-871: The compound has a CAS number of 1392809-08-3. Its molecular formula is C₂₂H₁₂ClF₃N₂O₃ and molecular weight is 444.79 g/mol. It is a potent and allosteric RORγt inverse agonist. It binds to an allosteric pocket in the RORγt LBD. It reduces IL-17a mRNA production in EL4 cells. Purity is typically ≥98%. It is for research use only and is not approved for clinical applications. No FDA approvals exist.

C22H12CLF3N2O3

444.790494918823

444.048

C, 59.41; H, 2.72; Cl, 7.97; F, 12.81; N, 6.30; O, 10.79

1392809-08-3

68159303

White to off-white solid powder

5.8

1

7

3

31

683

0

ClC1=CC=CC(C(F)(F)F)=C1C(N1C2C=CC=CC=2C(C2C=CC(C(=O)O)=CC=2)=N1)=O

DANLZOIRUUHIIX-UHFFFAOYSA-N

InChI=1S/C22H12ClF3N2O3/c23-16-6-3-5-15(22(24,25)26)18(16)20(29)28-17-7-2-1-4-14(17)19(27-28)12-8-10-13(11-9-12)21(30)31/h1-11H,(H,30,31)

4-[1-[2-chloro-6-(trifluoromethyl)benzoyl]indazol-3-yl]benzoic acid

MRL871; MRL 871; 1392809-08-3; 4-(1-(2-chloro-6-(trifluoromethyl)benzoyl)-1H-indazol-3-yl)benzoic acid; 4-{1-[2-Chloro-6-(Trifluoromethyl)benzoyl]-1h-Indazol-3-Yl}benzoic Acid; Benzoic acid, 4-[1-[2-chloro-6-(trifluoromethyl)benzoyl]-1H-indazol-3-yl]-; MRL-871

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~56.21 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)