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MRL-871 (MRL871) is a novel and potent allosteric RORγt inhibitor binding to a previously unidentified allosteric pocket in the RORγt LBD. As an allosteric retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonist, it exhibits an IC50 of 12.7 nM and reduces IL-17a mRNA production in EL4 cells.
| Targets |
Retinoic acid receptor-related orphan receptor γt (RORγt) [1]
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|---|---|
| ln Vitro |
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] In EL4 cells, MRL-871 (Compound 3; 10 μM, 24 hours) dramatically decreased IL-17a mRNA expression by a factor of 48 [1]. |
| Enzyme Assay |
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] |
| Cell Assay |
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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| ADME/Pharmacokinetics |
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] |
| References | |
| Additional Infomation |
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] |
| Molecular Formula |
C22H12CLF3N2O3
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|---|---|
| Molecular Weight |
444.790494918823
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| Exact Mass |
444.048
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| Elemental Analysis |
C, 59.41; H, 2.72; Cl, 7.97; F, 12.81; N, 6.30; O, 10.79
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| CAS # |
1392809-08-3
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| PubChem CID |
68159303
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| Appearance |
White to off-white solid powder
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| LogP |
5.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
31
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| Complexity |
683
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=CC=CC(C(F)(F)F)=C1C(N1C2C=CC=CC=2C(C2C=CC(C(=O)O)=CC=2)=N1)=O
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| InChi Key |
DANLZOIRUUHIIX-UHFFFAOYSA-N
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| InChi Code |
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)
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| Chemical Name |
4-[1-[2-chloro-6-(trifluoromethyl)benzoyl]indazol-3-yl]benzoic acid
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| Synonyms |
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
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~56.21 mM)
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2483 mL | 11.2413 mL | 22.4825 mL | |
| 5 mM | 0.4497 mL | 2.2483 mL | 4.4965 mL | |
| 10 mM | 0.2248 mL | 1.1241 mL | 2.2483 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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