Retinoic acid receptor-related orphan receptor γt (RORγt) [1, 2].
IC₅₀: 0.005 μM (in FRET assay) [2]; 0.02 μM (in IL-17F promoter assay) [1].
IC₅₀: 0.017 μM (in RORγt/Gal4 cell-based reporter assay) [2].

It was shown that TMP778, at concentrations more than 2.5 μM, started to have harmful effects on cell development. However, this was not dependent on RORγt, since it also decreased the proliferation of RORγt-deficient T cells cultivated under Th17 cell polarizing conditions. Furthermore, these inhibitors significantly suppressed the generation of IL-17 while having no effect on RORγt expression, nuclear translocation, or cell proliferation. TMP778, exhibiting a greater binding affinity for RORγt, demonstrated efficacy in decreasing IL-17 production over a wider dose range. According to these findings, TMP778 is the most efficient RORγt inhibitor at lowering the synthesis of IL-17 [2].

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In a FRET-based molecular screening assay, TMP778 inhibited the interaction between RORγt ligand-binding domain and the SRC1 cofactor peptide with an IC₅₀ of 0.005 μM [2].
In an IL-17F promoter-driven luciferase reporter assay in Jurkat cells stably expressing RORγt, TMP778 inhibited transcriptional activity with an IC₅₀ of 0.02 μM [1].
In a RORγt/Gal4 cell-based reporter assay in HEK293 cells, TMP778 inhibited RORγt transcriptional activity with an IC₅₀ of 0.017 μM, showing approximately 100-fold selectivity over RORα (IC₅₀ = 1.24 μM) and RORβ (IC₅₀ = 1.39 μM). It showed no activity (IC₅₀ > 10 μM) against 22 other nuclear receptors tested [2].
In primary naive CD4⁺ T cells cultured under Th17-polarizing conditions, TMP778 potently inhibited IL-17A secretion with an IC₅₀ of 0.005 μM. It did not affect the differentiation of Th1 or Th2 cells, nor the production of IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-13 under those conditions [1].
In human primary memory CD4⁺ T cells stimulated with anti-CD3/anti-CD28, TMP778 inhibited IL-17A production with an average IC₅₀ of 0.1 μM from five experiments. The inactive diastereomer TMP776 had no effect [1].
In human PBMCs stimulated with anti-CD3/anti-CD28, TMP778 (1 μM) significantly inhibited IL-17A production, but not Th1/Th2 cytokines. The IC₅₀ for IL-17A inhibition in this assay was 0.04 μM [1].
In human γδ T cells differentiated and restimulated with IL-1β, IL-6, and IL-23, TMP778 (1 μM) significantly inhibited IL-17A production without affecting IFN-γ or TNF-α production [1].
In mouse γδ T cells isolated from IMQ-treated mice and stimulated with IL-1β and IL-23, TMP778 (1 μM) inhibited IL-17A production. TNF-α production was also reduced, while IFN-γ expression was slightly elevated [1].
In naive CD4⁺ T cells transduced with RORγt-expressing lentivirus, TMP778 (0.1 μM) impaired the generation of IL-17A- and IL-17F-producing Th17 cells and Tc17 cells [1].
In established Th17 cells (from RORγt-transduced naive CD4⁺ T cells), TMP778 inhibited acute IL-17A secretion upon restimulation in a dose-dependent manner with an IC₅₀ of 0.03 μM [1].
In established Tc17 cells, TMP778 inhibited acute IL-17A secretion with an IC₅₀ of 0.005 μM [1].
At a concentration of 2.5 μM (used for maximal IL-17 inhibition without toxicity), TMP778 did not affect cell proliferation or RORγt expression or nuclear translocation, but efficiently inhibited IL-17 production [2].
In a panel of 45 kinases, GPCRs, transporters, and ion channels, TMP778 showed >1000-fold selectivity over all these targets [1].

In comparison to mice administered with a control, all three drugs (e.g., TMP778) considerably reduced the severity of illness development and delayed the beginning of disease. According to the in vitro findings, the TMP778 therapy had the biggest effect on the illness phenotype. The percentage of IL-17+ T cells (including IL-17+ IFNγ+) in the central nervous system (CNS) was significantly decreased by this treatment, in addition to lowering the number of monocytes invading the system. No group's CNS IFNγ+ IL-17-T cell percentage changed considerably, suggesting that no inhibitor had an impact on Th1 responses. TMP778 dramatically slows the development of EAE and dramatically suppresses the production of IL-17 by differentiated Th17 cells as well as the substantial inhibition of Th17 cell formation [2].

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In an imiquimod (IMQ)-induced psoriasis-like skin inflammation model in BALB/c mice, subcutaneous administration of TMP778 (20 mg/kg, twice daily for 10 days) significantly reduced ear thickness compared to vehicle-treated controls. Histological analysis showed reduced epidermal hyperplasia and inflammatory cell influx [1].
In the IMQ model, TMP778 treatment significantly reduced the number of IL-17A-producing γδ T cells in vivo and inhibited Th17 signature gene expression (Ccl20, Il23r, Ccr6, Il17f, Il22, Il17a) in skin-infiltrating cells [1].
In a mouse experimental autoimmune encephalomyelitis (EAE) model induced by MOG₃₅₋₅₅/CFA immunization, subcutaneous administration of TMP778 (200 μg per injection, twice daily starting from day 0) delayed disease onset and substantially reduced the severity of disease progression compared to control-treated mice [2].
In the EAE model, TMP778 treatment decreased the number of mononuclear cells infiltrating the central nervous system (CNS) and significantly reduced the percentage of IL-17⁺ T cells (including IL-17⁺IFNγ⁺) in the CNS [2].

FRET Assay: The fluorescence resonance energy transfer (FRET) assay was used to identify inverse agonists of RORγt. Biotinylated human RORγt protein and a biotinylated steroid receptor coactivator 1 (SRC1) peptide were used. Streptavidin-labeled allophycocyanin and europium were added to the reaction mixture. Compounds were incubated with the FRET mixture containing SRC1-europium and human RORγt-allophycocyanin for 1 hour. Emissions at 516 nm and 665 nm were read on a plate reader in Lance mode for europium/allophycocyanin. The percent activation at each dose was calculated and plotted to determine the IC₅₀ [1].

IL-17F Promoter Assay: A Jurkat cell line stably expressing RORγt and an IL-17F promoter-luciferase reporter gene was used. Cells were cultured in RPMI 1640 with 10% FBS, diluted to 5 × 10⁵ cells/mL, and stimulated with anti-CD3 antibody (10 μg/mL) in the presence of compound for 20 hours. Luciferase activity was measured after adding a detection mix and incubating for 30 minutes. Percent inhibition at each concentration was calculated to determine the IC₅₀ [1].
Nuclear Receptor Reporter Assay (GAL4): HEK293 cells were transiently transfected with a vector containing the GAL4 DNA-binding domain fused to the RORγt, RORα, or RORβ ligand-binding domain, along with a pG5 Luc reporter (containing 5 GAL4 binding sites upstream of a minimal TATA box). Twenty-four hours post-transfection, compounds were added for an additional 18 hours before luciferase activity was measured [2].
Naive CD4⁺ T Cell Th17 Differentiation: Naive CD4⁺ T cells were isolated and stimulated with anti-CD3/anti-CD28 Dynabeads in Th17-polarizing conditions (IL-6, TGF-β, IL-23, IL-1β, anti-IL-4, anti-IFN-γ) in the presence of compound or DMSO. After 6 days, supernatants were harvested for cytokine measurement by MSD. For Th1 or Th2 differentiation, cells were cultured with IL-12/anti-IL-4 or IL-4/anti-IL-12, respectively [1].
Memory CD4⁺ T Cell Assay: Memory CD4⁺ T cells were purified and stimulated with anti-CD3/anti-CD28 Dynabeads with or without IL-23 (50 ng/mL) for 2 days, and cytokines in supernatants were measured by MSD [1].
Human PBMC Assay: PBMCs were stimulated with soluble anti-CD3 and anti-CD28 antibodies with or without IL-23 (50 ng/mL) for 5 days, and cytokines in supernatants were measured by MSD [1].
γδ T Cell Assay: Human γδ T cells were purified from PBMCs by negative selection and stimulated with anti-CD3/anti-CD28 beads plus IL-1β, IL-6, and IL-23 for 2 weeks to differentiate into IL-17A-producing cells. Cells were then restimulated with the same cytokines in the presence of compounds for 5 days, and cytokine titers were determined by MSD [1].
Intracellular Cytokine Staining: Cells were stimulated with PMA (10-30 ng/mL) and ionomycin (500-1000 ng/mL) in the presence of brefeldin A for the last 3-4 hours. After surface staining, cells were fixed, permeabilized, and stained with fluorescence-conjugated cytokine antibodies before analysis by flow cytometry [1, 2].
RNA Extraction and qPCR: Total RNA was extracted using RNeasy kits with DNase I digestion. cDNA was synthesized, and TaqMan real-time PCR was performed to quantify gene expression [1].
Lentiviral Transduction of RORγt: Naive CD4⁺ or CD8⁺ T cells were transduced with RORγt-expressing lentivirus and stimulated with anti-CD3/anti-CD28 beads. Compounds were added at the time of transduction for some experiments [1].
ChIP-PCR: Th17 cells were cultured in the presence of compounds for 96 hours. ChIP was performed with anti-RORγt antibody, followed by real-time PCR analysis to confirm RORγt binding at selected loci [2].

Imiquimod (IMQ)-Induced Skin Inflammation Model: Female BALB/c mice (10-12 weeks old) were used. TMP778 was dissolved in a vehicle consisting of 3% dimethylacetamide, 10% Solutol, and 87% saline. IMQ was formulated at 50 mg/mL in ethanol/PBS/lactic acid (54:36:10%). TMP778 (20 mg/kg) or vehicle was administered subcutaneously twice daily (morning and afternoon, with 8 hours between doses) for 10 days, starting at day 0. Ear thickness was measured daily before IMQ application [1].
Experimental Autoimmune Encephalomyelitis (EAE) Model: Female C57BL/6 mice (8-12 weeks old) were immunized subcutaneously with an emulsion containing MOG₃₅₋₅₅ (100 μg/mouse) and Mycobacterium tuberculosis H37Ra extract (3 mg/mL) in CFA (100 μL/mouse). Pertussis toxin (100 ng/mouse) was administered intraperitoneally on days 0 and 2. TMP778 (200 μg per injection) was administered subcutaneously twice daily starting from day 0 throughout the experiment [2].

At concentrations >2.5 μM, TMP778 started to show toxic effects on T cell growth. This effect was not RORγt-dependent, as proliferation of RORγt-deficient T cells was also decreased at this concentration [2].
In a panel of 45 kinases, GPCRs, transporters, and ion channels, TMP778 showed >1000-fold selectivity over all these targets, indicating low off-target toxicity risk [1].
TMP778 did not show detectable activity against broad panels of nuclear receptors, GPCRs, kinases, ion channels, transporters, hERG, and CYP panel, and was negative in genotoxicity assays (as mentioned in the text) [1].

[1]. Pharmacologic inhibition of RORγt regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo. J Immunol. 2014 Mar 15;192(6):2564-75.

[2]. Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms. Immunity. 2014 Apr 17;40(4):477-89.

TMP778 is a potent and selective RORgt inverse agonist.

C31H30N2O4

494.580908298492

494.22

C, 75.28; H, 6.11; N, 5.66; O, 12.94

1422053-04-0

TMP780;1422053-03-9

71247311

White to off-white solid powder

5.6

2

5

7

37

758

2

CC1=CC(=C(C=C1)[C@H](C2=CC=CC=C2)NC(=O)CC3=CC4=C(C=C3)OC(=C4)[C@H](C5=C(ON=C5C)C)O)C

DIURRJOJDQOMFC-IOWSJCHKSA-N

InChI=1S/C31H30N2O4/c1-18-10-12-25(19(2)14-18)30(23-8-6-5-7-9-23)32-28(34)16-22-11-13-26-24(15-22)17-27(36-26)31(35)29-20(3)33-37-21(29)4/h5-15,17,30-31,35H,16H2,1-4H3,(H,32,34)/t30-,31+/m0/s1

2-[2-[(S)-(3,5-dimethyl-1,2-oxazol-4-yl)-hydroxymethyl]-1-benzofuran-5-yl]-N-[(S)-(2,4-dimethylphenyl)-phenylmethyl]acetamide

TMP-778 TMP 778 TMP778

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 : ~240 mg/mL (~485.26 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.)