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| ln Vitro |
MS402 selectively inhibits the differentiation of naive murine CD4+ T cells into T-helper 17 (Th17) cells in a dose-dependent manner (e.g., IL-17+ cell percentage reduced from 18.6% to 8.0% at an unspecified concentration within tested range). It has a modest effect on Th1 cell differentiation (IFN-γ+ cells reduced from 49.7% to 38.6%) and little to no effect on Th2 (IL-4 expression) or regulatory T cell (Treg, Foxp3 expression) differentiation.
MS402 dose-dependently inhibits mRNA expression of Th17 signature genes (e.g., rorc, il17a), modestly affects Th1 genes (tbx21/T-bet, ifng), and has minimal effects on Th2 (gata3, il4) or Treg (foxp3, il10) genes. MS402 treatment reduces Brd4 and Cdk9 occupancy and RNA Polymerase II Ser2 phosphorylation at the il17a/f and rorc loci, key for transcription elongation. MS402 does not affect T-cell proliferation. Genomic analyses (ChIP-seq, RNA-seq) show MS402 effectively reduces Brd4 occupancy and downregulates transcription at Th17 signature gene loci and super-enhancers, with fewer effects on housekeeping genes compared to the pan-BET inhibitor JQ1. [1] |
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| ln Vivo |
After 4 weeks of being reconstituted with naive CD4+CD45RBhi cells obtained from C57BL/6 mice's lymph nodes and spleens, Rag1-/- animals started to lose weight. Significantly less weight loss is caused by MS402 (10 mg/kg; intraperitoneally injected twice weekly for 3 weeks) [1].
In a preventive model of T-cell transfer-induced colitis in Rag1−/− mice, intraperitoneal administration of MS402 (10 mg/kg, twice weekly for 7 weeks, starting at week 0) significantly reduced body weight loss, attenuated colon shortening and inflammation, and resulted in lower disease histopathology scores (0-1 vs. 3 in controls). In a therapeutic model (treatment initiated at week 5 post-cell transfer when colitis was established), MS402 (10 mg/kg, twice weekly for 3 weeks) reversed weight loss, improved colon pathology, and reduced inflammation scores. Flow cytometry analysis of colon tissues showed MS402 treatment significantly reduced the percentages of IL-17-producing and IFN-γ-producing CD4+ T cells, with a more profound effect on Th17 cells in the therapeutic model. mRNA levels of key cytokines (il17, il21, il22, il6, ifng) and transcription factors (rorc, T-bet) in colon were significantly lower in MS402-treated mice compared to disease controls. [1] |
| Enzyme Assay |
A binding assay was used to determine the inhibitory constant (Ki). The affinity of MS402 for various bromodomains was measured, likely using a fluorescence polarization or similar binding assay, comparing its binding to BRD4 BD1, BD2, and bromodomains from other proteins (BRD2, BRD3, CBP, PCAF, etc.). The reported Ki value for BRD4 BD1 is 77 nM. The selectivity over BD2 and other bromodomains was calculated from these measurements. [1]
A thermal shift assay may have been employed to assess compound binding and protein stability, contributing to the characterization of binding selectivity. [1] |
| Cell Assay |
Naive CD4+ T cells were purified from mouse spleen and lymph nodes using antibody-coated magnetic beads.
For Th17 differentiation, cells were activated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cytokines IL-6 and TGF-β. MS402 or other inhibitors were added daily to the culture medium at specified concentrations (e.g., 100 nM, 500 nM). Cells were cultured for 3-3.5 days. For intracellular cytokine staining, differentiated cells were stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A for 5 hours. Cells were fixed, permeabilized, and stained with fluorescently labeled antibodies against IL-17, IFN-γ, and CD4, followed by flow cytometry analysis. For mRNA expression analysis, total RNA was extracted from differentiated cells, reverse transcribed, and analyzed by quantitative real-time PCR (qPCR) using gene-specific primers. Expression levels were normalized to housekeeping genes (Actin/Gapdh). Chromatin immunoprecipitation (ChIP) assays were performed. Cells were cross-linked with formaldehyde, lysed, and sonicated to shear chromatin. Chromatin extracts were immunoprecipitated with antibodies against Brd4 or other proteins. Precipitated DNA was analyzed by qPCR (ChIP-qPCR) or used for sequencing library preparation (ChIP-seq). [1] |
| Animal Protocol |
Animal/Disease Models: C57BL/6 mice, Rag1-/- mice, T cell transfer-induced colitis model [1]
Doses: 10 mg/kg Route of Administration: intraperitoneal (ip) injection, twice a week for 3 weeks Experimental Results: Weight loss is Dramatically diminished. T-cell transfer-induced colitis model: Naive CD4+CD45RBhi T cells were isolated from spleens and lymph nodes of donor mice (C57BL/6). Approximately 5 × 105 cells in sterile PBS were injected intraperitoneally into each Rag1−/− recipient mouse. Preventive study: MS402 treatment (10 mg/kg, dissolved in PBS) was administered intraperitoneally twice a week, starting on the day of T-cell transfer (week 0) and continuing for 7 weeks. A control group received PBS vehicle. Therapeutic study: Treatment with MS402 (10 mg/kg, i.p., twice weekly) was initiated at week 5 after T-cell transfer, when mice showed signs of colitis (e.g., weight loss), and continued for 3 weeks. Mice were monitored weekly for body weight changes. At the endpoint (week 7 or 8), mice were euthanized. Colon tissues were excised, measured, and fixed for histopathological analysis (H&E staining). Sections were scored for inflammation severity by a pathologist blinded to the treatment. Colon lamina propria lymphocytes were isolated for flow cytometry analysis to determine the frequency of IL-17+ and IFN-γ+ CD4+ T cells. Colon tissue was also used for RNA extraction and qPCR analysis of cytokine and transcription factor mRNA levels. [1] |
| References | |
| Additional Infomation |
MS402 is a small molecule inhibitor based on cyclopentanone designed to selectively target the first bromine domain (BD1) of the BET protein.
The crystal structure of the MS402 complex with BRD4 BD1 (PDB: 5ULA) shows that the compound binds to the ZA channel, forming specific hydrogen bonds and van der Waals forces. Key interactions with Gln85 and Ile146 in BD1 make it selective for BD2. This study suggests that selective inhibition of the BET protein BD1 (primarily by disrupting BRD4 function) is an effective strategy to block pathogenic Th17 cell differentiation without extensively impairing other T helper cell lineages, providing a potential treatment for Th17-mediated inflammatory bowel diseases such as colitis. [1] |
| Molecular Formula |
C20H19CLN2O3
|
|---|---|
| Molecular Weight |
370.8295
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| Exact Mass |
370.108
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| CAS # |
1672684-68-2
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| PubChem CID |
90718189
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| Appearance |
White to off-white solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
26
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| Complexity |
569
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C(C(N([H])C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=O)C([H])=C([H])C=1N([H])C1=C(C([H])([H])[H])C(C([H])([H])C1([H])[H])=O
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| InChi Key |
VZTVTSICPINUNG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H19ClN2O3/c1-12-17(9-10-19(12)24)23-18-8-3-13(11-16(18)21)20(25)22-14-4-6-15(26-2)7-5-14/h3-8,11,23H,9-10H2,1-2H3,(H,22,25)
|
| Chemical Name |
3-chloro-N-(4-methoxyphenyl)-4-[(2-methyl-3-oxocyclopenten-1-yl)amino]benzamide
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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 : ~100 mg/mL (~269.67 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.74 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6967 mL | 13.4833 mL | 26.9665 mL | |
| 5 mM | 0.5393 mL | 2.6967 mL | 5.3933 mL | |
| 10 mM | 0.2697 mL | 1.3483 mL | 2.6967 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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