CDK9- Cyclin T1 (IC50 = 5 nM); CDK1-Cyclin B (IC50 = 138 nM); cdk2-cyclin A (IC50 = 233 nM); cdk2-cyclin E (IC50 = 367 nM); CDK3-Cyclin E (IC50 = 399 nM); CDK4-Cyclin D (IC50 = 112 nM); cdk5-p35 (IC50 = 159 nM); cdk5-p25 (IC50 = 186 nM); cdk6-cyclin D3 (IC50 = 712 nM); CDK7-CycH/MAT1 (IC50 = 555 nM)
Cyclin-Dependent Kinase 9 (CDK9)-Cyclin T1 (IC₅₀ = 5 nM) [1]
Other CDKs (selectivity ≥22-fold vs. CDK9): CDK1-Cyclin B (IC₅₀ = 138 nM), CDK2-Cyclin A (IC₅₀ = 233 nM), CDK2-Cyclin E (IC₅₀ = 367 nM), CDK3-Cyclin E (IC₅₀ = 399 nM), CDK4-Cyclin D (IC₅₀ = 112 nM), CDK5-P35 (IC₅₀ = 159 nM), CDK5-P25 (IC₅₀ = 186 nM), CDK6-Cyclin D3 (IC₅₀ = 712 nM), CDK7-CycH/MAT1 (IC₅₀ = 555 nM) [1]
GSK-3α and GSK-3β [1]

MC180295 is a highly effective and selective CDK9-Cyclin T1 inhibitor. With an IC50 of 5 nM, it exhibits at least a 22-fold increase in CDK9 selectivity over other CDKs, including CDK1-Cyclin B (IC50, 138 nM), CDK2-Cyclin A (IC50, 233 nM), CDK2-Cyclin E (IC50, 367 nM), CDK3-Cyclin E (IC50, 399 nM), CDK4-Cyclin D (IC50, 112 nM), CDK5-P35 (IC50, 159 nM), CDK5-P25 (IC50, 186 nM), CDK6-Cyclin D3 (IC50, 712 nM), and CDK7-CycH/MAT1 (IC50, 555nM). Moreover, MC180295 inhibits GSK-3α and GSK-3β[1]. MC180295 (500 nM) targets CDK9 to reactivate genes that have been epigenetically silenced while leaving DNA methylation alone[1]. MC180295 (0.1 μM) prevents the growth of cancer cells[1].

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1. Gene reactivation of epigenetically silenced genes: Treatment of YB5 cells (a SW48-derived reporter cell line with methylated CMV promoter-silenced GFP) with (rel)-MC180295 (500 nM, 24–72 h) reactivated GFP expression (measured by FACS and fluorescent microscopy), along with other silenced genes including SYNE1 and MGMT (detected by qPCR). This reactivation was independent of DNA methylation changes, as confirmed by genomic analysis [1]
2. Anti-proliferative activity: (rel)-MC180295 (0.1 μM, single dose, 4-day incubation) inhibited proliferation of multiple cancer cell lines (SW48, HCT116, HL60, MCF7, DU145, LNCaP) measured by trypan blue exclusion assay, while showing minimal effect on normal lung fibroblast IMR90 cells. It also suppressed anchorage-independent growth: in soft agar assays, (rel)-MC180295 (single dose, 4-day treatment) reduced colony formation of SW48 and HCT116 cells [1]
3. Induction of cell differentiation: (rel)-MC180295 (single dose, 4-day exposure) induced differentiation of HL60 leukemia cells, as evidenced by increased expression of the differentiation marker CD11b (measured by flow cytometry), with efficacy comparable to the positive control all-trans retinoic acid (ATRA, 1 μM) [1]
4. Modulation of protein phosphorylation: (rel)-MC180295 (2 h treatment) dose-dependently inhibited phosphorylation of RNA polymerase II (RNAPII) at Ser2 (pSer2, a direct CDK9 target) in cancer cells. At 500 nM, it specifically inhibited CDK9 without affecting phosphorylation of CDK4/6 targets (phosphor-Rb at T870/811, T826; p130) or CDK1/2 targets (phosphor-CDK Substrate Motif (K/H)pSP; phosphor-PRC1); at 5 μM, it additionally inhibited CDK4/6 [1]
5. Interaction with BRG1 and epigenetic regulation: (rel)-MC180295 (in combination with recombinant CDK9) reduced phosphorylation of the SWI/SNF protein BRG1 in an isotope kinase activity assay. Overexpression of wild-type BRG1 (but not BRG1 mutants with 5 serine residues substituted by alanine (5STOA) or deleted (NO5S)) synergized with (rel)-MC180295 to reactivate GFP in YB5 cells (measured by confocal microscopy), confirming BRG1 dephosphorylation contributes to gene reactivation [1]
6. Activation of endogenous retroviruses (ERVs): (rel)-MC180295 (single dose, 4-day treatment) upregulated ERV expression in YB5 cells (detected by qPCR), similar to the positive control decitabine (DAC), indicating broad chromatin decompaction [1]

MC180295 (20 mg/kg, i.p., qod) shows no inhibitory action against human T cell growth in vivo, but inhibits significant anti-tumor activity in mice carrying SW48 cells[1].

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1. Anti-tumor efficacy in SW48 xenograft model: NSG mice were subcutaneously inoculated with 2×10⁶ SW48 colon cancer cells. When tumors became palpable (day 11), (rel)-MC180295 (20 mg/kg) or vehicle was administered intraperitoneally (i.p.) every other day (qod). (rel)-MC180295 significantly reduced tumor volume (measured by caliper) and prolonged mouse survival (log-rank test, p<0.05) compared to vehicle [1]
2. Synergy with immune checkpoint inhibitor in ovarian cancer model: In the VEGF-DEF ID8 syngeneic ovarian cancer mouse model, treatment with a CDK9 inhibitor (SNS-032, used as a surrogate for (rel)-MC180295 due to mechanism homology) every 3 days reduced ascites volume (a measure of tumor burden) at weeks 4 and 5. Combination with α-PD-1 immune checkpoint inhibitor further decreased ascites at week 5 and extended survival, demonstrating immunosensitization [1]
3. No inhibition of human T-cell growth: NSG mice were intraperitoneally injected with 2×10⁷ human peripheral blood mononuclear cells (PBMCs) from a healthy donor. (rel)-MC180295 (20 mg/kg, i.p., qod) was administered for 12 days, and whole blood was collected on day 14. Flow cytometry (anti-CD45, anti-CD4, anti-CD8 antibodies) showed no significant reduction in CD45+ leukocytes, CD4+ T cells, or CD8+ T cells, indicating no toxic effect on human T cells in vivo [1]

1. Isotope kinase activity assay for CDK9 and BRG1: Prepare reaction mixtures containing recombinant active full-length CDK9, recombinant BRG1, and γ-³²P-ATP. Add (rel)-MC180295 (or flavopiridol as a positive control) at specified concentrations. Incubate the mixtures under optimal kinase activity conditions. Separate reaction products by SDS-PAGE, and detect phosphorylation of substrates (RNAPII CTD, BRG1, and CDK9 itself) via autoradiography. Verify equal loading of proteins by Coomassie blue staining of the gel [1]
2. CDK selectivity assay: Test (rel)-MC180295 at concentrations ranging from 0.1 nM to 10 μM against 10 CDK/cyclin complexes (CDK1-Cyclin B, CDK2-Cyclin A/E, CDK3-Cyclin E, CDK4-Cyclin D, CDK5-P35/P25, CDK6-Cyclin D3, CDK7-CycH/MAT1, CDK9-Cyclin T1) in cell-free kinase assays. Measure kinase activity using a substrate-specific detection method (e.g., phosphorylation of peptide substrates). Calculate IC₅₀ values for each CDK/cyclin complex by plotting activity against inhibitor concentration [1]
3. Kinase panel screening: Incubate (rel)-MC180295 (1 μM) with a panel of human kinases. Measure inhibition of each kinase using a high-throughput kinase activity assay. Generate a phylogenetic tree to visualize the distribution of inhibited kinases, confirming high selectivity for CDK9 [1]

1. GFP reactivation assay in YB5 cells: Seed YB5 cells (SW48-derived, GFP-silenced by methylated CMV promoter) in culture plates. Treat cells with (rel)-MC180295 (0.1–500 nM) or vehicle (DMSO) for 24–72 h. Measure GFP expression by flow cytometry (FACS) to quantify the percentage of GFP+ cells and mean fluorescence intensity. Confirm results by fluorescent microscopy, capturing images of DMSO- and (rel)-MC180295-treated cells [1]
2. qPCR for gene expression analysis: Treat cancer cells (YB5, SW48, HCT116) with (rel)-MC180295 (0.1–10 μM) for 24–72 h. Isolate total RNA using a standard RNA extraction method, reverse-transcribe to cDNA. Perform qPCR with primers specific for silenced genes (GFP, SYNE1, MGMT), ERVs, or housekeeping genes (e.g., GAPDH). Calculate relative gene expression using the 2⁻ΔΔCt method, with DMSO-treated cells as control [1]
3. Western blot for protein phosphorylation: Treat cancer cells with (rel)-MC180295 (0.1–5 μM) for 2 h. Lyse cells with RIPA buffer containing protease and phosphatase inhibitors. Extract total proteins, quantify by BCA assay. Separate proteins by SDS-PAGE, transfer to PVDF membranes. Incubate with primary antibodies against pSer2-RNAPII, total RNAPII, phosphor-Rb (T870/811, T826), p130, phosphor-CDK Substrate Motif, phosphor-PRC1, BRG1, or tubulin (loading control). Incubate with HRP-conjugated secondary antibodies, detect bands by chemiluminescence. Quantify band intensity using ImageJ software [1]
4. Cell proliferation assay (trypan blue exclusion): Seed normal lung fibroblasts (IMR90) and cancer cell lines (SW48, HCT116, HL60, MCF7, DU145, LNCaP) in 24-well plates (5×10³ cells/well). Treat with (rel)-MC180295 (0.1 μM) or vehicle for 4 days. Trypsinize cells, stain with trypan blue, and count viable cells using a hemocytometer. Calculate relative proliferation compared to vehicle-treated cells [1]
5. Soft agar colony formation assay: Prepare a bottom layer of 0.6% agar in complete medium in 6-well plates. Mix cancer cells (SW48, HCT116) with 0.3% agar and (rel)-MC180295 (0.1–1 μM) or vehicle, add as a top layer. Incubate for 4 days at 37°C, 5% CO₂. Stain colonies with 0.005% crystal violet, count colonies using ImageJ. Calculate colony formation efficiency relative to vehicle [1]
6. HL60 differentiation assay: Seed HL60 cells in 6-well plates. Treat with (rel)-MC180295 (0.1–1 μM), ATRA (1 μM, positive control), or 1.25% DMSO (negative control) for 4 days. Harvest cells, stain with anti-CD11b antibody (fluorescently conjugated). Measure CD11b expression by flow cytometry, calculating the percentage of CD11b+ cells [1]
7. ATAC-seq for chromatin accessibility: Treat SW48 cells with (rel)-MC180295 (10 μM) or DMSO for 4 days. Isolate nuclei, tag accessible chromatin with Tn5 transposase, extract DNA. Prepare sequencing libraries, perform high-throughput sequencing. Analyze reads to calculate ATAC-seq signal enrichment around transcription start sites (TSS) of all genes and genes induced by (rel)-MC180295. Generate genome browser tracks for representative induced genes (e.g., SPOCK2, CYP1B1) to visualize gained peaks in promoter regions [1]
8. Co-immunoprecipitation (Co-IP) of CDK9 and BRG1: For transfected cells (HEK293T, SW48), transfect with FLAG-tagged CDK9/BRG1 or GFP-tagged CDK9 plasmids. For endogenous Co-IP, use untransfected HEK293T or SW48 cells. Lyse cells with IP buffer, incubate lysates with anti-CDK9, anti-BRG1, anti-FLAG, anti-GFP, or IgG (negative control) antibodies overnight at 4°C. Add protein A/G agarose beads, incubate for 2 h. Wash beads, elute proteins. Perform Western blot with antibodies against CDK9, BRG1, FLAG, or GFP to detect interacting proteins [1]

NSG mice
5-20mg/kg
IP

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1. SW48 xenograft tumor model: Use 6–8-week-old female NSG mice. Subcutaneously (s.c.) inoculate 2×10⁶ SW48 cells into the right flank. Monitor tumor growth daily; when tumors reach a palpable size (~100 mm³, day 11), randomize mice into two groups (n=5–6 per group). Administer (rel)-MC180295 (20 mg/kg, dissolved in a suitable vehicle e.g., DMSO/cremophor/normal saline) or vehicle via intraperitoneal (i.p.) injection every other day (qod). Measure tumor volume twice weekly using a caliper (volume = length × width² / 2). Monitor mouse survival daily, plot survival curves using the Kaplan-Meier method [1]
2. Human PBMC transfer model: Use 6–8-week-old female NSG mice. Intraperitoneally inject 2×10⁷ human PBMCs (from a healthy donor) on day 0. On day 1, start administering (rel)-MC180295 (20 mg/kg, i.p., qod) or vehicle for 12 days. On day 14, collect whole blood via retro-orbital bleeding. Lyse red blood cells, stain leukocytes with fluorescently conjugated antibodies against CD45 (pan-leukocyte), CD4 (T helper cells), and CD8 (cytotoxic T cells). Analyze cell populations by flow cytometry, calculating the percentage of CD45+, CD4+, and CD8+ cells [1]
3. VEGF-DEF ID8 syngeneic ovarian cancer model: Use 6–8-week-old female C57BL/6 mice. Intravenously inject VEGF-DEF ID8 ovarian cancer cells to induce ascites. When ascites is detectable (week 3), randomize mice into three groups: vehicle, CDK9 inhibitor (SNS-032, 10 mg/kg, i.p. every 3 days), or SNS-032 + α-PD-1 (200 μg/mouse, i.p. twice weekly). Measure ascites volume at weeks 4 and 5 by abdominal palpation and fluid aspiration. Monitor mouse survival daily, plot survival curves. (Note: SNS-032 is used as a mechanistically similar CDK9 inhibitor to (rel)-MC180295 to demonstrate immunosensitization) [1]

1. No human T cell toxicity: In the human peripheral blood mononuclear cell (PBMC) transfer model, (rel)-MC180295 (20 mg/kg, intraperitoneal injection, every other day for 12 days) did not reduce the number of CD45+ leukocytes, CD4+ T cells or CD8+ T cells in NSG mice, indicating that it has no inhibitory effect on the growth of human T cells in vivo [1]

[1]. Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer. Cell. 2018 Nov 15;175(5):1244-1258.e26.

1. Background and Development: (rel)-MC180295 is a novel, highly selective CDK9 inhibitor developed through structure-activity relationship (SAR) optimization and gene expression-guided screening. It belongs to the aminothiazole class of compounds and has been patented (PCT/US18/14465). The authors of this study are co-inventors [1].
2. Mechanism of Action: (rel)-MC180295 inhibits CDK9-mediated transcriptional elongation by blocking RNAPII Ser2 phosphorylation. It also dephosphorylates the SWI/SNF chromatin remodeling factor BRG1, thereby helping to reactivate epigenetically silenced tumor suppressor genes and endogenous retroviruses (ERVs). ERV activation can induce an interferon response, making cancer cells more sensitive to immune checkpoint inhibitors (such as α-PD-1) [1]
3. Therapeutic potential:(rel)-MC180295 is a promising epigenetic therapy for cancer, with broad anticancer activity against a variety of malignancies (colon cancer, leukemia, breast cancer, prostate cancer) in vitro and in vivo efficacy in xenograft models. Its synergistic effect with immune checkpoint inhibitors further supports its development in combination cancer therapy [1]
4. Structural features:(rel)-MC180295's aminothiazole core interacts with the CDK9 hinge region and forms hydrogen bonds with the conserved Lys48-Glu66 amino acid pair. Its norborneol can induce a unique conformation at the C-terminus of the CDK9 hinge, thereby giving it high selectivity for other CDKs [1]

C17H18N4O3S

358.414822101593

358.11

C, 56.97; H, 5.06; N, 15.63; O, 13.39; S, 8.94

2237942-08-2

2237942-08-2

137333456

White to off-white solid powder

4.7

2

7

4

25

543

3

C1C[C@H]2C[C@@H]1C[C@@H]2NC3=NC(=C(S3)C(=O)C4=CC=CC=C4[N+](=O)[O-])N

JRNXAQINDCOHGS-SCVCMEIPSA-N

InChI=1S/C17H18N4O3S/c18-16-15(14(22)11-3-1-2-4-13(11)21(23)24)25-17(20-16)19-12-8-9-5-6-10(12)7-9/h1-4,9-10,12H,5-8,18H2,(H,19,20)/t9-,10+,12+/m1/s1

[4-amino-2-[[(1S,2S,4R)-2-bicyclo[2.2.1]heptanyl]amino]-1,3-thiazol-5-yl]-(2-nitrophenyl)methanone

MC180295; MC 180295; MC-180295

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: 72~100 mg/mL (200.9~279.0 mM)
Ethanol: ~72 mg/mL (200.9 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.)