CDK8 (Kd = 2 nM by SPR [2]; Kd = 36 nM by competition binding [2]; IC50 for GSK3α = 0.462 μM, GSK3β = 0.690 μM [1])
CDK19 (Kd = 14-102 nM by competition binding [2]; IC50 for TNKS2 = 15.0 ± 0 μM [1])
TNKS1 (IC50 >10 μM [1])

CCT251545 substantially suppresses WNT pathway activity in COLO205-F1756 clone 4, an APC mutant human colorectal cancer cell line that is designed to express a luciferase-based WNT reporter construct, with an IC50 of 0.035 μM [1]. Tankyrase is weakly inhibited by CCT251545 (TNKS1 IC50 > 10 μM, TNKS2 IC50 = 15.0) [1]. With more than 100-fold selectivity over 291 other kinases, CCT251545 is an efficient and specific chemical probe for identifying human mediator complex-associated protein kinases CDK8 and CDK19 [2]. In addition to modulating additional on-target effects of CDK8 and CDK19, such as gene expression regulated by STAT1, CCT251545 modifies the expression of genes regulated by the WNT pathway [2]. Additionally, CCT251545, with an IC50 of 9 nM, lowers the levels of phospho-STAT1SER727 in SW620 cells [2]. Cell-based action is strong in CCT251545 [2].

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Inhibition of WNT pathway luciferase reporter in 7dF3 cells: IC50 = 0.005 ± 0.002 μM [1]
Inhibition of basal WNT pathway activity in LS174T (β-catenin mutant) cells: IC50 = 0.023 ± 0.011 μM [1]
Inhibition in SW480 (APC mutant) cells: IC50 = 0.190 ± 0.030 μM [1]
Inhibition of WNT3a ligand-dependent reporter in PA-1 cells: IC50 = 0.007 μM [1]
Inhibition in COLO205-F1756 clone 4 cells (APC mutant with WNT reporter): IC50 = 0.035 ± 0.003 μM [1]
Inhibition of endogenous cMYC transcription (RT-PCR) in 7dF3 cells: 12-fold reduction, IC50 = 2.3 nM [1]
No inhibition of EF1-α promoter-driven luciferase in RKO-F1522 cells (IC50 >10 μM) [1]
Reduction of phospho-STAT1 Ser727 levels in SW620 cells: IC50 = 9 nM by bead-based ELISA and immunoblotting [2]
Inhibition of TCF-dependent reporter activity in Colo205 cells with inducible shRNA against CDK8 or CDK19 phenocopied by compound treatment [2]
In 7dF3 cells, esiRNA knockdown of CDK8 or CDK19 reduced TCF-dependent transcription similarly to compound [2]
Overexpression of wild-type CDK8 (but not kinase-dead D173A) increased basal TCF-reporter expression in HEK293 cells, which was blocked by compound [2]
Gene expression microarray in mouse β-catenin-dependent intestinal organoids treated with analog 2 or 6 showed altered expression of WNT target genes (Axin2, Ascl2, Myc) and stress response/STAT-regulated genes, while inactive analog 8 had no effect [2]
Minimal inhibition across 291 non-mutated kinases (only GSK3α/β hits) and no significant inhibition of CDK1-7 or CDK9 with cyclin partners [1][2]

Tumor growth is inhibited in NCr athymic mice bearing established SW620 human colorectal cancer xenografts by CCT251545 (70 mg/kg; oral; twice daily) [2].
In Tet-O-ΔN-β-catenin mouse model of intestinal hyperplasia: oral administration of CCT-251545 (75, 37.5, 18.75 mg/kg twice daily for 2 days) dose-dependently reduced hyperplastic crypt length, decreased BrdU-positive proliferating cells, and increased Alcian Blue-stained goblet cells (P < 0.0003 by Kruskal-Wallis, P < 0.01 after Dunn's post-hoc test) [1][2]
In MMTV-WNT-1 breast cancer allograft model in CD1(nu/nu) mice: treatment with CCT-251545 (75 mg/kg twice daily or 37.5 mg/kg twice daily for 10 days) decreased tumor growth rate and final tumor weight (P < 0.01), reduced expression of WNT target genes Axin2 and Lbh, and induced the involution marker Igfbp5 (P < 0.05 after Dunn's post-hoc test for Igfbp5) [2]
In SW620 human colorectal cancer xenograft model in athymic mice: oral dosing of CCT-251545 (70 mg/kg once daily for 14 days) inhibited tumor growth with 70% reduction in final tumor weight compared to vehicle; concomitant reduction of phospho-STAT1 Ser727 in tumor tissue was observed [2]
In COLO205-F1756 clone 4 human colorectal cancer xenograft model: treatment with CCT-251545 (70 mg/kg twice daily for 9 days) significantly inhibited WNT-driven luciferase reporter activity (80% reduction normalized to tumor weight, P = 0.0003) and reduced tumor weights by 37.5% (P = 0.0379, Mann-Whitney U test) [1]

CDK8-cyclin C SPR: Full-length CDK8-cyclin C complex was immobilized onto sensor chips via standard amine coupling. Compounds were diluted in running buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% P20, 0.1 mM EGTA, 2% DMSO) and analyzed using a Biacore S51 with a twofold dilution series. Interaction cycles consisted of a 120 s sample injection (30 μL/min) followed by 350 s buffer flow. Data were fitted to a 1:1 interaction model, giving Kd = 2 nM for CCT-251545 [2]
Reporter displacement assay for CDK8/19: A probe targeting the ATP binding site of CDK8 or CDK19 with fast binding kinetics was used. Binding of the probe to the target produces an optical signal. Competitive displacement of the probe by test compounds results in loss of signal quantified with increasing compound concentrations. IC50 values and kinetic parameters were determined. For CCT-251545, Kd values against CDK8 and CDK19 were derived from competition experiments with analogs 4 and 6 [2]
TNKS autoparsylation assay: GST-tagged TNKS1 or TNKS2 (250 nM) and biotinylated NAD (5 μM) were incubated with test compound in 50 mM HEPES, 4 mM MgCl2, 0.05% Pluronic F-68, 1.4 mM DTT, 0.5% DMSO, pH 7.7 for 90 min at 30°C. Reaction was stopped with EDTA, and detection solution containing SA-Xlent and anti-GST-K was added. HTRF signal was measured at 615 and 665 nm after 1 h. IC50 values: TNKS1 >10 μM, TNKS2 = 15.0 ± 0 μM for CCT-251545 [1]

7dF3 luciferase reporter assay: 7dF3 cells (HEK293 with ER-DSH and TCF-luciferase-IRES-GFP) were seeded at 5000 cells/well in 384-well plates. After overnight incubation, compounds were added (final concentration range 90 μM to 0.3 nM). After 2 h, β-oestradiol (10 μM) was added to induce DSH expression. Following 24 h incubation, SteadyGlo luciferase reagent was added and luminescence read. IC50 was calculated using curve-fitting [1]
Tet-O-WNT reporter cell lines: HEK293 Tet-O cells with TCF-luciferase reporter and tetracycline-inducible cDNAs (ΔN-LRP6, Dvl2, Axin-GID, ΔN-β-catenin, VP16-TCF4) were used. Cells were seeded, and 24 h later compounds were added. After 2 h, doxycycline (0.05 μg/mL) was added to induce expression. After 24 h, BrightGlo was added and luminescence measured [1]
Quantitative RT-PCR (Taqman) for cMYC: 7dF3 cells were treated with CCT-251545 (9.1 μM to 0.068 nM) for 2 or 6 h. Cells were lysed, DNase treated, and cDNA synthesized using High-Capacity cDNA Reverse Transcription Kit. Taqman reactions were run with FAM-labeled primer/probe for cMYC and VIC-labeled for LRP0 as internal control. Ct values were normalized to LRP0 and expressed relative to vehicle control [1]
Immunoblotting for phospho-STAT1 Ser727 and other proteins: Cells were plated, treated with compound, lysed in RIPA buffer, and sonicated. Proteins (20 μg) were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 5% BSA, and incubated with primary antibodies (anti-p-STAT1 Ser727, anti-total STAT1, anti-GAPDH, etc.) overnight at 4°C, followed by HRP-conjugated secondary antibodies and ECL detection [2]
CETSA (cellular thermal shift assay): SW620 cells were treated with compound for 2 h, then lysed. Lysates were split: one kept at 4°C, the other heated at 50°C for 3 min. After centrifugation, CDK8 and CDK19 levels in supernatants were measured by bead-based ELISA. CCT-251545 showed stabilization at 0.30 μM [2]
shRNA/esiRNA knockdown: Colo205 cells transduced with doxycycline-inducible shRNA against CDK8 or stable shRNA against CDK19 were used. 7dF3 cells were transfected with esiRNA pools (10 ng each) against CDK8, CDK19, or control using Hiperfect reagent. After 24 h, estradiol was added to activate WNT pathway, and luciferase activity measured after another 24 h [2]

Animal/Disease Models: 6-8 week female NCr athymic mice with established SW620 xenografts [2]
Doses: 70mg/kg
Route of Administration: Oral;
Route of Administration: twice (two times) daily; Days 0-7 And the results on days 10-14: tumor growth was inhibited, and the final tumor weight was diminished by 70% relative to the control.

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Tet-O-ΔN-β-catenin mouse intestinal hyperplasia model: Female mice with doxycycline-inducible mutant β-catenin transgene received doxycycline (2 mg/mL) in drinking water for 6-9 days to induce hyperplastic crypts. CCT-251545 was administered orally by gavage at 75, 37.5, or 18.75 mg/kg twice daily for 2 days (formulation: 10% DMSO, 5% Tween 20, 85% saline). BrdU (2 mg/mL in PBS) was injected i.p. 2 h before euthanasia. Duodenal sections were stained for BrdU and Alcian Blue. Crypt length, % BrdU-positive cells, and % goblet cells were quantified [1][2]
MMTV-WNT-1 breast cancer allograft model: MMTV-WNT-1 tumor fragments (<1 mm3) were transplanted subcutaneously into CD1(nu/nu) mice. Once tumors >5 mm3, mice received CCT-251545 (37.5 or 75 mg/kg) orally twice daily for 10 days (formulation: 10% DMSO, 5% Tween 20, 85% saline). Tumor dimensions measured daily by caliper, volume = (width2 × length)/2. Tumors were harvested for RNA extraction and qRT-PCR [2]
SW620 human colorectal cancer xenograft model: 3 million SW620 cells were injected subcutaneously into female athymic mice. When tumors reached ~60 mm3 (day 13), mice received CCT-251545 (70 mg/kg) orally once daily for 14 days (formulation: 10% DMSO, 5% Tween 20, 85% saline). Tumor volumes and body weights measured three times weekly. Plasma and tumor samples collected for PK and p-STAT1 Ser727 analysis [2]
COLO205-F1756 clone 4 xenograft model: 3 million COLO205-F1756 clone 4 cells (APC mutant with TCF-luciferase reporter) injected subcutaneously into female athymic mice. When tumors reached ~100 mm3, mice received CCT-251545 (70 mg/kg) orally twice daily for 9 days (formulation: 10% DMSO, 5% Tween 20, 85% saline). WNT pathway activity measured by whole-body bioluminescent imaging after D-luciferin injection. Tumors excised and weighed [1]

Mouse PK after single intravenous (0.2 mg/kg) and oral (0.5 mg/kg) administration: IV clearance = 31 mL/min/kg, Vss = 1.1 L/kg, t1/2 = 0.55 h; Oral Tmax = 0.25 h, AUC0-inf = 143 h·ng/mL, bioavailability = 54% [1]
Rat PK after single intravenous (0.2 mg/kg) and oral (0.5 mg/kg): IV clearance = 26 mL/min/kg, Vss = 1.8 L/kg, t1/2 = 0.97 h; Oral Tmax = 0.25 h, AUC0-inf = 287.6 h·ng/mL, bioavailability = 88% [1]
In vitro metabolic stability (intrinsic clearance Clint): Mouse liver microsomes = 141 μL/min/mg, rat liver microsomes = 54 μL/min/mg, human liver microsomes = 84 μL/min/mg [1]
Caco-2 permeability: Papp = 24 × 10-6 cm/s, efflux ratio = 2.5 [1]
Solubility: Kinetic solubility = 94 μM, thermodynamic solubility = 0.006 mg/mL in phosphate buffer pH 7.4 [1]
Plasma stability: No plasma instability observed [1]
In vivo plasma and tumor concentrations in SW620 xenograft model (70 mg/kg oral, after last dose): at 2 h, plasma = 2525 ± 921 nM, tumor = 1598 ± 324 nM; at 6 h, plasma = 188 ± 64 nM, tumor = 639 ± 115 nM [2]

hERG potassium channel inhibition: IC50 >10 μM [1]
Weak CYP450 inhibition observed against CYP2C8, 2C9, and 3A4 (specific IC50 values not reported) [1]

[1]. Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen. J Med Chem. 2015 Feb 26;58(4):1717-35.

[2]. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat Chem Biol. 2015 Dec;11(12):973-980.

CCT251545 is a chloropyridine compound with the structure 3-chloropyridine, substituted at positions 4 and 5 with 1-oxo-2,8-diazaspiro[4.5]decane-8-yl and 4-(1-methyl-1H-pyrazol-4-yl)phenyl, respectively. It is a highly bioavailable, orally bioavailable Wnt signaling pathway inhibitor (IC50 = 5 nM) and a highly efficient and selective chemical probe for cyclin-dependent kinases CDK8 and CDK19. It exhibits activities as a Wnt signaling pathway inhibitor, an antitumor drug, and an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor. It is a chloropyridine compound belonging to the pyrazole class and azaspirocyclic compounds.

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CCT-251545 is a selective chemical probe for CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. It acts at or below TCF in the canonical WNT pathway, inhibiting β-catenin-mediated transcription. X-ray crystallography of CDK8-cyclin C complex with the compound reveals a type 1 binding mode with insertion of the CDK8 C-terminal Arg356 into the hinge region. This compound has been used to establish phospho-STAT1 Ser727 as a pharmacodynamic biomarker of CDK8/19 inhibition. It shows context-dependent modulation of WNT and STAT-regulated gene expression. Potential therapeutic applications include cancers with WNT pathway activation (e.g., colorectal, breast) and other diseases involving CDK8/19. [1][2]

C23H24CLN5O

421.9226

421.166

1661839-45-7

77050682

White to off-white solid powder

1.4±0.1 g/cm3

709.7±60.0 °C at 760 mmHg

383.0±32.9 °C

0.0±2.3 mmHg at 25°C

1.701

2.53

1

4

3

30

617

0

CN1C=C(C=N1)C2=CC=C(C=C2)C3=CN=CC(=C3N4CCC5(CCNC5=O)CC4)Cl

LBFYQISQYCGDDW-UHFFFAOYSA-N

InChI=1S/C23H24ClN5O/c1-28-15-18(12-27-28)16-2-4-17(5-3-16)19-13-25-14-20(24)21(19)29-10-7-23(8-11-29)6-9-26-22(23)30/h2-5,12-15H,6-11H2,1H3,(H,26,30)

8-(3-chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

CCT251545; CCT 251545; CCT-251545;

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 : ≥ 50 mg/mL (~118.51 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 16.7 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.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (3.96 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (3.96 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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