CCT251545

Reagent Authentication Certificate:

Chemical Probes Portal CCT251545.pdf

Protein target name:

CDK8, CDK19

Mechanism of action:

ATP-competitive inhibitor

Recommended Concentration for use in cells:

35-350 nM

Probe Availability

MedChem Express (1661839-45-7)

Cayman (1661839-45-7)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

CMGC

HUGO ID:

HGNC:1779 HGNC:19338

Entrez gene ID:

1024 23097

UNIPROT ID:

Q9BWU1 P49336

Target alias:

Cyclin dependent kinase 8, K35, Cyclin dependent kinase 19, CDC2L6, CDK11, bA346C16.3

Probe Details

Chemical Probes Portal ID:

CPP768

Probe alias:

CCT251545

PubChem CID:

77050682

ChEMBL ID:

CHEMBL3408213

SMILES string:

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

InChi Key:

LBFYQISQYCGDDW-UHFFFAOYSA-N

Control compound:

Compound 7 and 9 (Nat. Chem. Biol. 2015, 11, 973-980)

Orthogonal probe:

MSC2530818Cortistatin A

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

Concentrations up to 10 mM in DMSO

Potential reactivities:

None known

NMR:

1H NMR (500 MHz, CDCl3) δ 1.33−1.39 (m, 2H), 1.94−2.01 (m, 4H), 2.71−2.79 (m, 2H), 3.14−3.19 (m, 2H), 3.28 (t, J = 6.8 Hz, 2H), 3.97 (s, 3H), 5.58 (br s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.68 (s, 1H), 7.82 (s, 1H), 8.22 (s, 1H), 8.44 (s, 1H)

13C NMR (126 MHz, CDCl3) δ 31.6, 32.4, 38.7, 39.3, 41.8, 47.8, 122.7, 125.6, 127.2, 128.3, 129.8, 132.3, 133.7, 135.7, 136.9, 149.6, 150.9, 152.9, 181.6

Storage Recommendations

Storage (dry powder):

Room temperature

Storage (solution):

-20 oC for a year

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

CDK8: 5 nM, CDK19: 6 nM

Potency assay:

Reporter Displacement Assay for CDK8 and CDK19 provided by Proteros Biostructures GmbH. The assay is based upon the competitive displacement of a reporter probe designed to selectively target the ATP binding site of CDK8 or CDK19 with a fast binding kinetic signature. Binding of the probe to its target results in the emission of an optical signal. Competitive displacement of the probe by the corresponding compounds results in a loss of the optical signal that can be quantified with increasing compound concentrations. Methods Enzymol. 493, 299–320 (2011)

PDB ID for probe-target interaction (3D structure):

5BNJ

Structure-activity relationship:

Structure-activity relationship is available in J. Med. Chem. 2015, 58, 1717-1735, Nat. Chem. Biol. 2015, 11, 973-980 and J. Med. Chem. 2016, 59, 1078-1101. The SAR was determined using the 7dF3, LS174T reporter assays and a CDK8 biochemical assay.

Off target activity

Potency (off target):

IC50

Off target protein and potency:

GSK3alpha: 462 nMGSK3beta: 690 nMPRKCQ: 122 nM

Potency assay (off target):

The probe was tested at 1 uM across 293 kinases in the Millipore panel (activity assay); the probe inhibited 6 out the 293 kinases tested with more than 50%. MKK7beta was inhibited at 68%, LCK and PKG1beta were determined to have IC50 >10 uM and 3 off target kinases (GSK3alpha, GSK3beta and PRKCQ) were confirmed by IC50 determination (462 nM, 690 nM and 122 nM respectively).

Probe Selectivity in Vitro:

The probe was tested at 10 uM across a panel of 55 receptors, ion channels and enzymes (CEREP). The probe inhibited 2 of the 55 proteins with >50%. k(KOP) and 5-HT transporter(h) were inhibited with 72 and 70 % respectively. IC50 were determined k(KOP) IC50=4.4 uM, 5-HT transporter (h) IC50=3.6 uM

In cell validation

On target activity in cells

Potency in cells:

IC50

7dF3 5 nM, LS174T 23 nM

Potency assay (cells):

Cell-based potency was assessed in 7dF3 cells harbouring a WNT-dependent luciferase reporter (7dF3 is a variant of HEK293 cells containing both estrogen receptor-DSH and TCF-luciferase-IRES-GFP constructs). Cell-based potency was also assessed in LS174T cells bearing a WNT-dependent luciferase reporter.

Target engagement assay (cells):

Concentration-dependent inhibition of CDK8-dependent phosphorylation of STAT1 in SW620 colorectal cancer cells (phospho-STAT1 IC50=23nM)

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

CCT251545 is not cytotoxic (GI50 >30 uM) in 2D cell proliferation assays (96h) in 9 out of 10 colon cancer cell lines. (See Clarke, P. et al, eLife 2016;10.7554/eLife.20722 for full data set)

In vivo validation

Organism:

Female NMRI miceMale Wistar Rat

Dose:

0.2 mg/kg (IV)0.5 mg/kg (oral)

Route of delivery:

OralIntravenous

Plasma half life:

Mice: 0.55 hRat: 0.97 h

Systemic clearance:

mice: 31 mL/min/kg, rat : 26 mL/min/kg

Ratio of probe in organ to plasma (O:P):

0.63 after 2 h, 3.90 after 6 h

Fraction of probe bound to plasma protein:

mice ppb: 95rat ppb: 97

Cmax:

mice: 935 ng/mL (IV); 276 ng/mL (oral)rat: 735 ng/mL (IV); 223 ng/mL (oral)

Tmax:

mice: 0.1 h (IV); 0.25 (oral)rat: 0.1 h (IV); 0.25 (oral)

Organ of Interest

Organ (O):

SW620 human colon cancer xenograft

Target engagement assay:

NCr athymic mice bearing established SW620 xenografts were treated with CCT251545 at 70 mg/kg bid for days 0-7 and 10-14. Target engagement was demonstrated by clear reduction of phospho-STAT1(SER727) level at 2 and 6 h after the last dose.

Primary Reference

Reference (doi):

10.1038/nchembio.1952 10.1021/jm501436m 10.1021/acs.jmedchem.5b01685

Reference (PMID):

26502155 25680029 26796641

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells and in model organisms.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

CCT251545 was optimized from a phenotypic screening hit for inhibitors of WNT pathway activity. Impressively, this optimization was done without the benefit of structure-based design. CCT251545 has potent cell-based activity and good oral pharmokinetics (PK). The cellular targets of CCT251545 were subsequently determined to be CDK8/19 using SILAC-based affinity capture and rationalized by X-ray crystallography. The affinity-capture experiment suggests very high specificity for CDK8/19. However, direct kinase selectivity profiling experiments did not include the positive controls of CDK8 or CDK19. Therefore, a specificity score cannot be determined nor can it be shown that the compound was active in the assay. Efforts to connect CDK8/19 binding to cellular activity (e.g., WNT reporter gene-assay inhibition) would benefit from the use of a dominant drug resistant allele of CDK8/19. Such an allele could be expressed in cells to circumvent CDK8/19 inhibitory activity of CCT251545 and provide strong evidence that modulation of CDK8/19 by CCT251545 accounts for the observed cellular activity. Regardless, CCT251545 is a potent inhibitor of CDK8/19, has good PK properties and is expected to be highly specific for CDK8/19.

Mar 13 2017 - 9:58am

CCT251545 is a high-affinity ligand of CDK8 and CDK19 with biochemical IC50s for these targets of 7 and 6 nM, respectively. The compound exhibits high selectivity compared with other targets, as demonstrated by proteomics data (SILAC), and suggested by testing in an expanded kinase panel. CCT251545 exhibits potent target engagement in cells (reduction of phospho-STAT1-Ser727; IC50=9 nM) at concentrations consistent with those that reduced WNT-reporter activity in the same cell lines. In vivo pharmacokinetic properties were investigated in mice, rats, and dogs and found adequate for in vivo testing (moderate clearance and F >50% at a low oral dose of 0.5 mg/kg). CCT25145 inhibits the growth of MMTV-WNT-1 breast cancers transplanted from genetically engineered mice as well as APC-mutant SW620 human colorectal cancer xenografts, consistent with in vivo pharmacodynamic data (reduction of p-STATSer727) and at exposure levels that exceed cellular GI50's. Taken together, CCT251545 is a well suited and selective probe for CDK8/19 with proven cellular and in vivo target engagement and efficacy.

Mar 18 2017 - 8:40pm

CCT251545

Probe Availability

Probe Information

Target Details

Cyclin dependent kinase 8, K35, Cyclin dependent kinase 19, CDC2L6, CDK11, bA346C16.3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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CCT251545

Probe Availability

Probe Information

Target Details

Cyclin dependent kinase 8, K35, Cyclin dependent kinase 19, CDC2L6, CDK11, bA346C16.3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?