CCT244747

Protein target name:

CHEK1

Mechanism of action:

ATP-competitive inhibitor

Recommended Concentration for use in cells:

30 - 1000 nM

Probe Availability

MedChem Express (1404095-34-6)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

CAMK

HUGO symbol:

CHEK1

Entrez gene ID:

1111

Target alias:

Checkpoint kinase 1, CHK1

Probe Details

Chemical Probes Portal ID:

CPP693

Probe alias:

CCT244747, CHEMBL2203843, GTPL8215, SCHEMBL14948526, SYN1216, C20H24N8O2

PubChem CID:

54758482

ChEMBL ID:

CHEMBL2203843

SMILES string:

CC(CN(C)C)OC1=NC(=CN=C1C#N)NC2=NC=C(C(=C2)OC)C3=CN(N=C3)C

InChi Key:

IENLGMOXAQMNEH-CYBMUJFWSA-N

Control compound:

Not available

Orthogonal probe:

MK-8776 (SCH 900776)

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 8.23 (br s, 1H), 7.85 (s, 1H), 7.58 (s, 1H), 7.47 (s, 1H), 7.03 (s, 1H), 5.48−5.37 (m, 1H), 4.72 (q, J = 5 Hz, 1H), 3.98 (s, 3H), 2.90 (d, J = 5 Hz, 3H), 2.74 (dd, J = 13.4, 7.2 Hz, 1H), 2.51 (dd, J = 13.4, 4.4 Hz, 1H), 2.31 (s, 6H), 1.41 (d, J = 6.3 Hz, 3H).

Storage Recommendations

Storage (dry powder):

room temperature

Storage (solution):

-20 oC for storage over periods greater than 2 days.

Storage sensitivities:

Potentially sensitive to prolonged exposure to bright light.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

7.7 nM

Potency assay:

Inhibition of recombinant full-length CHK1 kinase activity was measured in a microfluidic assay that monitored the separation of a fluorescently labelled, phosphorylated product peptide from the unphosphorylated substrate (5-FAM-KKKVSRSGLYRSPSMPENLNRPR-COOH).

Structure-activity relationship:

Structure-activity data for inhibition of CHK1 in vitro were determined using a recombinant, full-length CHK1 in a kinase activity assay that monitored the separation of a fluorescently labelled, phosphorylated product peptide from the unphosphorylated substrate and are described in doi: 10.1021/jm3012933. Structure-activity data for selectivity for inhibition of CHK1 versus CHK2 and CDK1 in vitro are also described.

Off target activity

Potency (off target):

IC50

Off target protein and potency:

CHK2 >10,000 nMCDK1 >10,000 nM

Potency assay (off target):

Inhibition of recombinant, full-length CHK2 and CDK1 kinase activity was determined in a DELFIA assay measuring peptide substrate phosphorylation. Broad kinase activity screening was carried out using a radiometric assay format against 140 kinases, with CCT244747 tested at a single concentration of 1000 nM. 127 of the 140 kinases showed <50% inhibition at 1000 nM; RSK1/2, AMPK, BRSK1, IRAK1, TRKA were inhibited >80% at 1000 nM.

Selectivity against non-family targets:

We assessed binding/inhibition of eight cardiac ion channels (Millipore Ion Channel CardiacProfilerTM Panel). At 10,000 nM, the probe inhibited the following targets <25%: Nav1.5, hKv4.3/hKChIP2, hCav1.2, hKv1.5, hKCNQ1/hminK, hHCN4, and hKir2.1. HERG IC50 = 5000 nM. In a broad in vitro pharmacology screen of 80 non-family targets (Cerep), the probe inhibited 76 of the 80 proteins <80% at 10,000 nM,

In cell validation

On target activity in cells

Potency in cells:

IC50

29 nM

Potency assay (cells):

CCT244747 was assessed in HT29 human colon cancer cells for the ability to abrogate a G2 checkpoint, cell-cycle arrest induced by the DNA-damaging agent etoposide. The number of cells reaching mitosis was quantified following treatment with etoposide or etoposide + CCT244747.

Target engagement assay (cells):

CCT244747 inhibits SN38- and gemcitabine-induced CHK1 autophosphorylation on serine 296 in HT29 and SW620 human colon cancer cells at 50 nM concentration (measured by immuno-blot). Inhibition of SN38- and gemcitabine-induced phosphorylation of CDK1 tyrosine 15, a downstream component in the CHK1 signalling cascade, is observed from 50-100 nM concentration in these cell lines. Concomitant increases in gamma-H2Ax and C-PARP, indicative of increases in DNA damage and apoptosis, respectively, due to CHK1 inhibition are observed from 50-100 nM concentrations in these cell lines.

Off target activity in cells

Off target protein and potency (cells):

none reported

Selectivity against nonfamily targets (cells):

CHK1 inhibition alone is not expected to be cytotoxic in HT29 and SW620 colon cancer cell lines. Therefore, the ratio of single agent cytotoxicity (GI50; SRB proliferation assay) to the on-target potency for CHK1 inhibition (IC50; G2 checkpoint abrogation) in these cells provides a measure of the compound cellular selectivity. HT29 cellular selectivity = 21-fold; SW620 cellular selectivity = 18-fold.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

CHK1 inhibition alone is not expected to be potently cytotoxic to cancer cell lines except where a specific synthetic lethality with the cell genotype is observed. For cell lines where no synthetic lethality is observed, cytotoxicity (measured by SRB proliferation assay) is low: HT29 colon cancer GI50 = 620 nM; SW20 colon cancer GI50 = 3000 nM; MiaPaCa-2 pancreatic cancer GI50 = 1000 nM; Calu6 non-small cell lung cancer GI50 = 330 nM

In vivo validation

Organism:

CRTac:Ncr-Fox1(nu) athymic miceTH-MYCN hemizygotic transgenic mouse model of neuroblastoma

Dose:

25 - 150 mg/kg

Route of delivery:

Oral

Plasma half life:

BALB/c mice IV (10 mg/kg): 0.67 hBALB/c mice oral (10 mg/kg): 0.92 h

Systemic clearance:

BALB/c mice: 0.125 L/h

Fraction of probe bound to plasma protein:

human PPB: 82%mouse PPB: 76%

Cmax:

BALB/c mice IV: 3620 nM (10 mg/kg)BALB/c mice oral: 980 nM (10 mg/kg)

Tmax:

BALB/c mice IV: 5 min (10 mg/kg)BALB/c mice oral: 30 min (10 mg/kg PO)

Organ of Interest

Organ (O):

HT29 human colon cancer xenograft

Target engagement assay:

Target engagement was assessed in vivo by measuring inhibition of gemcitabine-induced CHK1 autophosphorylation on serine 296 (immuno-blot) in HT29 human colon cancer xenografts in CRTac:Ncr-Fox1(nu) athymic mice. At 24 h after gemcitabine +/- CCT244747 treatment, pS296 CHK1 was reduced ~4-fold (p < 0.05) by 100 and 150 mg/kg CCT244747.

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

47 at 12 h following 100 mg/kg oral dose

Primary Reference

Reference (doi):

doi: 10.1158/1078-0432.CCR-12-1322 doi: 10.1021/jm3012933 doi: 10.1158/1535-7163.MCT-15-0998

Reference (PMID):

PMID: 2292 PMID: 2308 PMID: 2742

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

The IC50 for the target kinase, CHK1, is 7.7 nM. The inhibitor was tested versus panel of 140 kinases and showed 50% inhibition against 127 of them and 80% inhibition against 5 at 1,000 nM; no IC50s for these off-target kinases were reported.

In vivo pharmacokinetic (PK) studies show good oral availability, with a more extended half-life compared to iv dosing. Accumulation in tumors was measured. Efficacy of gemcitabine and irinotecan was enhanced in several tumour xenograft models, while CCT244747 was active as a single agent in MYCN-driven neuroblastoma (significant shrinkage after 7 days at 100 mg/kg oral administration). Activity is likely due to on-target activity, but 5 other kinases in a panel of 140 were inhibited >50% at 1 uM; at the high concentrations dosed (75-150 mg/kg PO), possible effects through the inhibition of additional targets can not be ruled out.

CCT2444747 is a potent CHK1 inhibitor (IC50=7.7 nM) with high selectivity over CHK2 and CDK1 (IC50 >10 uM). Depending on the cell line, off-target antiproliferative effects can be seen at fairly low concentrations (e.g., HT29 620 nM; Calu6 330 nM; 21-35-fold window with respect to on-target effects). The compound exhibits >50% inhibition at 1 uM against RSK1, RSK2, AMPK, BRSK1, IRAK1 and TRKA; therefore, these kinases could be inhibited when the compound is dosed in mouse models at the higher 150 mg/kg dose (plasma Cmax likely to be >>1 uM; only 12 h data available for higher doses) . The compound is moderately bound to mouse plasma proteins (24% unbound) and has very high iv clearance (higher than liver blood flow when dosed at 10 mg/kg), yet has good oral exposure, suggesting saturation of metabolism and non-linear PK.

CCT244747

Probe Availability

Probe Information

Target Details

Checkpoint kinase 1, CHK1

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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CCT244747

Probe Availability

Probe Information

Target Details

Checkpoint kinase 1, CHK1

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?