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.
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.
Probe Selectivity in Vitro:
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
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 protein and potency (cells):
none reported
Probe Selectivity in Cell:
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
BALB/c mice IV: 5 min (10 mg/kg)BALB/c mice oral: 30 min (10 mg/kg PO)
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.