Purpose: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity.Experimental Design: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay.Results: CCT244747 inhibited cellular CHK1 activity (IC 50 29-170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G 2 arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma.Conclusion: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine–piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

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CHK1 is a serine/threonine kinase that is activated in response to single strand breaks (SSBs) in DNA caused by either direct DNA damage (e.g. by genotoxic chemotherapeutic agents) or replication stress. Activation of CHK1 initiates a signaling cascade culminating in cell cycle arrest leading to DNA repair, senescence or death. Inhibition of CHK1 abrogates cell cycle arrest, inhibits DNA repair and enhances tumor cell death following DNA damage by a range of chemotherapeutic agents. Cells lacking intact G1 checkpoints through inactivation of p53 are particularly dependent on S and G2/M checkpoints and therefore expected to be more sensitive to genotoxic treatment in the presence of a CHK1 inhibitor, whereas normal cells with functional G1 checkpoints would be predicted to undergo less cell death. Thus CHK1 is a validated therapeutic target for cancer treatment. In a collaborative program with Sareum Ltd (Cambridge, UK) we used structure-based design to progress fragment hits to potent inhibitors of CHK1, including our recently published inhibitor SAR-020106. Guided by both biochemical and molecular pharmacological studies, we identified a lead series with pM to nM activity against the CHK1 enzyme in vitro and high selectivity vs CHK2 (200- to >500-fold) and CDK1. The lead series compounds have both good in vitro ADME and in vivo pharmacokinetic (PK) properties. From this lead series we identified CCT244747 as a potent and selective CHK1 inhibitor with oral efficacy. Specifically, CCT244747 is an 8nM inhibitor of CHK1 with >1000-fold selectivity versus CDK1 and CHK2 and excellent selectivity in a kinase panel screen of 120 kinases (only 8/120 with > 80% inhibition at 10μM). CCT244747 shows sub-micromolar activity in a cell-based checkpoint abrogation assay and potentiates the cytotoxicity of a selection of genotoxic chemotherapeutics including gemcitabine in colon, pancreatic and lung human tumor cell lines. CCT244747 has oral bioavailability of 61% in mice with a plasma half-life of approximately 1 hour. Further PK/PD evaluation demonstrated biomarker modulation in human tumor xenografts (pSer296 CHK1) at 6 and 24 hours post CCT244747 administration, in combination with gemcitabine. Efficacy studies of CCT244747 in combination with both irinotecan and gemcitabine, showed significant oral antitumor activity. For example, in the HT29 human colon tumor xenograft model the average percentage treated/control (%T/C) tumor weights were 15.4% for gemcitabine +CCT244747 combined, versus 62.5% and 88.4%, for gemcitabine and CCT244747 alone, respectively as measured on day 18 of therapy. There is now also evidence that CHK1 inhibitors may have single agent activity in certain cancers. In particular, CHK1 was recently identified in an RNAi screen as a therapeutic target in MYCN amplified neuroblastoma and we have shown significant efficacy of CCT244747 in a MYCN-driven transgenic mouse model of neuroblastoma (TH-MYCN) using MRI tumor volume measurements and explant tumor weights as measures of outcome. In summary, we have identified CCT244747 as a novel, potent and selective inhibitor of CHK1, which demonstrates biomarker modulation and efficacy both alone in neuroblastoma and in combination with genotoxic chemotherapeutic agents in common solid tumors, when delivered by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A228.