Mark Joseph Chicarelli

Single-agent inhibition of Chk1 is antiproliferative in human cancer cell lines in vitro and inhibits tumor xenograft growth in vivo.

Chk1 is a serine/threonine kinase that plays several important roles in the cellular response to genotoxic stress. Since many current standard-of-care therapies for human cancer directly damage DNA or inhibit DNA synthesis, there is interest in using small molecule inhibitors of Chk1 to potentiate their clinical activity. Additionally, Chk1 is known to be critically involved in cell cycle progression of unperturbed cells. Therefore, it is plausible that treatment with a Chkl inhibitor alone could also be an efficacious cancer therapy. Here we report that Chk1-A, a potent and highly selective small molecule inhibitor of Chk1, is antiproliferative as a single agent in a variety of human cancer cell lines in vitro. The inhibition of proliferation is associated with collapse of DNA replication and apoptosis. Rapid decreases in inhibitory phosphorylation of CDKs and a concomitant increase in CDK kinase activity and chromatin loading of Cdc45 suggest that the antiproliferative and proapoptotic activity of Chk1-A is at least in part due to deregulation of DNA synthesis. We extend these in vitro studies by demonstrating that Chk1-A inhibits the growth of tumor xenografts in vivo in a treatment regimen that is well tolerated. Together, these results suggest that single-agent inhibition of Chk1 may be an effective treatment strategy for selected human malignancies.

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

Identification of a New Class of Glucokinase Activators through Structure-Based Design.

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high glucose concentrations. Glucose flux through GK also contributes to reducing hepatic glucose output. Because many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, compounds that can activate GK may serve as effective treatments for type 2 diabetes. Herein we report the identification and initial optimization of a novel series of allosteric glucokinase activators (GKAs). We discovered an initial thiazolylamino pyridine-based hit that was optimized using a structure-based design strategy and identified 26 as an early lead. Compound 26 demonstrated a good balance of in vitro potency and enzyme kinetic parameters and demonstrated blood glucose reductions in oral glucose tolerance tests in both C57BL/6J mice and high-fat fed Zucker diabetic fatty rats.

C5-Alkyl-2-methylurea-Substituted Pyridines as a New Class of Glucokinase Activators

Glucokinase (GK) activators represent a class of type 2 diabetes therapeutics actively pursued due to the central role that GK plays in regulating glucose homeostasis. Herein we report a novel C5-alkyl-2-methylurea-substituted pyridine series of GK activators derived from our previously reported thiazolylamino pyridine series. Our efforts in optimizing potency, enzyme kinetic properties, and metabolic stability led to the identification of compound 26 (AM-9514). This analogue showed a favorable combination of in vitro potency, enzyme kinetic properties, acceptable pharmacokinetic profiles in preclinical species, and robust efficacy in a rodent PD model.

Abstract 3874: Single-agent Chk1 inhibition is anti-proliferative in leukemia cells in vitro and in vivo

Chk1 is a serine/threonine kinase that plays important roles in the cellular response to genotoxic stress. For this reason, there is a great deal of interest in using inhibitors of Chk1 to potentiate the effects of DNA-damaging chemotherapeutics. In addition, multiple studies have demonstrated that Chk1 activity is essential during an unperturbed cell cycle to ensure proper DNA replication and maintain genomic integrity. Therefore, it is plausible that a Chk1 inhibitor could also be efficacious as a single-agent therapeutic for human cancer. Here we show that treatment with Chk1-A, a potent and selective inhibitor of Chk1, alone is anti-proliferative against a wide array of cancer cell lines with varying degrees of potency. We sought to understand the mechanisms by which Chk1 inhibition derives the observed anti-proliferative effect. Employing the human leukemia cell line HEL92.1.7, a line particularly sensitive to Chk1 inhibition in terms of proliferation, we characterized the biochemical and functional effects of Chk1-A treatment. We observed concentration-dependent increases in phosphorylation of H2A. X, Chk1, and Chk2, which are markers of DNA damage and cell-cycle checkpoint activation. These biochemical events correlated with S-phase accumulation and eventual apoptosis. In vivo, we found that HEL92.1.7 tumor xenografts were sensitive to oral administration of Chk1-A at a dose that was well tolerated. Together, these studies suggest that inhibition of Chk1 results in DNA damage that induces apoptosis and that use of a Chk1 inhibitor as a single-agent could be an effective strategy to treat certain types of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3874.

Abstract B254: Extended target‐coverage by selective Chk1 inhibitors enhances pharmacodynamic inhibition of Chk1 signaling and antitumor activity in vivo

Loss of coordination between cell cycle checkpoints and DNA damage repair is a fundamental feature tumor cells rely on for unregulated growth and developing chemotherapeutic resistance. The protein kinase Checkpoint kinase 1 (Chk1) is a sentinel molecule essential for cell cycle arrest at the S and G2M checkpoints, as well as regulating homologous recombination DNA repair. In tumor cells exposed to chemotherapy, Chk1 inhibition overrides cell cycle arrest and DNA repair functions, effectively driving tumor cells into a state of mitotic catastrophe and, ultimately, cell death. We have previously reported in schedule‐dependence studies, using Chk1 inhibitors and irinotecan (CPT‐11), that oral administration of Chk1 inhibitors allows for multi‐day target‐coverage, and thus continuous inhibition of Chk1 for a finite period of time, which maximizes anti‐tumor efficacy. Here, we extend these studies and investigate the pharmacodynamic relationship to efficacy, as well as the specific biomarkers that are predictive of an anti‐tumor effect, when Chk1 inhibitors are administered on a multi‐day dosing schedule. Utilizing potent (IC50=24–27nM), selective, and orally bio‐available small molecule Chk1 inhibitors of which Chk1‐A and Chk1‐C are representative, we find only modest inhibition of the functional biomarker phospho‐cdc2, following a single dose of a Chk1 inhibitor. Alternatively, on multi‐day Chk1 inhibitor dose schedules, we find dose‐related pharmacodynamic inhibition of Chk1 signaling that is maximized at doses where we see significant tumor growth inhibition in efficacy experiments. Furthermore, multi‐day dosing of Chk1 inhibitors induces marked inhibition of phospho‐cdc2 and Rad51 protein levels, suggesting tumoricidal activity related to Chk1 inhibition is due to both checkpoint override and impairment of DNA damage repair. In human tumor xenografts administered combination therapy with gemcitabine, an orally‐delivered Chk1 inhibitor dosed on a multi‐day schedule shows superior efficacy over an IV administered compound. Taken together, our findings show a clear correlative relationship between pharmacodynamic target inhibition and anti‐tumor activity that is exclusively achieved on multi‐day dose schedules. These results demonstrate the need for prolonged Chk1 inhibition to provide robust pharmacodynamic inhibition and maximal anti‐tumor efficacy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B254.