[show abstract][hide abstract] ABSTRACT: Checkpoint kinase 1 (Chk1) inhibition sensitizes pancreatic cancer cells and tumors to gemcitabine. We hypothesized that Chk1 inhibition would sensitize pancreatic cancer stem cells to gemcitabine. We tested this hypothesis by using two patient-derived xenograft models (designated J and F) and the pancreatic cancer stem cell markers CD24, CD44, and ESA. We determined the percentage of marker-positive cells and their tumor-initiating capacity (by limiting dilution assays) after treatment with gemcitabine and the Chk1 inhibitor, AZD7762. We found that marker-positive cells were significantly reduced by the combination of gemcitabine and AZD7762. In addition, secondary tumor initiation was significantly delayed in response to primary tumor treatment with gemcitabine + AZD7762 compared with control, gemcitabine, or AZD7762 alone. Furthermore, for the same number of stem cells implanted from gemcitabine- versus gemcitabine + AZD7762-treated primary tumors, secondary tumor initiation at 10 weeks was 83% versus 43%, respectively. We also found that pS345 Chk1, which is a measure of DNA damage, was induced in marker-positive cells but not in the marker-negative cells. These data demonstrate that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic cancer stem cells. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in stem cells than in non-stem cells suggests that Chk1 inhibition may selectively sensitize pancreatic cancer stem cells to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy.
Neoplasia (New York, N.Y.) 06/2012; 14(6):519-25. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.
Journal of Medicinal Chemistry 05/2012; 55(11):5130-42. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinase 1 (Chk1, CHEK1) is a Ser/Thr protein kinase that plays a key role in mediating the cellular response to DNA-damage. Synthesis and evaluation of a previously described class of Chk1 inhibitors, triazoloquinolones/triazolones (TZs) is further described herein. Our investigation of structure-activity relationships led to the identification of potent inhibitors 14c, 14h and 16e. Key challenges included modulation of physicochemical properties and pharmacokinetic (PK) parameters to enable compound testing in a Chk1 specific hollow fiber pharmacodynamic model. In this model, 16e was shown to abrogate topotecan-induced cell cycle arrest in a dose dependent manner. The demonstrated activity of TZs in this model in combination with a chemotherapeutic agent as well as radiotherapy validates this series of Chk1 inhibitors. X-ray crystal structures (PDB code: 2YEX and 2YER) for an initial lead and an optimized analog are also presented.
[show abstract][hide abstract] ABSTRACT: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models.
We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1.
AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism.
AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1.
Clinical Cancer Research 06/2011; 17(11):3706-15. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint Kinase-1 (Chk1, CHK1, CHEK1) is a Ser/Thr protein kinase that mediates cellular responses to DNA-damage. A novel class of Chk1 inhibitors, triazoloquinolones/triazolones (TZ's) was identified by high throughput screening. The optimization of these hits to provide a lead series is described.
[show abstract][hide abstract] ABSTRACT: The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer.
Cancer Research 06/2010; 70(12):4972-81. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inhibition of checkpoint kinase 1 has been shown to enhance the cytotoxicity of DNA-damaging targeted chemotherapy through cell cycle checkpoint abrogation and impaired DNA damage repair. A novel checkpoint kinase 1/2 inhibitor, AZD7762, was evaluated for potential enhancement of radiosensitivity for human tumor cells in vitro and in vivo xenografts.
Survival of both p53 wild-type and mutant human cell lines was evaluated by clonogenic assay. Dose modification factors (DMF) were determined from survival curves (ratio of radiation doses for control versus drug treated at 10% survival). Flow cytometry, Western blot, and radiation-induced tumor regrowth delay assays were conducted.
AZD7762 treatment enhanced the radiosensitivity of p53-mutated tumor cell lines (DMFs ranging from 1.6-1.7) to a greater extent than for p53 wild-type tumor lines (DMFs ranging from 1.1-1.2). AZD7762 treatment alone exhibited little cytotoxicity to any of the cell lines and did not enhance the radiosensitivity of normal human fibroblasts (1522). AZD7762 treatment abrogated radiation-induced G(2) delay, inhibited radiation damage repair (assessed by gamma-H2AX), and suppressed radiation-induced cyclin B expression. HT29 xenografts exposed to five daily radiation fractions and to two daily AZD7762 doses exhibited significant radiation enhancement compared with radiation alone.
AZD7762 effectively enhanced the radiosensitivity of mutated p53 tumor cell lines and HT29 xenografts and was without untoward toxicity when administered alone or in combination with radiation. The results of this study support combining AZD7762 with radiation in clinical trials.
Clinical Cancer Research 03/2010; 16(7):2076-84. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinase 1 (Chk1) regulates cell cycle checkpoints and DNA damage repair in response to genotoxic stress. Inhibition of Chk1 is an emerging strategy for potentiating the cytotoxicity of chemotherapeutic drugs. Here, we demonstrate that AZD7762, an ATP -competitive Chk1/2 inhibitor induces gammaH2AX in gemcitabine-treated cells by altering both dynamics and stability of replication forks, allowing the firing of suppressed replication origins as measured by DNA fiber combing and causing a dramatic increase in DNA breaks as measured by comet assay. Furthermore, we identify ATM and DNA-PK, rather than ATR, as the kinases mediating gammaH2AX induction, suggesting AZD7762 converts stalled forks into double strand breaks (DSBs). Consistent with DSB formation upon fork collapse, cells deficient in DSB repair by lack of BRCA2, XRCC3 or DNA-PK were selectively more sensitive to combined AZD7762 and gemcitabine. Checkpoint abrogation by AZD7762 also caused premature mitosis in gemcitabine-treated cells arrested in G(1)/early S-phase. Prevention of premature mitotic entry via Cdk1 siRNA knockdown suppressed apoptosis. These results demonstrate that chemosensitization of gemcitabine by Chk1 inhibition results from at least three cellular events, namely, activation of origin firing, destabilization of stalled replication forks and entry of cells with damaged DNA into lethal mitosis. Additionally, the current study indicates that the combination of Chk1 inhibitor and gemcitabine may be particularly effective in targeting tumors with specific DNA repair defects.
[show abstract][hide abstract] ABSTRACT: Pharmacokinetic/pharmacodynamic (PK/PD) models have been shown to be useful in predicting tumor growth rates in mouse xenografts. We applied novel PK/PD models to the published anticancer combination therapies of tumor growth inhibition to simulate synergistic changes in tumor growth rates. The parameters from the PK/PD model were further used to estimate clinical doses of the combination.
A PK/PD model was built that linked the dosing regimen of a compound to the inhibition of tumor growth in mouse xenograft models. Two subsequent PK/PD models were developed to simulate the published tumor growth profiles of combination treatments. Model I predicts the tumor growth curve assuming that the effect of two anticancer drugs, AZD7762 and irinotecan, is synergistic when given in combination. Model II predicts the tumor growth curve assuming that the effect of co-administering flavopiridol and irinotecan is maximally synergistic when dosed at an optimal interval.
Model I was able to account for the synergistic effects of AZD7762 following the administration of irinotecan. When Model II was applied to the antitumor activity of irinotecan and flavopiridol combination therapy, the modeling was able to reproduce the optimal dosing interval between administrations of the compounds. Furthermore, Model II was able to estimate the biologically active dose of flavopiridol recommended for phase II studies.
The timing of clinical combination therapy doses is often selected empirically. PK/PD models provide a theoretical structure useful in the design of the optimal clinical dose, frequency of administration and the optimal timing of administration between anticancer agents to maximize tumor suppression.
Cancer Chemotherapy and Pharmacology 10/2009; 66(2):245-54. · 2.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinase 1 (Chk1), a serine/threonine kinase, functions as a regulatory kinase in cell cycle progression and is a critical effector of the DNA-damage response. Inhibitors of Chk1 are known to sensitise tumours to a variety of DNA-damaging agents and increase efficacy in preclinical models.
The most advanced agents are now in Phase I clinical trials; the preclinical profiles of these drugs are compared and contrasted, together with a discussion of some of the opportunities and challenges facing this potentially revolutionary approach to cancer therapy.
A review of the publications and presentations on XL-844, AZD7762 and PF-477736.
Chk kinases are part of the DNA damage recognition and response pathways and as such represent attractive targets. Agents that target checkpoint kinases have demonstrated impressive evidence preclinically that this approach will provide tumour-specific potentiating agents and may have broad therapeutic utility.
[show abstract][hide abstract] ABSTRACT: Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings.
Molecular Cancer Therapeutics 10/2008; 7(9):2955-66. · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.
[show abstract][hide abstract] ABSTRACT: Insights from cell cycle research have led to the hypothesis that tumors may be selectivity sensitized to DNA-damaging agents, resulting in improved antitumor activity and a wider therapeutic margin. The theory relies primarily on the observation that the majority of tumors are deficient in the G(1)-DNA damage checkpoint pathway, resulting in reliance on S and G(2) phase checkpoints for DNA repair and cell survival. The S and G(2) phase checkpoints are predominantly regulated by checkpoint kinase 1; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G(1) checkpoint signaling pathway that allows for DNA repair and cell survival. There is now a large body of preclinical evidence showing that checkpoint kinase inhibitors do indeed enhance the efficacy of both conventional chemotherapy and radiotherapy, and several agents have recently entered clinical trials. Excitingly, additional therapeutic opportunities for checkpoint kinase inhibitors continue to emerge as biology outside their pivotal role in cell cycle arrest is further elucidated.
Clinical Cancer Research 08/2008; 14(13):4032-7. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint kinase 1 (CHK1) is a member of the serine/ threonine kinase family. CHK1 functions as a regulatory kinase in cell-cycle progression and is the main effector of theDNA-damage response within the cell. Over the past few years, a large number of novel inhibitors of CHK1 have been discovered that encompass an enormous area of chemical space and diversity and, in more recent reports, many of these inhibitors have been demonstrated preclinically to sensitize tumors to a wide variety of DNA-damaging agents. This review focuses on advances reported both in the literature and at conferences from 2005 to date concerning the chemical design and optimization of checkpoint kinase inhibitors for the treatment of cancer.
Current opinion in drug discovery & development 08/2007; 10(4):473-86. · 5.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: N-terminal methionine removal is an important cellular process required for proper biological activity, subcellular localization, and eventual degradation of many proteins. The enzymes that catalyze this reaction are called Methionine Aminopeptidases (MAPs). To date, only two MAP family members, MAP1A and MAP2, have been well characterized and studied in mammals. In our studies, we have cloned a full length MAP1D gene. Expression and purification of full length recombinant protein shows that the sequence encodes an enzyme with MAP activity. MAP1D is overexpressed in colon cancer cell lines and in colon tumors as compared to matched normal tissue samples. Downregulation of MAP1D expression by shRNA in HCT-116 colon carcinoma cells reduces anchorage-independant growth in soft agar. These data suggest that MAP1D is a potentially oncogenic, novel member of the MAP gene family that may play an important role in colon tumorigenesis.
[show abstract][hide abstract] ABSTRACT: Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
Cancer Cell 08/2004; 6(1):33-43. · 24.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: A virtual screen of a subsection of the AstraZeneca compound collection was performed for checkpoint kinase-1 (Chk-1 kinase) using a knowledge-based strategy. This involved initial filtering of the compound collection by application of generic physical properties followed by removal of compounds with undesirable chemical functionality. Subsequently, a 3-D pharmacophore screen for compounds with kinase binding motifs was applied. A database of approximately 200K compounds remained for docking into the active site of Chk-1 kinase, using the FlexX-Pharm program. For each compound that docked successfully into the binding site, up to 100 poses were saved. These poses were then postfiltered using a customized consensus scoring scheme for a kinase, followed by visual inspection of a selection of the docked compounds. This resulted in 103 compounds being ordered for testing in the project assay, and 36 of these (corresponding to four chemical classes) were found to inhibit the enzyme in a dose-response fashion with IC(50) values ranging from 110 nM to 68 microM.
Journal of Medicinal Chemistry 05/2004; 47(8):1962-8. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.
Journal of Biological Chemistry 01/2004; 278(52):52964-71. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bengamide B, a novel sponge-derived marine natural product with broad spectrum antitumor activity, was not suitable for further preclinical development because of its difficult synthesis and very poor water solubility. Bengamide B produced a 31% T/C at its solubility-limited maximum intravenous dose of 33 micromol/kg in MDA-MB-435 breast carcinoma implanted subcutaneously as a xenograft in nude mice. Compound 8a, a bengamide B analogue with three structural changes (t-Bu alkene substituent, unsubstituted lactam nitrogen, and inverted lactam 5'-myristoyloxy group), was as potent as bengamide B in vitro and more efficacious than bengamide B in vivo. A series of ester-modified analogues based on 8a were synthesized and tested in vitro and in vivo (MDA-MB-435). The cyclohexyl- and phenethyl-substituted esters, 8c and 8g, respectively, had in vitro and in vivo activities similar to that of 8a and enhanced water solubility (ca. 1 mg/mL). Consequently, 8c and 8g were tested in the MDA-MB-435 xenograft model at 100 micromol/kg and produced 29% and 57% tumor regression, respectively.
Journal of Medicinal Chemistry 11/2001; 44(22):3692-9. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Total syntheses of the cytotoxic marine natural products bengamides B and E are described. Both bengamides are prepared via amide coupling of a protected polyhydroxylated lactone intermediate 9 with a suitably substituted aminocaprolactam intermediate. Lactone 9 is prepared in five steps from commercially available alpha-D-glucoheptonic gamma-lactone. The key reactions are a selective deprotection of a 1,2-acetonide in the presence of a 1,3-acetonide and an (E)-selective olefination of an unstable aldehyde using a gem-dichromium reagent. The bengamide B lactam intermediate 10 is prepared in seven steps from commercially available (5R)-5-hydroxy-L-lysine (12). The desired S-configuration at the gamma-OH lactam position is established using the Mitsunobu reaction.
The Journal of Organic Chemistry 04/2001; 66(6):2118-22. · 4.56 Impact Factor