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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 (IC50 29–170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G2 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. Clin Cancer Res; 18(20); 5650–61. ©2012 AACR.

PDF file, 79K, Supplementary Table 3 - Mouse body weights for data in Figure 4A Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with gemcitabine (100mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 4 - Mouse body weights for data in Figure 4C Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with irinotecan (25mg/kg ip), CCT244747 (150mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 5 - Mouse body weights for data in Figure 4D Body Weights expressed as a percentage of the weight on day 0 for mice bearing Calu6 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 6 - Mouse body weights for data in Supplementary Table 2 Body Weights expressed as a percentage of the weight on day 0 for mice bearing SW620 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6.

PDF file, 92K, Supplementary Figure 2. A constrained, scaffold docking model of CCT244747 in the ATP pocket of human CHK1 realized using the SAR-020106 X-ray crystal structure (PDB 2ym8).

PDF file, 63KB, Kinase inhibitory profile of SAR-020106 at 10 μMa.

PDF file, 118K, Supplementary Table 2 Summary of Effects of Irinotecan, Gemcitabine and CCT244747 alone or in combination on human tumor xenografts growth delay.

PDF file, 810K, Supplementary Figure 3 Characterisation of the effects of minimally toxic concentrations of CCT244747 alone or in combination with genotoxic agents (+) in HT29 and SW620 colon cancer cell lines. A, HT29 cells were treated with SN38 (100nM) or CCT244747 alone or in combination for 24h. B, SW620 cells were treated with gemcitabine (200nM) or CCT244747 alone or in combination for 24h. Cells were pre-treated with CCT244747 alone 1h prior to cytotoxic exposure. Protein expression was assessed by western blotting (40μg per lane) as described in Figure 2 and Materials and Methods.

PDF file, 76K, Supplementary Table 1 Kinome selectivity profile of CCT244747 CCT244747 was tested at 1 μM (140 enzymes) and 10 μM (121 enzymes) for inhibition of human kinases in a radiometric protein phosphorylation assay (MRC Protein Phosphorylation Unit, Dundee University, UK). Assays were conducted in the presence of ATP at the approximate Km,ATP for each kinase. Results are quoted as % control kinase activity remaining (100% = no inhibition, 0% = complete inhibition). Kinases inhibited by >50% at each concentration are highlighted. n.d. = not determined.

PDF file, 67K, Supplementary Figure 4. Effects of scheduling and exposure times on the ability of two different CHK1 inhibitors to potentiate gemcitabine cytotoxicity in SW620 cells in vitro. A, Effect of different contact times on the capacity of SAR-020106 to enhance gemcitabine cytotoxicity in SW620 cells. Gemcitabine was continuously present (96h) at an IC50 concentration and SAR- 020106 was added at the start of treatment over a range of concentrations for the intervals shown (i.e. 0-T1, 0-T2, 0-T3, etc). Growth delay and potentiation were measured using an SRB assay as described in Materials and Methods. Potentiation Index (PI) was expressed as a percentage of the maximum PI. Values are mean�SE, for n=3-5 independent experiments. B, Treatment of synchronized SW620 cells with gemcitabine and SAR-020106. Cells were synchronized with nocodazole (100ng/ml x 16h). Eight hours following release (at G1/S boundary) cells were treated with a fixed concentration of gemcitabine (GI50) for 96h combined with different concentrations of SAR-020106 for 0-96, 0-48 or 0-24h. Values are mean�SE for 3 independent determinations. C, Effects of contact time on the potentiation of gemcitabine cytotoxicity in synchronized compared to asynchronous SW620 cells. See B and Materials and Methods for further details. Values are mean�SE, n=3-5. D, Effects of different contact times and treatment schedules on the ability of CCT244747 to enhance gemcitabine cytotoxicity in SW620 cells. Values are mean�SE, for 3-7 independent experiments.

PDF file, 36K, Supplementary Figure 5. Effects of acute CCT244747 administration on non-tumor bearing GEMM mouse body weights. CCT244747 was administered at a dose of 150mg/kg p.o. x 7days and mouse body weights were measured daily. Results are mean�SD body weights for 5 mice. There was no apparent body weight loss, toxicity or morbidity following this treatment and hemizygous MYC-N tumor bearing GEMM mice were therefore treated with 100mg/kg CCT244747 p.o. x 7 days (see Figure 5).

PDF file, 58K, Supplementary Figure 1. Comparison of assays for measuring CHK1 potentiation of cytotoxic agents in vitro. A, Determination of CCT244747 GI50 alone or in combination with a fixed concentration (GI50) of gemcitabine or SN38 in SW620 colon tumor cells in vitro. The potentiation index (PI, ratio of SRB GI50 of CCT244747 alone : Combination GI50) was 4.0 and 14.8 for SN38 and gemcitabine, respectively (see Table 1 for mean�SD values). Survival curves for combinations were corrected to 100%. B, Determination of SN38 or gemcitabine GI50 alone or in combination with a fixed concentration of CCT244747 (1μM) in SW620 colon tumor cells in vitro. The potentiation index (PI, ratio of SRB GI50 of genotoxic alone : genotoxic + 1μM CCT244747) was 1.4 and 3.2 for gemcitabine and SN38, respectively. PI values for several independent experiments as shown in B were 1.2�0.058 and 2.9�0.33 (mean�SD, n=4) for SN38 and gemcitabine, respectively.

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Mol Cancer Ther; 9(1); 89–100

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Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was generated by hybridization of two lead scaffolds derived from fragment-based drug design and optimized for CHK1 potency and high selectivity using a cell-based assay cascade. Efficient in vivo pharmacokinetic assessment was used to identify compounds with prolonged exposure following oral dosing. The optimized compound (CCT244747) was a potent and highly selective CHK1 inhibitor, which modulated the DNA damage response pathway in human tumor xenografts and showed antitumor activity in combination with genotoxic chemotherapies and as a single agent.

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1 bound to ADP, revealing the phosphoryl-transfer competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase active conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1 is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1 as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.

Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses.

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Mol Cancer Ther; 9(1); 89–100

High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP–responsive element binding protein–binding protein–associated factor (PCAF) and p300 (1 to >50 μmol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 μmol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2–48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of α-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

There is presently enormous interest in the function and regulatory roles of histone acetyltransferase enzymes. Along with deacetylases it is now evident that these enzymes play a key role in many cellular processes including chromatin remodeling and gene transcription. As such, effective small molecule enzyme inhibitors would be useful tools for molecular pharmacology and may also be suitable for further development into agents for the treatment of diseases such as cancer. A high-throughput assay based on the use of scintillating microplates (FlashPlates) suitable for screening libraries of compounds for inhibitors of acetylase activity is described here. Confirmation of activity of selected compounds is achieved with a conventional filter assay, the details of which are also described. In addition, an assay suitable for confirming that cellular protein acetylation has been altered by inhibition of acetylases or deacetylases is also presented. On the same plate, cells are grown, exposed to compound, fixed, and permeabilized, and protein acetylation is determined using standard ELISA methodology and a europium-labeled second antibody. This latter method provides a medium-throughput alternative to the use of immunoblotting for mechanistic studies.

The role of histone acetyltransferases (HATs) in the regulation of crucial cellular functions, e.g., gene transcription, differentiation, and proliferation, has recently been documented and there is increasing evidence that aberrant expression of these enzymes may have a role to play in the development of the malignant phenotype. The availability of potent and selective small molecule inhibitors of HATs would provide useful proof of principle probes for further validation of these enzymes as drug discovery targets and may also provide lead molecules for clinical drug development. We have developed a microplate assay for HAT activity suitable for high-throughput screening. In the assay, following incubation of histone H3, [3H]acetylCoA, and enzyme (recombinant p300/CBP-associated factor expressed as a glutathione S-transferase fusion protein), radiolabeled histone was captured onto the walls of a scintillating microplate (FlashPlate) generating a scintillation signal. The assay was reproducible, amenable to automation, and generated a wide signal to noise ratio. Although antiacetylated histone antibodies were initially used to capture the radiolabeled product, it was subsequently shown that a signal was effectively produced by histone passively binding to the walls of the FlashPlate. This resulted in a simple "mix and measure" assay that is currently being used for the identification of HAT inhibitors.

Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidin-biotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5,10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.

Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors.

Immunoassays for detecting and measuring organic microcontaminants in water supplies are gaining acceptance and are becoming more widely available. A number of assays for pesticides have been described. Immunoassays especially those utilising enzyme labels are ideally suited for rapid and low cost monitoring of water quality. One of the major attractions of immunoassays for water analyses is the sensitivity that can be achieved without sample preparation. Limits of detection below the EC Maximum Admissible Concentration (0.1 μg/l) are regularly obtained with acceptable assay performance in terms of accuracy and precision. The selectivity of analysis depends on the specificity of the antibody used. Generally antisera are compound or group specific and unlike conventional analyses only one analyte can be measured at one time. Enhanced luminescence has provided an alternative sensitive and robust endpoint for immunoassays of herbicides and can be adapted to produce semi-quantitative results away from the laboratory.

Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure-activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46 (IC(50) 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).

We report herein the first systematic exploration of inhibitors of the mitotic kinase Nek2. Starting from HTS hit aminopyrazine 2, compounds with improved activity were identified using structure-based design. Our structural biology investigations reveal two notable observations. First, 2 and related compounds bind to an unusual, inactive conformation of the kinase which to the best of our knowledge has not been reported for other types of kinase inhibitors. Second, a phenylalanine residue at the center of the ATP pocket strongly affects the ability of the inhibitor to bind to the protein. The implications of these observations are discussed, and the work described here defines key features for potent and selective Nek2 inhibition, which will aid the identification of more advanced inhibitors of Nek2.

A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold.

5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure-activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic omega-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.

Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells.

The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.

Histone deacetylase inhibitors are novel anticancer agents in clinical development that target the family of histone deacetylase (HDAC) enzymes responsible for deacetylating core nucleosomal histones and other proteins. The precise mechanisms resulting in the antiproliferative biologic effects of these agents are not yet known, but there are several proposed mechanistic models, including transcriptional and nontranscriptional processes. Clinical experience with these agents indicates that they are generally well tolerated, and anticancer activity has been observed in early clinical trials in several tumor types including non-small-cell lung cancer. The development of these agents continues, with an emphasis on the discovery of HDAC isoform-selective compounds. Successful future development relies on clearer understanding of the dominant mechanisms involved in the observed antiproliferative effects.

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.

ZD9331 is an antifolate drug that potently and specifically inhibits thymidylate synthase (TS). In contrast with TS inhibitors such as raltitrexed, it cannot be polyglutamated, leading to antitumour activity independent of folylpolyglutamyl synthetase (FPGS) activity. The growth inhibition IC 50 values for ZD9331 and raltitrexed were determined for a panel of 18 human tumour cell lines, that included six colon and six ovarian. The colon lines largely displayed overlapping sensitivities to both drugs with only one of the six lines being drug resistant. In contrast, the ovarian cell lines displayed non-overlapping sensitivities with four being highly resistant to raltitrexed and only one was cross-resistant to ZD9331. Studies were undertaken to explain these results. The colon and ovarian cell lines were characterised for TS activity, and TS and FPGS mRNA expression. TS activity correlated with sensitivity to ZD9331 ( r =0.50; p =0.097) and raltitrexed ( r =0.74; p =0.0063). Provided the data from the highly drug-resistant cell lines (BE and 41 M) were omitted, TS mRNA expression levels also correlated with ZD9331 ( r =0.77; p =0.013) and raltitrexed IC 50 ( r =0.84; p =0.0031). FPGS mRNA expression correlated with higher sensitivity to raltitrexed relative to ZD9331 (higher ZD9331/raltitrexed IC 50 ratios) ( r =0.62; p =0.048). Similarly, cell lines with IC 50 ratios>median expressed a 1.8-fold higher median level of FPGS mRNA ( p =0.0087) compared with those with ratios≤median. The four ovarian and one colon cell line that were relatively more sensitive to ZD9331 expressed FPGS mRNA≤median ( p =0.061). Thus, ZD9331 overcomes resistance to raltitrexed in ovarian tumour cell lines expressing low levels of FPGS. These data, and others demonstrating a lack of cross-resistance between cisplatin and ZD9331, support the clinical evaluation of ZD9331 in platinum-refractory ovarian cancer.

A previously described radioimmunoassay (RIA) method for the measurement of 4-hydroxyandrostenedione (4-OHA) was used to investigate the serum drug levels attained after a single oral dose in male and female rats. Marked variability of serum drug concentrations and their time course were evident in male animals at all dose levels. In the female rat, in contrast, serum 4-OHA showed fewer individual differences, rose more rapidly and was sustained at substantially higher concentrations. In all animals, 4-OHA appeared in the serum within 0.5 h following the oral dose and persisted for at least 48 h. Doubling the dose from 8 mg/kg produced a disproportionately large elevation in serum drug levels, but a further increase to 32 mg/kg did not further increase serum levels.

The biochemical consequences of thymidylate synthase (TS) inhibition are well known and shown diagramatically in Fig. 1. TS catalyzes the reductive methylation of dUMP to form thymidylate (dTMP), which is a rate-limiting step for the de novo synthesis of dTTP the only deoxynucleotide required exclusively for DNA synthesis. The methyl group involved in the reaction is donated by 5,10-methylene tetrahydrofolic acid. When the enzyme is inhibited dTMP pools (and subsequently dTTP pools) are depleted and dUMP accumulates behind the enzyme block because of loss of feedback inhibition by dTTP on three enzymes, ribonucleotide reductase, deoxycytidylate deaminase, and thymidine kinase (1).

The feasibility of using an enhanced chemiluminescent immunoassay for screening water samples was adapted to a Dynatech Microlite camera luminometer and assessed to provide a semiquantitative assay based on a photographic record of the luminescent end-point. An enhanced chemiluminescent immunoassay of the herbicide chlortoluron was chosen in this instance as an example, although other pesticide compounds could equally well have been used provided that suitable antisera were available. This luminescent assay has shown considerable potential by providing a rapid, simple and portable means of monitoring multiple water samples for the presence of chlortoluron. The assay was able to identify samples containing the herbicide at or above the European limit for individual pesticides in drinking water [the EU Maximum Admissible Concentration (MAC) = 0.1 microgram l-1]. A 100% accuracy was obtained in the analysis of samples containing chlortoluron at concentrations above the range of 0.07-0.12 microgram l-1; however, for concentrations within this range the results were 65% accurate. Consequently, the method could greatly facilitate increased monitoring for the presence of chlortoluron in water supplies and in other areas of environmental analysis.

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.

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromized G1/S checkpoint control. This has led to the hypothesis that S and G2/M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53 deficient background, as normal cells would be rescued at the G1/S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage linked S and G2/M checkpoint control. SAR-020106 (the structure will be shown on the poster) is an ATP competitive, potent and selective CHK1 inhibitor with an IC50 of 13.3nM on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55nM in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0 to 29-fold in a number of colon tumor lines in vitro and in a p53 dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug induced autophosphorylation of CHK1 at S296 and blocks phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and PARP cleavage consistent with SAR-020106 enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. Orally bioavailable analogues of SAR-02106 are currently under development. In conclusion SAR-020106 represents a novel class of CHK1 inhibitors which can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3518.

A radioimmunoassay has been used to determine levels of the anticancer drug bleomycin in sewage treatment works effluent, river and potable water samples. Samples were concentrated 100-fold by lyophilisation and a final limit of detection of 5 ng L−1 was achieved. Concentrations of immunoreactive bleomycin of between 11 and 19 ng L−1 were found in the effluents but a lower concentration range < 5–17 ng L−1 was found in river and potable water samples. The risk to human health of ingesting water (in SE England) with such low levels of this cytotoxic drug appears to be minimal in relation to the normal chemotherapeutic doses administered (20–30 mg m−2).

A competitive ELISA method suitable for the monitoring of the herbicide chlortoluron [N-(3-chloro-4-methylphenyl)-N'-dimethylurea] in different types of water and biological fluids was developed. The production of the immunogen utilized in this work was achieved by covalently coupling bovine thryroglobulin with the synthesized hapten (N'-3-chloro-4-methylphenyl-N-carboxypropyl urea) using the N-hydroxysuccinimide active ester method. The chlortoluron antibody, raised in sheep after immunization with the immunogen, showed no cross-reactivity with a large range of pesticides, although some cross-reactivity was displayed with various phenylurea herbicides (i.e., chlorbromuron, isoproturon and metoxuron). The limit of detection of the chlortoluron ELISA method was 0.015 microgram l-1, well below the legal European limit for individual pesticides in drinking water (the EC maximum admissible concentration, 0.1 microgram l-1). In addition, reproducible and quantitative recovery of chlortoluron from water, obtained from various sources, and biological fluids was possible without any sample preparation. The ELISA technique for chlortoluron developed and described here proved to be rapid, sensitive and specific, fulfilling the needs of present legislation relating to the use and levels of pesticides in the environment.