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

Supplementary Figures - SF1: GI50 determinations for CHK1 inhibitor SRA737 in A549 and SW620 cells. SF2: Knockdown of POLA1, POLE or POLE2 sensitises A549 and SW620 cells to the CHK1 inhibitor MK-8776. SF3: Knockdown of CHK1 sensitises A549 cells to the DNA polymerase inhibitor aphidicolin. SF4: POLA1, POLE or POLE2 knockdown is synthetically lethal with the CHK1 inhibitor SRA737 in additional NSCLC and colorectal cancer cell lines. SF5: Western blot analysis of POLA1, POLE and POLE2 knockdown in additional NSCLC cell lines. Western blot analysis of POLA1, POLE and POLE2 knockdown in additional colorectal cancer cell lines. SF7: SRA737 and gemcitabine synergistically inhibit the proliferation of 4 NSCLC cell lines. SF8: An additive interaction between SRA737 and aphidicolin in the HBEC3-KT cell line. SF9: Effect of CHK1 or B-family DNA polymerase pharmacological inhibition alone or in combination on RPA32 phosphorylation in A549 cells. SF10: Effect of CHK1 or B-family DNA polymerase pharmacological inhibition alone or in combination on ï§H2AX foci in A549 cells. SF 11: Correlation between POLA1, POLE and POLE2 basal protein expression in NSCLC and colorectal cancer cell lines. SF12: Lack of correlation between POLA, POLE and POLE2 basal protein expression and SRA737 or aphidicolin combination indices in NSCLC and colorectal cancer cell lines.

Supplementary Materials from CCT241533 Is a Potent and Selective Inhibitor of CHK2 that Potentiates the Cytotoxicity of PARP Inhibitors

Supplementary Tables 1-3 from CCT241533 Is a Potent and Selective Inhibitor of CHK2 that Potentiates the Cytotoxicity of PARP Inhibitors

Supplementary Tables - ST1: Significant siRNA hits identified from the large-scale synthetic lethal siRNA screen combined with SRA737 in A549 cells. ST2: Significant siRNA hits identified from the large-scale synthetic lethal siRNA screen combined with SRA737 in SW620 cells. ST3: CI (exclusive) values for combination treatment with SRA737 plus either aphidicolin, CD437 or gemcitabine in a panel of NSCLC and colorectal cancer cell lines. ST4: Key genetic aberrations in the total panel of NSCLC and colorectal cancer cell lines used. ST5: GI50 values for SRA737, aphidicolin, CD437 and gemcitabine as single agents in the total panel of NSCLC and colorectal cancer cell lines used.

Abstract Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non–small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. Significance: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

A series of imidazo[1,2-b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.

Deregulation of the cyclin‐dependent kinases ( CDK s) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDK s have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT 068127, a novel inhibitor of CDK 2 and CDK 9. Optimized from the purine template of seliciclib, CCT 068127 exhibits greater potency and selectivity against purified CDK 2 and CDK 9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X‐ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK 2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT 068127 treatment results in decreased retinoblastoma protein ( RB ) phosphorylation, reduced phosphorylation of RNA polymerase II , and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT 068127 shows greatest similarity to other small‐molecule CDK and also HDAC inhibitors. CCT 068127 caused a dramatic loss in expression of DUSP 6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL 1 protein levels are rapidly decreased by CCT 068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT 068127 and ABT 263, a BCL 2 family inhibitor. These findings support the rational combination of this series of CDK 2/9 inhibitors and BCL 2 family inhibitors for the treatment of human cancer.

The corresponding author of this article has informed us of concerns about the immunoblots in Fig. 2 which were carried out in the collaborating laboratory of Professor Ann Jackman.

Background The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. Results HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo . HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo . Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro . APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. Conclusion These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically.

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.

Abstract The PI3K/AKT pathway is a key driver of cell growth, proliferation and survival, and deregulation at many levels of the pathway is observed in numerous cancers. Given the importance of PI3K/AKT signaling in cancer, a number of compounds targeting this pathway are currently in clinical development. Several AKT-targeted compounds, such as the ATP-competitive inhibitor AZD5363 and the allosteric inhibitor MK2206, have entered clinical trial. Whilst resistance to AKT inhibition has not yet been well defined, the development of acquired resistance has been a major barrier to the success of a number of molecularly targeted therapies, such as those which target V600E mutant BRAF. Identifying resistance mechanisms to AKT inhibition prior to the development of resistance in the clinic may assist in selecting patient populations and generating combination treatments to overcome resistance, potentially increasing the clinical utility of these inhibitors. The aims of this study were therefore to generate and characterize cell lines resistant to AKT inhibition. CCT129254, a precursor of AZD5363, is a novel ATP-competitive AKT inhibitor identified in a drug discovery program at The Institute of Cancer Research, London UK, in collaboration with Astex Therapeutics and AstraZeneca. CCT129254 is a potent and selective inhibitor of the three AKT isoforms (IC50 values between 13 and 66 nM), and treatment of cells with CCT129254 reduced phosphorylation of PRAS40, GSK3β and rpS6, downstream biomarkers of AKT activity. By exposing PTEN-deficient A2780 ovarian carcinoma cells to escalating doses of inhibitor, we have successfully generated a cell line with acquired resistance to CCT129254 (A2780 254R). We obtained approximately 4.8-fold resistance to CCT129254 in A2780 254R cells compared to parental A2780 (GI50: A2780 = 2.9 μM; A2780 254R = 14 μM). Interestingly, testing against a panel of signal transduction inhibitors revealed a 10-fold resistance to MK2206 in A2780 254R cells (GI50: A2780 = 0.30 μM; A2780 254R = 3.1 μM). Minimal cross-resistance was observed to the PI3K inhibitor GDC-0941 (1.9-fold, GI50: A2780 = 99 nM; A2780 254R = 185 nM), the mTORC1 inhibitor RAD001 (1.9-fold, GI50: A2780 = 1.65 nM; A2780 254R = 3.18 nM), and the dual PI3K/mTOR inhibitor PI-103 (1.7-fold, GI50: A2780 = 112 nM; A2780 254R = 188 nM). In addition, we have observed changes in PI3K/AKT pathway signaling biomarkers using western blot analysis. We have also utilized gene expression microarray analysis and exome sequencing to identify candidate genes involved in the resistance phenotype. We are currently validating potential resistance mechanisms identified in our candidate and global based approaches, and are investigating inhibitor combinations to overcome resistance. Citation Format: Denis T. Akan, Michael I. Walton, Michelle D. Garrett. Generation of an acquired resistance model to the novel AKT inhibitor CCT129254. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4447. doi:10.1158/1538-7445.AM2013-4447

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.

Abstract The AGC kinase AKT is a key component of the phosphatidylinositol 3-kinase (PI3K) pathway, which is frequently deregulated in cancer, making AKT a target of major therapeutic interest. However, PI3K signaling through both AKT-dependent and AKT-independent mechanisms involving other AGC kinases, such as p70S6K, PKA, SGK and ROCK, is important in a range of cancers. Hence, the pharmacological inhibition of these multiple AGC kinases may increase response rates and minimize clinical resistance compared with targeting AKT alone. The clinical drug candidate AT13148 is a multi-AGC kinase, ATP-competitive inhibitor, identified utilizing high-throughput X-ray crystallography and fragment-based lead discovery techniques. Screening of this oral small molecule against a panel of kinases at 10μM revealed >80% inhibition of the structurally related AGC kinases AKT, PKA, ROCK2, p70S6K, MSK, RSK1/2, and SGK. We demonstrate that AT13148 has antiproliferative activity in a range of in vitro models harboring relevant genetic abnormalities, including PTEN, KRAS, PIK3CA and HER2 aberrations. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induction of apoptosis in both a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects both in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP competitive inhibitors of AKT, is not a therapeutically relevant reactivation step for this compound. We used gene expression microarray studies to characterize the underlying molecular mechanisms of action of AT13148 and the selective AKT inhibitor CCT128930, and observed the induction of upstream regulators including insulin receptor substrate-2 and PIK3IP1 due to compensatory feedback loops, consistent with blockade of AKT signaling. These studies also showed that AT13148 and CCT128930 have distinct molecular effects in cancer cells: AT13148 had a predominant effect on apoptosis genes and caused a greater apoptotic phenotype, while CCT128930 modulated genes in the network regulating cell cycle. This finding emphasizes the functional differences of AT13148 as a multi-AGC kinase inhibitor in contrast to a more AKT-selective inhibitor. In view of the potential mechanistic advantages detailed above, and the potent antitumor activity observed at well tolerated doses against established human tumor xenografts with clinically relevant genetic drivers, the clinical utility of such an AGC kinase inhibitor strategy will now be assessed in a first-in-human Phase I trial of AT13148. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 928. doi:1538-7445.AM2012-928

Abstract The DNA damage response network ensures the fidelity of DNA replication and controls the repair of damage arising during cellular replication or from exogenous agents such as genotoxic drugs. Checkpoint Kinase 1 (CHK1) is a serine/threonine kinase occupying a central position in this complex network of cell regulatory and DNA repair mechanisms. G1/S, S or G2/M cell cycle checkpoints are activated in response to genotoxic antitumor drugs to provide an opportunity for repair of damaged DNA or to activate apoptotic pathways. Unlike normal cells, human cancer cells frequently have functional defects in the tumor suppressor p53 with consequent loss of G1/S checkpoint control and greater reliance on S and G2/M checkpoints. Thus CHK1 inhibitors which abrogate the S and G2/M checkpoints will selectively sensitize p53 deficient cancer cells to DNA damaging agents. CHK1 inhibition by siRNA and several small molecule inhibitors have confirmed this in preclinical studies. The challenges of improving the CHK1 potency and selectivity of our initial, fragment derived pyrazolopyridine inhibitors, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps. Initial hit compounds were optimised into potent inhibitors of CHK1 using iterative cycles of design, synthesis, assay and crystallography, progressing through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and isoquinolines. The potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, and potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts when dosed i.p. in nude mice. Further lead optimisation led to orally bioavailable analogues with good in vitro ADME and in vivo pharmacokinetic properties, exemplified by CCT244747. CCT244747 has demonstrated both in vivo pharmacodynamic modulation of signaling through CHK1 and potentiation of cytotoxic drugs in human tumor xenografts. In summary, we show how a fragment derived compound with weak, micromolar activity against CHK1 evolved through a scaffold hopping strategy to give the selective CHK1 isoquinoline inhibitor SAR-020106, from which optimisation of pharmacokinetic properties led to potent, selective and orally bioavailable CHK1 inhibitors such as CCT244747. 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 A235.

Abstract CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC50 of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 μmol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors. Cancer Res; 71(2); 463–72. ©2011 AACR.

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.