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.

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimised from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 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 CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, treatment results in phosphorylation, reduced phosphorylation of RNA polymerase II and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2-family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer. are representative of 3 independent

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.

Following the recent discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin dependent kinase (CDK) inhibitors, a program was initiated to evaluate related ring-constrained analogues, specifically, 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines for inhibition of CDKs. Here we report the rational design, synthesis, structure-activity relationships (SARs), and cellular mode-of-action profile of these second generation CDK inhibitors. Many of the analogues from this chemical series inhibit CDKs with very low nanomolar K-i values. The most potent compound reported in this study inhibits CDK2 with an IC50 of 0.7 nM ([ATP] = 100 mu M). Furthermore, an X-ray crystal structure of 2-methyl-N-(3-(nitro)phenyl)-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine (11g), a representative from the chemical series in complex with cyclin A-CDK2, is reported, confirming the design rationale and expected binding mode within the CDK2 ATP binding pocket.

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models.

Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.

2069 Multiple myeloma is a disease of malignant B-cells that have extended survival and a low proliferation rate which accumulate in the bone marrow causing osteolytic bone lesions. Treatment with current chemotherapeutic agents initially reduces tumour burden but the disease remains incurable, with the average patient survival time being three years. Therefore, there is an urgent need for novel therapies ideally based on an understanding of the biology of the disease. One area of interest is that of cyclin dependent kinases that regulate two main cellular processes essential for malignant cell survival; cell cycle progression and transcription. These kinases are good cellular targets for anti-neoplastic drugs. Seliciclib (CYC202, R-roscovitine) is a CDK inhibitor, currently in clinical trials, that principally targets CDK2, CDK7 and CDK9 and shows potent activity against multiple myeloma cells (MacCallum et al., 2005. Cancer Res. 65 pp5399). Selicliclib acts in part by inhibiting the kinases that phosphorylate the C-terminal domain (CTD) of RNA polymerase II resulting in the inhibition of transcription and down regulation of mRNAs and proteins with short half-lives. One such protein is the anti-apoptotic Mcl-1 that is essential for multiple myeloma cell survival. This study characterises three novel CDK inhibitors, two tri-substituted purines and a 2-anilino-4-(thiazol-5-yl)-pyrimidine that have greater potency in both in vitro kinase assays and against cells in culture. Upon treatment of multiple myeloma cells, all three compounds rapidly inhibit phosphorylation of the CTD of RNA polymerase II at phospho-serine 2, accompanied by an equivalent effect on gene expression to that seen with seliciclib treatment. These changes include down regulation of Mcl-1 at both the mRNA and protein level resulting in induction of apoptosis; detectable by PARP cleavage and TUNEL. The results demonstrate that these CDK inhibitors work by a similar mechanism to seliciclib but with increased potency. Furthermore, they provide scientific rationale for their continued pre-clinical development.

Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online's data policy on reuse of materials please consult the policies page. SUMMARY The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription , i.e. CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mi-totic inhibitor groups. We describe the characterisation of the transcriptional in-hibitor class in terms of kinase inhibition profile, cellular mode of action, and se-lectivity for transformed cells. A structural selectivity rationale was used to opti-mise potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor,