RESUMO
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 αSßR-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.
Assuntos
Adenosina/análogos & derivados , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Purinas/química , Adenosina/síntese química , Adenosina/química , Adenosina/farmacocinética , Adenosina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Purinas/síntese química , Purinas/farmacocinética , Purinas/farmacologia , Roscovitina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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. Optimized 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, CCT068127 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 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.
Assuntos
Antineoplásicos/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Melanoma/metabolismo , Purinas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/genética , Células HCT116 , Células HT29 , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Purinas/química , Proteína do Retinoblastoma/metabolismo , Sulfonamidas/farmacologiaRESUMO
A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.
Assuntos
Trifosfato de Adenosina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ligação Competitiva/efeitos dos fármacos , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K(i)s against CDK2, are reported. Furthermore, X-ray crystal structures of four representative analogues from our chemical series in complex with CDK2 are presented, and these structures are used to rationalize the observed biochemical SARs. Finally results are reported that show, using the most potent CDK2 inhibitor compound from the current series, that the observed antiproliferative and proapoptotic effects are consistent with cellular CDK2 and CDK9 inhibition.
Assuntos
Compostos de Anilina/síntese química , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Pirimidinas/síntese química , Tiazóis/síntese química , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Humanos , Estrutura Molecular , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologiaRESUMO
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 IC(50) of 0.7 nM ([ATP] = 100 microM). 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.
Assuntos
Aminoquinolinas/química , Aminoquinolinas/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Tiazóis/química , Tiazóis/farmacologia , Aminoquinolinas/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Tiazóis/síntese químicaRESUMO
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.
Assuntos
Antineoplásicos/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Animais , Antineoplásicos/uso terapêutico , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Leucemia/tratamento farmacológico , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Tiazóis/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Aurora Quinase A , Aurora Quinases , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Mitose/efeitos dos fármacos , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Especificidade por Substrato , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca(2+)-ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca(2+)-ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca(2+)-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca(2+)-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca(2+)-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca(2+)-ATPase is not a significant regulatory feature of cardiac Ca(2+) homeostasis.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/fisiologia , Serina/química , Sequência de Aminoácidos , Animais , Western Blotting , Calibragem , Membrana Celular/metabolismo , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Immunoblotting , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Peptídeos/química , Fosforilação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A series of 2-anilino-4-(1H-pyrrol-3-yl)pyrimidines were prepared and evaluated for their ability to inhibit cyclin-dependent kinases (CDKs). A number of analogues were found to be potent CDK2 and CDK4 inhibitors and to exhibit anti-proliferative activity against human tumour cell lines. Structure-activity relationships and biochemical characterization are presented.