RESUMO
The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.
Assuntos
Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Piridinas/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Caspase 3 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Desenho de Fármacos , Inibidores Enzimáticos/química , Células HCT116 , Humanos , Imidazóis/química , Concentração Inibidora 50 , Fosforilação/efeitos dos fármacos , Piridinas/química , Proteína do Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
Novel substituted indolocarbazoles were synthesized, and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles 4 were found to be potent and selective D1/CDK4 inhibitors. 4d and 4h exhibited potent and ATP-competitive D1/CDK4 activities with IC50 values of 76 and 42 nM, respectively. Both compounds had high selectivity against the other kinases. These D1/CDK4 inhibitors inhibited tumor cell growth, arrested tumor cells at the G1 phase, and inhibited pRb phosphorylation.
Assuntos
Antineoplásicos/síntese química , Carbazóis/síntese química , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Indóis/síntese química , Proteínas Proto-Oncogênicas , Antineoplásicos/química , Antineoplásicos/farmacologia , Carbazóis/química , Carbazóis/farmacologia , Divisão Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Humanos , Indóis/química , Indóis/farmacologia , Fosforilação , Proteína do Retinoblastoma/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.
Assuntos
Células Epiteliais/citologia , Inibidores do Crescimento/química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mesoderma/citologia , Inibidores de Proteínas Quinases/química , Pirazóis/química , Fator de Crescimento Transformador beta/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Inibidores do Crescimento/metabolismo , Células HeLa , Humanos , Cinética , MAP Quinase Quinase Quinases/metabolismo , Espectrometria de Massas , Mesoderma/química , Mesoderma/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Pirazóis/metabolismo , Serina/metabolismo , Proteína Smad2 , Proteína Smad3 , Especificidade por Substrato/efeitos dos fármacos , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Structure-based design approach was successfully used to guide the evolution of imidazopyridine scaffold yielding new structural class of highly selective inhibitors of cyclin dependent kinases that were able to form a new interaction with an identified residue of the protein, Lys89. Compounds from this series have shown no detectable effect when tested against a representative set of other serine/threonine kinases such as GSK3beta, CAMKII, PKA, PKC-alpha,beta,epsilon,gamma. Compound 2i inhibits proliferation in HCT 116 cells in tissue culture. Synthesis, co-crystal structure of CDK2 in complex with compound 2i, and preliminary SAR study are disclosed.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Piridinas/síntese química , Células Cultivadas , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Lisina/química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
A novel series of pyrrolo[3,4-c] carbazoles fused with a quinolinyl/isoquinolinyl moiety were synthesized and their D1/CDK4 inhibitory and antiproliferative activity were evaluated. Compound 8H, 14H-isoquinolinyl[6,5-a]-pyrrolo[3,4-c]carbazole-7,9-dione (1d) was found to be a highly potent D1/CDK4 inhibitor with an IC(50) of 69 nM. Compound 1d also inhibited tumor cell growth, arrested tumor cells in G1 phase and inhibited pRb phosphorylation.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carbazóis/síntese química , Carbazóis/farmacologia , Ciclina D1/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas , Ciclo Celular , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina , Ciclização , Dimetil Sulfóxido , Humanos , Cinética , Fosforilação , Células Tumorais CultivadasRESUMO
We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Imidazóis , Piridinas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The synthesis of a novel series of 1,7-annulated indolocarbazoles 2 and 16 is described. These compounds were found to be potent cyclin dependent kinase inhibitors with good antiproliferative activity against two human carcinoma cell lines. These inhibitors also arrested tumor cells at the G1 phase and inhibited pRb phosphorylation.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Indóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Inibidores do Crescimento/síntese química , Inibidores do Crescimento/farmacologia , Humanos , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The synthesis and kinase inhibitory activity of a series of novel 1,7-annulated indolocarbazoles 6 and 16 is described. These compounds exhibited potent inhibitory activity against cyclin-dependent kinase 4 and good antiproliferative activity in a human colon carcinoma cell line.
Assuntos
Carbazóis/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Indóis/química , Inibidores de Proteínas Quinases/química , Carbazóis/farmacologia , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good antiproliferative agents.
Assuntos
Compostos Aza/síntese química , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Indóis/síntese química , Compostos Aza/farmacologia , Sítios de Ligação , Quinases relacionadas a CDC2 e CDC28/química , Ciclo Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A series of indolo[2,3-a]pyrrolo[3,4-c]carbazoles and their bis-indolylmaleimides precursors have been prepared in order to compare their activity as D1-CDK4 inhibitors. Both enzymatic and antiproliferative assays have shown that the structurally more constrained indolo[2,3-a]pyrrolo[3,4-c]carbazoles are consistently more active (8-42-fold) in head-to-head comparison with their bis-indolylmaleimides counterparts. Cell-cycle analysis using flow cytometry have also shown that the indolocarbazoles are selective G1 blockers while the bis-indolylmaleimides arrest cells in the G2/M phase.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carbazóis/síntese química , Carbazóis/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/CDK4.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carbazóis/síntese química , Carbazóis/farmacologia , Ciclina D1/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Proto-Oncogênicas , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Quinase 4 Dependente de Ciclina , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Fosforilação , Rubídio/metabolismo , Relação Estrutura-AtividadeRESUMO
The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.