RESUMO
The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).
Assuntos
Neoplasias/tratamento farmacológico , Medicina de Precisão , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Transforming growth factor-ß (TGFß) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFß receptor I (TGFßRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFßRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFß-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFß-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects. Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFß-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFß pathway inhibitors.
RESUMO
In our continuing effort to expand the SAR of the quinoline domain of dihydropyrrolopyrazole series, we have discovered compound 15d, which demonstrated the antitumor efficacy with oral bioavailability. This effort also demonstrated that the PK/PD in vivo target inhibition paradigm is an effective approach to assess potential for antitumor efficacy. The dihydropyrrolopyrazole inhibitor 15d (LY2109761) is representative of a novel series of antitumor agents.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Pirróis/síntese química , Pirróis/química , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo I , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel dihydropyrrolopyrazole-substituted benzimidazoles were synthesized and evaluated in vitro as inhibitors of transforming growth factor-beta type I receptor (TGF-beta RI), TGF-beta RII, and mixed lineage kinase-7 (MLK-7). These compounds were found to be potent TGF-beta RI inhibitors and selective versus TGF-beta RII and MLK-7 kinases. Benzimidazole derivative 8b was active in an in vivo target (TGF-beta RI) inhibition assay.
Assuntos
Benzimidazóis/síntese química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Pirróis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Vison , Estrutura Terciária de Proteína , Pirazóis/química , Pirazóis/farmacologia , Pirróis/química , Pirróis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
We have expanded our previously reported series of pyrazole-based inhibitors of the TGF-beta type I receptor kinase domain (TbetaR-I) to now include new 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole analogues. Limited examination of the SAR of this new series in both enzyme and cell based in vitro assays has revealed selectivity differences with respect to p38 MAP kinase (p38 MAPK) depending on the nature of the 'warhead' group on the dihydropyrrolopyrazole ring. As with our original pyrazole series, phenyl substituents tended to show greater selectivity against p38 MAPK than those comprised of the quinoline-4-yl moiety. We have also achieved co-crystallization and X-ray analysis of compounds 3 and 15, two potent examples of this new series, with the TbetaR-I receptor kinase domain.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pirazóis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Proteínas Serina-Treonina Quinases , Pirazóis/metabolismo , Pirazóis/farmacologia , Quinolinas/química , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pyrazole-based inhibitors of the transforming growth factor-beta type I receptor kinase domain (TbetaR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TbetaR-I receptor kinase domain.