RESUMO
We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.
Assuntos
Acetatos/farmacologia , Descoberta de Drogas , Piperidonas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Sulfonamidas/química , Proteína Supressora de Tumor p53/antagonistas & inibidores , Acetatos/química , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Conformação Molecular , Piperidonas/química , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Ratos , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/químicaRESUMO
Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel ß-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.
Assuntos
Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Cristalografia por Raios X , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ciclina B/antagonistas & inibidores , Ciclina B/metabolismo , Ciclina B1 , Ciclina E/antagonistas & inibidores , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Descoberta de Drogas , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.
Assuntos
Acetatos/síntese química , Acetatos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Morfolinas/síntese química , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/química , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Indicadores e Reagentes , Camundongos , Modelos Moleculares , Conformação Molecular , Morfolinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.
Assuntos
Acetatos/farmacologia , Antineoplásicos/farmacologia , Ácidos Carboxílicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Piperidonas/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Acetatos/química , Animais , Neoplasias Ósseas/tratamento farmacológico , Ácidos Carboxílicos/química , Células Cultivadas , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Humanos , Ligação de Hidrogênio , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Osteossarcoma/tratamento farmacológico , Piperidonas/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).
Assuntos
Acetatos/farmacologia , Antineoplásicos/farmacologia , Piperidonas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Acetatos/química , Administração Oral , Antineoplásicos/química , Disponibilidade Biológica , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Piperidonas/química , Conformação ProteicaRESUMO
Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 µM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 µM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Dicroísmo Circular , Cristalografia , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Nus , Modelos Moleculares , Morfolinas/síntese química , Morfolinas/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.
Assuntos
Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Sequência de Aminoácidos , Inibidores Enzimáticos/metabolismo , Imunoensaio/métodos , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Peptídeos/metabolismoRESUMO
Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.
Assuntos
Acetatos/síntese química , Antineoplásicos/síntese química , Piperidonas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetatos/farmacocinética , Acetatos/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Hepatócitos/metabolismo , Humanos , Macaca fascicularis , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Transplante de Neoplasias , Piperidonas/farmacocinética , Piperidonas/farmacologia , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Transplante Heterólogo , Proteínas rho de Ligação ao GTP/biossínteseRESUMO
Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.
Assuntos
Antineoplásicos/síntese química , Benzotiazóis/síntese química , Inibidores de Fosfoinositídeo-3 Quinase , Sulfonamidas/síntese química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzotiazóis/química , Benzotiazóis/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Transplante HeterólogoRESUMO
Tumor protein 53 (p53) is a critical regulator of cell cycle and apoptosis that is frequently disabled in human tumors. In many tumor types, p53 is deleted or mutated, but in others p53 is inactivated by overexpression or amplification of its negative regulator mouse double minute 2 (MDM2). A high-throughput screening effort identified 6,7-bis(4-bromophenyl)-7,12-dihydro-6H-chromeno[4,3-d][1,2,4]triazolo[1,5-a]pyrimidine as a potent inhibitor of the MDM2-p53 protein-protein interaction. This screening hit was found to be chemically unstable and difficult to handle due to poor DMSO solubility. Co-crystallization with the target protein helped to direct further optimization and provided a tractable lead series of novel MDM2-p53 inhibitors. In cellular assays, these compounds were shown to upregulate p53 protein levels and p53 signaling and to cause p53-dependent inhibition of proliferation and apoptosis.