RESUMO
In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.
Assuntos
Indóis/farmacologia , Indóis/uso terapêutico , Linfoma Folicular/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Mutação/genética , Complexo Repressor Polycomb 2/antagonistas & inibidores , Piridonas/farmacologia , Piridonas/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Linfoma Folicular/enzimologia , Linfoma Folicular/genética , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Metilação/efeitos dos fármacos , Camundongos , Transplante de Neoplasias , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transplante HeterólogoRESUMO
The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.
RESUMO
Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.
Assuntos
Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Feminino , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Camundongos SCID , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Especificidade por SubstratoRESUMO
Small molecule antagonists of protein-protein interactions represent a particular challenge for pharmaceutical discovery. One approach to finding molecules that can disrupt these interactions is to seek mimics of common protein structure motifs. We present an analysis of how molecules based on the 1,4-benzodiazepine-2,5-dione scaffold serve to mimic the side-chains presented by the hydrophobic face of two turns of an alpha-helix derived from the tumor suppressor protein p53, and thus antagonize the HDM2-p53 protein-protein binding interaction.
Assuntos
Benzodiazepinas/química , Desenho de Fármacos , Mimetismo Molecular , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Benzodiazepinas/farmacologia , Humanos , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/químicaRESUMO
Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).
Assuntos
Benzodiazepinas/química , Desenho de Fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Alquilação , Animais , Benzodiazepinas/síntese química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Ácidos Pentanoicos/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/químicaRESUMO
The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.
Assuntos
Benzodiazepinonas/farmacologia , Doxorrubicina/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Benzodiazepinonas/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Nus , Complexos Multiproteicos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Assuntos
Benzodiazepinas/síntese química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2 , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/biossínteseRESUMO
A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.