RESUMO
A molecular systems architecture is presented for acute myeloid leukemia (AML) to provide a framework for organizing the complexity of biomolecular interactions. AML is a multifactorial disease resulting from impaired differentiation and increased proliferation of hematopoietic precursor cells involving genetic mutations, signaling pathways related to the cancer cell genetics, and molecular interactions between the cancer cell and the tumor microenvironment, including endothelial cells, fibroblasts, myeloid-derived suppressor cells, bone marrow stromal cells, and immune cells (e.g., T-regs, T-helper 1 cells, T-helper 17 cells, T-effector cells, natural killer cells, and dendritic cells). This molecular systems architecture provides a layered understanding of intra- and inter-cellular interactions in the AML cancer cell and the cells in the stromal microenvironment. The molecular systems architecture may be utilized for target identification and the discovery of single and combination therapeutics and strategies to treat AML.
RESUMO
BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Neoplasias Hematológicas/tratamento farmacológico , Metionina/química , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/química , Proteínas de Ciclo Celular , Cristalografia por Raios X , Neoplasias Hematológicas/patologia , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Bromodomain (BD) and extra-terminal domain containing proteins (BET) are chromatin adapters that bind acetylated histone marks via two tandem BDs, BD1 and BD2, to regulate gene transcription. BET proteins are involved in transcriptional reprogramming in response to inflammatory stimuli. BET BD inhibitors (BETis) that are nonselective for BD1 or BD2 have recognized anti-inflammatory properties in vitro and counter pathology in models of inflammation or autoimmune disease. Although both BD1 and BD2 bind acetylated histone residues, they may independently regulate the expression of BET-sensitive genes. Here we characterized the ability of RVX-297, a novel orally active BETi with selectivity for BD2, to modulate inflammatory processes in vitro, in vivo, and ex vivo. RVX-297 suppressed inflammatory gene expression in multiple immune cell types in culture. Mechanistically, RVX-297 displaced BET proteins from the promoters of sensitive genes and disrupted recruitment of active RNA polymerase II, a property shared with pan-BETis that nonselectively bind BET BDs. In the lipopolysaccharide model of inflammation, RVX-297 reduced proinflammatory mediators assessed in splenic gene expression and serum proteins. RVX-297 also countered pathology in three rodent models of polyarthritis: rat and mouse collagen-induced arthritis, and mouse collagen antibody-induced arthritis. Further, RVX-297 prevented murine experimental autoimmune encephalomyelitis (a model of human multiple sclerosis) disease development when administered prophylactically and reduced hallmarks of pathology when administered therapeutically. We show for the first time that a BD2-selective BETi maintains anti-inflammatory properties and is effective in preclinical models of acute inflammation and autoimmunity.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Proteínas/antagonistas & inibidores , Quinazolinonas/uso terapêutico , Doença Aguda , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos/imunologia , Artrite/induzido quimicamente , Artrite/imunologia , Artrite/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Colágeno/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Ratos Endogâmicos Lew , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Células U937RESUMO
Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208.
RESUMO
Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. We demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains.
Assuntos
Quinazolinonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Termodinâmica , Fatores de Transcrição/químicaRESUMO
The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Luciferases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low µM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).
Assuntos
Desenho de Fármacos , Ligantes , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Proteínas de Ciclo Celular , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Quinoxalinas/química , Quinoxalinas/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Despite the benefit of statins in reducing cardiovascular risk, a sizable proportion of patients still remain at risk. Since HDL reduces CVD risk through a process that involves formation of pre-beta particles that facilitates the removal of cholesterol from the lipid-laden macrophages in the arteries, inducing pre-beta particles, may reduce the risk of CVD. A novel BET bromodomain antagonist, RVX-208, was reported to raise apoA-I and increase preß-HDL particles in non-human primates and humans. In the present study, we investigated the effect of RVX-208 on aortic lesion formation in hyperlipidemic apoE(-/-) mice. Oral treatments of apoE(-/-) mice with 150 mg/kg b.i.d RVX-208 for 12 weeks significantly reduced aortic lesion formation, accompanied by 2-fold increases in the levels of circulating HDL-C, and â¼50% decreases in LDL-C, although no significant changes in plasma apoA-I were observed. Circulating adhesion molecules as well as cytokines also showed significant reduction. Haptoglobin, a proinflammatory protein, known to bind with HDL/apoA-I, decreased >2.5-fold in the RVX-208 treated group. With a therapeutic dosing regimen in which mice were fed Western diet for 10 weeks to develop lesions followed by switching to a low fat diet and concurrent treatment with RVX-208 for 14 weeks, RVX-208 similarly reduced lesion formation by 39% in the whole aorta without significant changes in the plasma lipid parameters. RVX-208 significantly reduced the proinflammatory cytokines IP-10, MIP1(®) and MDC. These results show that the antiatherogenic activity of BET inhibitor, RVX-208, occurs via a combination of lipid changes and anti-inflammatory activities.
Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Betaína-Homocisteína S-Metiltransferase/antagonistas & inibidores , Hiperlipidemias/tratamento farmacológico , Quinazolinas/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Apolipoproteína A-I/sangue , Apolipoproteínas E/deficiência , Aterosclerose/sangue , Aterosclerose/etiologia , Aterosclerose/patologia , Linhagem Celular , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Citocinas/sangue , Dieta com Restrição de Gorduras , Dieta Ocidental/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais , Perfilação da Expressão Gênica , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/complicações , Hiperlipidemias/dietoterapia , Hiperlipidemias/genética , Inflamação/sangue , Inflamação/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinazolinas/farmacologia , Quinazolinonas , RNA Mensageiro/análise , Células U937RESUMO
The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Quinazolinonas/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Sinergismo Farmacológico , Linfoma , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismoRESUMO
Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.
Assuntos
Apolipoproteína A-I/biossíntese , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Apolipoproteína A-I/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Cristalografia por Raios X , Epigênese Genética/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Quinazolinas/química , Quinazolinonas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Increases in p53 protein levels after DNA damage have largely been attributed to an increase in the half-life of p53 protein. Here we demonstrate that increased translation of p53 mRNA is also a critical step in the induction of p53 protein in irradiated cells. Ribosomal protein L26 (RPL26) and nucleolin were found to bind to the 5' untranslated region (UTR) of p53 mRNA and to control p53 translation and induction after DNA damage. RPL26 preferentially binds to the 5'UTR after DNA damage, and its overexpression enhances association of p53 mRNA with heavier polysomes, increases the rate of p53 translation, induces G1 cell-cycle arrest, and augments irradiation-induced apoptosis. Opposite effects were seen when RPL26 expression was inhibited. In contrast, nucleolin overexpression suppresses p53 translation and induction after DNA damage, whereas nucleolin downregulation promotes p53 expression. These findings demonstrate the importance of increased translation of p53 in DNA-damage responses and suggest critical roles for RPL26 and nucleolin in affecting p53 induction.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Regiões 5' não Traduzidas/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Genes cdc/fisiologia , Camundongos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética , NucleolinaRESUMO
DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD(+) binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B(3)) increases the rate of intracellular NAD(+) synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD(+) on p53, as a molecule structurally related to part of NAD(+), TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B(1)), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53.
Assuntos
DNA/metabolismo , NAD/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Linhagem Celular , DNA/genética , Dano ao DNA , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Niacinamida/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiamina/farmacologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization.