RESUMO
Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.
Assuntos
Neuroblastoma , Sequências Reguladoras de Ácido Nucleico , Acetilação , Criança , Proteína p300 Associada a E1A/genética , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , OncogenesRESUMO
Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.
Assuntos
Lisina , Mieloma Múltiplo , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Genes cdc , Humanos , Metilação , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II , Proteína 2 de Ligação ao Retinoblastoma , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The use of epigenetic bromodomain inhibitors as anticancer therapeutics has transitioned from targeting bromodomain extraterminal domain (BET) proteins into targeting non-BET bromodomains. The two most relevant non-BET bromodomain oncology targets are cyclic AMP response element-binding protein (CBP) and E1A binding protein P300 (EP300). To explore the growing CBP/EP300 interest, we developed a highly efficient two-step synthetic route for dimethylisoxazole-attached imidazo[1,2-a]pyridine scaffold-containing inhibitors. Our efficient two-step reactions enabled high-throughput synthesis of compounds designed by molecular modeling, which together with structure-activity relationship (SAR) studies facilitated an overarching understanding of selective targeting of CBP/EP300 over non-BET bromodomains. This led to the identification of a new potent and selective CBP/EP300 bromodomain inhibitor, UMB298 (compound 23, CBP IC50 72 nM and bromodomain 4, BRD4 IC50 5193 nM). The SAR we established is in good agreement with literature-reported CBP inhibitors, such as CBP30, and demonstrates the advantage of utilizing our two-step approach for inhibitor development of other bromodomains.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína p300 Associada a E1A/antagonistas & inibidores , Isoxazóis/química , Piridinas/química , Sítios de Ligação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína p300 Associada a E1A/metabolismo , Humanos , Simulação de Acoplamento Molecular , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Transcription is epigenetically regulated by the orchestrated function of chromatin-binding proteins that tightly control the expression of master transcription factors, effectors, and supportive housekeeping genes required for establishing and propagating the normal and malignant cell state. Rapid advances in chemical biology and functional genomics have facilitated exploration of targeting epigenetic proteins, yielding effective strategies to target transcription while reducing toxicities to untransformed cells. Here, we review recent developments in conventional active site and allosteric inhibitors, peptidomimetics, and novel proteolysis-targeted chimera (PROTAC) technology that have deepened our understanding of transcriptional processes and led to promising preclinical compounds for therapeutic translation, particularly in cancer.
Assuntos
Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Neoplasias/genética , Animais , Antineoplásicos/farmacologia , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/fisiologia , Epigenômica/métodos , Humanos , Neoplasias/terapia , Proteólise/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mieloma Múltiplo/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Bortezomib/farmacologia , Sistemas CRISPR-Cas , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Dendríticas/citologia , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Humanos , Lenalidomida/farmacologia , Camundongos , Camundongos SCID , Mieloma Múltiplo/terapia , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ubiquitina/químicaRESUMO
Although the exact cause or causes of Parkinson's disease (PD) are not fully understood, it is believed that environmental factors play a major role. The discovery that a synthetic chemical, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-derived N-methyl-4-phenylpyridinium (MPP+), recapitulates major pathophysiological characteristics of PD in humans has provided the strongest support for this possibility. While the mechanism of the selective dopaminergic toxicity of MPP+ has been extensively studied and is, in most respects, well accepted, several key aspects of the mechanism are still debatable. In the present study, we use a series of structurally related, novel, and lipophilic MPP+ derivatives [ N-(2-phenyl-1-propene)-4-phenylpyridinium] to probe the mechanism of action of MPP+ using dopaminergic MN9D and non-neuronal HepG2 cells in vitro. Here we show that effective mitochondrial complex I inhibition is necessary and that the specific uptake through plasma membrane dopamine transporter is not essential for dopaminergic toxicity of MPP+ and related toxins. We also provide strong evidence to support our previous proposal that the selective vulnerability of dopaminergic cells to MPP+ and similar toxins is likely due to the high inherent propensity of these cells to produce excessive reactive oxygen species as a downstream effect of complex I inhibition. Based on the current and previous findings, we propose that MPP+ is the simplest of a larger group of unidentified environmental dopaminergic toxins, a possibility that may have major public health implications.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Complexo I de Transporte de Elétrons/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/análogos & derivados , Animais , Neurônios Dopaminérgicos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Células Hep G2 , Humanos , Camundongos , Doença de Parkinson/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Inhibition of the Hsp90 C-terminus is an attractive therapeutic approach for the treatment of cancer. Novobiocin, the first Hsp90 C-terminal inhibitor identified, contains a synthetically complex noviose sugar that has limited the generation of structure-activity relationships for this region of the molecule. The work described herein utilizes various ring systems as noviose surrogates to explore the size and nature of the surrounding binding pocket.