RESUMO
BACKGROUND: iPSC reprogramming technology exhibits significant promise in the realms of clinical therapeutics, disease modeling, pharmaceutical drug discovery, and various other applications. However, the extensive utilization of this technology has encountered impediments in the form of inefficiency, prolonged procedures, and ambiguous biological processes. Consequently, in order to improve this technology, it is of great significance to delve into the underlying mechanisms involved in iPSC reprogramming. The BET protein BRD4 plays a crucial role in the late stage of reprogramming; however, its precise function in the early stage remains unclear. RESULTS: Our study aims to investigate BRD4's role in the early stages of iPSC reprogramming. Our investigation reveals that early inhibition of BRD4 substantially enhances iPSC reprogramming, whereas its implementation during the middle-late stage impedes the process. During the reprogramming, ribosome DNA expression initially increases before decreasing and then gradually recovers. Early inhibition of BRD4 improved the decline and restoration of rDNA expression in the early and middle-late stages, respectively. Additionally, we uncovered the mechanism of BRD4's regulation of rDNA transcription throughout reprogramming. Specifically, BRD4 interacts with UBF and co-localizes to both the rDNA promoter and enhancer regions. Ultimately, BRD4 facilitates rDNA transcription by promoting the enrichment of histone H3 lysine 27 acetylation in the surrounding chromatin. Moreover, we also discovered that early inhibition of BRD4 facilitates cells' transition out of the somatic cell state and activate pluripotent genes. CONCLUSIONS: In conclusion, our results demonstrate that early inhibition of BRD4 promotes sequential dynamic expression of rDNA, which improves iPSC reprogramming efficiency.
Assuntos
Reprogramação Celular , DNA Ribossômico , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição , Células-Tronco Pluripotentes Induzidas/metabolismo , Reprogramação Celular/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , DNA Ribossômico/genética , Animais , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas que Contêm BromodomínioRESUMO
Small molecule inhibitors targeting the bromodomain and extra-terminal domain (BET) family proteins have emerged as a promising class of anti-cancer drugs. Nevertheless, the clinical advancement of these agents has been significantly hampered by challenges related to their potency, oral bioavailability, or toxicity. In this study, virtual screening approaches were employed to discover novel inhibitors of the bromodomain-containing protein 4 (BRD4) by analyzing their comparable chemical structural features to established BRD4 inhibitors. Several of these compounds exhibited inhibitory effects on BRD4 activity ranging from 60 % to 70 % at 100 µM concentrations, while one compound also exhibited an 84 % inhibition of Sirtuin 2 (SIRT2) activity. Furthermore, a subset of structurally diverse compounds from the BRD4 inhibitors was selected to investigate their anti-cancer properties in both 2D and 3D cell cultures. These compounds exhibited varying effects on cell numbers depending on the specific cell line, and some of them induced cell cycle arrest in the G0/G1 phase in breast cancer (MDA-MB-231) cells. Moreover, all the compounds studied reduced the sizes of spheroids, and the most potent compound exhibited a 90 % decrease in growth at a concentration of 10 µM in T47D cells. These compounds hold potential as epigenetic regulators for future studies.
Assuntos
Antineoplásicos , Neoplasias da Mama , Fatores de Transcrição , Feminino , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Domínios Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Flavonoides/química , Flavonoides/farmacologiaRESUMO
Progressive supranuclear palsy (PSP) is an incurable neurodegenerative disease characterized by 4-repeat (0N/4R)-Tau protein accumulation in CNS neurons. We generated transgenic zebrafish expressing human 0N/4R-Tau to investigate PSP pathophysiology. Tau zebrafish replicated multiple features of PSP, including: decreased survival; hypokinesia; impaired optokinetic responses; neurodegeneration; neuroinflammation; synapse loss; and Tau hyperphosphorylation, misfolding, mislocalization, insolubility, truncation, and oligomerization. Using automated assays, we screened 147 small molecules for activity in rescuing neurological deficits in Tau zebrafish. (+)JQ1, a bromodomain inhibitor, improved hypokinesia, survival, microgliosis, and brain synapse elimination. A heterozygous brd4+/- mutant reducing expression of the bromodomain protein Brd4 similarly rescued these phenotypes. Microglial phagocytosis of synaptic material was decreased by (+)JQ1 in both Tau zebrafish and rat primary cortical cultures. Microglia in human PSP brains expressed Brd4. Our findings implicate Brd4 as a regulator of microglial synaptic elimination in tauopathy and provide an unbiased approach for identifying mechanisms and therapeutic targets in PSP.
Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Microglia , Paralisia Supranuclear Progressiva , Sinapses , Fatores de Transcrição , Peixe-Zebra , Proteínas tau , Animais , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Microglia/metabolismo , Microglia/patologia , Sinapses/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/patologia , Azepinas/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Triazóis/farmacologia , Ratos , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Encéfalo/metabolismo , Encéfalo/patologia , Fagocitose , Neurônios/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo CelularRESUMO
Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma, associated with poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is limited. Bromodomain and extra-terminal (BET) proteins are involved in both physiological and pathological events. However, the role of BET proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BET protein family members, BRD2, BRD3, and BRD4, are aberrantly overexpressed in uLMS tissues compared to the myometrium, with a significant change by histochemical scoring assessment. Furthermore, inhibiting BET proteins with their small, potent inhibitors (JQ1 and I-BET 762) significantly inhibited the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-sequencing analysis revealed that the inhibition of BET proteins with JQ1 and I-BET 762 altered several critical pathways, including the hedgehog pathway, EMT, and transcription factor-driven pathways in uLMS. In addition, the targeted inhibition of BET proteins altered several other epigenetic regulators, including DNA methylases, histone modification, and m6A regulators. The connections between BET proteins and crucial biological pathways provide a fundamental structure to better understand uterine diseases, particularly uLMS pathogenesis. Accordingly, targeting the vulnerable epigenome may provide an additional regulatory mechanism for uterine cancer treatment.
Assuntos
Leiomiossarcoma , Fatores de Transcrição , Neoplasias Uterinas , Humanos , Feminino , Leiomiossarcoma/metabolismo , Leiomiossarcoma/patologia , Leiomiossarcoma/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Neoplasias Uterinas/genética , Fatores de Transcrição/metabolismo , Proliferação de Células , Azepinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Triazóis/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Epigênese Genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Pessoa de Meia-Idade , Proteínas que Contêm Bromodomínio , Benzodiazepinas , ProteínasRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) poses significant challenges in treatment due to its aggressive nature and immune escape mechanisms. Despite recent advances in immune checkpoint blockade therapies, GBM prognosis remains poor. The role of bromodomain and extraterminal domain protein 4 (BRD4) in GBM, especially its interaction with immune checkpoints, is not well understood. METHODS: Bioinformatic gene expression and survival analysis for BRD4 was utilized in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Clone formation assay, Transwell, Cell Counting Kit-8 (CCK8), and wound healing assay were utilized to validate BRD4's promotion of glioma cell proliferation, invasion, and migration. Chromatin immunoprecipitation (ChIP) assay was conducted to confirm BRD4 binding to the programmed death ligand 1 (PD-L1) promoter. A co-culture model was utilized with activated cluster of differentiation 8 (CD8)+ T cells and glioma cells. GL261 cells with BRD4 short hairpin RNA (shRNA) and/or PD-L1 cDNA were intracranially injected into mice to investigate tumor growth and survival time. Tumor tissue characteristics were analyzed using hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining and immune cell infiltration were assessed by flow cytometry. RESULTS: Bioinformatics analyses reveal elevated BRD4 expression in high-grade gliomas, correlating with poor patient survival. In vitro studies confirm that BRD4 promotes proliferation, invasion, and migration in GBM cells. BRD4 is a regulator of PD-L1 at the transcriptional level, implying its involvement in GBM's immune escape mechanisms. Co-culture experiments with CD8+ T cells demonstrate that BRD4 inhibition enhances tumor cell apoptosis. In vivo studies indicate that BRD4 knockout reduces immunosuppression, improves prognosis. Simultaneous manipulation of BRD4 and PD-L1 levels provides insights into their intertwined roles in shaping the immune landscape of GBM. CONCLUSIONS: BRD4 has the capability to regulate the growth of glioblastoma and enhance immune suppression by promoting PD-L1 expression. Targeting BRD4 represents a promising direction for future research and treatment.
Assuntos
Antígeno B7-H1 , Proteínas de Ciclo Celular , Glioma , Fatores de Transcrição , Antígeno B7-H1/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Humanos , Camundongos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Glioma/imunologia , Glioma/genética , Glioma/patologia , Linhagem Celular Tumoral , Regulação para Cima , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Evasão Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas que Contêm BromodomínioRESUMO
The calculation of absolute binding free energies (ABFEs) for protein-ligand systems has long been a challenge. Recently, refined force fields and algorithms have improved the quality of the ABFE calculations. However, achieving the level of accuracy required to inform drug discovery efforts remains difficult. Here, we present a transferable enhanced sampling strategy to accurately calculate absolute binding free energies using OneOPES with simple geometric collective variables. We tested the strategy on two protein targets, BRD4 and Hsp90, complexed with a total of 17 chemically diverse ligands, including both molecular fragments and drug-like molecules. Our results show that OneOPES accurately predicts protein-ligand binding affinities with a mean unsigned error within 1 kcal mol-1 of experimentally determined free energies, without the need to tailor the collective variables to each system. Furthermore, our strategy effectively samples different ligand binding modes and consistently matches the experimentally determined structures regardless of the initial protein-ligand configuration. Our results suggest that the proposed OneOPES strategy can be used to inform lead optimization campaigns in drug discovery and to study protein-ligand binding and unbinding mechanisms.
Assuntos
Proteínas de Choque Térmico HSP90 , Ligação Proteica , Termodinâmica , Ligantes , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Sítios de Ligação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas que Contêm BromodomínioRESUMO
In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-KrasG12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis.
Assuntos
Heme Oxigenase-1 , Macrófagos , Fator 2 Relacionado a NF-E2 , Neoplasias Pancreáticas , Fatores de Transcrição , Animais , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas/efeitos dos fármacos , Humanos , Pancreatite/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/genética , Camundongos Knockout , Microambiente Tumoral/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Proteínas que Contêm Bromodomínio , Proteínas de Membrana , Proteínas NuclearesRESUMO
INTRODUCTION: Bromodomain-containing protein 4 (BRD4), an important epigenetic reader, is closely associated with the pathogenesis and development of many diseases, including various cancers, inflammation, and infectious diseases. Targeting BRD4 inhibition or protein elimination with small molecules represents a promising therapeutic strategy, particularly for cancer therapy. AREAS COVERED: The recent advances of patented BRD4 degraders were summarized. The challenges, opportunities, and future directions for developing novel potent and selective BRD4 degraders are also discussed. The patents of BRD4 degraders were searched using the SciFinder and Cortellis Drug Discovery Intelligence database. EXPERT OPINION: BRD4 degraders exhibit superior efficacy and selectivity to BRD4 inhibitors, given their unique mechanism of protein degradation instead of protein inhibition. Excitingly, RNK05047 is now in phase I/II clinical trials, indicating that selective BRD4 protein degradation may offer a viable therapeutic strategy, particularly for cancer. Targeting BRD4 with small-molecule degraders provides a promising approach with the potential to overcome therapeutic resistance for treating various BRD4-associated diseases.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular , Desenvolvimento de Medicamentos , Neoplasias , Patentes como Assunto , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Proteólise/efeitos dos fármacos , Descoberta de Drogas , Proteínas que Contêm BromodomínioRESUMO
A 3D structure-based pharmacophore model built for bromodomain-containing protein 4 (BRD4) is reported here, specifically developed for investigating and identifying the key structural features of the (+)-JQ1 known inhibitor within the BRD4 binding site. Using this pharmacophore model, 273 synthesized and purchased compounds previously considered for other targets but yielding poor results were screened in a drug repositioning campaign. Subsequently, only six compounds showed potential as BRD4 binders and were subjected to further biophysical and biochemical assays. Compounds 2, 5, and 6 showed high affinity for BRD4, with IC50 values of 0.60 ± 0.25 µM, 3.46 ± 1.22 µM, and 4.66 ± 0.52 µM, respectively. Additionally, these compounds were tested against two other bromodomains, BRD3 and BRD9, and two of them showed high selectivity for BRD4. The reported 3D structure-based pharmacophore model proves to be a straightforward and useful tool for selecting novel BRD4 ligands.
Assuntos
Proteínas de Ciclo Celular , Fatores de Transcrição , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Humanos , Ligação Proteica , Ligantes , Reposicionamento de Medicamentos , Sítios de Ligação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Triazóis/química , Triazóis/farmacologia , Azepinas/química , Azepinas/farmacologia , Simulação de Acoplamento Molecular , Modelos Moleculares , Relação Estrutura-Atividade , Avaliação Pré-Clínica de Medicamentos , Farmacóforo , Proteínas que Contêm BromodomínioRESUMO
The bromodomain-containing protein 4 (BRD4), which is a key epigenetic regulator in cancer, has emerged as an attractive target for the treatment of melanoma. In this study, we investigate 7-phenoxy-benzimidazole derivative 12, which is a novel BRD4 inhibitor for the treatment of melanoma, by performing scaffold hopping on the previously reported benzimidazole derivative 1. Despite their good oral and intravenous exposure, the compounds obtained by modifying derivate 1 exhibit mutagenicity, which was confirmed by the positive Ames test results. Based on our hypothesis that the cause of the Ames test positivity is the metabolic intermediates generated from those chemical series, we implemented a scaffold hopping strategy to avoid the N-benzyl moiety by relocating the substituent groups to preserve the essential interaction. Based on this strategy, we successfully obtained compound 12; the Ames test results of this compound were negative. Notably, compound 12 not only exhibited a favorable pharmacokinetic (PK) profile but also significant tumor growth inhibition in a mouse melanoma xenograft model, indicating its potential as a therapeutic agent for the treatment of melanoma.
Assuntos
Antineoplásicos , Benzimidazóis , Proteínas de Ciclo Celular , Melanoma , Fatores de Transcrição , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzimidazóis/síntese química , Camundongos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Melanoma/tratamento farmacológico , Melanoma/patologia , Humanos , Administração Oral , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Relação Estrutura-Atividade , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas que Contêm BromodomínioRESUMO
JQ1 is a wonder therapeutic molecule that selectively inhibits the BRD4 signaling pathway and is thus widely used in the anticancer drug discovery program. Due to its unique selective BRD4 binding property, its applications are further extended in the design and synthesis of bi-functional PROTAC molecules. This BRD4 targeting PROTAC molecule selectively degrades the protein by proteolysis. There are several modifications of JQ1 known to date and extensively explored for their applications in PROTAC technology by several research groups in academia as well as industry for targeting oncogenic genes. In this review, we have covered the discovery and synthesis of the JQ1 molecule. The SAR of the JQ1 analogs will help researchers develop potent JQ1 compounds with improved inhibitory properties against malignant cells. Furthermore, we explored the potential application of JQ1 analogs in PROTAC technology. The brief history of the bromodomain family of proteins, as well as the obstacles connected with PROTAC technology, can help comprehend the context of the current research, which has the potential to improve the drug development process. Overall, this review comprehensively appraises JQ1 molecules and their prior implementation in PROTAC technology and cancer therapy.
Assuntos
Antineoplásicos , Azepinas , Neoplasias , Triazóis , Humanos , Azepinas/farmacologia , Azepinas/química , Azepinas/síntese química , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Estrutura Molecular , Proteólise/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimera de Direcionamento de Proteólise , Proteínas que Contêm BromodomínioRESUMO
Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Fatores de Transcrição , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linhagem da Célula/genética , Células Cultivadas , Análise de Célula Única , Proteínas que Contêm BromodomínioRESUMO
The bromodomain and extraterminal domain (BET) family proteins serve as primary readers of acetylated lysine residues and play crucial roles in cell proliferation and differentiation. Dysregulation of BET proteins has been implicated in tumorigenesis, making them important therapeutic targets. BET-bromodomain (BD) inhibitors and BET-targeting degraders have been developed to inhibit BET proteins. In this study, we found that the BET inhibitor MS645 exhibited superior antiproliferative activity than BET degraders including ARV771, AT1, MZ1 and dBET1 in triple-negative breast cancer (TNBC) cells. Treatment with MS645 led to the dissociation of BETs, MED1 and RNA polymerase II from the E2F1-3 promoter, resulting in the suppression of E2F1-3 transcription and subsequent inhibition of cell growth in TNBC. In contrast, while ARV771 displaced BET proteins from chromatin, it did not significantly alter E2F1-3 expression. Mechanistically, ARV771 induced BRD4 depletion at protein level, which markedly increased EGR1 expression. This elevation of EGR1 subsequently recruited septin 2 and septin 9 to E2F1-3 promoters, enhancing E2F1-3 transcription and promoting cell proliferation rate in vitro and in vivo. Our findings provide valuable insights into differential mechanisms of BET inhibition and highlight potential of developing BET-targeting molecules as therapeutic strategies for TNBC.
Assuntos
Proliferação de Células , Fator de Transcrição E2F1 , Proteína 1 de Resposta de Crescimento Precoce , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Proliferação de Células/efeitos dos fármacos , Animais , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F1/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Linhagem Celular Tumoral , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição E2F/genética , Antineoplásicos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Camundongos Nus , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular , Fator de Transcrição E2F3RESUMO
Bromodomain-containing protein 9 (BRD9) is a key player in chromatin remodeling and gene expression regulation, and it is closely associated with the development of various diseases, including cancers. Recent studies have indicated that inhibition of BRD9 may have potential value in the treatment of certain cancers. Molecular dynamics (MD) simulations, Markov modeling and principal component analysis were performed to investigate the binding mechanisms of allosteric inhibitor POJ and orthosteric inhibitor 82I to BRD9 and its allosteric regulation. Our results indicate that binding of these two types of inhibitors induces significant structural changes in the protein, particularly in the formation and dissolution of α-helical regions. Markov flux analysis reveals notable changes occurring in the α-helicity near the ZA loop during the inhibitor binding process. Calculations of binding free energies reveal that the cooperation of orthosteric and allosteric inhibitors affects binding ability of inhibitors to BRD9 and modifies the active sites of orthosteric and allosteric positions. This research is expected to provide new insights into the inhibitory mechanism of 82I and POJ on BRD9 and offers a theoretical foundation for development of cancer treatment strategies targeting BRD9.
Assuntos
Cadeias de Markov , Simulação de Dinâmica Molecular , Ligação Proteica , Fatores de Transcrição , Regulação Alostérica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/antagonistas & inibidores , Humanos , Sítios de Ligação , Análise de Componente Principal , Termodinâmica , Proteínas que Contêm BromodomínioRESUMO
In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.
Assuntos
Proteínas de Ciclo Celular , Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Fatores de Transcrição , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Separação de Fases , Proteínas que Contêm BromodomínioRESUMO
The antiapoptotic protein BCL2A1 is highly, but very heterogeneously expressed in Diffuse Large B-cell Lymphoma (DLBCL). Particularly in the context of resistance to current therapies, BCL2A1 appears to play an important role in protecting cancer cells from the induction of cell death. Reducing BCL2A1 levels may have therapeutic potential, however, no specific inhibitor is currently available. In this study, we hypothesized that the signaling network regulated by epigenetic readers may regulate the transcription of BCL2A1 and hence that inhibition of Bromodomain and Extra-Terminal (BET) proteins may reduce BCL2A1 expression thus leading to cell death in DLBCL cell lines. We found that the mechanisms of action of acetyl-lysine competitive BET inhibitors are different from those of proteolysis targeting chimeras (PROTACs) that induce the degradation of BET proteins. Both classes of BETi reduced the expression of BCL2A1 which coincided with a marked downregulation of c-MYC. Mechanistically, BET inhibition attenuated the constitutively active canonical nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway and inhibited p65 activation. Furthermore, signal transducer of activated transcription (STAT) signaling was reduced by inhibiting BET proteins, targeting another pathway that is often constitutively active in DLBCL. Both pathways were also inhibited by the IκB kinase inhibitor TPCA-1, resulting in decreased BCL2A1 and c-MYC expression. Taken together, our study highlights a novel complex regulatory network that links BET proteins to both NFκB and STAT survival signaling pathways controlling both BCL2A1 and c-MYC expression in DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas que Contêm Bromodomínio , Proteínas , Antígenos de Histocompatibilidade MenorRESUMO
Interest in the use of proteolysis-targeting chimeras (PROTACs) in cancer therapy has increased in recent years. Targeting bromodomain and extra terminal domain (BET) proteins, especially bromodomain-containing protein 4 (BRD4), has shown inhibitory effects on basal-like breast cancer (BLBC). However, the bioavailability of BRD4 PROTACs is restricted by their non-selective biodegradability and low tumor-targeting ability. We demonstrated that 6b (BRD4 PROTAC) suppresses BLBC cell growth by targeting BRD4, but not BRD2 and BRD3, for cereblon (CRBN)-mediated ubiquitination and proteasomal degradation. Compound 6b also inhibited expression of Krüppel-like factor 5 (KLF5) transcription factor, a key oncoprotein in BLBC, controlled by BRD4-mediated super-enhancers. Moreover, 6b inhibited HCC1806 tumor growth in a xenograft mouse model. The combination of 6b and KLF5 inhibitors showed additive effects on BLBC. These results suggest that BRD4-specific PROTAC can effectively inhibit BLBC by downregulating KLF5, and that 6b has potential as a novel therapeutic drug for BLBC.
Assuntos
Neoplasias da Mama , Proteínas de Ciclo Celular , Fatores de Transcrição Kruppel-Like , Proteólise , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Camundongos , Feminino , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Camundongos Nus , Proteínas que Contêm BromodomínioRESUMO
The precise and safe treatment of bioorthogonal prodrug system is hindered by separate administration of prodrug and its activator, which often results in poor therapeutic effects and severe side effects. To address above issues, we herein construct a single bioorthogonal-activated co-delivery system for simultaneous PROTAC prodrug (proPROTAC) delivery and controlled, site-specific activation for tumor-specific treatment. In this co-delivery system (termed AuPLs), prodrug (proPROTAC) and water-soluble Pd-catalyst are first encapsulated by gold nanocubes (AuNCs), which are further coated with a layer of phase-change material (lauric acid/stearic acid, LA/SA). Below 39 °C, the solid state of LA/SA prevents the activation of Pd-mediated bioorthogonal reaction due to the solidification of Pd-catalyst and proPROTAC. Nevertheless, once over 42 °C, the phase change of LA/SA into liquid state, enabled by the photothermal effect of AuNCs, triggers the simultaneous release of proPROTAC and Pd-catalyst and initiates the in situ bioorthogonal reaction for proPROTAC activation. In the tumor-bearing mouse models, the systemic administration of AuPLs results in the accumulation in tumor region, where the photothermal effect activates and controls the tumor-specific bioorthogonal reaction to degrade BRD4 protein, leading to anti-tumor effects with minimized side effects. Overall, the co-delivery proPROTAC and Pd-catalyst and controlled activation by photothermal effects provide a precise way for biorthogonal-based anticancer prodrugs.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular , Ouro , Camundongos Nus , Neoplasias , Paládio , Pró-Fármacos , Animais , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Humanos , Ouro/química , Ouro/administração & dosagem , Neoplasias/tratamento farmacológico , Paládio/química , Paládio/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição , Sistemas de Liberação de Medicamentos , Camundongos Endogâmicos BALB C , Linhagem Celular Tumoral , Catálise , Feminino , Camundongos , Proteínas Nucleares , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Proteínas que Contêm BromodomínioRESUMO
We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular , Bibliotecas de Moléculas Pequenas , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proliferação de Células/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Azepinas/farmacologia , Azepinas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Indolizinas/química , Indolizinas/farmacologia , Linhagem Celular Tumoral , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ligantes , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/síntese química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas que Contêm Bromodomínio , Óxidos N-Cíclicos , Compostos de PiridínioRESUMO
Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.