RESUMO
Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/metabolismo , Embrião não Mamífero/citologia , Heterocromatina , Código das Histonas , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Embrião não Mamífero/metabolismo , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.
Assuntos
Metilases de Modificação do DNA/metabolismo , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Metiltransferases/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas de Transporte/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/normas , Humanos , Metiltransferases/antagonistas & inibidores , Nucleotídeos/metabolismo , Oxirredutases N-Desmetilantes/antagonistas & inibidores , RNA/metabolismoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer mortality. HCC has high morbidity, high mortality, and low survival rates. Screening is one of the most significant methods of lowering incidence and death while also increasing survival. OBJECTIVES: The aim of this study was to identify the facilitators and barriers to participation in HCC screening among high-risk populations. METHODS: A comprehensive and systematic search was undertaken in PubMed, Web of Science, MEDLINE, EMBACE, EBSCOhost and the Cochrane Library. A combination of synonyms of the keywords including HCC, screening, factors and adherence were used for searching. Studies addressing the facilitators and barriers to HCC screening compliance in at-risk individuals were included. Data were synthesized using Review Manager version 5.4. A random/fixed effects model meta-analysis was performed to estimate the pooled data and expressed with odds ratio (OR) and 95% confidence interval (CI). RESULTS: A total of seven articles met the inclusion criteria. Qualitative (n = 1) and quantitative (n = 6) studies using various types of surgery were conducted. The most commonly mentioned barriers were insufficient knowledge and awareness of HCC screening, unawareness of the necessity for early detection of HCC and lack of physician recommendation. A meta-analysis of seven studies showed that individuals with a family history of HCC increased screening uptake by nearly three times (OR: 2.69, 95% CI: 1.93, 3.75). Other most frequently reported facilitators include age, education level, and perceived risk et al. CONCLUSIONS: Many barriers to HCC screening were found. Meanwhile, this review points out that improving the awareness of high-risk populations toward HCC screening is expected to enhance compliance, thereby promoting early diagnosis of liver cancer, reducing mortality, and alleviating the burden of HCC.
Assuntos
Carcinoma Hepatocelular , Detecção Precoce de Câncer , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Cooperação do Paciente/estatística & dados numéricos , Programas de Rastreamento/métodos , Fatores de RiscoRESUMO
Autophagy related 4B (ATG4B) which regulates autophagy by promoting the formation of autophagosome through reversible modification of LC3, is closely related to cancer cell growth and drug resistance, and therefore is an attractive therapeutic target. Recently, ATG4B inhibitors have been reported, yet with drawbacks including weak potency. To discover more promising ATG4B inhibitors, we developed a high-throughput screening (HTS) assay and identified a new ATG4B inhibitor named DC-ATG4in. DC-ATG4in directly binds to ATG4B and inhibits its enzyme activity with an IC50 of 3.08 ± 0.47 µM. We further confirmed that DC-ATG4in is an autophagy inhibitor and blocks autophagy induced by Sorafenib in Hepatocellular Carcinoma (HCC) cells. More importantly, combination of DC-ATG4in with Sorafenib synergized the cancer cell killing effect and proliferation inhibition activities on HCC cells. Our data suggested that inactivation of autophagy via ATG4B inhibition may be a viable strategy to sensitize existing targeted therapy such as Sorafenib in the future.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Sorafenibe , Humanos , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Sorafenibe/farmacologia , Sorafenibe/uso terapêuticoRESUMO
O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 µM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 µM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.
Assuntos
Ensaios de Triagem em Larga Escala , Processamento de Proteína Pós-Traducional , GlicosilaçãoRESUMO
This paper studies the use of MUltiple SIgnal Classification (MUSIC) as a super-resolution algorithm to improve demodulation results for intrinsic Fabry-Perot interferometer (IFPI) sensor arrays. Through distinction between noise and signal subspaces in an observation matrix, this paper shows that a 38-fold improvement in the full width at half maximum (FWHM) estimation of IFPI optical path differences (OPD) can be achieved using this algorithm. Based on this improved method, this paper demonstrates that a tunable laser with a 1.3-nm tuning range can achieve the same sensor demodulation performance as a tunable laser with a 50-nm tuning range if a conventional Fourier transform-based algorithm is used. This paper presents a new approach to analyzing optical signals produced by multiple multiplexed interferometers with similar OPDs with potential applications for both single-mode and multiple-mode devices.
RESUMO
Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Taninos Hidrolisáveis/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/antagonistas & inibidores , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos Organometálicos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Modern drug design aims to discover novel lead compounds with attractable chemical profiles to enable further exploration of the intersection of chemical space and biological space. Identification of small molecules with good ligand efficiency, high activity, and selectivity is crucial toward developing effective and safe drugs. However, the intersection is one of the most challenging tasks in the pharmaceutical industry, as chemical space is almost infinity and continuous, whereas the biological space is very limited and discrete. This bottleneck potentially limits the discovery of molecules with desirable properties for lead optimization. Herein, we present a new direction leveraging posttranslational modification (PTM) protein isoforms target space to inspire drug design termed as "Post-translational Modification Inspired Drug Design (PTMI-DD)." PTMI-DD aims to extend the intersections of chemical space and biological space. We further rationalized and highlighted the importance of PTM protein isoforms and their roles in various diseases and biological functions. We then laid out a few directions to elaborate the PTMI-DD in drug design including discovering covalent binding inhibitors mimicking PTMs, targeting PTM protein isoforms with distinctive binding sites from that of wild-type counterpart, targeting protein-protein interactions involving PTMs, and hijacking protein degeneration by ubiquitination for PTM protein isoforms. These directions will lead to a significant expansion of the biological space and/or increase the tractability of compounds, primarily due to precisely targeting PTM protein isoforms or complexes which are highly relevant to biological functions. Importantly, this new avenue will further enrich the personalized treatment opportunity through precision medicine targeting PTM isoforms.
Assuntos
Desenho de Fármacos , Processamento de Proteína Pós-Traducional , Humanos , Isoformas de Proteínas , UbiquitinaçãoRESUMO
Tumor suppressor p53-binding protein 1 (53BP1), a tantem tudor domain (TTD) protein, takes part in DNA Damage Repair (DDR) pathways through the specific recognition of lysine methylation on histones. The dysregulation of 53BP1 is closely related to the development of many diseases including cancer. Moreover, recent studies found that deficiency of 53BP1 could increase the efficiency of precise CRISPR/Cas9 genome editing. Thus, discovery of inhibitor is beneficial to the study of biological functions of 53BP1 and the application of CRISPR/Cas9 genome editing. UNC2170 and its derivatives have been reported as 53BP1 targeted small molecular inhibitors with modest activities. Hence, to discover better 53BP1 inhibitors, we conducted an AlphaScreen assay based high-throughput screening (HTS) and identified a novel and effective 53BP1-TTD inhibitor DP308 which disrupts the binding between 53BP1 and H4K20me2 peptide with an IC50 value of 1.69 ± 0.73 µM. Both Microscale Themophoresis (MST) and Surface Plasmon Resonance (SPR) assays confirmed the direct binding between DP308 and 53BP1-TTD protein with binding affinity (Kd) of about 2.7 µM. Molecular docking studies further suggested that DP308 possibly occupies the H4K20me2 binding pocket of the 53BP1-TTD aromatic cage. These results demonstrated that DP308 is a promising small molecule inhibitor for further optimization towards a more potent chemical probe of 53BP1. Additionally, it could be a potential valuable tool for applying to gene editing therapy by increasing the efficiency of CRISPR/Cas9 genome editing.
Assuntos
Descoberta de Drogas/métodos , Canal de Potássio ERG1/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Canal de Potássio ERG1/genética , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Técnicas de Patch-Clamp , RatosRESUMO
This paper presents a method of using femtosecond laser inscribed nanograting as low-loss- and high-temperature-stable in-fiber reflectors. By introducing a pair of nanograting inside the core of a single-mode optical fiber, an intrinsic Fabry-Perot interferometer can be created for high-temperature sensing applications. The morphology of the nanograting inscribed in fiber cores was engineered by tuning the fabrication conditions to achieve a high fringe visibility of 0.49 and low insertion loss of 0.002 dB per sensor. Using a white light interferometry demodulation algorithm, we demonstrate the temperature sensitivity, cross-talk, and spatial multiplexability of sensor arrays. Both the sensor performance and stability were studied from room temperature to 1000°C with cyclic heating and cooling. Our results demonstrate a femtosecond direct laser writing technique capable of producing highly multiplexable in-fiber intrinsic Fabry-Perot interferometer sensor devices with high fringe contrast, high sensitivity, and low-loss for application in harsh environmental conditions.
RESUMO
Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
Assuntos
Antineoplásicos/farmacologia , Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonas/química , Sulfonas/farmacologia , Triazóis/química , Triazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Histonas/química , Humanos , Lisina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Relação Estrutura-Atividade , Sulfonas/metabolismo , Triazóis/metabolismo , Células Tumorais CultivadasRESUMO
The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Cromatina/genética , Cristalografia por Raios X , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Lisina/química , Metilação , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteína 4 de Ligação ao Retinoblastoma/química , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Transcrição GênicaRESUMO
The EED (embryonic ectoderm development) subunit of the Polycomb repressive complex 2 (PRC2) plays an important role in the feed forward regulation of the PRC2 enzymatic activity. We recently identified a new class of allosteric PRC2 inhibitors that bind to the H3K27me3 pocket of EED. Multiple assays were developed and used to identify and characterize this type of PRC2 inhibitors. One of them is a genetically encoded EED biosensor based on the EED[G255D] mutant and the split firefly luciferase. This EED biosensor can detect the compound binding in the transfected cells and in the in vitro biochemical assays. Compared to other commonly used cellular assays, the EED biosensor assay has the advantage of shorter compound incubation with cells. The in vitro EED biosensor is much more sensitive than other label-free biophysical assays (e.g. DSF, ITC). Based on the crystal structure, the DSF data as well as the biosensor assay data, it's most likely that compound-induced increase in the luciferase activity of the EED[G255D] biosensor results from the decreased non-productive interactions between the EED subdomain and other subdomains within the biosensor construct. This new insight of the mechanism might help to broaden the use of the split luciferase based biosensors.
Assuntos
Bioensaio/métodos , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Mutação de Sentido Incorreto , Complexo Repressor Polycomb 2/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Humanos , Luciferases de Vaga-Lume/genética , Complexo Repressor Polycomb 2/genética , Ligação Proteica , Domínios ProteicosRESUMO
Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.
Assuntos
Linfoma Difuso de Grandes Células B/patologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião de Mamíferos/citologia , Proteína Potenciadora do Homólogo 2 de Zeste , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Linfoma Difuso de Grandes Células B/genética , Metilação/efeitos dos fármacos , Camundongos , Mutação/genética , Fenótipo , Complexo Repressor Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell-active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 =31±2â nM, KD =53±2â nM) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well-characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.
Assuntos
Inibidores Enzimáticos/química , Isoquinolinas/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Regulação Alostérica , Sítios de Ligação , Calorimetria , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Células HEK293 , Histonas , Humanos , Isoquinolinas/metabolismo , Metilação , Simulação de Dinâmica Molecular , Mutagênese , Ligação Proteica , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual 'hit-and-run' (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.
Assuntos
Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Dimerização , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/síntese química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Fatores de Transcrição de p300-CBP/síntese químicaRESUMO
While lysine methylation is well-known for regulating gene expression transcriptionally, its implications in translation have been largely uncharted. Trimethylation at lysine 22 (K22me3) on RPL40, a core ribosomal protein located in the GTPase activation center, was first reported 27 years ago. Yet, its methyltransferase and role in translation remain unexplored. Here, we report that SMYD5 has robust in vitro activity toward RPL40 K22 and primarily catalyzes RPL40 K22me3 in cells. The loss of SMYD5 and RPL40 K22me3 leads to reduced translation output and disturbed elongation as evidenced by increased ribosome collisions. SMYD5 and RPL40 K22me3 are upregulated in hepatocellular carcinoma (HCC) and negatively correlated with patient prognosis. Depleting SMYD5 renders HCC cells hypersensitive to mTOR inhibition in both 2D and 3D cultures. Additionally, the loss of SMYD5 markedly inhibits HCC development and growth in both genetically engineered mouse and patient-derived xenograft (PDX) models, with the inhibitory effect in the PDX model further enhanced by concurrent mTOR suppression. Our findings reveal a novel role of the SMYD5 and RPL40 K22me3 axis in translation elongation and highlight the therapeutic potential of targeting SMYD5 in HCC, particularly with concurrent mTOR inhibition. This work also conceptually broadens the understanding of lysine methylation, extending its significance from transcriptional regulation to translational control.
Assuntos
Carcinoma Hepatocelular , Histona-Lisina N-Metiltransferase , Neoplasias Hepáticas , Lisina , Metiltransferases , Proteínas Ribossômicas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Lisina/metabolismo , Metilação , Camundongos Nus , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.
Assuntos
Cromatografia Líquida/métodos , Descoberta de Drogas/métodos , Histona-Lisina N-Metiltransferase/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios Enzimáticos/métodos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , S-Adenosil-Homocisteína/análise , S-Adenosil-Homocisteína/metabolismoRESUMO
Oncogenic transcription activation is associated with tumor development and resistance derived from chemotherapy or target therapy. The super elongation complex (SEC) is an important complex regulating gene transcription and expression in metazoans closely related to physiological activities. In normal transcriptional regulation, SEC can trigger promoter escape, limit proteolytic degradation of transcription elongation factors and increase the synthesis of RNA polymerase II (POL II), and regulate many normal human genes to stimulate RNA elongation. Dysregulation of SEC accompanied by multiple transcription factors in cancer promotes rapid transcription of oncogenes and induce cancer development. In this review, we summarized recent progress in understanding the mechanisms of SEC in regulating normal transcription, and importantly its roles in cancer development. We also highlighted the discovery of SEC complex target related inhibitors and their potential applications in cancer treatment.
Assuntos
Neoplasias , Fator B de Elongação Transcricional Positiva , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel ß7 and ß8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.