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Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.
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Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Peptídeos/farmacologia , beta Catenina/antagonistas & inibidores , Compostos Bicíclicos Heterocíclicos com Pontes/química , Modelos Moleculares , Peptídeos/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a. By using the hydrocarbon peptide-stapling approach, cell-permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metabolismo dos Lipídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Células HeLa , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Peptídeos/química , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
RFX transcription factors are key regulators of ciliogenesis in vertebrates. In Xenopus and zebrafish embryos, knockdown of Rfx2 causes defects in neural tube closure and in left-right axis patterning. To determine the essential role of the Rfx2 gene in mammalian development, we generated Rfx2-deficient mice using an embryonic stem cell clone containing a lacZ gene trap reporter inserted into the first intron of the Rfx2 gene. We found that the Rfx2 lacZ reporter is expressed in ciliated tissues during mouse development including the node, the floor plate and the dorsal neural tube. However, mice homozygous for the Rfx2 gene trap mutation did not have defects in neural tube closure or in organ situs. The gene trap insertion appears to create a null allele as Rfx2 mRNA was not detected in Rfx2gt/gt embryos. Although Rfx2-deficient mice do not have an obvious embryonic phenotype, we found that Rfx2gt/gt males are infertile because of a defect in spermatid maturation at or before the round and elongating spermatid stage. Our results indicate that Rfx2 is not essential for embryonic development in the mouse but is required for spermatogenesis. genesis 53:604-611, 2015. © 2015 Wiley Periodicals, Inc.
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UNLABELLED: Susceptibility to develop nonalcoholic fatty liver disease (NAFLD) has genetic bases, but the associated variants are uncertain. The aim of the present study was to identify genetic variants that could help to prognose and further understand the genetics and development of NAFLD. Allele frequencies of 3,072 single-nucleotide polymorphisms (SNPs) in 92 genes were characterized in 69 NAFLD patients and 217 healthy individuals. The markers that showed significant allele-frequency differences in the pilot groups were subsequently studied in 451 NAFLD patients and 304 healthy controls. Besides this, 4,414 type 2 diabetes mellitus (T2DM) cases and 4,567 controls were genotyped. Liver expression of the associated gene was measured and the effect of its potential role was studied by silencing the gene in vitro. Whole genome expression, oxidative stress (OS), and the consequences of oleic acid (OA)-enriched medium on lipid accumulation in siSLC2A1-THLE2 cells were studied by gene-expression analysis, dihydroethidium staining, BODIPY, and quantification of intracellular triglyceride content, respectively. Several SNPs of SLC2A1 (solute carrier family 2 [facilitated glucose transporter] member 1) showed association with NAFLD, but not with T2DM, being the haplotype containing the minor allele of SLC2A1 sequence related to the susceptibility to develop NAFLD. Gene-expression analysis demonstrated a significant down-regulation of SLC2A1 in NAFLD livers. Enrichment functional analyses of transcriptome profiles drove us to demonstrate that in vitro silencing of SLC2A1 induces an increased OS activity and a higher lipid accumulation under OA treatment. CONCLUSIONS: Genetic variants of SLC2A1 are associated with NAFLD, and in vitro down-regulation of this gene promotes lipid accumulation. Moreover, the oxidative response detected in siSLC2A1-THLE2 cells corroborated the antioxidant properties previously related to this gene and linked the most representative clinical characteristics of NAFLD patients: oxidative injury and increased lipid storage.
Assuntos
Fígado Gorduroso/genética , Transportador de Glucose Tipo 1/genética , Adolescente , Adulto , Idoso , Diabetes Mellitus Tipo 2/genética , Feminino , Frequência do Gene , Inativação Gênica , Predisposição Genética para Doença , Transportador de Glucose Tipo 1/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica , Ácido Oleico/farmacologia , Estresse Oxidativo/genética , Polimorfismo de Nucleotídeo Único , TranscriptomaRESUMO
The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.
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Neoplasias , Humanos , Entropia , Metionina Adenosiltransferase/metabolismoRESUMO
PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.
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Inibidores Enzimáticos , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Humanos , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Camundongos , Descoberta de Drogas , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese químicaRESUMO
The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.
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Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sirtuína 1/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Guanilato Ciclase/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Estabilidade de RNA , Sirtuína 1/deficiência , Sirtuína 1/genéticaRESUMO
Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.
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The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.
Assuntos
Interferon Tipo I , Neoplasias , DNA , Dano ao DNA , Células Dendríticas/metabolismo , Exodesoxirribonucleases , Indóis , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Morfolinas , Neoplasias/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Pirimidinas , SulfonamidasRESUMO
The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10-15 µM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pK a, to identify compound 18, which combines low hERG activity (IC50 = 75 µM) with excellent kinome selectivity and favorable pharmacokinetic properties.
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BACKGROUND & AIMS: Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine are tightly regulated by 2 genes: methionine adenosyltransferase 1A (MAT1A) and methionine adenosyltransferase 2A (MAT2A). MAT1A is expressed in the adult liver, whereas MAT2A expression primarily is extrahepatic and is associated strongly with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. METHODS: In silico analysis of the 3' untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AU-rich RNA binding factor 1 (AUF1) and HuR, respectively. We investigated the posttranscriptional regulation of MAT1A and MAT2A by AUF1, HuR, and methyl-HuR in the aforementioned biological processes. RESULTS: During hepatic de-differentiation, the switch between MAT1A and MAT2A coincided with an increase in HuR and AUF1 expression. S-adenosylmethionine treatment altered this homeostasis by shifting the balance of AUF1 and methyl-HuR/HuR, which was identified as an inhibitor of MAT2A messenger RNA (mRNA) stability. We also observed a similar temporal distribution and a functional link between HuR, methyl-HuR, AUF1, and MAT1A and MAT2A during fetal liver development. Immunofluorescent analysis revealed increased levels of HuR and AUF1, and a decrease in methyl-HuR levels in human livers with hepatocellular carcinoma (HCC). CONCLUSIONS: Our data strongly support a role for AUF1 and HuR/methyl-HuR in liver de-differentiation, development, and human HCC progression through the posttranslational regulation of MAT1A and MAT2A mRNAs.
Assuntos
Antígenos de Superfície/metabolismo , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Hepatócitos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina Adenosiltransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Superfície/genética , Sítios de Ligação , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/genética , Meia-Vida , Hepatócitos/patologia , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Metionina Adenosiltransferase/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , S-Adenosilmetionina/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
UNLABELLED: LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient [SAMe-D]) from MAT1A knockout (MAT1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein-activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. CONCLUSION: The SAMe-D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies.
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Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Divisão Celular , Ativação Enzimática , Inativação Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metionina Adenosiltransferase/deficiência , Metionina Adenosiltransferase/genética , Camundongos , Camundongos Knockout , Fosforilação , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificaçãoRESUMO
MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.
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Desenho de Fármacos , Inibidores Enzimáticos/química , Metionina Adenosiltransferase/antagonistas & inibidores , Sítio Alostérico , Animais , Proliferação de Células , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Técnicas de Inativação de Genes , Células HCT116 , Meia-Vida , Humanos , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ratos , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.
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Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/sangue , Glicina N-Metiltransferase/genética , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Análise Multivariada , Hepatopatia Gordurosa não Alcoólica , Análise de Componente PrincipalRESUMO
UNLABELLED: S-adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA-binding protein that increases the half-life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)-mediated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. CONCLUSION: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy.
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Proteínas Quinases Ativadas por AMP/metabolismo , Hepatócitos/citologia , Hepatócitos/enzimologia , Regeneração Hepática/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Divisão Celular , Replicação do DNA , Inativação Gênica , Hepatectomia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , FosforilaçãoRESUMO
UNLABELLED: Hepatic S-adenosylmethionine (SAMe) is maintained constant by the action of methionine adenosyltransferase I/III (MATI/III), which converts methionine into SAMe and glycine N-methyltransferase (GNMT), which eliminates excess SAMe to avoid aberrant methylation reactions. During liver regeneration after partial hepatectomy (PH) MATI/III activity is inhibited leading to a decrease in SAMe. This injury-related reduction in SAMe promotes hepatocyte proliferation because SAMe inhibits hepatocyte DNA synthesis. In MATI/III-deficient mice, hepatic SAMe is reduced, resulting in uncontrolled hepatocyte growth and impaired liver regeneration. These observations suggest that a reduction in SAMe is crucial for successful liver regeneration. In support of this hypothesis we report that liver regeneration is impaired in GNMT knockout (GNMT-KO) mice. Liver SAMe is 50-fold higher in GNMT-KO mice than in control animals and is maintained constant following PH. Mortality after PH was higher in GNMT-KO mice than in control animals. In GNMT-KO mice, nuclear factor kappaB (NFkappaB), signal transducer and activator of transcription-3 (STAT3), inducible nitric oxide synthase (iNOS), cyclin D1, cyclin A, and poly (ADP-ribose) polymerase were activated at baseline. PH in GNMT-KO mice was followed by the inactivation of STAT3 phosphorylation and iNOS expression. NFkappaB, cyclin D1 and cyclin A were not further activated after PH. The LKB1/AMP-activated protein kinase/endothelial nitric oxide synthase cascade was inhibited, and cytoplasmic HuR translocation was blocked despite preserved induction of DNA synthesis in GNMT-KO after PH. Furthermore, a previously unexpected relationship between AMPK phosphorylation and NFkappaB activation was uncovered. CONCLUSION: These results indicate that multiple signaling pathways are impaired during the liver regenerative response in GNMT-KO mice, suggesting that GNMT plays a critical role during liver regeneration, promoting hepatocyte viability and normal proliferation.
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Glicina N-Metiltransferase/metabolismo , Regeneração Hepática , S-Adenosilmetionina/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Ciclo Celular , Células Cultivadas , Hepatectomia , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
DNA-PK is a key component within the DNA damage response, as it is responsible for recognizing and repairing double-strand DNA breaks (DSBs) via non-homologous end joining. Historically it has been challenging to identify inhibitors of the DNA-PK catalytic subunit (DNA-PKcs) with good selectivity versus the structurally related PI3 (lipid) and PI3K-related protein kinases. We screened our corporate collection for DNA-PKcs inhibitors with good PI3 kinase selectivity, identifying compound 1. Optimization focused on further improving selectivity while improving physical and pharmacokinetic properties, notably co-optimization of permeability and metabolic stability, to identify compound 16 (AZD7648). Compound 16 had no significant off-target activity in the protein kinome and only weak activity versus PI3Kα/γ lipid kinases. Monotherapy activity in murine xenograft models was observed, and regressions were observed when combined with inducers of DSBs (doxorubicin or irradiation) or PARP inhibition (olaparib). These data support progression into clinical studies (NCT03907969).
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Proteína Quinase Ativada por DNA/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/uso terapêutico , Piranos/uso terapêutico , Triazóis/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Cães , Descoberta de Drogas , Humanos , Camundongos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Purinas/síntese química , Purinas/farmacocinética , Piranos/síntese química , Piranos/farmacocinética , Ratos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.
Assuntos
Peptidomiméticos , Fatores de Transcrição/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Via de Sinalização Hippo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
UNLABELLED: Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice. CONCLUSION: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.
Assuntos
Carcinoma Hepatocelular/enzimologia , Fígado Gorduroso/enzimologia , Glicina N-Metiltransferase/metabolismo , Neoplasias Hepáticas/enzimologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Metilação de DNA , Epigênese Genética/fisiologia , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Glicina N-Metiltransferase/genética , Histonas/metabolismo , Janus Quinases/metabolismo , Cirrose Hepática/sangue , Cirrose Hepática/enzimologia , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Metionina/sangue , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , S-Adenosilmetionina/sangue , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Transaminases/sangue , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismoRESUMO
DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.