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2.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
3.
Nat Commun ; 11(1): 3958, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769971

RESUMO

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.


Assuntos
Dimetilaliltranstransferase/metabolismo , Liases Intramoleculares/metabolismo , Alternaria/enzimologia , Domínio Catalítico , Dimetilaliltranstransferase/química , Ensaios Enzimáticos , Escherichia coli/metabolismo , Fusarium/enzimologia , Indóis/química , Indóis/metabolismo , Liases Intramoleculares/química , Cinética , Ligantes , Modelos Moleculares , Prenilação , Terpenos/metabolismo
4.
Food Chem ; 331: 127192, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32569963

RESUMO

Melatonin and serotonin are bioactive compounds present in foods and beverages and related to neuroprotection and anti-angiogenesis, among other activities. They have been described in wines and the role of yeast in their formation is clear. Thus, this study evaluates the content of these bioactives and other related indolic compounds in beer. For this purpose, commercial beers were analyzed by a validated UHPLC-HRMS method and sample treatment optimized due to the low concentrations expected. Moreover, a wort was fermented with different commercial beer yeast (Abbaye, Diamond, SafAle, SafLager) in order to monitor the formation of these bioactives during the elaboration process. Results show that indolic compounds such as N-acetylserotonin and 3-indoleacetic acid are produced during the alcoholic fermentation of wort. Moreover, the occurrence of four indolic compounds (5-hydroxytryptophan, N-acetylserotonin, 3-indoleacetic acid, l-tryptophan ethyl ester) in commercial beers is reported for the first time.


Assuntos
Cerveja/análise , Cerveja/microbiologia , Indóis/metabolismo , Melatonina/metabolismo , Triptofano/metabolismo , Bebidas , Cromatografia Líquida de Alta Pressão/métodos , Fermentação , Análise de Alimentos/métodos , Armazenamento de Alimentos , Indóis/análise , Melatonina/análise , Saccharomyces cerevisiae/metabolismo , Serotonina/análogos & derivados , Temperatura , Triptofano/análogos & derivados
5.
Ecotoxicol Environ Saf ; 200: 110736, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450438

RESUMO

As an effective neonicotinoid insecticide, imidacloprid (IMI) has been widely used in crop production, but its residue affects normal plant growth. Selenium (Se) is a non-essential mineral nutrient in higher plants, that acts as the active centre of glutathione peroxidase (GSH-Px), which removes harmful peroxides. In this study, we investigated the mechanism by which selenium improves the growth status of IMI-treated garlic plants through analyses of apparent morphology and antioxidant enzyme activity as well as the dynamic changes in nutrients and metabolites in the plants. The results showed that 80 µg/kg Na2SeO3 had a strong effect on alleviating the damage in garlic plants exposed to IMI (1.2 mg/kg) by increasing the absorption of mineral elements to enhance the synthesis of chlorophyll and antioxidant enzymes. A nontarget metabolomics analysis based on gas chromatography-mass spectrometry (GC-MS) indicated that the addition of Na2SeO3 to IMI-treated garlic could reconstruct the plant metabolic distribution by enhancing the nitrogen and indole metabolism, maintaining lower concentrations of secondary metabolites and maintaining the balance of the plant energy metabolism. Our study provides novel insights into the molecular mechanisms by which garlic plants responds to IMI exposure and suggests the use of selenium with IMI-contaminated plants as a solution for the advancement of sustainable agricultural pesticide use.


Assuntos
Alho/efeitos dos fármacos , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Selenito de Sódio/farmacologia , Antioxidantes/metabolismo , Clorofila/metabolismo , Metabolismo Energético/efeitos dos fármacos , Alho/enzimologia , Alho/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Indóis/metabolismo , Nitrogênio/metabolismo , Metabolismo Secundário/efeitos dos fármacos
6.
PLoS One ; 15(4): e0232461, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348373

RESUMO

AST-120 (Kremezin) is used to treat progressive chronic kidney disease (CKD) by adsorbing uremic toxin precursors produced by gut microbiota, such as indole and phenols. In this study, we propose that AST-120 reduces indole level, consequently suppresses indole effects on induction of drug tolerance and virulence in Escherichia coli including enterohaemorrhagic strains. In experiments, AST-120 adsorbed both indole and tryptophan, a precursor of indole production, and led to decreased expression of acrD and mdtEF which encode drug efflux pumps, and elevated glpT, which encodes a transporter for fosfomycin uptake and increases susceptibility to aztreonam, rhodamine 6G, and fosfomycin. AST-120 also decreased the production of EspB, which contributes to pathogenicity of enterohaemorrhagic E. coli (EHEC). Aztreonam, ciprofloxacin, minocycline, trimethoprim, and sulfamethoxazole were also adsorbed by AST-120. However, fosfomycin, in addition to rifampicin, colistin and amikacin were not adsorbed, thus AST-120 can be used together with these drugs for therapy to treat infections. These results suggest another benefit of AST-120, i.e., that it assists antibacterial chemotherapy.


Assuntos
Antibacterianos/farmacologia , Carbono/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Indóis/metabolismo , Óxidos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Humanos , Virulência/efeitos dos fármacos
7.
PLoS One ; 15(3): e0229868, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163448

RESUMO

The purpose of this study was to examine the influence of medium-chain fatty acid-containing triglycerides (MCT), long-chain polyunsaturated fatty acid-containing triglycerides, and their combination on the plasma metabolome of cats (Felis catus), including circulating microbiome-derived postbiotics. After a 14-day lead-in on the control food, cats were randomized to one of four foods (control, with 6.9% MCT, with fish oil [FO; 0.14% eicosapentaenoate, 1.0% docosahexaenoate], or with FO+MCT; n = 16 per group) for 28 days. Analysis of plasma metabolites showed that the addition of FO and MCT led to synergistic effects not seen with either alone across a number of lipid classes, including fatty acids, acylcarnitines, and acylated amines including endocannabinoids. Notably, the FO+MCT group had an increase in ketone body production relative to baseline and beyond that seen with MCT alone. N-acyl taurines, the accumulation of which has been implicated in the onset of type 2 diabetes, were significantly decreased in the FO+MCT group. Significant decreases in the gut microbiome-derived postbiotic classes of indoles/indolic sulfates and phenols/phenolic sulfates were observed only the FO+MCT group. Overall, the combination of MCT and FO led to number of changes in plasma metabolites that were not observed with either oil alone, particularly in postbiotics.


Assuntos
Ração Animal , Ácidos Docosa-Hexaenoicos/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Triglicerídeos/administração & dosagem , Animais , Gatos , Feminino , Indóis/sangue , Indóis/metabolismo , Corpos Cetônicos/sangue , Corpos Cetônicos/metabolismo , Lipidômica , Lipídeos/sangue , Masculino , Fenóis/sangue , Fenóis/metabolismo
8.
Xenobiotica ; 50(9): 1032-1042, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32129697

RESUMO

1. The absorption, distribution, metabolism, elimination, and drug-drug interaction (DDI) potential of the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib was characterised in vitro.2. Rucaparib showed moderate cellular permeability, moderate human plasma protein binding (70.2%), and slow metabolism in human liver microsomes (HLMs). In HLMs, cytochrome P450 (CYP) 1A2 and CYP3A contributed to the metabolism of rucaparib to its major metabolite M324 with estimated fractions of metabolism catalysed by CYP (fm,CYP) of 0.27 and 0.64, respectively. Rucaparib reversibly inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3As (IC50, 3.55, 12.9, 5.42, 41.6, and 17.2-22.9 µM [2 substrates], respectively), but not CYP2B6 or CYP2C8 (>190 µM). No time-dependent inhibition of any CYP was observed. In cultured human hepatocytes, rucaparib showed concentration-dependent induction of CYP1A2 mRNA and downregulation of CYP3A4 and CYP2B6 mRNA. In transfected cells expressing drug transporters, rucaparib was a substrate for P-gp and BCRP, but not for OATP1B1, OATP1B3, OAT1, OAT3, or OCT2. Rucaparib inhibited P-gp and BCRP (IC50, 169 and 55 µM, respectively) and slightly inhibited OATP1B1, OATP1B3, OAT1, and OAT3 (66%, 58%, 58%, and 42% inhibition, respectively) at 300 µM. Rucaparib inhibited OCT1, OCT2, MATE1, and MATE2-K (IC50, 4.3, 31, 0.63, and 0.19 µM, respectively).3. DDI risk assessment using static models suggested potential CYP-related DDIs, with rucaparib as a perpetrator. Caution is advised when co-administering rucaparib with sensitive substrates of MATEs, OCT1, and OCT2.


Assuntos
Indóis/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/metabolismo , Transporte Biológico , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Humanos , Indóis/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos , Proteínas de Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo
9.
J Med Chem ; 63(6): 3215-3226, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32142284

RESUMO

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.


Assuntos
Inibidores Enzimáticos/química , Indóis/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Tiofenos/química , Sítio Alostérico , Cristalografia por Raios X , Descoberta de Drogas , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Indóis/síntese química , Indóis/metabolismo , Estrutura Molecular , NAD/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Espermidina/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(11): 6114-6120, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123098

RESUMO

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposed Escherichia coli to a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response-wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.


Assuntos
Quimiotaxia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Microbioma Gastrointestinal/fisiologia , Indóis/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Receptores de Superfície Celular/metabolismo , Adaptação Fisiológica
11.
Proc Natl Acad Sci U S A ; 117(12): 6910-6917, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152121

RESUMO

Auxin is a class of plant hormone that plays a crucial role in the life cycle of plants, particularly in the growth response of plants to ever-changing environments. Since the auxin responses are concentration-dependent and higher auxin concentrations might often be inhibitory, the optimal endogenous auxin level must be closely controlled. However, the underlying mechanism governing auxin homeostasis remains largely unknown. In this study, a UDP-glycosyltransferase (UGT76F1) was identified from Arabidopsis thaliana, which participates in the regulation of auxin homeostasis by glucosylation of indole-3-pyruvic acid (IPyA), a major precursor of the auxin indole-3-acetic acid (IAA) biosynthesis, in the formation of IPyA glucose conjugates (IPyA-Glc). In addition, UGT76F1 was found to mediate hypocotyl growth by modulating active auxin levels in a light- and temperature-dependent manner. Moreover, the transcription of UGT76F1 was demonstrated to be directly and negatively regulated by PIF4, which is a key integrator of both light and temperature signaling pathways. This study sheds a light on the trade-off between IAA biosynthesis and IPyA-Glc formation in controlling auxin levels and reveals a regulatory mechanism for plant growth adaptation to environmental changes through glucosylation of IPyA.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Hipocótilo/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Indóis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Hipocótilo/efeitos dos fármacos , Hipocótilo/metabolismo , Hipocótilo/efeitos da radiação , Indóis/química , Luz , Reguladores de Crescimento de Planta/farmacologia , Plântula , Temperatura
12.
Nat Commun ; 11(1): 855, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071312

RESUMO

Cognitive decline is one of the complications of type 2 diabetes (T2D). Intermittent fasting (IF) is a promising dietary intervention for alleviating T2D symptoms, but its protective effect on diabetes-driven cognitive dysfunction remains elusive. Here, we find that a 28-day IF regimen for diabetic mice improves behavioral impairment via a microbiota-metabolites-brain axis: IF enhances mitochondrial biogenesis and energy metabolism gene expression in hippocampus, re-structures the gut microbiota, and improves microbial metabolites that are related to cognitive function. Moreover, strong connections are observed between IF affected genes, microbiota and metabolites, as assessed by integrative modelling. Removing gut microbiota with antibiotics partly abolishes the neuroprotective effects of IF. Administration of 3-indolepropionic acid, serotonin, short chain fatty acids or tauroursodeoxycholic acid shows a similar effect to IF in terms of improving cognitive function. Together, our study purports the microbiota-metabolites-brain axis as a mechanism that can enable therapeutic strategies against metabolism-implicated cognitive pathophysiologies.


Assuntos
Disfunção Cognitiva/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Jejum , Microbioma Gastrointestinal/fisiologia , Animais , Encéfalo/metabolismo , Cognição , Biologia Computacional , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Metabolismo Energético/genética , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal/genética , Regulação da Expressão Gênica , Hipocampo/metabolismo , Indóis/metabolismo , Resistência à Insulina , Masculino , Metaboloma , Camundongos , Propionatos/metabolismo , RNA Ribossômico 16S , Serotonina/metabolismo , Sinapses/ultraestrutura , Ácido Tauroquenodesoxicólico/metabolismo
13.
Chem Commun (Camb) ; 56(20): 3089-3092, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32052805

RESUMO

Reaction of FeIII(O2˙-)(TPP) with 2,3-dimethylindole at -40 °C gives the ring-opened, dioxygenated N-(2-acetyl-phenyl)-acetamide product. The reaction was monitored in situ by low-temperature UV-vis and 1H NMR spectroscopies. This work demonstrates that a discrete iron(iii)(superoxo) porphyrin is competent to carry out indole oxidation, as proposed for the tryptophan and indoleamine 2,3-dioxygenases.


Assuntos
Compostos Férricos/química , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indóis/química , Metaloporfirinas/química , Superóxidos/química , Triptofano Oxigenase/química , Compostos Férricos/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indóis/metabolismo , Metaloporfirinas/metabolismo , Estrutura Molecular , Oxirredução , Superóxidos/metabolismo , Triptofano Oxigenase/metabolismo
14.
Toxicol Lett ; 324: 104-110, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32092453

RESUMO

Dietary and microbial indoles can act as ligands and activators of pregnane X receptor (PXR), with implications in human intestinal health. In the current study, we examined the effects of simple mono-methylated indoles (MMIs) on the activity and function of PXR, using a series of human hepatic and intestinal cell models. Indoles 1-MMI and 2-MMI strongly induced CYP3A4 and MDR1 mRNAs in human intestinal adenocarcinoma cells LS180, but not in primary human hepatocytes. The levels of CYP3A4 mRNA were increased by 1-MMI and 2-MMI in wild type, but not in PXR-knock-out human hepatic progenitor HepaRG cells, implying the involvement of PXR in CYP3A4 induction by MMIs. Utilizing reporter gene assay, we observed dose-dependent activation of PXR by all MMIs, and their efficacies and potencies were comparable. Tested MMIs also displayed moderate antagonist effects on PXR, revealing about partial agonist effects of these compounds. As demonstrated using the Chromatin immunoprecipitation assay (ChIP),1-MMI increased PXR occupancy of the CYP3A4 promoter. Time-Resolved Fluorescence Resonance Energy Transfer revealed that MMIs are weak ligands of human PXR. Collectively, we show that MMIs are ligands and partial agonists of human PXR, which induce PXR-regulated genes in human intestinal cells.


Assuntos
Hepatócitos/efeitos dos fármacos , Indóis/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Receptor de Pregnano X/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiologia , Hepatócitos/metabolismo , Humanos , Indóis/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos
15.
Biochem Pharmacol ; 174: 113845, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32032581

RESUMO

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC expresses AHR and AHR ligands have anti-cancer activity in TNBC. The aggressiveness of TNBC is due in part to JAG1-NOTCH1 signaling. ITE is a putative endogenous AHR ligand. We show that ITE reduces the expression of JAG1 the amount of Notch 1 intracellular domain (NICD1) and the phosphorylation of STAT3 (at tyrosine 705) in TNBC MDA-MB-231 cells. The STAT3 inhibitor STATTIC also reduced JAG1. STAT3, thus, mediates regulation of JAG1 in MDA-MB-231 cells. Reducing the expression of JAG1 with short interfering RNA decreases the growth, migration and invasiveness of MDA-MB-231 cells. JAG1, therefore, has cellular effects in MDA-MB-231 cells under basal conditions. We consequently evaluated if exposing cells to greater amounts of JAG1 would counteract ITE cellular effects in MDA-MB-231 cells. The results show that JAG1 does not counteract the cellular effects of ITE. JAG1, thus, has no effect on growth or invasiveness in MDA-MB-231 cells treated with ITE. JAG1, therefore, has context dependent roles in MDA-MB-231 cells (basal versus ITE treatment). The results also show that other pathways, not inhibition of the JAG1-NOTCH1 pathway, are important for mediating the growth and invasive inhibitory effect of ITE on MDA-MB-231 cells.


Assuntos
Antineoplásicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Indóis/metabolismo , Proteína Jagged-1/metabolismo , Receptor Notch1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Tiazóis/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Proteína Jagged-1/antagonistas & inibidores , Ligantes , Células MCF-7 , Receptor Notch1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
16.
J Med Chem ; 63(6): 3131-3141, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32105468

RESUMO

Structure-based stabilization of protein-protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.


Assuntos
Alcaloides/farmacologia , Antivirais/farmacologia , Indóis/farmacologia , Proteínas do Nucleocapsídeo/metabolismo , Multimerização Proteica/efeitos dos fármacos , Alcaloides/química , Alcaloides/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/química , Ligação Proteica , Domínios Proteicos , Alinhamento de Sequência , Células Vero
17.
Comput Biol Chem ; 84: 107189, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31891900

RESUMO

A series of indole-derived methoxylated chalcones were described as anti-dermatophyte agents. The in vitro antifungal susceptibility testing against different dermatophytes revealed that most of compounds had potent activity against the dermatophyte strains. In particular, the 4-ethoxy derivative 4d with MIC values of 0.25-2 µg/ml was the most potent compound against Trichophyton interdigitale, Trichophyton veruccosum and Microsporum fulvum. Moreover, the 4-butoxy analog 4i displaying MIC values in the range of 1-16 µg/ml had the highest inhibitory activity against Trichophyton mentagrophytes, Microsporum canis, and Arthroderma benhamiae. To predict whether the synthesized compounds interact with tubulin binding site of dermatophytes, the 3D-structure of target protein was modeled by homology modeling and then used for molecular docking and molecular dynamics (MD) simulation studies. Docking simulation revealed that the promising compound 4d can properly bind with tubulin. The molecular dynamics analysis showed that interactions of compound 4d with the active site of target protein have binding stability throughout MD simulation. The results of this study could utilize in the design of more effective antifungal drugs with tubulin inhibition mechanism against keratinophilic fungi.


Assuntos
Antifúngicos/farmacologia , Chalconas/farmacologia , Indóis/farmacologia , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/metabolismo , Arthrodermataceae/efeitos dos fármacos , Sítios de Ligação , Chalconas/química , Chalconas/metabolismo , Humanos , Indóis/química , Indóis/metabolismo , Testes de Sensibilidade Microbiana , Fungos Mitospóricos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
18.
Am J Clin Nutr ; 111(5): 1087-1099, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31942927

RESUMO

BACKGROUND: Indoxyl sulfate (IS) and p-cresyl sulfate (pCS), 2 important protein-bound uremic toxins, are independent risk factors for cardiovascular disease in patients with end-stage renal disease. Indole and p-cresol are gut microbiome-generated precursors of IS and pCS. OBJECTIVE: The aim of the present study was to determine whether inulin-type fructans (ITFs) reduce the production of indole and p-cresol by altering their producing bacteria in patients with peritoneal dialysis. METHODS: Patients receiving peritoneal dialysis for >3 mo without diabetes and not using antibiotics were recruited to a randomized, double-blind, placebo-controlled, crossover trial of ITF intervention over 36 wk (12-wk washout). The primary outcomes were gut microbiome, fecal indole and p-cresol, indole-producing bacteria, p-cresol-producing bacteria, and serum IS and pCS. The secondary outcomes were fecal pH, 24-h urine, and dialysis removal of IS and pCS. RESULTS: Of 21 individuals randomly assigned, 15 completed the study. The daily nutrient intakes, including protein, tryptophan, and tyrosine, were isostatic during the prebiotic, washout, and placebo intervention. There were no baseline differences in the outcomes of interest between treatments. For fecal indole, its concentrations did not change significantly in either treatment. However, there was a trend toward the treatment-by-time effect (P = 0.052), with a quantitative reduction in the ITF treatment and an increase in the control. The difference in the changes between the 2 treatments was significant (-10.07 ± 7.48 µg/g vs +13.35 ± 7.66 µg/g; P = 0.040). Similar to Bacteroides thetaiotaomicron, there was a difference over time between the 2 treatments, with a significant treatment and time interaction effect (P = 0.047). There were no treatment, time, or interaction effects for fecal p-cresol, serum IS and pCS, 24-h urine, and dialysis removal of IS and pCS. CONCLUSIONS: Our results suggested that ITFs restricted the increase in gut microbiome-generated indole in patients with peritoneal dialysis. This trial was registered at http://www.chictr.org.cn/showproj.aspx?proj=21228 as ChiCTR-INR-17013739.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Indóis/metabolismo , Inulina/administração & dosagem , Falência Renal Crônica/tratamento farmacológico , Falência Renal Crônica/microbiologia , Prebióticos/administração & dosagem , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Cresóis , Estudos Cross-Over , Fezes/microbiologia , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal
19.
Infect Immun ; 88(4)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31964747

RESUMO

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.


Assuntos
Adaptação Fisiológica , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Triptofano/metabolismo , Linhagem Celular , Chlamydia trachomatis/citologia , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/enzimologia , Chlamydophila pneumoniae/citologia , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/enzimologia , Inibidores Enzimáticos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Indóis/metabolismo , Leucina/metabolismo , Modelos Biológicos , Transcrição Genética
20.
Biochem Biophys Res Commun ; 524(1): 64-69, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980178

RESUMO

PGRMC1 is a protein from the MAPR family with a range of cellular functions. PGRMC1 has been described to play a role in fertility, neuroprotection, steroidogenesis, membrane trafficking and in cancer cell biology. PGRMC1 represents a likely key regulator of cell metabolism and proliferation, as well as a potential target for anti-cancer therapies. To further understand the functions of PGRMC1 and the mechanism of the small molecule inhibitor of PGRMC1, AG-205, proteins differentially bound to PGRMC1 were identified following AG-205 treatment of MIA PaCa-2 cells. Our results suggest that AG-205 influences PGRMC1 interactions with the actin cytoskeleton. The binding of two PGRMC1-associated proteins that support this, RACK1 and alpha-Actinin-1, was reduced following AG-205 treatment. The biology associated with PGRMC1 binding partners identified here merits further investigation.


Assuntos
Actinas/metabolismo , Indóis/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Receptores de Progesterona/antagonistas & inibidores , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Ligação Proteica , Receptores de Quinase C Ativada/metabolismo
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