RESUMO
Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 µM and 3.6 µM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.
Assuntos
Espectrometria de Massas em Tandem , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Células HeLa , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Simulação de Acoplamento Molecular , Inibidores Enzimáticos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de MembranaRESUMO
In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.
Assuntos
Acinetobacter baumannii , Antibacterianos , Helicobacter pylori , Hemiterpenos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Compostos Organofosforados/farmacologia , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).
Assuntos
Proteínas de Bactérias/metabolismo , Guanidina/análogos & derivados , Mycobacterium tuberculosis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Ureia/análogos & derivados , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Guanidina/química , Guanidina/metabolismo , Guanidina/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Conformação Molecular , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Ureia/química , Ureia/metabolismo , Ureia/farmacologiaRESUMO
Translocase MraY is the target for bacteriophage ÏX174 lysis protein E, which interacts via a protein-protein interaction mediated by Phe-288 and Glu-287 of E. coli MraY, and an Arg-Trp-x-x-Trp motif on protein E, also found in several cationic antimicrobial peptides. Analogues of Arg-Trp-octyl ester, found previously to show antimicrobial activity, were tested for antimicrobial activity, with Lys-Trp-oct (MIC50P. fluorescens 5 µg/mL) and Arg-Trp-decyl ester (MIC50P. fluorescens 3 µg/mL) showing enhanced antimicrobial activity. Synthesis and testing of α-helix peptidomimetic analogues for this motif revealed improved antibacterial activity (MIC50E. coli 4-7 µg/mL) for analogues containing two aromatic substituents, mimicking the Arg-Trp-x-x-Trp motif, and MraY inhibition (IC50 140 µM) by one such peptidomimetic. Investigation of mechanism of action using the Alamar Blue membrane permeabilisation assay revealed bacteriostatic and bacteriocidal mechanisms in different members of this set of compounds, raising the possibility of more than one biological target. The observed antimicrobial activity and MraY inhibition shown by peptidomimetic compounds confirms that this site could be targeted by drug-like molecules.
Assuntos
Antibacterianos/farmacologia , Peptídeos Antimicrobianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Peptidomiméticos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Antimicrobianos/síntese química , Peptídeos Antimicrobianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas Virais/metabolismoRESUMO
Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Oligopeptídeos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Uridina/análogos & derivados , Animais , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Interações Hidrofóbicas e Hidrofílicas , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oligopeptídeos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Uridina/química , Uridina/farmacologia , Peixe-ZebraRESUMO
Sphingolipids (SLs), such as ceramide, glucosylceramide (GlcCer), and sphingomyelin, play important roles in the normal development/functions of the brain and peripheral tissues. Disruption of SL homeostasis in cells/organelles, specifically up-regulation of ceramide, is involved in multiple diseases including Alzheimer's disease (AD). One of the pathological features of AD is aggregates of amyloid beta (Aß) peptides, and SLs regulate both the formation/aggregation of Aß and Aß-induced cellular responses. Up-regulation of ceramide levels via de novo and salvage synthesis pathways is reported in Aß-treated cells and brains with AD; however, the effects of Aß on ceramide decomposition pathways have not been elucidated. Thus, we investigated the effects of the 25-35-amino acid Aß peptide (Aß25-35), the fundamental cytotoxic domain of Aß, on SL metabolism in cells treated with the fluorescent nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide). Aß25-35 treatment reduced the formation of NBD-GlcCer mediated by GlcCer synthase (GCS) without affecting the formation of NBD-sphingomyelin or NBD-ceramide-1-phosphate, and reduced cell viability. Aß25-35-induced responses decreased in cells treated with D609, a putative inhibitor of sphingomyelin synthases. Aß25-35-induced cytotoxicity significantly increased in GCS-knockout cells and pharmacological inhibition of GCS alone demonstrated cytotoxicity. Our study revealed that Aß25-35-induced cytotoxicity is at least partially mediated by the inhibition of GCS activity.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Glucosiltransferases/antagonistas & inibidores , Norbornanos/farmacologia , Fragmentos de Peptídeos/metabolismo , Tiocarbamatos/farmacologia , Doença de Alzheimer/patologia , Linhagem Celular , Glucosiltransferases/metabolismo , Humanos , Norbornanos/uso terapêutico , Tiocarbamatos/uso terapêutico , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.
Assuntos
Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Pirimidinonas/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Ureia/química , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium abscessus/enzimologia , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
High infiltration of M2-polarized macrophages in the primary tumor indicates unfavorable prognosis and poor overall survival in the patients with triple-negative breast cancer (TNBC). Thus, reversing M2-polarized tumor-associated macrophages in the tumors has been considered as a potential therapeutic strategy for TNBC. Sphingomyelin synthase 2 (SMS2) is the key enzyme for sphingomyelin production, which plays an important role in plasma membrane integrity and function. In this study we investigated whether SMS2 inhibitor or SMS2 gene knockout could reduce macrophages M2 polarization and tumor progression in a mouse model of TNBC. We showed that SMS2 mRNA expression was linked to immunosuppressive tumor microenvironment and poor prognosis in TNBC patients. The knockout of SMS2 or application of 15w (a specific SMS2 inhibitor) markedly decreased the generation of M2-type macrophages in vitro, and reduced the tumor weight and lung metastatic niche formation in a 4T1-TNBC mouse model. We further demonstrated that the in vivo antitumor efficacy of 15w was accompanied by a multifaceted remodeling of tumor immune environment reflecting not only the suppression of M2-type macrophages but also diminished levels of regulatory T cells and myeloid-derived suppressor cells leading to a dramatically improved infiltration of antitumor CD8+ T lymphocytes. Collectively, our results reveal a novel and important role of SMS2 in the protumorigenic function and may offer a new strategy for macrophage-targeted anticancer therapy.
Assuntos
Macrófagos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Neoplasias de Mama Triplo Negativas/fisiopatologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Técnicas de Inativação de Genes , Humanos , Imunidade Celular/efeitos dos fármacos , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
Species of the genus Rhododendron have been used in traditional Chinese medicine, with the medicinal herb "Manshanfong" used as an expectorant and for the treatment of acute bronchitis. Daurichromenic acid (DCA), a constituent of Rhododendron dauricum, is a meroterpenoid with antibacterial, anti-HIV, and anti-inflammatory activities. However, the mechanisms underlying these pharmacologic activities are poorly understood. To develop new drugs based on DCA, more information is required regarding its interactions with biomolecules. The present study showed that DCA inhibits the activity of the enzyme sphingomyelin synthase, with an IC50 of 4 µM. The structure-activity relationships between DCA and sphingomyelin synthase were evaluated using derivatives and cyclized hongoquercin A. In addition, DCA was found to inhibit amyloid ß aggregation. These results may help in the design of effective drugs based on DCA.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Cromanos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Plantas Medicinais/química , Agregados Proteicos/efeitos dos fármacos , Rhododendron/química , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Cromanos/química , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Estrutura MolecularRESUMO
Sphingomylin participates in sperm function in animals, and also regulates the Akt and ERK signaling pathways, both of which are associated with the asthenospermia. Sphingomyelin synthase 2 (SMS2) is involved in the biosynthesis of sphingomylin. To determine the relationship between SMS2 and human sperm function, we analyzed the distribution of SMS2 in human sperm and testes, and SMS2 expression in patients with asthenospermia and normozoospermia; human sperm were treated with anti-SMS2, and the sperm motility, penetration ability into methylcellulose, capacitation and acrosome reaction, and sperm [Ca2+]i imaging were evaluated, while the Akt and ERK pathway and cleaved caspase 3 were also analyzed. Results showed that SMS2 was localized in the testis and human sperm, and the protein levels of normozoospermia were higher than asthenospermia. Inhibition of SMS2 activity significantly decreased sperm motility and penetration ability into methylcellulose, but had no influence on capacitation and acrosome reaction, or on intracellular [Ca2+]i compared to IgG-treated control groups. Moreover, the phosphorylation level of Akt was decreased, whereas the phosphorylation of ERK and cleaved-caspase 3 levels were significantly increased. Taken together, SMS2 can affect sperm motility and penetration ability into methylcellulose, and participate in apoptosis associated with the Akt and ERK signaling pathways.
Assuntos
Apoptose , Espermatozoides/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Caspase 3/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Metilcelulose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Motilidade dos Espermatozoides , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidoresRESUMO
With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Candida albicans/enzimologia , Reposicionamento de Medicamentos , Proteínas Fúngicas/antagonistas & inibidores , Testes de Sensibilidade MicrobianaRESUMO
Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.
Assuntos
Éxons , Fibroblastos/metabolismo , Mucolipidoses/terapia , Mutação , Oligonucleotídeos Antissenso/genética , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Sequência de Aminoácidos , Estudos de Casos e Controles , Humanos , Mucolipidoses/genética , Mucolipidoses/patologia , Fenótipo , Homologia de Sequência , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Sphingomyelin synthase 2 (SMS2) has attracted attention as a drug target for the treatment of various cardiovascular and metabolic diseases. The modification of a high throughput screening hit, 2-quinolone 10, enhanced SMS2 inhibition at nanomolar concentrations with good selectivity against SMS1. To improve the pharmaceutical properties such as passive membrane permeability and aqueous solubility, adjustment of lipophilicity was attempted and 1,8-naphthyridin-2-one 37 was identified as a potent and selective SMS2 inhibitor. A significant reduction in hepatic sphingomyelin levels following repeated treatment in mice suggested that compound 37 could be an effective in vivo tool for clarifying the role of SMS2 enzyme and developing the treatment for SMS2-related diseases.
Assuntos
Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Linhagem Celular , Descoberta de Drogas , Inibidores Enzimáticos , Humanos , Masculino , CamundongosRESUMO
Enantiomers or diastereomers of chiral bioactive compounds often exhibit different biological and toxicological properties. Here, we report the efficient synthesis of four stereoisomers of sphingosine and derivatization of unique chiral ceramides through a combinatorial chemistry by solid-phase activated resin ester. In addition, to test the effectivity of stereochemistry of ceramide, we demonstrated a cell-based assay of sphingomyelin synthase inhibition in the presence ofchiral unique ceramides, which suggested that libraries of this sort will be a rich source of biologically active synthetic molecules.
Assuntos
Ceramidas/química , Ceramidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Ceramidas/síntese química , Inibidores Enzimáticos/síntese química , Fibroblastos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Esfingosina/química , Estereoisomerismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Bacterial lipopolysaccharide (LPS) is recognized by CD14 protein and the Toll-like receptor (TLR)4/MD2 complex localized in the plasma membrane of immune cells. TLR4 triggers two signaling pathways engaging the MyD88 and TRIF adaptor proteins which lead to production of various pro-inflammatory cytokines. These processes are likely to be modulated by sphingomyelin, as the CD14 - TLR4 interaction takes place in plasma membrane rafts enriched in this lipid. To verify this assumption, we analyzed the influence of tricyclodecane-9-yl xanthogenate (D609), which was proven here to be an SMS inhibitor, and silencing of sphingomyelin synthase (SMS) 1 and/or SMS2 on LPS-induced signaling in macrophages. LPS up-regulated the expression and activity of SMS while exposure to D609 or silencing of SMS1 and SMS2 counteracted this action and led (except for SMS2 silencing) to a depletion of sphingomyelin in cells. Concomitantly, the MyD88- and TRIF-dependent signaling pathways of TLR4 were inhibited with the latter being especially sensitive to the reduction of the SMS1 and/or SMS2 activity. The D609 treatment and SMS1 and/or SMS2 depletion all reduced the level of CD14 protein in cells, which likely was an important determinant of the reduction of the LPS-induced pro-inflammatory responses.
Assuntos
Transdução de Sinais/imunologia , Esfingomielinas/metabolismo , Receptor 4 Toll-Like/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Norbornanos , Cultura Primária de Células , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos , Tionas/farmacologia , Receptor 4 Toll-Like/genética , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) has been proven to be the most common liver disease in the world, which is a sterile liver disease and is characterized by chronic hepatic steatosis and inflammation. The first step of the spectrum of the disease is the non-alcoholic fatty liver (NAFL). Based on hepatocellular necrosis and inflammation, NAFL will progress to non-alcoholic steatohepatitis (NASH), which may have the potential to progress cirrhosis, and even hepatocellular carcinoma (HCC) in a few years. Kupffer cells (KCs) are liver-resident macrophages and have been proven to play a crucial role in NAFLD development. Cardiolipin is reported to be effective to trigger the activation of NLRP3 inflammasome through a ROS-independent signaling pathway. However, the exact mechanism of NLRP3 inflammasome activated by cardiolipin in KCs is still unclear. MATERIALS AND METHODS: To make clear of the specific mechanism mentioned above, we firstly used a MCD-induced NASH mice model to demonstrate that CLS1 suppression reduced hepatic steatosis and inflammation. Secondly, the results of IHC staining indicated that the expressions of CLS1 and NLRP3 in liver tissues were significantly upregulated in the NASH group compared to the ND group. On the contrary, CLS1 inhibition significantly downregulated NLRP3 expression in liver tissues, which indicated that CLS1 probably regulated the level of NLRP3 expression. Furthermore, we demonstrated that CLS1 suppression significantly ameliorated the liver function and decreased the TG level, and interleukin-1ß (IL-1ß) and IL-18 were markedly reduced upon CLS1 inhibition. RESULTS: In this work, we reported that cardiolipin is involved in the development of NASH, and the suppression of the cardiolipin synthesis by shRNA-CLS1 could ameliorate the hepatic pathogenic manifestations, as well as the serum inflammatory biomarkers. We further showed that the protein expressions of CLS1, NLRP3, ASC, and Caspase-1 were all upregulated in the NASH liver tissues and palmitic stimulated KCs. CONCLUSIONS: Our study showed that the upregulation of NLRP3 inflammasome activated by cardiolipin is crucial in NASH pathogenesis, which might provide a novel potential role of cardiolipin blockade in the treatment of NASH.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cardiolipinas/metabolismo , Caspase 1/metabolismo , Inflamassomos/metabolismo , Células de Kupffer/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Inflamação , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , RNA Interferente Pequeno , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Triglicerídeos/metabolismoRESUMO
Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1-3, which converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.
Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Azepinas/química , Mycobacterium/efeitos dos fármacos , Relação Estrutura-Atividade , Antituberculosos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Estrutura Molecular , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidoresRESUMO
Insulin resistance is a typical precursor and primary feature of type 2 diabetes mellitus (T2DM). Sphingomyelin (SM) is a kind of sphingolipid located in animal brain, liver, kidney and muscle. Sphingomyelin synthase 2 (SMS2) is the key enzyme in the synthesis of sphingomyelin, inhibition of which shows protective effects on cardiovascular and glucose metabolism. We used Ly93, a selective sphingomyelin synthase 2 inhibitor, to investigate the effect of SMS2 inhibitor on insulin resistance in vitro and in vivo. Our previous studies have shown that Ly93 is able to dose-dependently inhibit the SMS activity and attenuate the atherosclerotic lesions in apoE knock out mice. In this present study, we found that high fat diet (HFD) induced insulin-resistant C57BL/6 mice treated with Ly93 were more sensitive to insulin than untreated mice, and presented lower blood insulin levels and improved insulin tolerance. Furthermore, insulin signal pathway related protein levels were detected by western blot, which indicated that SMS2 inhibitor significantly upregulated the phosphorylation of IRS-1, Akt and GSK-3ß, thus enhanced the insulin signaling. In vitro, Ly93 enhanced the phosphorylation of Akt in HepG2 cells, which was reversed by exogenous sphingomyelin. These results suggest that SMS2 inhibitor could ameliorate insulin resistance via regulating the insulin signaling. Our findings support that SMS2 is a potential target for insulin resistance.
Assuntos
Inibidores Enzimáticos/farmacologia , Resistência à Insulina , Insulina/sangue , Proteínas de Membrana/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Compostos Orgânicos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Dieta Hiperlipídica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Coenzima A/metabolismo , Guanidina/análogos & derivados , Hidrolases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Ureia/análogos & derivados , Proteína de Transporte de Acila/metabolismo , Animais , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Feminino , Guanidina/farmacologia , Hidrolases/genética , Metabolismo dos Lipídeos , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Óperon , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas , Ureia/farmacologiaRESUMO
There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, which encodes the α and ß subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.