RESUMO
Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of â¼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.
Assuntos
Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Animais , Humanos , Bacteriófago M13 , Técnicas de Visualização da Superfície Celular , Células HEK293 , Ligação ProteicaRESUMO
Micropterus salmoides rhabdovirus (MSRV) is a significant pathogen that causes high morbidity and mortality in largemouth bass, leading to enormous economic losses for largemouth bass aquaculture in China. The aim of this study was to investigate the efficacy of four disinfectants (potassium permanganate, glutaraldehyde, trichloroisocyanuric acid and povidone iodine) on MSRV, to control the infection and transmission of MSRV in largemouth bass aquaculture. The disinfectants were tested at different concentrations (5, 25, 50, 100 and 500 mg/L) prepared with distilled water for 30 min contact time, and the viral nucleic acid was quantified using qPCR and the infectivity was tested by challenge experiment. Potassium permanganate at 5-500 mg/L, glutaraldehyde at 500 mg/L, trichloroisocyanuric acid at 50-500 mg/L and povidone iodine at 500 mg/L concentration could effectively decrease the virus nucleic acid, and the survival rate of largemouth bass juveniles after challenge experiment increased significantly from 3.7% ± 6.41% to 33.33 ± 11.11% - 100%. Moreover, the minimum effective time of 5 mg/L potassium permanganate was further studied at 2, 5, 10 and 20 min contact time. The viral nucleic acid decreased significantly at 5-20 min exposure time, and the survival rate increased significantly from 7.41% ± 6.41% to 77.78 ± 11.11% - 100%. The median lethal concentration (LC50 ) values of potassium permanganate were 10.64, 6.92 and 3.7 mg/L at 24, 48 and 96 h, respectively. Potassium permanganate could be used for the control of MSRV in the cultivation process; the recommended concentration is 5 mg/L and application time should be less than 24 h. The results could be applied to provide a method to control the infection and transmission of MSRV in water, and improve the health status of largemouth bass.
Assuntos
Bass , Desinfetantes , Doenças dos Peixes , Ácidos Nucleicos , Rhabdoviridae , Animais , Desinfetantes/farmacologia , Glutaral , Permanganato de Potássio , Povidona-Iodo , Doenças dos Peixes/prevenção & controle , ÁguaRESUMO
Decapod iridescent virus 1 (DIV1), White spot syndrome virus (WSSV), and Enterocytozoon hepatopenaei (EHP) pose serious threats to the shrimp farming. To date, early detection remains an important way to control the occurrence and diffusion of these pathogens. Here, we developed for the first time, a loop-mediated isothermal amplification (LAMP)-based microfluidic chip detection system, which could detect DIV1, WSSV, and EHP simultaneously. The limits of detection (LoD) of the system were 10 copies/reaction for EHP and DIV1, and 102 copies/reaction for WSSV. The entire detection procedure could be completed rapidly in 40 min at 63°C with 100% specificity and had no cross-reaction with other common shrimp pathogens. This newly established method was further validated using 94 Penaeus vannamei clinical samples, which were comparable to a typical qPCR assay and revealed good stability and reproducibility. These results illustrate that this LAMP microfluidic chip detection system allows rapid triplex pathogen analysis and could satisfy the demands of the field and routine diagnoses in aquaculture.
Assuntos
Doenças dos Peixes , Penaeidae , Viroses , Animais , Microfluídica , Técnicas de Amplificação de Ácido Nucleico/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Escherichia coli is the most common Gram-negative bacillary organism causing neonatal meningitis. Escherichia coli meningitis remains an important cause of mortality and morbidity, but the pathogenesis of E. coli penetration of the blood-brain barrier remains incompletely understood. Escherichia coli entry into the brain occurs in the meningeal and cortex capillaries, not in the choroid plexus, and exploits epidermal growth factor receptor (EGFR) and cysteinyl leukotrienes (CysLTs) for invasion of the blood-brain barrier. The present study examined whether EGFR and CysLTs are inter-related in their contribution to E. coli invasion of the blood-brain barrier and whether counteracting EGFR and CysLTs is a beneficial adjunct to antibiotic therapy of E. coli meningitis. We showed that (a) meningitis isolates of E. coli exploit EGFR and CysLTs for invasion of the blood-brain barrier, (b) the contribution of EGFR is upstream of that of CysLTs, and (c) counteracting EGFR and CysLTs as an adjunctive therapy improved the outcome (survival, neuronal injury and memory impairment) of animals with E. coli meningitis. These findings suggest that investigation of host factors contributing to E. coli invasion of the blood-brain barrier will help in enhancing the pathogenesis and development of new therapeutic targets for E. coli meningitis in the era of increasing resistance to conventional antibiotics.
Assuntos
Acetatos/uso terapêutico , Barreira Hematoencefálica/microbiologia , Ciclopropanos/uso terapêutico , Cisteína/metabolismo , Receptores ErbB/metabolismo , Escherichia coli/patogenicidade , Gefitinibe/uso terapêutico , Leucotrienos/metabolismo , Meningite devida a Escherichia coli/microbiologia , Quinolinas/uso terapêutico , Sulfetos/uso terapêutico , Animais , Antibacterianos/uso terapêutico , Barreira Hematoencefálica/fisiopatologia , Encéfalo/irrigação sanguínea , Ceftriaxona/uso terapêutico , Células Cultivadas , Quimioterapia Combinada , Células Endoteliais , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Recém-Nascido , Antagonistas de Leucotrienos/uso terapêutico , Masculino , Meningite devida a Escherichia coli/tratamento farmacológico , Camundongos , Permeabilidade , Fosfolipases A2 Citosólicas/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMO
Sirtuin 1 (SIRT1) is an NAD+-dependent protein deacetylase that plays a critical role in controlling energy metabolism, stress response and aging. Hence, enhancing SIRT1 activity could be a potential therapeutic strategy to treat metabolic diseases such as diabetes. However, pharmacological activators for SIRT1 are scarce to date. In this study, using the optimized high throughput screening, we identified E6155, a piperazine 1, 4- diamide compound, as a new small molecular activator of SIRT1. We further found that E6155 significantly upregulated glucose uptake in cultured normal liver cells and skeletal muscle cells through increasing SIRT1 deacetylase activity. In type 2 diabetic KKAy mice, E6155 treatment markedly decreased the level of fasting glucose. Moreover, E6155 improved oral glucose tolerance and insulin tolerance. Euglycemic clamp and the homeostasis model assessment of insulin resistance index showed that E6155 ameliorated the insulin resistance and increased insulin sensitivity in diabetic mice. Mechanistically, we observed that the antidiabetic effects of E6155 were involved in SIRT1 dependent activation of LKB1/AMPK and IRS1/AKT pathways. In conclusion, our findings identified E6155 as a novel SIRT1 activator and suggested that E6155 could be a promising drug candidate for treating insulin resistance and diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Piperazinas/uso terapêutico , Sirtuína 1/metabolismo , Animais , Glicemia/análise , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Células Hep G2 , Humanos , Hipoglicemiantes/química , Insulina/metabolismo , Camundongos , Piperazinas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
Activation of liver X receptor (LXR) is associated with cholesterol metabolism and anti-inflammatory processes, which makes it beneficial to antiatherosclerosis therapy. Nevertheless, existing agonists that target LXR, for example TO901317, are related to unwanted side effects. In the present study, using a screening method we identified IMB-808, which displayed potent dual LXRα/ß agonistic activity. In vitro, IMB-808 effectively increased the expressing quantity of genes related to reverse cholesterol transport process as well as those associated with cholesterol metabolism pathway in multiple cell lines. Additionally, IMB-808 remarkably promoted cholesterol efflux from RAW264.7 as well as THP-1 macrophage cells and reduced cellular lipid accumulation accordingly. Interestingly, compared with TO901317, IMB-808 almost did not increase the expressing quantity of genes related to lipogenesis in HepG2 cells, which indicated that IMB-808 could exhibit fewer internal lipogenic side effects with a characteristic of selective LXR agonist. Furthermore, in comparison with the full LXR agonist TO901317, IMB-808 recruits coregulators differently and possesses a distinct predictive binding pattern for the LXR ligand-binding domain. In summary, our study demonstrated that IMB-808 could act as an innovative partial LXR agonist that avoids common lipogenic side effects, providing insight for the design of novel LXR modulators. Our data indicate that this compound might be used as a promising therapeutic agent for the prospective treatment of atherosclerosis in the future.
Assuntos
Benzodiazepinas/farmacologia , Colesterol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/genética , Receptores X do Fígado/agonistas , Animais , Benzodiazepinas/química , Transporte Biológico/efeitos dos fármacos , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Perfilação da Expressão Gênica , Células Hep G2 , Homeostase/efeitos dos fármacos , Humanos , Hidrocarbonetos Fluorados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipoproteínas LDL/farmacologia , Receptores X do Fígado/química , Receptores X do Fígado/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Mutação/genética , Domínios Proteicos , Células RAW 264.7 , Sulfonamidas/farmacologiaRESUMO
OBJECTIVES: Drug-resistant Mycobacterium tuberculosis poses a great threat to human health. Tyrosyl-tRNA synthetase (TyrRS) is one of the aminoacyl tRNA synthetases that catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. There are several distinctive differences between bacterial and human TyrRS and therefore it could be a potential target for developing antimicrobial agents. This study aimed to identify a new anti-TB agent targeting M. tuberculosis TyrRS (MtTyrRS). METHODS: We first used Mycobacterium smegmatis for a phenotypic screening of 20â000 compounds. The hit compounds were then screened with MtTyrRS. The interaction between hit compound IMB-T130 and the target protein was analysed by surface plasmon resonance (SPR) assay and molecular docking experiments. The target of IMB-T130 was further confirmed by the overexpression of the target protein. The antibacterial activity of IMB-T130 against various standard and clinical drug-resistant M. tuberculosis strains was evaluated using the microplate Alamar blue assay. RESULTS: Compound IMB-T130 was identified as a hit compound that inhibits the growth of M. smegmatis and the in vitro activity of MtTyrRS. The interaction between IMB-T130 and MtTyrRS was confirmed by SPR assay and molecular docking analysis. The higher MIC for a strain overexpressing the target protein also suggests that MtTyrRS is likely to be the target of IMB-T130. IMB-T130 shows excellent anti-TB activity and low cytotoxicity. CONCLUSIONS: IMB-T130 inhibits the growth of MDR-TB and XDR-TB by targeting MtTyrRS. Because of its low cytotoxicity against mammalian cells, IMB-T130 is a promising new agent against drug-resistant M. tuberculosis.
Assuntos
Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Tirosina-tRNA Ligase/antagonistas & inibidores , Antituberculosos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
A high-throughput screening effort on 45,000 compounds resulted in the discovery of a disubstituted oxazole as a new structural class inhibitor of Mycobacterium tuberculosis (Mtb). In order to improve the activity and investigate the SAR of this scaffold, a series of disubstituted azole analogues have been designed and synthesized. The newly synthesized compounds 1a-y were evaluated for their in vitro anti-TB activity versus replicating, multi- and extensive drug resistant Mtb strains. All the compounds, except 1o, 1p and 1q, showed potent anti-TB activity with MIC of 1-64 mg/L. The test of broad spectrum panel revealed that this series are specific to Mtb. The cytotoxicity assessment indicated that the compounds were not cytotoxic against HEK 293 cells. The compounds could have a novel mechanism to anti-Mtb as they can inhibit drug sensitive and drug resistant Mtb.
Assuntos
Antituberculosos/farmacologia , Dioxanos/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Oxazóis/farmacologia , Antituberculosos/síntese química , Dioxanos/síntese química , Farmacorresistência Bacteriana Múltipla , Células HEK293 , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazóis/síntese química , Relação Estrutura-AtividadeRESUMO
ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE(-/-)) mice treated with RUT for 8 weeks showed â¼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE(-/-) mice. Meanwhile, RUT treatment significantly increased the fecal (3)H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE(-/-) mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/biossíntese , Apolipoproteínas E/genética , Aterosclerose/prevenção & controle , Alcaloides Indólicos/farmacologia , Fígado/metabolismo , Quinazolinas/farmacologia , Receptores Depuradores Classe B/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Fígado/patologia , Camundongos , Camundongos Knockout , Células RAW 264.7 , Receptores Depuradores Classe B/genéticaRESUMO
Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Capreomicina/farmacologia , Proteínas Ribossômicas/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The ability to survive and proliferate in acidic environments is a prerequisite for the infection of Listeria monocytogenes. The glutamate decarboxylase (GAD) system is responsible for acid resistance, and three GAD homologs have been identified in L. monocytogenes: gadD1, gadD2, and gadD3. To examine whether GAD genes are specific to lineage, serovar, or certain subpopulation, we performed a systematic investigation on the prevalence of GAD genes in 164 L. monocytogenes. In contrast to gadD2 and gadD3 conserved in all L. monocytogenes strains, gadD1 was identified in 36.6% (60/164) of L. monocytogenes strains, including all serovar 1/2c and 68.5% (37/54) of serovar 1/2a strains, as well as a small fraction of serovar 1/2b (3.4%, 1/29) and lineage III (13.8%, 4/29) strains. All serovar 4b and lineage IV strains lacked this gene. According to the ascB-dapE structure, L. monocytogenes strains were classified into four subpopulations, carrying inlC2DE, inlGC2DE, inlGHE, or no internalin cluster, respectively. All L. monocytogenes strains with inlGC2DE or inlGHE pattern harbored gadD1, whereas those bearing inlC2DE or no internalin cluster between ascB and dapE lacked gadD1. In addition, other five non-monocytogenes Listeria species lacking ascB-dapE internalin cluster were gadD1-negative. Overall, the presence of gadD1 is not fully dependent on lineages or serovars but correlates with ascB-dapE internalin profiles, suggesting gadD1 might have co-evolved with the ascB-dapE internalin cluster in the primitive L. monocytogenes before divergence of serovars.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Glutamato Descarboxilase/genética , Listeria monocytogenes/genética , DNA Bacteriano/genética , Listeria monocytogenes/enzimologia , Especificidade da EspécieRESUMO
The immune response of brain cells to invading bacteria in vivo and the mechanism used by pathogenic bacteria to escape brain immune surveillance remain largely unknown. It is believed that microglia eliminate bacteria by phagocytosis based on in vitro data. Here we find that a small percentage of microglia in the brain engulf neonatal meningitis-causing Escherichia coli (NMEC), but more microglia are activated to produce tumor necrosis factor alpha (TNFα), which activates astrocytes to secrete complement component 3 (C3) involved in anti-bacterial activity. To evade anti-bacterial activity of the immune system, NMEC senses low concentration of threonine in cerebrospinal fluid (CSF) to down-modulate the expression of flagellin and reduce microglial TNFα and astrocyte C3 production. Our findings may help develop strategies for bacterial meningitis treatment.
Assuntos
Astrócitos , Microglia , Astrócitos/metabolismo , Bactérias/metabolismo , Encéfalo/metabolismo , Flagelina/metabolismo , Flagelina/farmacologia , Humanos , Recém-Nascido , Microglia/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Group B Streptococcus (GBS) remains the most common Gram-positive bacterium causing neonatal meningitis and GBS meningitis continues to be an important cause of mortality and morbidity. In this study, we showed that GBS penetration into the brain occurred initially in the meningeal and cortex capillaries, and exploits a defined host cell signaling network comprised of S1P2 , EGFR, and CysLT1. GBS exploitation of such network in penetration of the blood-brain barrier was demonstrated by targeting S1P2 , EGFR, and CysLT1 using pharmacological inhibition, gene knockout and knockdown cells, and gene knockout animals, as well as interrogation of the network (up- and downstream of each other). More importantly, counteracting such targets as a therapeutic adjunct to antibiotic therapy was beneficial in improving the outcome of animals with GBS meningitis. These findings indicate that investigating GBS penetration of the blood-brain barrier provides a novel approach for therapeutic development of GBS meningitis.
Assuntos
Meningites Bacterianas , Infecções Estreptocócicas , Animais , Receptores ErbB/metabolismo , Meningites Bacterianas/tratamento farmacológico , Transdução de Sinais , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus agalactiae/metabolismoRESUMO
The demand for novel antibiotics is imperative for drug-resistant Gram-negative bacteria which causes diverse intractable infection disease in clinic. Here, a comprehensive screening was implemented to identify potential agents that disrupt the assembly of ß-barrel outer-membrane proteins (OMPs) in the outer membrane (OM) of Gram-negative bacteria. The assembly of OMPs requires ubiquitous ß-barrel assembly machinery (BAM). Among the five protein subunits in BAM, the interaction between BamA and BamD is essential for the function of this complex. We first established a yeast two-hybrid (Y2H) system to confirm the interaction between BamA and BamD, and then screened agents that specifically disrupt this interaction. From this screen, we identified a compound IMB-H4 that specially blocks BamA-BamD interaction and selectively inhibits the growth of Escherichia coli and other Gram-negative bacteria. Moreover, our results suggest that IMB-H4 disrupts BamA-BamD interaction by binding to BamA. Strikingly, E. coli cells having been treated with IMB-H4 showed impaired OM integrity and decreased the abundance of OMPs. Therefore, an antibacterial agent was identified successfully using Y2H system, and this compound likely blocks the assembly of OMPs by targeting BamA-BamD interaction in Gram-negative bacteria.
RESUMO
Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria Escherichia coli, which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against E. coli. We used glutathione-S-transferase (GST) pull-down and surface plasmon resonance (SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction in vitro and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.
RESUMO
The global pandemic of multidrug-resistant (MDR) Mycobacterium tuberculosis (TB) drives for more effective anti-TB drugs with new drug target. Filamentous temperature sensitive protein Z (FtsZ), a GTP dependent prokaryotic cell division protein, forms a dynamic Z-ring in the center of the cell. Differences between bacterial FtsZ and eukaryotic tubulin make FtsZ a highly attractive drug target. In this study, we used phenotype screening of M. smegmatis and model screening targeting M. tuberculosis FtsZ (Mtb-FtsZ) to identify a hit compound TB-E12. TB-E12 was found to prevent the growth of M. smegmatis by inhibiting the GTPase activity of Mtb-FtsZ. Molecular docking and site-directed mutagenesis analyses identified Asn22 of Mtb-FtsZ as the key amino site. The higher MIC of TB-E12 for M. smegmatis strain overexpressing Mtb-FtsZ confirmed that Mtb-FtsZ is likely the target. Importantly, TB-E12 exhibits excellent anti-TB activity, but had no anti-bacterial activity to other strains. In vitro, the proliferation of Mycobacterium smegmatis was inhibited by TB-E12. All these results indicate TB-E12 is a promising lead compound against drug-resistant tuberculosis.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Desenho de Fármacos , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Humanos , Testes de Sensibilidade Microbiana/métodos , Simulação de Acoplamento Molecular/métodos , Terapia de Alvo Molecular/métodos , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Especificidade da EspécieRESUMO
The anti-tuberculosis (TB) agent IMB-T130 was speculated to be a multi-target compound. In this research, we found that IMB-T130 inhibits the catalytic activity of Mycobacterium tuberculosis 3-dehydroquinate synthase (MtDHQS), the enzyme in the second step of the shikimate pathway. IMB-T130 was identified as a selective inhibitor of MtDHQS with an IC50 value of 0.87 µg/mL. The interaction between the compound and protein was analysed by surface plasmon resonance and circular dichroism. Based on the in silico molecular docking results, the essential amino acids in the binding pocket were then confirmed by site-directed mutagenesis. Overexpression of DHQS reduced the antibacterial activity of IMB-T130 in cells, verifying that DHQS is the target of IMB-T130. IMB-T130 inhibited standard and drug-resistant M. tuberculosis strains by targeting DHQS. Our findings improve our understanding of MtDHQS and make it to be a potential target for new anti-TB drug discovery.
Assuntos
Antituberculosos/química , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Fósforo-Oxigênio Liases/química , Animais , Antituberculosos/farmacocinética , Catálise , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacocinética , Concentração Inibidora 50 , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/genética , Fósforo-Oxigênio Liases/antagonistas & inibidores , Fósforo-Oxigênio Liases/genética , Ligação Proteica , Ratos , Proteínas Recombinantes/químicaRESUMO
BACKGROUND AND AIMS: Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent protein deacetylase. Recent studies have demonstrated that enhancing SIRT1 expression or activity may modulate cholesterol and lipid metabolism. However, pharmacological and molecular regulators for SIRT1 are scarce. Here, we aimed to find novel small molecule modulators of SIRT1 to regulate cholesterol and lipid metabolism. METHODS: A high-throughput screening assay was established to identify SIRT1 activators. Surface plasmon resonance and immunoprecipitation were performed to confirm the interaction of E1231 with SIRT1. Cholesterol assay was performed to demonstrate the in vitro effect of E1231. The in vivo effect of E1231 was evaluated in experimental models. RESULTS: E1231, a piperazine 1,4-diamide compound, was identified as a SIRT1 activator with EC50 value of 0.83⯵M. E1231 interacted with recombinant human SIRT1 protein and deacetylated liver X receptor-alpha (LXRα). E1231 increased ATP-binding cassette transporter A1 (ABCA1) expression in RAW 264.7â¯cells dependent on SIRT1 and LXRα. E1231 promoted cholesterol efflux and inhibited lipid accumulation in RAW 264.7â¯cells via SIRT1 and ABCA1. In the golden hamster hyperlipidemia model, E1231 treatment decreased total cholesterol and triglyceride levels in both serum and the liver, while increased cholesterol content in feces. Moreover, E1231 increased ABCA1 and SIRT1 protein expression in the liver. In ApoE-/- mice, E1231 treatment reduced atherosclerotic plaque development compared with untreated ApoE-/- mice. CONCLUSIONS: We identified a novel SIRT1 activator E1231 and elucidated its beneficial effects on lipid and cholesterol metabolism. Our study suggests that E1231 might be developed as a novel drug for treating atherosclerosis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aterosclerose/prevenção & controle , Colesterol/sangue , Diaminas/farmacologia , Ativadores de Enzimas/farmacologia , Hipolipemiantes/farmacologia , Macrófagos/efeitos dos fármacos , Piperazinas/farmacologia , Sirtuína 1/metabolismo , Triglicerídeos/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/patologia , Biomarcadores/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Ativação Enzimática , Células Hep G2 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Mesocricetus , Camundongos , Camundongos Knockout para ApoE , Células RAW 264.7 , Sirtuína 1/genética , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Tuberculosis remains one of the deadliest infectious diseases in humans. It has caused more than 100 million deaths since its discovery in 1882. Currently, more than 5 million people are infected with TB bacterium each year. The cell wall of Mycobacterium tuberculosis plays an important role in maintaining the ability of mycobacteria to survive in a hostile environment. Therefore, we report a virtual screening (VS) study aiming to identify novel inhibitors that simultaneously target RmlB and RmlC, which are two essential enzymes for the synthesis of the cell wall of M. tuberculosis. METHODS: A hybrid VS method that combines drug-likeness prediction, pharmacophore modeling and molecular docking studies was used to indentify inhibitors targeting RmlB and RmlC. RESULTS: The pharmacophore models HypoB and HypoC of RmlB inhibitors and RmlC inhibitors, respectively, were developed based on ligands complexing with their corresponding receptors. In total, 20 compounds with good absorption, distribution, metabolism, excretion, and toxicity properties were carefully selected using the hybird VS method. DISCUSSION: We have established a hybrid VS method to discover novel inhibitors with new scaffolds. The molecular interactions of the selected potential inhibitors with the active-site residues are discussed in detail. These compounds will be further evaluated using biological activity assays and deserve consideration for further structure-activity relationship studies.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Carboidratos Epimerases/antagonistas & inibidores , Parede Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Parede Celular/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/citologia , Conformação ProteicaRESUMO
Nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the regulation of glucose homeostasis and lipid metabolism. However, current PPARγ-targeting drugs such as thiazolidinediones (TZDs) are associated with undesirable side effects. We identified a small molecular compound, F12016, as a selective PPARγ agonist by virtual screening, which showed moderate PPARγ agonistic activity and binding ability for PPARγ. F12016 did not activate other PPAR subtypes at 30⠵M and selectively modulated PPARγ target gene expression. In diabetic KKAy mice, F12016 had insulin-sensitizing and glucose-lowering properties, and suppressed weight gain. In vitro, F12016 effectively increased glucose uptake and blocked cyclin-dependent kinase 5-mediated phosphorylation of PPARγ at Ser273, but slightly triggered adipogenesis and less inhibited osteoblastogenesis than rosiglitazone. Moreover, compared with the full agonist rosiglitazone, F12016 had a distinct group of coregulators and a different predicted binding mode for the PPARγ ligand-binding domain. A site mutation assay confirmed the key epitopes, especially Tyr473 in AF-2. In summary, our study shows that F12016 is a non-TZD, novel selective PPARγ agonist without the classical lipogenic side effects, which may provide a new structural strategy for designing PPARγ ligands with advantages over TZDs.