RÉSUMÉ
The rapid proliferation and colonization of probiotics in the intestines are essential for human health. Quorum sensing (QS) is a communication mechanism among bacteria, which can regulate various bacterial crowd behavior. This study aimed to enhance the viability of Lactobacillus reuteri 1-12 by regulating QS. Herein, we built a database containing 72 natural products (previously reported) that can improve intestinal flora. Virtual screening (VS) was subsequently conducted to screen four potential active compounds. After that, molecular docking was conducted to analyze the binding mode of the four natural products to S-Ribosylhomocysteinase (LuxS). The results showed that norathyriol, mangiferin, baicalein, and kaempferol had good binding ability to LuxS. The validation experiment showed that norathyriol, mangiferin, baicalein, and kaempferol could inhibit the production of autoinducer-2 (AI-2). Moreover, mangiferin significantly increased L. reuteri 1-12 biomass and promoted L. reuteri 1-12 biofilm formation and structure. Besides, only mangiferin inhibited luxS expression, thus increasing L. reuteri 1-12 biomass. This research indicated that mangiferin may be a potential inhibitor of LuxS, promoting the probiotic properties of L. reuteri and human health.
Sujet(s)
Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Limosilactobacillus reuteri , Probiotiques/usage thérapeutique , Xanthones/usage thérapeutique , Produits biologiques , Humains , Simulation de docking moléculaire , Phytothérapie , Probiotiques/composition chimique , Xanthones/composition chimiqueRÉSUMÉ
Fragment-based screening using 19F NMR (19F-FS) is an efficient method for exploring seed and lead compounds for drug discovery. Here, we demonstrate the utility and merits of using 19F-FS for methionine γ-lyase-binding fragments, together with a 19F NMR-based competition and mutation assay, as well as enzymatic and in silico methods. 19F NMR-based assays provided useful information on binding between 19F-FS hit fragments and target proteins. Although the 19F-FS and enzymatic assay were weakly correlated, they show that the 19F-FS hit fragments contained compounds with inhibitory activity. Furthermore, we found that in silico calculations partially account for the differences in activity levels between the 19F-FS hits as per NMR analysis. A comprehensive approach combining the 19F-FS and other methods not only identified fragment hits but also distinguished structural differences in chemical groups with diverse activity levels.
Sujet(s)
Carbon-sulfur lyases/antagonistes et inhibiteurs , Dosages enzymatiques , Antienzymes/composition chimique , Résonance magnétique nucléaire biomoléculaire/méthodes , Bibliothèques de petites molécules/composition chimique , Simulation numérique , Antienzymes/pharmacologie , Fluor , Ligands , Bibliothèques de petites molécules/pharmacologieRÉSUMÉ
The enzyme L-methionine-γ-lyase is commonly found in a wide range of bacteria and catalyzes the α-elimination and γ-elimination of L-methionine to produce methyl mercaptan, α-ketobutyrate and ammonia. Black cumin seed essential oil (BC oil) reportedly exhibits deodorizing activity against methyl mercaptan. Therefore, we hypothesized that BC oil may also suppress methyl mercaptan production. In this study, we aimed to evaluate the inhibitory effect of BC oil on L-methionine-γ-lyase activity in Fusobacterium nucleatum. Recombinant L-methionine-γ-lyase was incubated under appropriate conditions with BC oil and its constituent thymoquinone. To analyze L-methionine-γ-lyase activity, α-ketobutyric acid and ammonia concentrations were determined. The concentrations of α-ketobutyric acid and ammonia were significantly decreased by 10 µg mL-1 of BC oil (P < 0.01) and 16.4 µg/mL of thymoquinone (P < 0.05). An enzyme kinetic assay showed a mixed inhibition pattern between L-methionine-γ-lyase and thymoquinone. In conclusion, BC oil not only had a deodorizing effect against methyl mercaptan but also an inhibitory effect on methyl mercaptan production through the suppression of L-methionine-γ-lyase activity. Thymoquinone may be mainly responsible for these effects of BC oil. Thus, application of natural BC oil may be adapted not only for medical use but also in other areas of life.
Sujet(s)
Antibactériens/pharmacologie , Carbon-sulfur lyases/antagonistes et inhibiteurs , Fusobacterium nucleatum/effets des médicaments et des substances chimiques , Nigella sativa/composition chimique , Huile essentielle/pharmacologie , Ammoniac/métabolisme , Benzoquinones/pharmacologie , Butyrates/métabolisme , Carbon-sulfur lyases/métabolisme , Fusobacterium nucleatum/enzymologie , Fusobacterium nucleatum/métabolisme , Méthionine/métabolisme , Tests de sensibilité microbienne , Protéines recombinantes/métabolisme , Graines/composition chimiqueRÉSUMÉ
Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.
Sujet(s)
Biofilms/effets des médicaments et des substances chimiques , Antienzymes/pharmacologie , Fluorouracil/pharmacologie , Tests de criblage à haut débit/méthodes , Staphylococcus aureus résistant à la méticilline/effets des médicaments et des substances chimiques , Détection du quorum/effets des médicaments et des substances chimiques , Animaux , Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Antienzymes/usage thérapeutique , Femelle , Fluorouracil/usage thérapeutique , Cellules HEK293 , Homosérine/analogues et dérivés , Homosérine/biosynthèse , Humains , Lactones , Staphylococcus aureus résistant à la méticilline/métabolisme , Souris de lignée C57BL , N-Glycosyl hydrolases/antagonistes et inhibiteurs , Bibliothèques de petites molécules , Infections à staphylocoques/prévention et contrôleRÉSUMÉ
Thiosulfinates, a natural antibiotic, existed in all parts of Allium, therefore might be accumulated in large amounts in food waste (FW). FW was often added into waste activated sludge (WAS) anaerobic digestion process as a kind of supplement for nutrition balance. However, the impact of thiosulfinates on methane production and the possible approach to mitigate its inhibition on the co-digestion process could be available in few literatures. This work was carried out in a series of batch experiment at pH 7.0⯱â¯0.2 and 35⯱â¯1.0â¯â to promote the further understanding of this process. The experimental results showed that the methane accumulation decreased from 270.6⯱â¯13.4 to 16.7⯱â¯7.0â¯mL/gâ¯VSS (volatile suspended solids) when the initial concentration of thiosulfinates increased from 0 to 2.5⯵g/gâ¯VSS. The activities of functional enzymes (F420 and CoM) were inhibited by 99.06% and 99.82% compared with control group when reactor contained 2.5⯵g/gâ¯VSS thiosulfinates. Furthermore, different temperature, pH, and combination pretreat were applied to impair the inhibition of thiosulfinate. Compared with no pretreatment group, methane yield was increased by 2.26, 32.18 and 42.2-fold, respectively which group was under pretreatment method of heat (100â¯â), alkali (pH 9) and combination.
Sujet(s)
Allium/composition chimique , Méthane/biosynthèse , Eaux d'égout/composition chimique , Déchets solides , Acides sulfiniques/pharmacologie , Élimination des déchets liquides/méthodes , Allium/métabolisme , Anaérobiose , Biocarburants/analyse , Bioréacteurs/microbiologie , Carbon-sulfur lyases/antagonistes et inhibiteurs , Disulfures , Fermentation , Modèles théoriques , Eaux d'égout/microbiologie , Acides sulfiniques/administration et posologie , Acides sulfiniques/métabolismeRÉSUMÉ
Streptococcus pyogenes is well documented as a multi-virulent and exclusively human pathogen. The LuxS-based signaling in these bacteria has a crucial role in causing several infections through pathways that are pathogenic. This study evaluated the individual and synergistic effects of citral and phloretin against S. pyogenes in relation to major virulence traits. The in vitro synergy of citral and phloretin was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values were calculated to determine the interactions between the inhibitors. The bacteria's virulence properties were tested in the presence of the molecules, individually as well as in combination. Molecules' cytotoxicity was tested using human tonsil epithelial cells. The synergistic effects of the molecules on the expression of biofilm and quorum sensing genes were tested using quantitative real-time polymerase chain reaction (qRT-PCR). The molecules were also tested for their impact on LuxS protein by molecular docking, modeling, and free-energy calculations. When the two molecules were assessed in combination (synergistic effect, FIC Index of 0.5), a stronger growth inhibitory activity was exhibited than the individual molecules. The cell surface hydrophobicity, as well as genes involved in quorum sensing and biofilm formation, showed greater suppression when the molecules were tested in combination. The in silico findings also suggest the inhibitory potential of the two molecules against LuxS protein. The binding orientation and the binding affinity of citral and phloretin well support the notion that there is a synergistic effect of citral and phloretin. The data reveal the combination of citral and phloretin as a potent antibacterial agent to combat the virulence of S. pyogenes.
Sujet(s)
Monoterpènes acycliques/pharmacologie , Antibactériens/pharmacologie , Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Phlorétine/pharmacologie , Infections à streptocoques/traitement médicamenteux , Streptococcus pyogenes/effets des médicaments et des substances chimiques , Virulence/effets des médicaments et des substances chimiques , Biofilms/effets des médicaments et des substances chimiques , Biofilms/croissance et développement , Cellules cultivées , Association médicamenteuse , Cellules épithéliales/cytologie , Cellules épithéliales/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes bactériens/effets des médicaments et des substances chimiques , Humains , Simulation de docking moléculaire , Tonsille palatine/cytologie , Tonsille palatine/effets des médicaments et des substances chimiques , Conformation des protéines , Détection du quorum , Infections à streptocoques/microbiologieRÉSUMÉ
Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.
Sujet(s)
Antibactériens/pharmacologie , Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Antienzymes/pharmacologie , Homocystéine/analogues et dérivés , Antibactériens/synthèse chimique , Antibactériens/composition chimique , Protéines bactériennes/composition chimique , Carbon-sulfur lyases/composition chimique , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Homocystéine/synthèse chimique , Homocystéine/pharmacologie , Détection du quorum/effets des médicaments et des substances chimiquesRÉSUMÉ
luxS is conserved in several bacterial species, including A. hydrophila, which causes infections in prawn, fish, and shrimp, and is consequently a great risk to the aquaculture industry and public health. luxS plays a critical role in the biosynthesis of the autoinducer-2 (AI-2), which performs wide-ranging functions in bacterial communication, and especially in quorum sensing (QS). The prediction of a 3D structure of the QS-associated LuxS protein is thus essential to better understand and control A. hydrophila pathogenecity. Here, we predicted the structure of A. hydrophila LuxS and characterized it structurally and functionally with in silico methods. The predicted structure of LuxS provides a framework to develop more complete structural and functional insights and will aid the mitigation of A. hydrophila infection, and the development of novel drugs to control infections. In addition to modeling, the suitable inhibitor was identified by high through put screening (HTS) against drug like subset of ZINC database and inhibitor ((-)-Dimethyl 2,3-O-isopropylidene-l-tartrate) molecule was selected based on the best drug score. Molecular docking studies were performed to find out the best binding affinity between LuxS homologous or predicted model of LuxS protein for the ligand selection. Remarkably, this inhibitor molecule establishes agreeable interfaces with amino acid residues LYS 23, VAL 35, ILE76, and SER 90, which are found to play an essential role in inhibition mechanism. These predictions were suggesting that the proposed inhibitor molecule may be considered as drug candidates against AI-2 biosynthesis of A. hydrophila. Therefore, (-)-Dimethyl 2,3-O-isopropylidene-l-tartrate inhibitor molecule was studied to confirm its potency of AI-2 biosynthesis inhibition. The results shows that the inhibitor molecule had a better efficacy in AI-2 inhibition at 40 µM concentration, which was further validated using Western blotting at a protein expression level. The AI-2 bioluminescence assay showed that the decreased amount of AI-2 biosynthesis and downregulation of LuxS protein play an important role in the AI-2 inhibition. Lastly, these experiments were conducted with the supplementation of antibiotics via cocktail therapy of AI-2 inhibitor plus OXY antibiotics, in order to determine the possibility of novel cocktail drug treatments of A. hydrophila infection.
Sujet(s)
Aeromonas hydrophila/physiologie , Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Carbon-sulfur lyases/composition chimique , Carbon-sulfur lyases/métabolisme , Homosérine/analogues et dérivés , Bibliothèques de petites molécules/pharmacologie , Aeromonas hydrophila/métabolisme , Protéines bactériennes/antagonistes et inhibiteurs , Sites de fixation , Carbon-sulfur lyases/antagonistes et inhibiteurs , Simulation numérique , Évaluation préclinique de médicament , Antienzymes/composition chimique , Antienzymes/pharmacologie , Tests de criblage à haut débit , Homosérine/biosynthèse , Lactones , Modèles moléculaires , Simulation de docking moléculaire , Liaison aux protéines , Conformation des protéines , Détection du quorum , Bibliothèques de petites molécules/composition chimiqueRÉSUMÉ
Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.
Sujet(s)
Carbon-sulfur lyases/physiologie , Spectrométrie de masse/méthodes , Phytolaccaceae/physiologie , Carbon-sulfur lyases/antagonistes et inhibiteurs , Carbon-sulfur lyases/isolement et purification , Cystéine/analogues et dérivés , Cystéine/métabolisme , Rétrocontrôle physiologique , Cinétique , Phytolaccaceae/enzymologie , Racines de plante/enzymologie , Racines de plante/physiologie , Acide pyruvique/analyse , Acide pyruvique/métabolisme , Acides sulfiniques/analyse , Acides sulfiniques/métabolismeRÉSUMÉ
Dimethyl sulfide (DMS) is released at rates of >107 tons annually and plays a key role in the oceanic sulfur cycle and ecology. Marine bacteria, algae, and possibly other organisms release DMS via cleavage of dimethylsulfoniopropionate (DMSP). DMSP lyases have been identified in various organisms, including bacteria, coral, and algae, thus comprising a range of gene families putatively assigned as DMSP lyases. Metagenomics may therefore provide insight regarding the presence of DMSP lyases in various marine environments, thereby promoting a better understanding of global DMS emission. However, gene counts, and even mRNA levels, do not necessarily reflect the level of DMSP cleavage activity in a given environmental sample, especially because some of the families assigned as DMSP lyases may merely exhibit promiscuous lyase activity. Here, we describe a range of biochemical profiling methods that can assign an observed DMSP lysis activity to a specific gene family. These methods include selective inhibitors and DMSP substrate analogues. Combined with genomics and metagenomics, biochemical profiling may enable a more reliable identification of the origins of DMS release in specific organisms and in crude environmental samples.
Sujet(s)
Organismes aquatiques/métabolisme , Carbon-sulfur lyases/analyse , Surveillance de l'environnement/méthodes , Dosages enzymatiques/méthodes , Organismes aquatiques/génétique , Carbon-sulfur lyases/antagonistes et inhibiteurs , Carbon-sulfur lyases/génétique , Carbon-sulfur lyases/métabolisme , Climat , Métagénomique/méthodes , Sulfures/analyse , Sulfures/métabolisme , Composés de sulfonium/métabolismeRÉSUMÉ
The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.
Sujet(s)
Protéines bactériennes/composition chimique , Carbon-sulfur lyases/composition chimique , Citrobacter freundii/enzymologie , Mutation faux-sens , Norleucine/métabolisme , Mutation ponctuelle , Substitution d'acide aminé , Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/métabolisme , Carbon-sulfur lyases/antagonistes et inhibiteurs , Carbon-sulfur lyases/métabolisme , Domaine catalytique , Citrobacter freundii/génétique , Cristallographie aux rayons X , Modèles moléculaires , Liaison aux protéines , Conformation des protéinesRÉSUMÉ
LuxS (S-ribosylhomocysteinase; EC 4.4.1.21) is an enzyme that catalyzes the cleavage of the thioether linkage in the catalytic pathway of S-ribosylhomocysteine (SRH) which produces homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is the precursor of the signaling molecules known as autoinducer 2 (AI-2) responsible for the bacterial quorum sensing (QS) identified as cell to cell communication. Inhibitors of LuxS should be able to interfere with its catalytic pathway thus preventing the formation of the autoinducer molecules. In this work, the synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by the coupling of the corresponding 2-bromo-2-deoxypentafuranosyl sugars with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate group from C5 was observed leading to a novel isomeric analogue of SRH in which Hcy moiety is attached to a ribose ring via C2-sulfur bond.
Sujet(s)
Bacillus subtilis/enzymologie , Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Antienzymes/composition chimique , Homocystéine/analogues et dérivés , Ribose/analogues et dérivés , Protéines bactériennes/métabolisme , Carbon-sulfur lyases/métabolisme , Cristallographie aux rayons X , Antienzymes/synthèse chimique , Antienzymes/pharmacologie , Homocystéine/synthèse chimique , Homocystéine/pharmacologie , Isomérie , Modèles moléculaires , Ribose/synthèse chimique , Ribose/pharmacologieRÉSUMÉ
Atmospheric dimethylsulfide (DMS) is massively produced in the oceans by bacteria, algae, and corals. To enable identification of DMS sources, we developed a potent mechanism-based inhibitor of the algal Alma dimethylsulfoniopropionate lyase family that does not inhibit known bacterial lyases. Its application to coral holobiont indicates that DMS originates from Alma lyase(s). This biochemical profiling may complement meta-genomics and transcriptomics to provide better understanding of the marine sulfur cycle.
Sujet(s)
Anthozoa/métabolisme , Haptophyta/métabolisme , Sulfures/métabolisme , Animaux , Anthozoa/effets des médicaments et des substances chimiques , Anthozoa/enzymologie , Bactéries/effets des médicaments et des substances chimiques , Bactéries/enzymologie , Bactéries/métabolisme , Carbon-sulfur lyases/antagonistes et inhibiteurs , Carbon-sulfur lyases/métabolisme , Antienzymes/métabolisme , Haptophyta/effets des médicaments et des substances chimiques , Haptophyta/enzymologie , Océans et mersRÉSUMÉ
Oldenlandia diffusa has been empirically used as a therapeutic adjunct for the treatment of respiratory infections. To establish the basic evidence of its clinical usefulness, antimicrobial and biofilm inhibitory activities of an O. diffusa extract were examined against clinical isolates of Haemophilus influenzae, a major causative pathogen of respiratory and sensory organ infections. No significant growth inhibitory activity was observed during incubation for more than 6 h after the extract addition into a culture of H. influenzae. On the other hand, biofilm formation by H. influenzae, evaluated by a crystal violet method, was significantly and dose-dependently inhibited by the O. diffusa extract. Furthermore, the mRNA level of the biofilm-associated gene luxS of H. influenzae significantly decreased soon after the extract addition, and the suppressive effect continued for at least 2 h. At 2 h after the addition of the O. diffusa extract, the autoinducer in the culture supernatant was also significantly reduced by the O. diffusa extract in a dose-dependent manner. These results revealed that O. diffusa extract shows inhibitory activity against luxS-dependent biofilm formation but has no antimicrobial activity against planktonic cells of H. influenzae. Thus, O. diffusa extract might be useful as an adjunctive therapy for the treatment of respiratory infections caused by H. influenzae.
Sujet(s)
Biofilms/effets des médicaments et des substances chimiques , Biofilms/croissance et développement , Haemophilus influenzae/effets des médicaments et des substances chimiques , Haemophilus influenzae/physiologie , Oldenlandia/composition chimique , Extraits de plantes/pharmacologie , Protéines bactériennes/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Haemophilus influenzae/croissance et développement , Haemophilus influenzae/métabolisme , HumainsRÉSUMÉ
Asiasarum root (roots and rhizome of Asiasarum sieboldii or A. heterotropoides var. mandshuricum) has been frequently used in traditional Chinese medicinal formulas for the management of oral malodor syndrome caused by periodontal disease. However, there are no scientific reports concerning these effects and the mechanism of action. The objective of this study was to examine the inhibitory effects of Asiasarum root and its constituents on oral malodor syndrome and periodontal disease. A 50% ethanolic extract of Asiasarum root (AR-ext) showed L-methionine γ-lyase (METase) inhibitory activity at a concentration of 200 µg/mL, and inhibited interleukin (IL)-1ß-stimulated matrix metalloproteinase (MMP)-1 secretion from human gingival fibroblasts (HGFs) at a concentration of 10 and 50 µg/mL without cytotoxic effects. Activity-guided fractionation of the AR-ext suggested that METase inhibitory activity was attributable to a mixture of linoleic and oleic acid, because these unsaturated fatty acids showed weak METase inhibitory activities. Similar fractionation using MMP-1 secretion inhibitory activity led to the isolation of two unsaturated fatty acid amides, (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (1) and (2E,4E,8Z,10Z)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), as active constituents with inhibitory activity on MMP-1 secretion from HGFs. To elucidate the inhibition mechanism on MMP-1 secretion, the effect of 2 on mitogen-activated protein kinase (MAPK) phosphorylation was examined. Western blotting analysis revealed that 2 (10 µM) reduced the phosphorylation of p38 and c-Jun-N-terminal kinase. These results suggested that 2 suppresses intracellular MMP-1 expression and MMP-1 secretion from IL-1ß-stimulated HGFs by down-regulation of MAPK phosphorylation.
Sujet(s)
Aristolochiaceae , Carbon-sulfur lyases/antagonistes et inhibiteurs , Fibroblastes/effets des médicaments et des substances chimiques , Gencive/cytologie , Matrix metalloproteinase 1/métabolisme , Extraits de plantes/pharmacologie , Carbon-sulfur lyases/métabolisme , Survie cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Fibroblastes/métabolisme , Halitose , Humains , Interleukine-1 bêta/pharmacologie , Mitogen-Activated Protein Kinases/métabolisme , Racines de plante , Porphyromonas gingivalis/effets des médicaments et des substances chimiquesRÉSUMÉ
Garlic (Allium sativum L.), which is a widely distributed plant, is globally used as both spice and food. This study identified five novel phenolic compounds, namely, 8-(3-methyl-(E)-1-butenyl)diosmetin, 8-(3-methyl-(E)-1-butenyl)chrysin, 6-(3-methyl-(E)-1-butenyl)chrysin, and Alliumones A and B, along with nine known compounds 6-14 from the ethanol extract of garlic. The structures of these five novel phenolic compounds were established via extensive 1D- and 2D-nuclear magnetic resonance spectroscopy experiments. The effects of the phenolic compounds isolated from garlic on the enzymatical or nonenzymatical formation of sulfur-containing compounds produced during garlic processing were examined. Compound 12 significantly reduced the thermal decomposition of alliin, whereas compound 4 exhibited the highest percentage of alliinase inhibition activity (36.6%).
Sujet(s)
Manipulation des aliments , Ail/composition chimique , Phénols/pharmacologie , Composés du soufre/antagonistes et inhibiteurs , Composés organiques volatils/antagonistes et inhibiteurs , Carbon-sulfur lyases/antagonistes et inhibiteurs , Cystéine/analogues et dérivés , Cystéine/composition chimique , Température élevée , Spectroscopie par résonance magnétique , Structure moléculaire , Phénols/analyse , Phénols/composition chimique , Extraits de plantes/composition chimique , Composés du soufre/analyse , Composés organiques volatils/composition chimiqueRÉSUMÉ
Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the ß-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.
Sujet(s)
Protéines bactériennes/composition chimique , Carbon-sulfur lyases/composition chimique , Citrobacter freundii/composition chimique , Imines/composition chimique , Phosphate de pyridoxal/composition chimique , Alanine/composition chimique , Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/génétique , Carbon-sulfur lyases/antagonistes et inhibiteurs , Carbon-sulfur lyases/génétique , Domaine catalytique , Citrobacter freundii/enzymologie , Cristallographie aux rayons X , Cyclosérine/composition chimique , Cystéine/composition chimique , Antienzymes/composition chimique , Escherichia coli/génétique , Escherichia coli/métabolisme , Expression des gènes , Glycine/composition chimique , Cinétique , Modèles chimiques , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Thermodynamique , Valine/analogues et dérivés , Valine/composition chimiqueRÉSUMÉ
The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS.
Sujet(s)
Apicoplastes/métabolisme , Carbon-sulfur lyases/antagonistes et inhibiteurs , Ferrosulfoprotéines/métabolisme , Plasmodium falciparum/métabolisme , Soufre/métabolisme , Antimétabolites/pharmacologie , Carbon-sulfur lyases/composition chimique , Carbon-sulfur lyases/métabolisme , Domaine catalytique , Cristallographie aux rayons X , Cyclosérine/pharmacologie , Cystéine/métabolisme , Concentration inhibitrice 50 , Ferrosulfoprotéines/antagonistes et inhibiteurs , Ferrosulfoprotéines/composition chimique , Modèles moléculaires , Maquettes de structure , Mutagenèse , Plasmodium falciparum/effets des médicaments et des substances chimiques , Plasmodium falciparum/croissance et développement , Cartographie d'interactions entre protéines , Protéines de protozoaire/antagonistes et inhibiteurs , Protéines de protozoaire/composition chimique , Protéines de protozoaire/métabolisme , Phosphate de pyridoxal/métabolisme , Sulfures/métabolismeRÉSUMÉ
The ability of foods and beverages to reduce allyl methyl disulfide, diallyl disulfide, allyl mercaptan, and allyl methyl sulfide on human breath after consumption of raw garlic was examined. The treatments were consumed immediately following raw garlic consumption for breath measurements, or were blended with garlic prior to headspace measurements. Measurements were done using a selected ion flow tube-mass spectrometer. Chlorophyllin treatment demonstrated no deodorization in comparison to the control. Successful treatments may be due to enzymatic, polyphenolic, or acid deodorization. Enzymatic deodorization involved oxidation of polyphenolic compounds by enzymes, with the oxidized polyphenols causing deodorization. This was the probable mechanism in raw apple, parsley, spinach, and mint treatments. Polyphenolic deodorization involved deodorization by polyphenolic compounds without enzymatic activity. This probably occurred for microwaved apple, green tea, and lemon juice treatments. When pH is below 3.6, the enzyme alliinase is inactivated, which causes a reduction in volatile formation. This was demonstrated in pH-adjusted headspace measurements. However, the mechanism for volatile reduction on human breath (after volatile formation) is unclear, and may have occurred in soft drink and lemon juice breath treatments. Whey protein was not an effective garlic breath deodorant and had no enzymatic activity, polyphenolic compounds, or acidity. Headspace concentrations did not correlate well to breath treatments.
Sujet(s)
Manipulation des aliments , Ail/composition chimique , Halitose/prévention et contrôle , Extraits de plantes/pharmacologie , Polyphénols/pharmacologie , Composés du soufre/métabolisme , Composés organiques volatils/métabolisme , Composés allyliques/métabolisme , Carbon-sulfur lyases/antagonistes et inhibiteurs , Citrus/composition chimique , Désodorisants , Disulfures/métabolisme , Fruit/composition chimique , Halitose/métabolisme , Humains , Concentration en ions d'hydrogène , Spectrométrie de masse , Oxydoréduction , Sulfures/métabolismeRÉSUMÉ
Methionine γ-lyase [EC 4.4.1.11] participates in a methionine catabolism at a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. Lack of this enzyme at mammals allows consider it as a perspective target for rational antibacterial drug design. Currently in medical practice there are no the preparations based on an inhibition of methionine γ-lyase activity. We present results of the search of potential inhibitors of the enzyme using the NMR screening techniques based on identification of compounds, which able to bind specifically to their biological target. Study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds, capable to interact with enzyme. Identification of binding was carried out by means of saturation transfer difference (STD) spectroscopy and WaterLOGSY technique. At the final stage the experimental assessment of inhibiting ability of the selected compounds in the reaction of γ-elimination of L-methionine catalyzed by methionine γ-lyase was carried out. Binding constants of two leading compounds were determined using the WaterLOGSY method. The research expands structural group of potential inhibitors of methionine γ-lyase and allows approach to the design of the inhibitors with higher efficacy.