ABSTRACT
INTRODUCTION: Cholera is a bacterial diarrheal disease caused by pathogen bacteria Vibrio cholerae, which produces the cholera toxin (CT). In addition to improving water sanitation, oral cholera vaccines have been developed to control infection. Besides, rehydration and antibiotic therapy are complementary treatment strategies for cholera. ToxT regulatory protein activates transcription of CT gene, which is enhanced by bicarbonate (HCO3-). AREAS COVERED: This review delves into the genomic blueprint of V. cholerae, which encodes for α-, ß-, and γ- carbonic anhydrases (CAs). We explore how the CAs contribute to the pathogenicity of V. cholerae and discuss the potential of CA inhibitors in mitigating the disease's impact. EXPERT OPINION: CA inhibitors can reduce the virulence of bacteria and control cholera. Here, we reviewed all reported CA inhibitors, noting that α-CA from V. cholerae (VchCAα) was the most effective inhibited enzyme compared to the ß- and γ-CA families (VchCAß and VchCAγ). Among the CA inhibitors, acyl selenobenzenesulfonamidenamides and simple/heteroaromatic sulfonamides were the best VchCA inhibitors in the nM range. It was noted that some antibacterial compounds show good inhibitory effects on all three bacterial CAs. CA inhibitors belonging to other classes may be synthesized and tested on VchCAs to harness cholera.
Subject(s)
Anti-Bacterial Agents , Carbonic Anhydrase Inhibitors , Carbonic Anhydrases , Cholera , Vibrio cholerae , Vibrio cholerae/enzymology , Carbonic Anhydrase Inhibitors/pharmacology , Cholera/drug therapy , Cholera/microbiology , Humans , Anti-Bacterial Agents/pharmacology , Carbonic Anhydrases/metabolism , Animals , Virulence , Cholera Toxin/pharmacology , Cholera Toxin/antagonists & inhibitors , Cholera Vaccines/pharmacology , Drug DevelopmentABSTRACT
Secretory diarrhea is one of the most common types of diarrhea with high morbidity and mortality. Previous studies showed that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels alleviated fluid loss in secretory diarrheas. This study aimed to identify novel CFTR inhibitors from fungal metabolites and explore its underlying mechanisms and potential utility in secretory diarrheas. Electrophysiological analyses in human intestinal epithelial (T84) cells were performed to investigate the effect and mechanism of fungal metabolites on CFTR-mediated Cl- secretion. Anti-diarrheal efficacy and the effect of compound on fluid absorption were investigated in mouse closed-loop models. We found that the screening identified arthropsolide A, a fungal metabolite from an endophytic fungus Roussoella sp. PSU-H51, as an inhibitor of CFTR-mediated Cl- secretion in T84 cells (IC50 ~0.8 µM). Arthropsolide A inhibited both CFTR and cAMP-activated basolateral K+ channels. Arthropsolide A had no effect on Na+-K+ ATPase activity. Interestingly, the inhibitory effect of arthropsolide A on CFTR was attenuated by cell depolarization and AMPK inhibition independent of multi-drug resistance protein 4, phosphodiesterases, and protein phosphatases. Importantly, arthropsolide A suppressed cholera toxin (CT)-induced Cl- secretion in T84 cells and CT-induced intestinal fluid secretion in mice by ~75% without affecting intestinal fluid absorption. Taken together, arthropsolide A represents a novel class of fungal metabolites that acts as a potent CFTR inhibitor. Further development of this class of compounds may provide a therapy for secretory diarrheas.
Subject(s)
Antidiarrheals/pharmacology , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Intestines/drug effects , Spiro Compounds/pharmacology , Animals , Antidiarrheals/therapeutic use , Cell Line , Cell Polarity/drug effects , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/pharmacology , Drug Resistance , Fungi/metabolism , Humans , KCNQ Potassium Channels/antagonists & inhibitors , Male , Mice , Mice, Inbred ICR , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Spiro Compounds/therapeutic useABSTRACT
A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose-fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose-fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.
Subject(s)
Cholera Toxin , Epithelial Cells/drug effects , Fucose/pharmacology , Galactose/pharmacology , Animals , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/metabolism , Humans , Intestine, Small/cytology , Mice , Mice, Inbred C57BL , Polymers/pharmacology , Protein BindingABSTRACT
Cholera is a potentially fatal bacterial infection caused by the cholera toxin (CT), an AB5 toxin secreted by Vibrio cholera. GM1 has long been known as the receptor of the cholera toxin in the intestine. However, increasing evidence is pointing towards the role of fucosylated conjugates as additional attachment options of the toxin. In the present paper we have synthesized a polymeric hybrid which can inhibit both modes of attachment.
Subject(s)
Cholera Toxin/antagonists & inhibitors , Fucose/pharmacology , Polymers/pharmacology , Cell Line , Cholera Toxin/metabolism , Enzyme-Linked Immunosorbent Assay , Fucose/chemistry , Humans , Molecular Structure , Polymers/chemical synthesis , Polymers/chemistryABSTRACT
Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.
Subject(s)
Cholera Toxin/antagonists & inhibitors , Protein Disulfide-Isomerases/metabolism , Rutin/pharmacology , Animals , Biological Transport , CHO Cells , Cholera Toxin/metabolism , Cholera Toxin/toxicity , Cricetulus , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Protein Conformation , Protein Disulfide-Isomerases/chemistry , Substrate SpecificityABSTRACT
Vibrio cholerae is a Gram-negative bacterium that causes secretory diarrhea and constitutes a major health threat in the industrialized world and even more in developing countries. Its main virulence factor is the cholera toxin, which is internalized by intestinal epithelial cells after binding to the glycosphingolipid receptor GM1a on their apical surface. A potential future solution to dampen complications of cholera infection is by scavenging the cholera toxin by presenting competitive binding motifs to diminish the in vivo toxicity of V. cholerae. Here, we generate GM1a-functionalized and biocompatible microgels with diameters of 20 µm using drop-based microfluidics. The microgels are designed to exhibit a mesoporous and widely meshed network structure, allowing diffusion of the toxin protein deep into the microgel scavengers. Flow cytometry demonstrates strong and multivalent binding at high capacity of these microgels to the binding domain of the cholera toxin. Cell culture-based assays reveal the ability of these microgels to scavenge and retain the cholera toxin in direct binding competition to colorectal cells. This ability is evidenced by suppressed cyclic adenosine monophosphate production as well as reduced vacuole formation in mucus-forming colorectal HT-29 cells. Therefore, glycan-functionalized microgels show great potential as a non-antibiotic treatment for toxin-mediated infectious disorders.
Subject(s)
Cholera Toxin , G(M1) Ganglioside , Microgels/chemistry , Vibrio cholerae/metabolism , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/chemistry , Cholera Toxin/metabolism , G(M1) Ganglioside/chemistry , G(M1) Ganglioside/pharmacology , HT29 Cells , HumansABSTRACT
For decades, the pathogenesis of a variety of human diseases has been attributed to increased intestinal paracellular permeability even though scientific evidence supporting this hypothesis has been tenuous. Nevertheless, during the past decade, there have been a growing number of publications focused on human genetics, the gut microbiome, and proteomics, suggesting that loss of mucosal barrier function, particularly in the gastrointestinal tract, may substantially affect antigen trafficking, ultimately causing chronic inflammation, including autoimmunity, in genetically predisposed individuals. The gut mucosa works as a semipermeable barrier in that it permits nutrient absorption and also regulates immune surveillance while retaining potentially harmful microbes and environmental antigens within the intestinal lumen. Celiac disease (CD), a systemic, immune-mediated disorder triggered by gluten in genetically susceptible individuals, is associated with altered gut permeability. Pre-clinical and clinical studies have shown that gliadin, a prolamine component of gluten that is implicated in CD pathogenesis, is capable to disassembling intercellular junctional proteins by upregulating the zonulin pathway, which can be inhibited by the zonulin antagonist larazotide acetate. In this review, we will focus on CD as a paradigm of chronic inflammatory diseases in order to outline the contribution of gut paracellular permeability toward disease pathogenesis; moreover, we will summarize current evidence derived from available clinical trials of larazotide acetate in CD.
Subject(s)
Celiac Disease/drug therapy , Gastrointestinal Agents/therapeutic use , Glutens/immunology , Intercellular Junctions/drug effects , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Oligopeptides/therapeutic use , Animals , Celiac Disease/immunology , Celiac Disease/metabolism , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/metabolism , Haptoglobins , Humans , Intercellular Junctions/immunology , Intercellular Junctions/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestine, Small/immunology , Intestine, Small/metabolism , Oligopeptides/adverse effects , Permeability , Protein PrecursorsABSTRACT
Cholera is a potentially fatal bacterial infection that affects a large number of people in developing countries. It is caused by the cholera toxin (CT), an AB5 toxin secreted by Vibrio cholera. The toxin comprises a toxic A-subunit and a pentameric B-subunit that bind to the intestinal cell surface. Several monovalent and multivalent inhibitors of the toxin have been synthesized but are too complicated and expensive for practical use in developing countries. Meta-nitrophenyl α-galactoside (MNPG) is a known promising ligand for CT, and here mono- and multivalent compounds based on MNPG were synthesized. We present the synthesis of MNPG in greatly improved yields and its use while linked to a multivalent scaffold. We used economical polymers as multivalent scaffolds, namely, polyacrylamide, dextran, and hyperbranched polyglycerols (hPGs). Copper-catalyzed alkyne azide cycloaddition reaction (CuAAC) produced the inhibitors that were tested in an ELISA-type assay and an intestinal organoid swelling inhibition assay. The inhibitory properties varied widely depending on the type of polymer, and the most potent conjugates showed IC50 values in the nanomolar range.
Subject(s)
Cholera Toxin/antagonists & inhibitors , Galactosides/metabolism , Polymers/pharmacology , Cholera Toxin/metabolism , Enzyme-Linked Immunosorbent Assay , Ligands , Vibrio cholerae/metabolismABSTRACT
Multivalency achieves strong, yet reversible binding by the simultaneous formation of multiple weak bonds. It is a key interaction principle in biology and promising for the synthesis of high-affinity inhibitors of pathogens. We present a molecular model for the binding affinity of synthetic multivalent ligands onto multivalent receptors consisting of n receptor units arranged on a regular polygon. Ligands consist of a geometrically matching rigid polygonal core to which monovalent ligand units are attached via flexible linker polymers, closely mimicking existing experimental designs. The calculated binding affinities quantitatively agree with experimental studies for cholera toxin ( n = 5) and anthrax receptor ( n = 7) and allow to predict optimal core size and optimal linker length. Maximal binding affinity is achieved for a core that matches the receptor size and for linkers that have an equilibrium end-to-end distance that is slightly longer than the geometric separation between ligand core and receptor sites. Linkers that are longer than optimal are greatly preferable compared to shorter linkers. The angular steric restriction between ligand unit and linker polymer is shown to be a key parameter. We construct an enhancement diagram that quantifies the multivalent binding affinity compared to monovalent ligands. We conclude that multivalent ligands against influenza viral hemagglutinin ( n = 3), cholera toxin ( n = 5), and anthrax receptor ( n = 7) can outperform monovalent ligands only for a monovalent ligand affinity that exceeds a core-size dependent threshold value. Thus, multivalent drug design needs to balance core size, linker length, as well as monovalent ligand unit affinity.
Subject(s)
Anthrax/drug therapy , Cholera Toxin/antagonists & inhibitors , Cholera/drug therapy , Influenza, Human/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Polymers/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Binding Sites/drug effects , Humans , Ligands , Microfilament Proteins , Models, Molecular , Polymers/chemistryABSTRACT
Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd ) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea.
Subject(s)
Cholera Toxin/antagonists & inhibitors , Electrophoresis, Capillary/methods , Enzyme Inhibitors/analysis , Cholera Toxin/chemistry , Cholera Toxin/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Formamides , G(M1) Ganglioside/chemistry , G(M1) Ganglioside/metabolism , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Protein BindingABSTRACT
Hydrogen sulphide (H2S) is a gasotransmitter that participates in various physiological and pathophysiological processes within the gastrointestinal tract. We studied the effects and possible mechanism of action of H2S in secretory diarrhoea caused by cholera toxin (CT). The possible mechanisms of action of H2S were investigated using an intestinal fluid secretion model in isolated intestinal loops on anaesthetized mice treated with CT. NaHS and Lawesson's reagent and l-cysteine showed antisecretory activity through reduction of intestinal fluid secretion and loss of Cl- induced by CT. Pretreatment with an inhibitor of cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG), reversed the effect of l-cysteine and caused severe intestinal secretion. Co-treatment with PAG and a submaximal dose of CT increased intestinal fluid secretion, thus supporting the role of H2S in the pathophysiology of cholera. CT increased the expression of CSE and the production of H2S. Pretreatment with PAG did not reverse the effect of SQ 22536 (an AC inhibitor), bupivacaine (inhibitor of cAMP production), KT-5720 (a PKA inhibitor), and AICAR (an AMPK activator). The treatment with Forskolin does not reverse the effects of the H2S donors. Co-treatment with either NaHS or Lawesson's reagent and dorsomorphin (an AMPK inhibitor) did not reverse the effect of the H2S donors. H2S has antisecretory activity and is an essential molecule for protection against the intestinal secretion induced by CT. Thus, H2S donor drugs are promising candidates for cholera therapy. However, more studies are needed to elucidate the possible mechanism of action.
Subject(s)
Cholera Toxin/antagonists & inhibitors , Diarrhea/chemically induced , Diarrhea/drug therapy , Hydrogen Sulfide/pharmacology , Signal Transduction , AMP-Activated Protein Kinases/metabolism , Adenylyl Cyclases/metabolism , Animals , Cholera Toxin/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Male , MiceABSTRACT
Vibrio cholerae causes cholera and is the leading cause of diarrhea in developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 µg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 µg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antidotes/pharmacology , Cholera Toxin/antagonists & inhibitors , Metal Nanoparticles , Receptors, Cell Surface/antagonists & inhibitors , Vibrio cholerae/drug effects , Zinc Oxide/pharmacology , Absorption, Physiological/drug effects , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/metabolism , Antidotes/adverse effects , Antidotes/metabolism , Cell Survival/drug effects , Cholera Toxin/biosynthesis , Cholera Toxin/metabolism , Cholera Toxin/toxicity , Drug Resistance, Multiple, Bacterial , Gene Expression Regulation, Bacterial/drug effects , HEK293 Cells , HT29 Cells , Humans , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Metal Nanoparticles/adverse effects , Metal Nanoparticles/chemistry , Microbial Viability/drug effects , Particle Size , Poisons/chemistry , Poisons/metabolism , Poisons/toxicity , Protein Structure, Secondary/drug effects , Receptors, Cell Surface/agonists , Receptors, Cell Surface/metabolism , Vacuoles/drug effects , Vacuoles/pathology , Vibrio cholerae/growth & development , Vibrio cholerae/metabolism , Zinc Oxide/adverse effects , Zinc Oxide/chemistry , Zinc Oxide/metabolismABSTRACT
Increased intestinal permeability has been implicated in various pathologies, has various causes, and can develop during vigorous athletic training. Colostrum bovinum is a natural supplement with a wide range of supposed positive health effects, including reduction of intestine permeability. We assessed influence of colostrum supplementation on intestinal permeability related parameters in a group of 16 athletes during peak training for competition. This double-blind placebo-controlled study compared supplementation for 20 days with 500 mg of colostrum bovinum or placebo (whey). Gut permeability status was assayed by differential absorption of lactulose and mannitol (L/M test) and stool zonulin concentration. Baseline L/M tests found that six of the participants (75%) in the colostrum group had increased intestinal permeability. After supplementation, the test values were within the normal range and were significantly lower than at baseline. The colostrum group Δ values produced by comparing the post-intervention and baseline results were also significantly lower than the placebo group Δ values. The differences in stool zonulin concentration were smaller than those in the L/M test, but were significant when the Δ values due to intervention were compared between the colostrum group and the placebo group. Colostrum bovinum supplementation was safe and effective in decreasing of intestinal permeability in this series of athletes at increased risk of its elevation.
Subject(s)
Biological Products/therapeutic use , Colostrum/chemistry , Dietary Supplements , Gastrointestinal Agents/therapeutic use , Gastrointestinal Diseases/prevention & control , Intestinal Mucosa/metabolism , Stress, Physiological , Adult , Animals , Athletes , Biological Products/adverse effects , Cattle , Cell Membrane Permeability/drug effects , Cholera Toxin/analysis , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/metabolism , Cholera Toxin/toxicity , Cross-Over Studies , Dietary Supplements/adverse effects , Double-Blind Method , Feces/chemistry , Freeze Drying , Gastrointestinal Agents/adverse effects , Gastrointestinal Diseases/chemically induced , Gastrointestinal Diseases/metabolism , Gastrointestinal Diseases/physiopathology , Haptoglobins , Humans , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/physiology , Intestinal Mucosa/physiopathology , Male , Martial Arts , Poisons/analysis , Poisons/chemistry , Poisons/metabolism , Poisons/toxicity , Poland , Protein Precursors , ToxicokineticsABSTRACT
Increased small intestinal permeability (IP) has been proposed to be an integral element, along with genetic makeup and environmental triggers, in the pathogenies of chronic inflammatory diseases (CIDs). We identified zonulin as a master regular of intercellular tight junctions linked to the development of several CIDs. We aim to study the role of zonulin-mediated IP in the pathogenesis of CIDs. Zonulin transgenic Hp2 mice (Ztm) were subjected to dextran sodium sulfate (DSS) treatment for 7 days, followed by 4-7 days' recovery and compared to C57Bl/6 (wild-type (WT)) mice. IP was measured in vivo and ex vivo, and weight, histology, and survival were monitored. To mechanistically link zonulin-dependent impairment of small intestinal barrier function with clinical outcome, Ztm were treated with the zonulin inhibitor AT1001 added to drinking water in addition to DSS. We observed increased morbidity (more pronounced weight loss and colitis) and mortality (40-70% compared with 0% in WT) at 11 days post-DSS treatment in Ztm compared with WT mice. Both in vivo and ex vivo measurements showed an increased IP at baseline in Ztm compared to WT mice, which was exacerbated by DSS treatment and was associated with upregulation of zonulin gene expression (fourfold in the duodenum, sixfold in the jejunum). Treatment with AT1001 prevented the DSS-induced increased IP both in vivo and ex vivo without changing zonulin gene expression and completely reverted morbidity and mortality in Ztm. Our data show that zonulin-dependent small intestinal barrier impairment is an early step leading to the break of tolerance with subsequent development of CIDs.
Subject(s)
Cholera Toxin/genetics , Colitis/genetics , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Animals , Body Weight/drug effects , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/metabolism , Colitis/chemically induced , Colitis/physiopathology , Dextran Sulfate , Female , Haptoglobins , Intestinal Mucosa/physiopathology , Intestine, Small/physiopathology , Kaplan-Meier Estimate , Male , Mice, Inbred C57BL , Mice, Transgenic , Oligopeptides/pharmacology , Permeability/drug effects , Protein Precursors , Time FactorsABSTRACT
Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.
Subject(s)
Biflavonoids/pharmacology , Catechin/analogs & derivatives , Cholera Toxin/antagonists & inhibitors , Phenols/pharmacology , Proanthocyanidins/pharmacology , ADP Ribose Transferases/antagonists & inhibitors , Animals , Bacterial Toxins/antagonists & inhibitors , Binding Sites/drug effects , CHO Cells , Catechin/pharmacology , Cell Membrane/metabolism , Cells, Cultured , Chlorocebus aethiops , Cholera Toxin/metabolism , Cricetulus , Diphtheria Toxin/antagonists & inhibitors , Exotoxins/antagonists & inhibitors , Fruit/chemistry , Grape Seed Extract/pharmacology , Molecular Docking Simulation , Plant Extracts/pharmacology , Ricin/antagonists & inhibitors , Vero Cells , Virulence Factors/antagonists & inhibitors , Vitis/chemistry , Pseudomonas aeruginosa Exotoxin AABSTRACT
Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of IC50 values over a wide range of potencies (15 pM to 9 mM). The results indicate for the first time that an organoid-based swelling assay is a useful preclinical method to evaluate inhibitor potencies of drugs that target pathogen-derived toxins.
Subject(s)
Antitoxins/pharmacology , Cholera Toxin/antagonists & inhibitors , Intestines/drug effects , Organoids/drug effects , Antitoxins/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Synthesis of a set of novel glycopeptide analogues as potential cholera/cholera-like toxin inhibitors in their protected form is described. They include di-, tri-, tetra- and pentavalent scaffolds. The synthetic steps were achieved using a combination of solvent-free mechanochemical as well as the conventional solution-phase reactions. During the conventional DIC-HOBt-mediated peptide coupling followed for the preparation of certain glycopeptide analogues an interesting in situ Fmoc deprotection was observed which has been demonstrated to hold potential for synthesiszing glycopeptides/neoglycopeptides with extended polyamide chains.
Subject(s)
Glycopeptides/chemical synthesis , Glycopeptides/pharmacology , Antitoxins/chemistry , Antitoxins/pharmacology , Cholera Toxin/antagonists & inhibitors , Glycopeptides/chemistry , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Molecular StructureABSTRACT
A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.
Subject(s)
Anti-Bacterial Agents/metabolism , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/biosynthesis , Foeniculum/chemistry , Gene Expression/drug effects , Plant Extracts/metabolism , Vibrio cholerae/drug effects , Anti-Bacterial Agents/isolation & purification , Methanol , Microbial Viability/drug effects , Plant Extracts/isolation & purification , Seeds/chemistry , Solvents , Vibrio cholerae/geneticsABSTRACT
A series of diseases, ranging from cholera via travelers' diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For example, the protein that causes cholera, cholera toxin (CT), has five identical, symmetrically placed binding pockets (B proteins), on top of which the toxic A protein resides. A promising strategy to counteract the devastating biological effects of this AB5 protein involves the development of inhibitors that can act as mimics of membrane-bound GM1 molecules, i.e., that can bind CT strongly and selectively. To reach this goal, two features are essential: First of all, the inhibitor should display oligosaccharides that resemble as much as possible the naturally occurring cell-surface pentasaccharide onto which CT normally binds, the so-called GM1 sugar (the oligosaccharide part of which is then labeled GM1os). Second, the inhibitor should be able to bind CT via multivalent interactions so as to bind CT as strongly as possible to allow for a real competition with the cell-membrane-bound GM1 molecules. In this Account, we present elements of the path that leads to strong CT inhibition by outlining the roles of multivalency and the development and use of GM1 mimics. First, multivalency effects were investigated using "sugar-coated" platforms, ranging from dendritic structures with up to eight oligosaccharides to platforms that mimicked the fivefold symmetry of CT itself. The latter goal was reached either via synthetic scaffolds like corannulene or calix[5]arene or via the development of a neolectin CT mimic that itself carries five GM1os groups. Second, the effect of the nature of the oligosaccharide appended to this platform was investigated via the use of oligosaccharides of increasing complexity, from galactose and lactose to the tetrasaccharide GM2os and eventually to GM1os itself. The combination of these threads gives rise to a series of inhibitors that can strongly bind CT, with IC50 values below 100 pM, and in some cases can even bind one-on-one.
Subject(s)
Anti-Bacterial Agents/chemistry , Cholera Toxin/antagonists & inhibitors , Cholera Toxin/chemistry , Dendrimers/chemistry , Galactose/analogs & derivatives , Galactose/chemistry , Oligosaccharides/chemistry , Cholera/drug therapy , Molecular MimicryABSTRACT
Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.