Functional Characterization of Cholera Toxin Inhibitors Using Human Intestinal Organoids

Ommen, Domenique D. Zomer-van; Pukin, Aliaksei V.; Fu, Ou; Ufford, Linda H.C. Quarles van; Janssens, Hettie M.; Beekman, Jeffrey M.; Pieters, Roland J.

doi:10.1021/acs.jmedchem.6b00770

Cited by 45 publications

(54 citation statements)

References 26 publications

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“…This way, a K d value of 600 ± 20 nM was obtained for carbohydrate 1 and a K d value of 90 ± 50 nM for carbohydrate 2. While we cannot directly compare K d values with IC 50 values of competition experiments or swelling inhibition, the differences between the two compounds of close to an order of magnitude are in reasonable agreement with those of our previously described IC 50 values using the common ELISA assay and the newly developed assay based on organoid swelling inhibition [10]. As such the method is capable of differentiating compounds of relatively similar binding or inhibitory potency, even though the assay was run at pH 4 rather than the usual neutral condition in, for example, the ELISA assay.…”

supporting

confidence: 80%

“…Of these, multivalent inhibitors based on the ganglioside GM1- Abbreviations: eff , electrophoretic mobility; CTB, cholera toxin B-subunit; GM1os, ganglioside GM1-oligosaccharide; H5, influenza hemagglutinin; K d , dissociation constant oligosaccharide (GM1os) have shown the highest inhibitory potency [8,9], however more development of cheaper and still effective compounds is expected, for which new assay methods are required. Recently we demonstrated that an assay based on the swelling of intestinal organoids provides a biorelevant alternative to the in vivo rabbit ileal loop assay [10], which is notoriously time-consuming, difficult, and stressful for the animals [11]. A drawback is the limited availability of the organoids.…”

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confidence: 99%

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Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

et al. 2017

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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 (K ) determination. The K 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 IC 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.

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Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

et al. 2017

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“…The combination of simple inhibitors into multivalent constructs has been proposed before as effective antagonists of CT action in Vibrio cholerae infections 42, 49 . One of the inhibitors shows the surprising capability to facilitate bridging to nearby toxins.…”

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confidence: 99%

Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

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Mackenzie

Martinsen

et al. 2017

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Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Here we report atomic-resolution crystal structures of three such compounds in complexes with the cholera toxin. The structures give unprecedented atomic details of the molecular interactions and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their relatively easy synthesis and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics.

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“…While enteroids are biologically relevant models for infectious diarrheal diseases (16,17,22,23), their suitability as a platform to study ST-induced intestinal secretion remains unknown (17). Since previous studies demonstrated that pharmacological agents, including complex multimeric proteins like cholera toxin, readily access the lumen of enteroids (25,26), we predicted that these organoids should respond to GUCY2C ligands. Here, we reveal the ability of mouse and human enteroids to respond to ST and its homologs in a GUCY2C-dependent fashion, providing a high-throughput biologically relevant model to explore the pathophysiology of ST and screen antisecretory agents to treat and prevent diarrheal disease.…”

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Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins

et al. 2016

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bEnterotoxigenic Escherichia coli (ETEC) causes ϳ20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease.

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Functional Characterization of Cholera Toxin Inhibitors Using Human Intestinal Organoids

Cited by 45 publications

References 26 publications

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins

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