title: Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.
abstract: 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.
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, 700054, India.
authors.forename: Tanaya
authors.initials: T
authors.lastname: Chatterjee
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera & Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India.
authors.forename: Irshad Ali
authors.initials: IA
authors.lastname: Sheikh
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, 700054, India.
authors.forename: Devlina
authors.initials: D
authors.lastname: Chakravarty
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, 700054, India.
authors.forename: Pinak
authors.initials: P
authors.lastname: Chakrabarti
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera & Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India.
authors.forename: Paramita
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera & Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India.
authors.forename: Tultul
authors.initials: T
authors.lastname: Saha
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera & Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Chakrabarti
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera & Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India.
authors.forename: Kazi Mirajul
authors.initials: KM
authors.lastname: Hoque
mesh_terms: D000818:Animals; D018118:Chloride Channels / Q000037:antagonists & inhibitors*; D002771:Cholera / Q000503:physiopathology; D002772:Cholera Toxin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002942:Circular Dichroism; D053630:Depsides / Q000494:pharmacology; D003967:Diarrhea / Q000503:physiopathology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D062105:Molecular Docking Simulation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013634:Tannins / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D014734:Vibrio cholerae / Q000502:physiology*
chemical_list: C000607369:6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxylic acid; C081530:Ace toxin; D018118:Chloride Channels; D053630:Depsides; D011994:Recombinant Proteins; D013634:Tannins; D013876:Thiophenes; C067648:digallic acid; D005707:Gallic Acid; D002772:Cholera Toxin