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.