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Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.

Chatterjee T, Sheikh IA, Chakravarty D, Chakrabarti P, Sarkar P, Saha T, Chakrabarti MK, Hoque KM - PLoS ONE (2015)

Bottom Line: 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.In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure.The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, 700054, India.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of CaCC inhibitors on Ace stimulated intestinal fluid accumulation.Representative mouse ileal loops 6 h after luminal injection with Ace in the presence or absence of (A) DGA and CaCCinh-A01 and (B) tannic acid in 1:1 molar ratio (left panel). The right panel provides the bar graph showing the quantified averaged fluid accumulation in the loop experiment (n = 5–10). Values are mean ± SEM.
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pone.0141283.g006: Effect of CaCC inhibitors on Ace stimulated intestinal fluid accumulation.Representative mouse ileal loops 6 h after luminal injection with Ace in the presence or absence of (A) DGA and CaCCinh-A01 and (B) tannic acid in 1:1 molar ratio (left panel). The right panel provides the bar graph showing the quantified averaged fluid accumulation in the loop experiment (n = 5–10). Values are mean ± SEM.

Mentions: Ussing chamber experiment was performed in mouse tissue to find out the inhibitory effect of small molecules, CaCCinh-A01 and tannic acid on chloride channel before being stimulated by Ace. To prove that low concentration of small molecules did not have any effect on chloride channels but was effective enough to cause alteration of toxin structure (as found in ileal loop experiment, given below), tissues were pre-incubated by 5 μM as well as 100 μM of CaCCinh-A01. Low concentration of CaCCinh-A01 did not have any effect either on basal or Ace stimulated short-circuit current (Isc) (Fig 5A). However, a high concentration (100 μM) of CaCCinh-A01, which is believed to inhibit chloride channel, effectively inhibited Ace stimulated Isc [28]. Similar effect was also found with high concentration of tannic acid, while low concentration of the same was not able to inhibit basal as well as Ace stimulated Isc (Fig 5B). These results demonstrate that low concentration of CaCCinh-A01 and tannic acid has nothing to do with CaCC inhibition, rather they altered the structure of Ace, which in turn affected the degree of fluid accumulation in mouse ileal loop experiments (Fig 6).


Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.

Chatterjee T, Sheikh IA, Chakravarty D, Chakrabarti P, Sarkar P, Saha T, Chakrabarti MK, Hoque KM - PLoS ONE (2015)

Effect of CaCC inhibitors on Ace stimulated intestinal fluid accumulation.Representative mouse ileal loops 6 h after luminal injection with Ace in the presence or absence of (A) DGA and CaCCinh-A01 and (B) tannic acid in 1:1 molar ratio (left panel). The right panel provides the bar graph showing the quantified averaged fluid accumulation in the loop experiment (n = 5–10). Values are mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634967&req=5

pone.0141283.g006: Effect of CaCC inhibitors on Ace stimulated intestinal fluid accumulation.Representative mouse ileal loops 6 h after luminal injection with Ace in the presence or absence of (A) DGA and CaCCinh-A01 and (B) tannic acid in 1:1 molar ratio (left panel). The right panel provides the bar graph showing the quantified averaged fluid accumulation in the loop experiment (n = 5–10). Values are mean ± SEM.
Mentions: Ussing chamber experiment was performed in mouse tissue to find out the inhibitory effect of small molecules, CaCCinh-A01 and tannic acid on chloride channel before being stimulated by Ace. To prove that low concentration of small molecules did not have any effect on chloride channels but was effective enough to cause alteration of toxin structure (as found in ileal loop experiment, given below), tissues were pre-incubated by 5 μM as well as 100 μM of CaCCinh-A01. Low concentration of CaCCinh-A01 did not have any effect either on basal or Ace stimulated short-circuit current (Isc) (Fig 5A). However, a high concentration (100 μM) of CaCCinh-A01, which is believed to inhibit chloride channel, effectively inhibited Ace stimulated Isc [28]. Similar effect was also found with high concentration of tannic acid, while low concentration of the same was not able to inhibit basal as well as Ace stimulated Isc (Fig 5B). These results demonstrate that low concentration of CaCCinh-A01 and tannic acid has nothing to do with CaCC inhibition, rather they altered the structure of Ace, which in turn affected the degree of fluid accumulation in mouse ileal loop experiments (Fig 6).

Bottom Line: 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.In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure.The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, 700054, India.

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.

No MeSH data available.


Related in: MedlinePlus