ith two hour cell culture assays utilizing 100 mM PBA. In contrast, PBA-induced alterations to chaperone expression require a 48 hour exposure to at least 0.5 mM PBA. Alterations to the unfolded protein response require a 24 hour exposure to 10 mM PBA or a 72 hour exposure to 2 mM PBA. Inhibition of the cystic fibrosis transmembrane regulator in polarized epithelial cells only occurs after a 24 hour exposure to 5 mM PBA. The low concentration of PBA required to stabilize CTA1, combined with the specific interaction between PBA and CTA1, thus minimizes off-target effects while still providing substantial cellular resistance to CT. Exposure to 100 mM PBA generated a 3-fold reduction in the efficiency of CTA1 translocation to the cytosol. This, in turn, promoted a greater than 25-fold level of cellular resistance to CT. PBA-induced toxin resistance was also demonstrated in the physiological ileal loop model of intoxication. Previous work has shown that high levels of toxin resistance can be achieved without a complete block of toxin access to the cytosol. Furthermore, as BIX-01294 web recently shown for the plant toxin ricin, moderate levels of drug-induced toxin resistance in cell culture can correspond to high levels of drug-induced toxin resistance in an animal model. We therefore expect that PBA will provide substantial protection against V. cholerae in an animal model of infection. The maximum FDA-approved dosage of PBA is 20 g/day, 23472002 which in clinical studies represented 10.6 to 13.8 g/M2/d. The drug was administered orally three to four times a day in the form of 500 mg tablets. With this regimen, patients attained serum PBA concentrations of 600,700 mM. It should therefore be possible to orally administer, at dosages well below the FDAapproved limit, a concentration of PBA that can inhibit CTA1 unfolding and CT intoxication in intestinal epithelial cells. One limitation of a PBA-based therapeutic strategy is the timing of the inhibitory effect: PBA must act on the holotoxin before dissociation and translocation of the CTA1 subunit. While this would be acceptable for a preventative measure, its utility in treating patients already exposed to CT could 12672252 be more limited. However, a number of observations suggest a post-exposure inhibition of CT might at least attenuate the effects of cholera. The rapid turnover of ADP-ribosylated Gsa and the removal of ADP-ribose modifications by ADP-ribosylprotein hydrolase suggest that the activated form of Gsa can be cleared from the intoxicated cell, provided that CTA1 is also removed from the cytosol. Experiments demonstrating a correlation between toxin persistence in the cytosol and the extent of intoxication further support this possibility: the diminished potency of CT and recombinant CT constructs which are rapidly degraded in the cytosol indicates the cytosolic pool of toxin must be maintained at a certain level for optimal intoxication. This precedent has been established for other virulence factors such as Shiga toxin and the SpvB effector protein from Salmonella. Thus, PBA application after the initial exposure to Vibrio cholerae/CT would prevent additional toxin from reaching the cytosol of an intoxicated cell, and this effect might attenuate the effects of intoxication. Attenuation of the disease state could also occur by the protective effect PBA would extend to newly differentiated enterocytes in the intestinal epithelium of an infected individual. Our collective data indicates that PBA binds to the hith two hour cell culture assays utilizing 100 mM PBA. In contrast, PBA-induced alterations to chaperone expression require a 48 hour exposure to at least 0.5 mM PBA. Alterations to the unfolded protein response require a 24 hour exposure to 10 mM PBA or a 72 hour exposure to 2 mM PBA. Inhibition of the cystic fibrosis transmembrane regulator in polarized epithelial cells only occurs after a 24 hour exposure to 5 mM PBA. The low concentration of PBA required to stabilize CTA1, combined with the specific interaction between PBA and CTA1, thus minimizes off-target effects while still providing substantial cellular resistance to CT. Exposure to 100 mM PBA generated a 3-fold reduction in the efficiency of CTA1 translocation to the cytosol. This, in turn, promoted a greater than 25-fold level of cellular resistance to CT. PBA-induced toxin resistance was also demonstrated in the physiological ileal loop model of intoxication. Previous work has shown that high levels of toxin resistance can be achieved without a complete block of toxin access to the cytosol. Furthermore, as recently shown for the plant toxin ricin, moderate levels of drug-induced toxin resistance in cell culture can correspond to high levels of drug-induced toxin resistance in an animal model. We therefore expect that PBA will provide substantial protection against V. cholerae in an animal model of infection. The maximum FDA-approved dosage of PBA is 20 g/day, which in clinical studies represented 10.6 to 13.8 g/M2/d. The drug was administered orally three to four times a day in the form of 500 mg tablets. With this regimen, patients attained serum PBA concentrations of 600,700 mM. It should therefore be possible to orally administer, at dosages well below the FDAapproved limit, a concentration of PBA that can inhibit CTA1 unfolding and CT intoxication in intestinal epithelial cells. One limitation of a PBA-based therapeutic strategy is the timing of the inhibitory effect: PBA must act on the holotoxin before dissociation and translocation of the CTA1 subunit. While this would be acceptable for a preventative measure, its utility in treating patients already exposed to CT could be more limited. However, a number of observations suggest a post-exposure inhibition of CT might at least attenuate the effects of cholera. The rapid turnover of ADP-ribosylated Gsa and the removal of ADP-ribose modifications by ADP-ribosylprotein hydrolase suggest that the activated form of Gsa can be cleared from the intoxicated cell, provided that CTA1 is also removed from the cytosol. Experiments demonstrating a correlation between 
toxin persistence in the cytosol and the extent of intoxication further support this possibility: the diminished potency of CT and recombinant CT constructs which are rapidly degraded in the cytosol indicates the cytosolic pool of toxin must be maintained at a certain level for optimal intoxication. This precedent has been established for other virulence factors such as Shiga toxin and the SpvB effector protein from Salmonella. Thus, PBA application after the initial exposure to Vibrio cholerae/CT would prevent additional toxin from reaching the cytosol of an intoxicated cell, and this effect might attenuate the effects of intoxication. Attenuation of the disease state could also occur by the protective effect PBA would extend to newly differentiated enterocytes in the intestinal epithelium of an infected individual. Our collective data indicates that PBA binds to the h
