Testing the Efficacy and Toxicity of Adenylyl Cyclase Inhibitors against Enteric Pathogens Using In Vitro and In Vivo Models of Infection

Abstract

). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxinproducing bacteria.Diarrheal diseases caused by enteric pathogens such as enterotoxigenic Escherichia coli (ETEC) or Vibrio cholerae remain a major cause of morbidity and mortality worldwide (25,31,34). ETEC, a pathogen of increasing frequency in the United States, is a leading cause of traveler's diarrhea (36). Prevention of diarrhea caused by these toxigenic organisms, by virtue of improved hygiene and provision of sanitation and water treatment, often is impractical in most developing countries, where the morbidity and mortality rates are highest (37). ETEC and V. cholerae produce the heat-labile toxin (LT) and cholera toxin (CT), respectively, and both toxins display ADP ribosylation activity, which results in increased chloride and water efflux into the intestinal lumen, leading to significant volumes of watery diarrhea (25). Interestingly, recent studies have confirmed prior observations indicating that enterotoxins, such as LT and CT, enhance enteric bacterial colonization and pathogenicity (reviewed in reference 8). Anti-toxigenic compounds have been shown to decrease morbidity and mortality of diseases caused by other toxin-producing bacteria (18, 29). Therapy using anti-toxigenic compounds is therefore an area of great interest. Identification of a new class of drugs that afford selective anti-toxigenic activities would constitute a highly desired compound useful for future therapy; however, these drugs need to be experimentally validated by first testing efficacy, bioavailability, and the absence of toxicity in relevant animal models.We have previously shown that prostaglandin E 2 -histidine (PGE 2 -L-histidine) and prostaglandin E 2 -imidazole (PGE 2 -imidazole) adducts significantly reduced CT-induced fluid loss and cyclic AMP (cAMP) accumulation in the murine ligated small intestinal loop model (21). These and other derived adducts have been shown to act on ETEC LT and on the edema factor (EF) produced by Bacillus anthracis (15). Our recent progress has resulted in the development of structurally stable compounds that inhibit toxin-induced accumulation of cAMP in in vitro cell culture assays (3). Our studies have shown that although some of these compounds are extremely active in vitro and showed reduced fluid accumulation in the murine model of experimental cholera, they were also toxic...

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes

“…Female ICR mice of 20-25-g (Charles River Laboratories) were used after 72 h of quarantine as described previously [76]. Briefly, food-restricted animals received streptomycin (5 g/L in drinking water supplemented with 7% fructose) for 48 h prior to oral inoculation with NRG857c or the iutA mutant.…”

Structure-based redesign of an edema toxin inhibitor

“…Their ability to reduce total extracellular cAMP production in mammalian cells treated with protective antigen (PA) and EF (which together 17 are called edema toxin, ET) was determined. This assay gives more variability than using isolated enzymes, but it is biologically relevant, as the concentrations needed to inhibit diarrhea and intestinal damage in the ETEC murine model (7.5–15 μg/mouse) were similar to those indicated by the cell culture IC 50 values for compound 1 8 . The IC 50 values for compound 1 in the assays shown here (Table 1) ranged from 1.64~12.6 μM, in part due to dilutions being done in 10-fold steps when large groups of inhibitors were assayed together.…”

“…Our previously identified lead compound 1 was active in bioassays in the low μM range, and the effective dose to reduce diarrhea in mice (from ETEC) was also low, on the order of 7.5–15 μg when given intraperitoneally or by gavage 8 . Before beginning additional animal studies, it was necessary to eliminate as many markers of toxicity as possible, and improve solubility.…”

“…Compound 1 consistently inhibited cAMP production induced by edema toxin (IC 50 2–9 μM in cells) 7 . Compound 1 also reduced diarrhea caused by enterotoxigenic E.coli (ETEC), at an oral gavage dose of 15 μg/mouse 8 . As we have previously shown that compound 1 inhibits cholera toxin 9 , which is 80% identical to lethal toxin of ETEC 10 , and human ETEC strains can also produce adenylyl cyclase toxins 11 , we attribute this activity to direct inhibition of the E.coli toxins.…”

Mycobacterial Adenylyl Cyclases: Potential Drug Targets

“…Previous reports have shown the feasibility of the design and testing of specific inhibitors against class III adenylyl cyclases, such as the heat-labile enterotoxin (LT) and CyaB, present in enterotoxigenic Escherichia coli and Pseudomona aeruginosa, respectively. The molecule KH7.148 inhibited CyaB in vitro [7], whereas the fluorenone-based molecule DC5 decreased colonization of ETEC to epithelial cells [8]. In light of these results, it would be worthwhile to test these molecules if a class III general inhibition mechanism exists, or develop, by Medicinal Chemistry approaches, new M. tuberculosis, AC-specific drug candidates and evaluate them for their capacity to halt bacterial replication and determine their potential side effects, so that the class III AC mycobacterial enzymes could be exploited as drug targets.…”