Abstract
The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.
Highlights
The C. perfringens ε-toxin is a potent bacterial protein toxin, with an LD50 in mice on the order of 100 ng per kg [1,2], and is categorized by the US Department of Health and Human Services as a select agent
To assess the suitability of the assay for use in a high-throughput screening (HTS), MDCK cells were dispensed into 384-well plates
Results indicated that compounds I and II did not interfere with the measurement of propidium iodide fluorescence; compound III interfered with the fluorescence of propidium iodide and was not evaluated for the ability to inhibit ε-toxin-induced dye uptake by MDCK cells (Figure 5a)
Summary
The C. perfringens ε-toxin is a potent bacterial protein toxin, with an LD50 in mice on the order of 100 ng per kg [1,2], and is categorized by the US Department of Health and Human Services as a select agent. The ε-toxin is responsible for a severe enterotoxemia primarily in livestock animals but may be toxic to humans [3,4,5,6,7]. As is true of many select agents and toxins, human exposure to ε-toxin appears to be rare. Pathologic changes are observed primarily in the brains and kidneys of animals intoxicated by ε-toxin [8,9,10], but edema of the heart and lungs is common. Following interaction with sensitive cells, ε-toxin assembles into heptameric oligomers [11,12]. Addition of purified toxin to cells is followed rapidly by efflux of intracellular K+
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