A Screen for Small Molecule Compounds that Inhibit Bacterial Toxins

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DESCRIPTION (provided by applicant): Shiga toxin (Stx), cholera toxin (Ctx) and ricin are important mediators of human disease and are potential agents of biowarfare. These toxins follow a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface molecules, these toxins are internalized and trafficked in retrograde fashion through the Golgi to the endoplasmic reticulum (ER). From the ER lumen, the toxins must gain access to the cytoplasm, where they carry out their cytotoxic activities. This recently discovered pathway likely represents a recycling and cellular quality control mechanism that has as yet been poorly characterized. The retrograde transport pathway, exploited by toxins of widely different origin, is an attractive target for therapeutic intervention. However, the mechanisms of retrograde traffic and the host molecules involved are largely unknown. We are employing a small molecule screen for inhibitors of Stx susceptibility as a tool to dissect the components of toxin transport pathways. We have developed several assays to examine the effects of inhibitory compounds on retrograde and forward (secretory) transport. The goal of this proposal is to identify several more compounds that, among them, inhibit toxins at multiple sites in their transport from plasma membrane to ER lumen. Our assay is based on luciferase activity as a reporter for toxin resistance. Cells are transduced with an adenoviral construct expressing luciferase that has been modified to be degraded rapidly by the proteasome. Exposure to Stx, which inhibits protein synthesis, results in markedly diminished luciferase content. Compounds that inhibit Stx transport or activity result in a rescue of the luciferase activity and are readily detected in a luminometer. A counterscreen, consisting of incubation with cycloheximide rather than Stx, was incorporated to exclude false-positive hits. The utility of the screen and counterscreen were validated at the ICCB facility, where we found z and z' scores to be > 0.6 and demonstrated the ability to exclude compounds with nonspecific effects on luciferase activity. Based on our previous round of screening we anticipate identifying at least 50 inhibitory compounds from a screen of 100,000 molecules. We will follow up on these compounds by determining their potency, specificity, toxicity, reversibility, and a preliminary assessment of their effect on toxin trafficking in vitro. Based on the results of this analysis, the compounds will be prioritized for their inclusion into studies that will identify the site of toxin inhibition and the intracellular targets of inhibitory compounds. Investigations proposed here will identify potent and specific inhibitors of toxin trafficking. These compounds will be used as probes to identify trafficking pathways and host molecules involved in various stages of toxin transport. In future experiments, some of these compounds will be optimized for potential use as therapeutic agents. Shiga toxin, cholera toxin and ricin are important agents of human disease and are recognized as potential agents of biowarfare. These and other toxins follow a complex intracellular pathway in order to kill susceptible cells. This transport pathway is an attractive target for therapeutic intervention. We are employing a small molecule screen for inhibitors of shiga toxin action. Compounds with activity against shiga toxin will be examined for their ability to inhibit ricin and cholera toxin. The compounds will then be characterized with respect to their potency, toxicity, reversibility, and mechanism of action. The resulting compounds will be valuable tools to dissect toxin and vesicle trafficking pathways. In the final aim, the host targets of inhibitory molecules will be identified. Over the long term, compounds that inhibit toxin trafficking and display minimal toxicity in vitro will be explored as potential therapeutic agents.