Identification and Characterization of Small Molecules That Inhibit Intracellular Toxin Transport

Abstract

Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.

Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model

“…Current screening methods for small molecule inhibitors of ricin and Shiga toxin include the use of high throughput cellbased assays using luciferase (Zhao and Haslam 2005;Saenz et al 2007;Wahome et al 2010) or radioactive translation assays (Stechmann et al 2010). The majority of compounds identified from the cell-based assays interfered with intracellular trafficking of the toxins (Saenz et al 2007;Stechmann et al 2010).…”

“…Current screening methods for small molecule inhibitors of ricin and Shiga toxin include the use of high throughput cellbased assays using luciferase (Zhao and Haslam 2005;Saenz et al 2007;Wahome et al 2010) or radioactive translation assays (Stechmann et al 2010). The majority of compounds identified from the cell-based assays interfered with intracellular trafficking of the toxins (Saenz et al 2007;Stechmann et al 2010). Structure-based screening has been employed where a chemical library is screened in silico against a resolved structure to identify small molecules that potentially provide a best ''fit'' into the active site and most likely disrupt enzymatic function (Yan et al 1997;Bai et al 2009Bai et al , 2010.…”

“…We failed to see significant inhibition of RTA depurination by 9OG at any concentration <1 mM (open triangle; large dashed line), and observed only 16% inhibition of depurination at 333 mM and 35% inhibition at 1 mM. In addition, we tested golgicide A, and compounds 75 and 134, which have been shown to inhibit toxin transport (Saenz et al 2007) and found essentially no effect in the qRT-PCR assay at levels up to 1 mM (data not shown). These results demonstrated the potential of the qRT-PCR assay in screening for small molecule inhibitors of catalytic activity of RIPs without interference by inhibitors that target other steps in ricin cytotoxicity.…”

“…Dissolving PTA in NaOH prior to dilution for the qRT-PCR analysis had no impact on the final pH of the buffered reaction. Compounds 74 and 134, obtained from ChemDiv, Inc, and golgicide A (gift of Dr. David Haslam) were dissolved in 100% DMSO (Saenz et al 2007). …”

“…This assay is labor intensive and suffers from high background noise and a low dynamic range. Nonradioactive luminescence based protein synthesis assays have also been developed using luciferase as a reporter (Saenz et al 2007;Wahome et al 2010). Because the luciferase-based assays do not measure the depurination activity directly, they are prone to false positives that interfere with the translation machinery or affect the turnover of the luciferase reporter (Saenz et al 2007).…”