## 3 Small Molecule Inhibitors to Study Shiga Toxin Trafficking  
One of the best-known facets of chemical biology is the use of small molecule bioprobes to perturb cellular processes and to determine the consequences, which may then lead to the discovery not only of novel molecular mechanisms, but also of more efficient therapeutic drug candidates [57; 58; 59; 60; 61]. Due to the possibility to select for survival, toxins have been the target for small molecule-based intervention strategies [62], including the prototypical GL-Lect cargo protein Shiga toxin [63].  
In one study, a small molecule screen on cells has led to the identification of two compounds, termed Retro-1 and Retro-2, which slowed the intracellular progression of Shiga toxin towards the ribosomes, its cellular target in the cytosol, specifically at the retrograde trafficking step between early endosomes and the Golgi apparatus [64]. Of note, Retro-2 was the first small molecule which had a protective effect in animal models against Shiga toxin [65] or the plant toxin ricin [64]. An optimized version of Retro-2, termed Retro-2.1, was obtained through a medicinal chemistry approach [66; 67; 68]. It was then shown that Retro-2.1 protected against a number of other pathogens and pathogenic factors, including several viruses, Leishman and Chlamydiales [69].  
In the initial study, it was found that Retro-1 and Retro-2 induced the redistribution of the trafficking factor syntaxin-5 from the Golgi apparatus to the ER [64]. How this was related to the accumulation of Shiga toxin in early endosomes under Retro compound treatment conditions had remained unexplained. In a recent study, a model for this has been proposed based on the identification of a Retro-2 interacting partner, the ER exit site protein Sec16A [70] (Figure 7). In this study, a clickable version of Retro-2 was synthesized, and Retro-2 interacting proteins were pulled down and identified by mass spectrometry. Sec16A was one of these. Through a yet-to-be-determined mechanism, the binding of Retro-2 to Sec16A was found to slow down the anterograde transport of COPII cargo syntaxin-5 from the ER to the Golgi apparatus. This then also led to the loss of the interaction of syntaxin-5 with another protein, termed GPP130, which was previously shown to be important for Shiga toxin trafficking from early endosomes to the Golgi apparatus [71]. Indeed, a mutant of GPP130 that failed to interact with syntaxin-5 also failed to enable the retrograde trafficking of Shiga toxin to the Golgi. With this model, it could be explained how an effect of Retro-2 on a cellular target in the ER could cause Shiga toxin accumulation in early endosomes (Figure 7). Using small molecules as tools, this study has provided  
Figure 6: Induction of tubular membrane invaginations by fluorescently labeled STxB on Gb3-containing GUVs. Note that less than 1 min after addition of STxB, tubular membrane invaginations (arrows) were observed on GUVs that contained a C22 Gb3 species with a single unsaturated acyl chain (top panel, C22:1). In contrast, no tubules were observed on GUVs that contained a C22 Gb3 species with fully saturated acyl chain (bottom panel, C22:0), even after dozens of minutes of incubation with STxB. Bars = 5 μm. Reproduced with permission from Ref. [14].  
novel cell biological insights on protein sorting at ER exit sites and at the level of the trans-Golgi network. It also helped to further qualify Retro-2 as a broad-spectrum drug candidate for several pathogenic viruses, bacteria, and toxins.