# Protection against Shiga Toxins
## 2 Compounds that Protect Cells against Stx
### Retro-2 Substances  
By performing a high-throughput screening of 16,480 drugs [PERSON] et al. [100] identified two low molecular weight substances that reduced sensitivity to Stx1, Stx2, and the plant toxin ricin (which also follows a retrograde route to the ER), when added 30 min prior to challenge with toxins in A459 and HeLa cells. These substances were called Retro-1 and Retro-2. They were reported to inhibit retrograde transport of Stx1B from endosomes to the Golgi apparatus without affecting compartment integrity and endogenous retrograde cargo transport [100]. Other compounds, which may have similarities to the Retro compounds, have also been shown to block trafficking and toxicity of Stx1 and ricin [147]. Both Retro-1 and Retro-2 have been found to relocalize the SNARE proteins syntaxin 5 and, to a lesser extent, syntaxin 6 from their normal site of accumulation on perinuclear Golgi membranes [100]. Thus, it has been suggested that the inhibition of Stx1B transport by these compounds could be mediated by the relocalization of syntaxin 5 and 6, although additional studies are required to confirm this. Retro-2 was found to be the most effective of these drugs, and an intraperitoneal injection of 200 mg/kg Retro-2 given 1 h prior to toxin challenge completely protected mice that were given a lethal nasal instillation of ricin (animal experiments with Stx were not performed) [100]. Later, this group and others published data for several substances similar to Retro-2, and showed that it was a cyclic form (Retro-2\({}^{\mathrm{cycl}}\)) and not Retro-2 that was active [148; 149; 150]. Cyclization and modification of Retro-2 resulted in a compound with approx. 100-fold increased efficacy in inhibiting Stx1, and only one enantiomer was found to be active with an EC50 value (the concentration of the drug that gives 50% of its full inhibitory effect against the toxin) of approx. 300 nM in HeLa cells [149]. The most active of the Retro substances in counteracting the cytotoxicity of Stx1 (named (S)-Retro-2.1) has been reported to have an EC50 value of 54 nM in HeLa cells [151].  
[PERSON] et al. has investigated whether Retro-2\({}^{\mathrm{cycl}}\) could protect mice against the toxic effects of infection with _E. coli_ O104:H4, the strain that was responsible for the deadly outbreak in Europe in 2011 [152; 153]. The bacteria were given to mice by oral gavage, and on days 16 and 26 the mice received intraperitoneal injections of 100 mg/kg of Retro-2\({}^{\mathrm{cycl}}\), which resulted in reduced mortality rate in the treated group (29 and 16 mice were dead in control and treated group, respectively, out of 40 mice per group) [101]. The same authors recently published a review article about the use of Retro-2 and similar compounds to protect against Stxs, ricin, multiple viruses, including different polyomaviruses, Ebola virus and poxviruses, and intracellular parasites, such as _Leishmania_[154], and we refer to this review for further discussions about these substances. The authors conclude that these lead compounds now need to be developed as drugs for human use. As these drugs, as far as we know, have not been given to humans or even been tested in formal preclinical drug safety studies, that task will be ongoing for many years. Furthermore, it will be interesting to see which compound will be selected for such a development as the most efficient compound may also be the most toxic. Finally, it should also be investigated whether all of these compounds have similar effects on the localization of syntaxin 5 as Retro-1 and Retro-2 [100], since targeting of a universal trafficking factor such as syntaxin 5 may prove challenging in a clinical setting.