# Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
## 4 Small-Molecule Inhibitors Targeting Early Endosome-to-Golgi Transport of STx, STx1, and STx2
### Tamoxifen and Derivatives  
Small molecules that increase the pH of the endolysosomal compartment inhibit the fusion of late endosomes with lysosomes [79; 80]. Several drugs that are currently approved by the Food and Drug Administration (FDA) for the treatment of other diseases alter endolysosomal pH. As late endosome-lysosome fusion is necessary for early endosome-to-Golgi trafficking of STx2B (Figure 4A,B), [PERSON] et al. hypothesized that FDA-approved drugs that affect endolysosomal pH may inhibit the trafficking and toxicity of STx2 [20]. A screen of such FDA-approved drugs identified the breast cancer chemotherapeutic tamoxifenfe to be a potent inhibitor of STx2 trafficking in HeLa cells (Figure 5) [20]. In tamoxifenfe-treated cells, trafficking of STx2B is inhibited at the early endosome-to-Golgi step, and the toxin is rerouted to late endosomes and degraded [20]. Treatment with 10 uM tamoxifenfe provides >100-fold protection against STx2-induced cell death, and the protective effect is evident at lower doses as well (2.5 or 5 uM) [20]. Similar levels of tamoxifen also block the trafficking of STx1 and protect cells against STx1-induced death [20]. Notably, in mouse experiments, treatment with 70 mg tamoxifenfe/kg, which corresponds to currently approved human dosing regimens, significantly improves survival after exposure to a lethal amount of STx1 or STx2 [20].  
Tamoxifen is a selective estrogen receptor modulator that antagonizes the effects of estrogen and is, therefore, used for breast cancer therapy [81]. However, as described in [PERSON] et al. [20], a less appreciated feature of tamoxifenfe is that it is a lysosometic weak base that accumulates within acidic endolysosomes and directly increases compartment pH [82; 83]. The capability of tamoxifenfe to change endolysosomal pH is independent of estrogen receptors and, instead, depends on a tertiary amine in its structure that makes it a weak base (Figure 5) [82; 83]. Based on this, [PERSON] et al. hypothesized that activity of tamoxifenfe against STx1/STx2 depended on its tertiary amine base, and not estrogen signaling [20]. Notably, the cell-based assays described above [20] were performed in HeLa cells, which do not express estrogen receptors [84], indicating that the protective effect of tamoxifen against STx2 is independent of estrogen receptor modulation [20]. Furthermore, three clinically approved tamoxifenfe derivatives with the tertiary amine base, toremifene, raoxifene, and bazedoxifene, provide as much protection as tamoxifen against STx2, but ospemifene, which does not have an amine group and is not a weak base, fails to protect all together (all compounds used at 10 uM) (Figure 5) [20]. The results, in totality, imply that tamoxifen exerts its protective effect against STx1/STx2 by acting as a weak base that directly increases endolysosomal pH, and not by acting as a selective estrogen receptor modulator [20]. As tamoxifen, toremifene, raoxifene, and bazedoxifene are already approved for human use, it may be feasible to translate their protective activity against STx1/STx2 in experimental systems to human therapy more rapidly than other drugs in development [20].