# Protection against Shiga Toxins
## 2 Compounds that Protect Cells against Stx  
### Chloroquine  
Chloroquine (CQ; \(N^{\prime}\)-(7-chloroquinoline-4-yl)-\(N\),\(N\)-dietyl-pentane-1\(A\)-diamine) is a weak base that in its unprotonated form easily can diffuse across membranes and accumulate in acidic compartments of the cell. There, CQ becomes protonated and trapped, leading to an elevated pH and swelling of the compartments. CQ (first named resochin) was developed in 1934 by Bayer laboratories as a synthetic antimalarial drug [113]. CQ was FDA-approved in 1949 and has proven to be one of the most effective and best tolerated agents against malaria [113; 114]. It was widely used throughout the world in the 1950s and 1960s, and also later, but due to the emergence of the CQ-resistant malaria parasites _Plasmodium falciparum_ and _Plasmodium vivax_, CQ has been abandoned as a prophylactic drug in most countries [115]. CQ and its analogues are also FDA-approved for the treatment of systemic lupus erythematosus and rheumatoid arthritis, and are in clinical trials as an adjuvant for anti-cancer chemotherapy and radiotherapy [115; 116]. In short-term 3 h toxicity experiments in HEp-2 cells, a 2 h preincubation with 100 uM CQ gave a 15-fold increase in the Shiga toxin concentration needed to inhibit protein synthesis by 50% (IC50) [98]. In the retrograde pathway, the acidification decreases in the direction from early endosomes to the Golgi and the ER, and one might therefore expect CQ to have the most prominent effect early in the retrograde pathway. However, Shiga toxin transport to the Golgi apparatus was not affected by CQ treatment, neither was release of the A\({}_{1}\)-moiety of the toxin in the ER, suggesting that transport to the ER was also normal [98]. Thus, CQ might interfere with StxA\({}_{1}\) translocation into the cytosol. Other compounds that disrupt the pH gradient, such as the V-ATPase inhibitors bafilomycin A1 (Baf) and concanamycin A (ConA) and the ionophore nigericin (Nig), also protect against Shiga toxin [98]. In contrast to CQ, the V-ATPase inhibitors interfere with Shiga toxin transport to the Golgi [98]. Even though all the compounds inhibit acidification, they interfere with different toxin transport steps, suggesting that also pH-independent processes are affected. The V-ATPase inhibitors show poor selectivity in vivo [117; 118], and are therefore not suitable for clinical application. Shiga toxin is shown to interact with the Sec61 channel [119; 120], and the knockdown of Sec61B has been shown to protect cells against Shiga toxin [119], suggesting that the translocation of StxA\({}_{1}\) into the cytosol is dependent on the Sec61 channel. Interestingly, CQ has previously been shown to block several channels, such as the inward rectifier potassium channel Kir2.1 [121], and the translocation pores of the pore-forming toxins anthrax and C2 toxin [122; 123]. One can speculate that CQ might also interact with the Sec61 channel to block the translocation of Shiga toxin. Importantly, as shown here, CQ also protects against Stx2 (see below). For acute treatment of malaria, the recommended dose of CQ is 10 mg/kg/day. CQ is rapidly distributed in the body and reaches a peak plasma concentration within 3-12 h after an oral dose [124]. In most cases, CQ is slowly eliminated with a half-life median value of 40 days, but there are big differences between individuals and the half-life of CQ has been reported to range from 1 to 157 days [124]. The plasma concentration is strongly dependent on the administered dose and the duration of the treatment [124]. The therapeutic concentration in blood of hydroxychloroquine (HCQ), a less toxic analogue of CQ with similar pharmacokinetics, has been reported to be between 0.03 and 15 mg/L; the toxic and lethal concentration ranges were 3-26 mg/L and 20-104 mg/L, respectively [125]. CQ at high concentrations has been reported to have a number of effects in cell cultures, but clinically safe and achievable doses are in the low micromolar range [116].