# Protection against Shiga Toxins
## 2 Compounds that Protect Cells against Stx
### The Glucose Analogues 2-Deoxy-D-glucose and 2-Fluoro-2-deoxy-D-glucose  
2-Deoxy-D-glucose (2 DG) is a structural analogue of glucose, and has been used in research as a glycolytic inhibitor since 1950s [126; 127]. 2 DG differs from glucose only by the absence of one oxygen atom at the second carbon. Similarly to glucose, 2 DG is taken up through the glucose transporters and phosphorylated by hexokinase to form 2 DG-6-phosphate (2 DG-6-P). However, 2 DG-6-P is not metabolized further and accumulates in the cells [127; 128; 129]. 2 DG-6-P inhibits glycolysis by competing with glucose-6-P for phosphoglucose isomerase [127], and by acting as a non-competitive inhibitor of hexokinase [130]. However, although the inhibition of glycolysis has been a commonly exploited effect of 2 DG, this compound has a much broader spectrum of activities. In addition to inhibiting glycolysis, 2 DG inhibits \(N\)-linked protein glycosylation [131; 132]. In turn, this leads to accumulation of misfolded proteins and triggers the unfolded protein response in the ER, leading to ER stress [133; 134]. Interestingly, [PERSON] et al. recently discovered that 2 DG inhibits the expression of the Gb3 synthase by yet unknown mechanisms, and thus reduces cellular Gb3 levels in the cells [135]. 2-Fluoro-2-deoxy-D-glucose (FDG) is a structural analogue of glucose where the hydroxyl group at the second carbon is replaced by a fluorine atom. Like glucose and 2 DG, FDG is transported into cells, where it is phosphorylated by hexokinase to yield FDG-6-P. However, FDG-6-P does not undergo isomerization to fructose and thus cannot be further catabolized, leading to accumulation of FDG-6-P in the cells [136]. Similarly to 2 DG, FDG also inhibits glycolysis, but because the binding energy of FDG-6-P for the allosteric site of the hexokinase is lower than that of 2 DG-6-P, and closely resembles the energy of glucose-6-P, FDG is a better inhibitor of glycolysis than 2 DG [137]. FDG also interferes with \(N\)-linked protein glycosylation [131; 138; 139], but in contrast to 2 DG, FDG does not become incorporated into dolichol-linked oligosaccharides [138]. FDG seems to slow down rather than prevent the assembly of the dolichol-linked oligosaccharidesaride, and thus is a weaker inhibitor of \(N\)-glycosylation than 2 DG. In addition, we have recently found that in contrast to 2 DG, FDG does  
Figure 4: Cell protection against Stx2 by CQ and HCQ. HEp-2 cells were treated with or without 25 uM chloroquine (CQ) or 25 uM hydroxychloroquine (HCQ) in complete growth medium for 1 h prior to incubation with 10-fold serial dilutions of Stx2 for 24 h in the presence or absence of the drugs. The cells were then incubated in the presence of [3H]leucine for 20 min, and protein synthesis was measured as described in [98]. The left panel shows relative protein synthesis as a percentage of the samples without Stx2 added. The right panel shows relative fold protection against Stx2. The protection was calculated as an increase in IC50 for treated samples compared to control. The error bars show SEM (\(n=4\)). One sample \(t\)-test was used for statistical analysis of the protection data, and obtained \(p\) values are given in the figure. not become incorporated into newly synthesized glycolipids [48]. [18F]FDG, which contains the 18F radioisotope, is a commonly used imaging agent for positron emission tomography (PET). [18F]FDG based PET is widely used for diagnosis and monitoring of oncological, neurological, and cardiological diseases (for review see [140; 141]). We have recently discovered that both 2 DG and FDG reduce cell sensitivity to both Shiga toxin and Stx2. Four hours preincubation with either 10 mM 2 DG or 1 mM FDG led to an increase in the IC50 for Shiga toxin by 13-fold in HEp-2 cells [48; 99]. In addition, 24 h preincubation with 10 mM 2 DG reduced cell sensitivity to Shiga toxin by 30-fold [99], while 24 h pretreatment with 1 mM FDG made HEp-2 cells fully resistant to both Shiga toxin and Stx2 (maximal concentration tested was 100 ng/mL with 3 h challenge) [48].