# Shiga Toxin Therapeutics: Beyond Neutralization
## 7 Targeting the UPR  
The most promising approach to the treatment of UPR-related diseases is to target the downstream effectors of chronic CHOP-ATF6 activity leading to the upregulation of apoptotic factors, the downregulation of anti-apoptotic factors, and the eventual activation of caspase 3 and caspase 8 to initiate apoptosis (Figure 2). A high-throughput therapeutic screening technique validated this theory in a variety of bacterial toxins including ricin, anthrax toxin, and diphthe toxin by identifying a universally protective compound called biothionol. Biothionol is a small molecule caspase 3, 6, and 7 inhibitor found to prevent cytotoxicity by ricin in RAW264.7 and C32 cells in the face of ribosomal inactivation, and did not show signs of toxicity in mice [101]. Biothionol has not been used in STX models to date. Ouabain is a cardiotonic steroid that decreases Ca\({}^{+2}\) fluxes intracellularly and increases Bcl-XL expression via the activation of Nf-KB p65 subunits, thus blunting the outcomes of chronic UPR activation. Treatment of rat proximal renal tubular cells exposed to STX2 with Ouabain in vitro prevented caspase 3 activation and cellular apoptosis. Ouabain was also found to protect human renal tubular epithelial cells and human glomerular endothelial cells from STX2-induced apoptosis in vitro [101]. Treatment of mice with continuous subcutaneous infusion of Ouabain prevented renal tubular epithelial apoptosis and a loss of podocytes 48 h after injection with STX2.  
An alternate protective strategy is to increase the cellular capacity to resolve the UPR in an attempt to avoid chronic ER stress (Figure 2). Small molecule protein chaperones that improve protein folding in cells undergoing ER stress improve outcomes in mouse models of diabetes and inflammatory bowel disease, and have shown positive results in human patients with insulin resistance or cirrhosis [102; 103; 104; 105]. Extendin-4 is an agonist of glucagon-like peptide agonist 1 that increases the expression of ATF4 in pancreatic beta cells to increase the protective effects of the early UPR [106]. Extendin-4 has been approved for use in human diabetics in the United States and Europe. To date,  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Disease** & **Cells Type Affected** & **Outcome** & **Model System(s)** & **Reference** \\ \hline Shiga toxicosis & Leukocytes, endothelial & Hemolytic uremic & Rodent, human monocyte, & \\ following & cells, renal epithelium, & syndrome? Inflammatory & renal epithelial, and & [34; 44; 46; 89; 90] \\ STEC infection & gastrointestinal epithelium & cytokine secretion? & endothelial cells in vitro & \\ \hline Diabetes mellitus & Pancreatic beta cells & Loss of insulin production & Rodent & [91; 92] \\ \hline Obesity & Hepatocytes & Hepatic lipidosis, insulin resistance & & \begin{tabular}{c} Rodent, various \\ hepatocyte cell lines \\ in vitro \\ \end{tabular} & [91; 92] \\ \hline Inflammatory & Intestinal Panel & Loss of Paneth cells, & \begin{tabular}{c} Rodent \\ gastrointestinal \\ inflammation \\ \end{tabular} & Rodent & [94; 95] \\ \hline Neurode & Neurons & Neuron dysfunction & \multirow{2}{*}{Rodent} & \multirow{2}{*}{[93]} \\ Diseases & & and degeneration & & \\ \hline Vascular & Retinal endothelial and & & & \\ retinopathies & pigmented epithelial cells & Aberrant angiogenesis & & \\ \hline Cardiac disease & Cardiomyocytes & \begin{tabular}{c} Cardiac hypertrophy, \\ arrhythmia, \\ cardiac fibrosis \\ \end{tabular} & \
\begin{tabular}{c} Rodent, rabbit, human \\ cardiomyocytes \\ \end{tabular} & [97] \\ \hline Neoplasia & Malignant cells & Inflammatory cytokine &
\begin{tabular}{c} Human-mouse xenografts, \\ neoplastic cells in vitro \\ \end{tabular} & [98; 99; 100] \\ \hline \hline \end{tabular} STEC: Shiga toxin-producing entero hemorrhagic _Escherichia coli_.  
\end{table}
Table 2: Diseases associated with Unfolded Protein Response (UPR) Activation.  
therapeutics that enhance UPR resolution have not been studied in the context of Shiga toxin-mediated disease in vitro or in vivo.