Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Abstract

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

Bacillus anthracis toxins

“…Experimentally, this difference can be appreciated by using ammonium chloride, an agent that neutralizes protons and increases the pH of late endosomes. Cells with CMG2 as the receptor can be completely protected from anthrax toxins by 10 mM ammonium chloride, whereas cells with TEM8 as the receptor remain sensitive to the toxins [171,191,192]. Therefore, CHO cells and RAW264.7, which use CMG2 as the receptor, could be completely protected from anthrax toxins by ammonium chloride, whereas HeLa cells that express only TEM8 receptor cannot.…”

“…Because CHO cells which express only CMG2 receptor were used in these studies, this model may be applicable only when CMG2 is used as the toxin receptor. The fact that the TEM8-expressing cells (such as HeLa cells) treated with anthrax toxins are insensitive to ammonium chloride suggests that LF or EF translocation can occur at early endosomes when TEM8 is used as the receptor [191,192].…”

Exploiting endocytic pathways to prevent bacterial toxin infection

“…The large number of drugs, which target endocytosis or the trafficking of toxins that have been identified through high-throughput screens demonstrates the potential for finding therapeutic targets in this manner. Other identified inhibitors [9] were more effective at blocking anthrax toxicity when the toxin was bound to one of its two receptors. These drugs are thought to act on the pH-driven formation of the endosomal pore, and the differences were attributed to the different pH requirements of the toxin when bound to different receptors [9].…”

“…Other identified inhibitors [9] were more effective at blocking anthrax toxicity when the toxin was bound to one of its two receptors. These drugs are thought to act on the pH-driven formation of the endosomal pore, and the differences were attributed to the different pH requirements of the toxin when bound to different receptors [9]. Therefore, although these drugs are not ideal therapeutic compounds, they provide a base for further research and show the advantage of being able to carry out high-throughput screening to select promising drugs.…”

Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays

“…The qHTS format also reduces scheduling of repeats due to plate-handling failures, identifies carryover effects by compounds that can occur from pin tool delivery, and enables characterization of a chemical series based on pharmacologic parameters in one HTS experiment. [3][4][5][6][7][8][9][10][11][12][13] However, when dealing with very large compound collections, (e.g., a million compounds), a qHTS approach becomes less practical because of very large reagent and time consumption. Still, if titration-based screening were enabled to be just as fast and cost-effective as the current single-concentration paradigm, this format would be become the obvious choice for HTS.…”