Inhibitors of the cellular trafficking of ricin

Abstract

Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress.

Keywords: chemical genetics; retrograde transport; ricin; small-molecule inhibitor.

Figure 1

High-throughput cell-based assays. These phenotypic assays measure the effects of small-molecule compounds on cellular cytotoxicity induced by ricin. (a) Chemical compounds are distributed into microplates for screening. A small volume of each compound is then added to a well of a microplate seeded with cells. (b) After addition of toxin and incubation, one of several possible methods is used to assess the effects of each compound on the toxin-mediated inhibition of protein biosynthesis. (c) The CellTiter-Glo luminescent cell-viability assay quantifies ATP, which signals the presence of metabolically active cells. Luminescent signal is generated by the luciferase (green star) reaction after cell lysis and is proportional to the amount of ATP present [14]. In luciferasereporter-gene assays, a modified luciferase is constitutively transcribed, and enzyme activity is used as a measure of ongoing protein biosynthesis. Inhibition of protein biosynthesis thus leads to diminish luciferase translation, and as existing luciferase protein is degraded there is a proportional decrease in light output [21]. In the third method of assessment, the inhibitory effect of ricin on protein biosynthesis in intact cells is measured through the incorporation of radioactive amino acids into neosynthesized polypeptides. Intact cells are able to concentrate the amino acid that strikes the incorporated scintillant molecules present in the bottom of each well (bold line) in the microplate [15].

Figure 2

Chemical structures of the known cellular inhibitors of ricin and Shiga toxins. (a) Structures of inhibitors with a known molecular target; (b) Structure of inhibitors identified by cellular high-throughput screening whose cellular targets are currently unidentified. The CID number is the Chemical IDentifier for molecular structures in PubChem. References for the molecules are given in the text.

Figure 3

Cellular targets of some inhibitors of ricin and/or Shiga toxins.