Cycloheximide Counterscreen for Small Molecule Inhibitors of Shiga Toxin
Shiga toxin (Stx) is released by certain strains of E. coli and is associated with food-borne gastroenteritis. In some patients, especially children, the toxin enters the bloodstream and causes hemolytic uremic syndrome, a condition that results in kidney, heart, and occasionally brain injury. The pathogenic effects of Stx arise by the toxin entering cells and inhibiting protein synthesis. To more ..
BioActive Compounds: 37013
Shiga toxin (Stx) is released by certain strains of E. coli and is associated with food-borne gastroenteritis. In some patients, especially children, the toxin enters the bloodstream and causes hemolytic uremic syndrome, a condition that results in kidney, heart, and occasionally brain injury. The pathogenic effects of Stx arise by the toxin entering cells and inhibiting protein synthesis. To identify inhibitors of Stx activity and transport, a cell-based assay was used that employed luciferase activity as a measure of protein synthesis in Stx-treated cells. Vero cells were transduced with an adenovirus expressing a form of luciferase that is degraded rapidly by the proteasome (pAd-luc). Exposure to Stx, which inhibits protein synthesis, results in markedly diminished luciferase translation (Zhao and Haslam, 2005; Saenz et al., 2007). A counterscreen assay was used to identify nonspecific compounds that inhibit protein synthesis independent of Stx action. Cycloheximide (Chx) readily diffuses into cells and immediately blocks protein synthesis in the absence of Stx. Stx actives that are active in the Chx assay are thus nonspecific compounds that stabilize luciferase activity.
Zhao L, Haslam DB. A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis. J Med Microbiol. 2005, 54:1023-30.
Saenz JB, Doggett TA, Haslam DB. Identification and characterization of small molecules that inhibit intracellular toxin transport. Infect Immun. 2007, 75:4552-61.
Six million Vero cells were seeded in a T225 flask and grown 24 hrs at 37 C and 5% CO2. The medium was replaced and the cells infected with 3 x109 plaque forming units of pAd-luc for 24 hrs at 37 C and 5% CO2. Cells were then suspended and dispensed at 750 cells/5 uL/well into white solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following a 24 hr incubation at 37 C and 5% CO2, 23 nL compound or DMSO vehicle was added to each well by a pin tool, the plates were incubated 30 min at 37 C and 5% CO2, and 1 uL/well cyclohemixide (1 ug/mL final concentration) was added. The plates were centrifuged 30 s at 1000 RPM and incubated 6 hr at 37 C and 5% CO2. After addition of 3 uL Photinus luciferase detection reagent, the plates were incubated 10 min at ambient temperature and then read by an ViewLux (Perkin Elmer) to detect luminescence
1. A compound was active if its signal was greater than or equal to 3 standard deviations above the mean DMSO signal on its plate
2. Otherwise a compound is marked inactive
Active compounds receive a score of 100 and inactive compounds receive a score of 0
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** Test Concentration.
Data Table (Concise)