A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein

Abstract

Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranose Inhibits Angiogenesis via Inhibition of Capillary Morphogenesis Gene 2

“…Further characterization of this hit demonstrated a complex inhibition curve and toxic effects upon administration in mice 19 , damping our enthusiasm for further study in the context of angiogenesis. However, during follow-up experiments at Clemson University we discovered that there were dramatic differences in the anti-CMG2 activity of separate lots of tannic acid, even though they were from the same supplier.…”

Designing inhibitors of anthrax toxin

“…To confirm this conclusion, the authors tested cisplatin against two other toxins translocated by PA, the anthrax edema toxin and the cytotoxin FP59, a fusion of LF and exotoxin A from Pseudomonas aeruginosa , and demonstrated that cisplatin inhibited both toxins. Note that the cisplatin’s receptor-binding inhibitory properties were reported (57) as discussed earlier in present review. Interestingly, the administration of cisplatin-pretreated lethal doses of LT resulted in total protection of BALB/cJ mice and Fisher 344 rats, whereas the administration of cisplatin to mice before or after toxin administration was not protective.…”

“…More recently, two pilot screen studies were reported where a sensitive high-throughput fluorescence resonance energy transfer (FRET) assay was used to identify potent small-molecule inhibitors of PA interaction with CMG2 (57) and TEM8 (58). The assay was based on FRET detected during the interaction of a fluorescently labeled CMG2 or TEM8 with a fluorescently labeled PA and allowed identification of tannic acid and cisplatin, and edselen and thimerosal as, respectively, CMG2/PA and TEM8/PA interaction inhibitors.…”

FRET-Based Enzyme Activity Reporter: Practical Hints for Kinases as Indicators of Virulence

“…Abovementioned advantages of KAR have thus made these tools amenable for high throughput [61] and led the kinase sensors to be cited as best biosensors in physiology [62]. …”