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

Department of Surgery, Vascular Biology Program, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 06/2012; 7(6):e39911. DOI: 10.1371/journal.pone.0039911
Source: PubMed


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.

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    ABSTRACT: CMG2 is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with anti-angiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of anti-tumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo anti-tumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.
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    ABSTRACT: Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.
    Preview · Article · Mar 2013 · Journal of Biomolecular Screening
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    ABSTRACT: The main feature of protein misfolding disorders is the presence of protein deposits in cells and tissues as a consequence of aberrant protein-protein interactions. Neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease belong to this class of disorders. The deposition of large amyloid deposits is a dynamic process that starts with the generation of small, soluble species known as dimers and oligomers. The identification of physical, chemical and genetic conditions that promote - or inhibit - the pathologic deposition of proteins is essential for understanding and preventing neurodegeneration. In this chapter, we explain the history, features, design, development and optimization of bimolecular fluorescence complementation (BiFC) assays for the study of protein oligomerization, aggregation and toxicity in neurodegenerative disorders. In BiFC assays protein chimeras containing the protein of interest fused to non-fluorescent halves of a fluorescent reporter protein are generated. When the proteins of interest interact, the halves of the fluorophore become close enough to reconstitute the fluorescence, which is therefore proportional to the production of dimers and oligomers of the proteins of interest. The fact that BiFC assays allow the direct visualization and measurement of the first steps of aggregation makes them a unique tool in the field of protein misfolding disorders. Additionally, BiFC assays require only basic cell and molecular biology materials and equipment, have a wide array of possible experimental settings/fluorophores, allow studies in living cells and organisms, and are especially suitable for large drug and genetic screenings.
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