title: 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.
authors: Department of Surgery, Vascular Biology Program, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Michael S
authors: MS
authors: Rogers
authors: Lorna M
authors: LM
authors: Cryan
authors: Kaiane A
authors: KA
authors: Habeshian
authors: Lauren
authors: L
authors: Bazinet
authors: Thomas P
authors: TP
authors: Caldwell
authors: P Christine
authors: PC
authors: Ackroyd
authors: Kenneth A
authors: KA
authors: Christensen
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002945:Cisplatin / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D055767:Immobilized Proteins / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D010865:Pilot Projects; D011485:Protein Binding / Q000187:drug effects; D018000:Receptors, Peptide; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D013634:Tannins / Q000494:pharmacology; D013997:Time Factors
chemical_list: C447150:ANTXR2 protein, human; D020533:Angiogenesis Inhibitors; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D055767:Immobilized Proteins; D008565:Membrane Proteins; D018000:Receptors, Peptide; D013634:Tannins; C030325:anthrax toxin; D002945:Cisplatin