A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein Michael S. Rogers 1 , Lorna M. Cryan 1 , Kaiane A. Habeshian 1 , Lauren Bazinet 1 , Thomas P. Caldwell 2 , P. Christine Ackroyd 2 , Kenneth A. Christensen 2 * 1 Department of Surgery, Vascular Biology Program, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Chemistry, Clemson University, Clemson, South Carolina, United States of America 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. Citation: Rogers MS, Cryan LM, Habeshian KA, Bazinet L, Caldwell TP, et al. (2012) A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein. PLoS ONE 7(6): e39911. doi:10.1371/journal.pone.0039911 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received June 24, 2010; Accepted June 3, 2012; Published June 29, 2012 Copyright: ß 2012 Rogers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health (grants 1R03NS053690-01 to KAC and 1R01EY018829-01 to MSR), and the Department of Defense (grant W81XWH-08-1-0710 to MSR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kchris@clemson.edu Introduction Angiogenesis is the process of blood vessel formation that occurs when new capillaries sprout from pre-existing vessels [1]. It is a biological process that is normally only seen in the female reproductive system, in fetal development, and in wound healing [1–4]. Angiogenesis is required for any process that results in the accumulation of more than a few microns of new tissue, as well as many processes involving tissue remodeling. As such, it is a characteristic of multiple common disease pathologies that involve inappropriate tissue development [5], including cancer [6,7], cardiovascular disease, arthritis, psoriasis, several rare genetic diseases [8], and a variety of eye disorders, including macular degeneration [9], diabetic retinopathy [10], herpetic keratitis, trachoma, and retinopathy of prematurity [11]. Therapies that target angiogenesis can thus be used to halt or slow the development of these disorders, and have been shown to be effective in a variety of diseases [12–15]. We have previously demonstrated that protective antigen (PA), a non-pathogenic component of the anthrax toxin which binds to endothelial cell surface receptors, can inhibit angiogenesis [16]. Treatment with a PA mutant (PA SSSR ), with three altered amino acids [17], increased inhibition of vessel growth in both VEGF- and bFGF-induced corneal neovascularization assays, inhibited migration of endothelial cells, and resulted in pronounced ($40%) reductions in tumor growth [16]. Anthrax toxin binds and co-opts two endothelial cell surface receptors, anthrax toxin receptor 1 (ANTXR1; also called tumor endothelial marker 8, TEM8) [18], and anthrax toxin receptor 2 (ANTXR2; also called capillary morphogenesis gene 2 protein, CMG2) [19]. Significantly, PA mutants that do not bind these receptors do not inhibit angiogenesis, and the binding affinity of individual PA mutants for the receptors correlates with their degree of inhibition [16]. These data strongly suggest that interaction with an anthrax receptor is responsible for the anti-angiogenic effects of PA SSSR . The normal biological function(s) of TEM8 and CMG2 have not been fully described, although the existing data indicates that these receptors are involved in angiogenic processes, consistent with the observed impact of PA SSSR binding on angiogenesis. Both receptors contain a von Willebrand A or integrin-like inserted I domain, with 60% identity in this region, and are the closest related proteins to integrins, which are involved in cell binding to a variety of extracellular matrix components. TEM8 was initially PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39911