This proposal describes the development of a fluorescence resonance energy transfer (FRET) assay to identify inhibitors of anthrax toxin receptor 1 (ATR1). Inhibition of this receptor has recently been shown to inhibit angiogenesis and tumor growth. As a result it is anticipated that inhibitors identified using the proposed assay will be valuable therapeutics against angiogenesis-dependant diseases such as cancer, macular degeneration, diabetic retinopathy, psoriasis, arthritis, and cardiovascular and cerebrovascular disease. The first step in anthrax intoxication involves the binding of protective antigen (PA), an otherwise non-toxic component of the multimeric anthrax toxin, to the cell surface receptors ATR1 or anthrax toxin receptor 2 (ATR2). As a result it is expected. The proposal describes the adaptation of a similar FRET-based assay for inhibitors of ATR2 for use with ATR1. Protein expression, purification and labeling will be optimized to produce homogenously labeled ATR1. Fluorescent label selection and placement will be optimized to maximize the FRET efficiency between ATR1 and PA in a cuvette-based assay. This assay will then be converted to 384-well format and optimized for reproducibility, robustness, stability, and sensitivity. The resulting assay will then be validated and piloted using a library of ~3000 bioactive compounds at the New England Research Center of Excellence (NERCE). Compounds showing activity in this assay can then be tested in various cell-based assays for their toxicity as well as their ability to inhibit endothelial cell migration and anthrax intoxication. Resulting compounds would avoid a key difficulty with further anthrax toxin inhibitor development, since they can be safety tested in the cancer context, thereby avoiding issues regarding testing for a rare indication (anthrax intoxication). Good inhibitors of ATR1 can also be used to probe the function and signaling mechanism of that receptor to identify further targets for inhibitors of angiogenesis and/or anthrax intoxication. The assay developed in this proposal will allow the discovery and development of inhibitors of anthrax toxin receptor 1 (ATR1). Such inhibitors may serve as treatments for angiogenesis-dependant diseases such as cancer, macular degeneration, diabetic retinopathy, psoriasis, arthritis, and cardiovascular and cerebrovascular disease. They may also be useful in treating toxicity associated with anthrax infection. ?
| Jia, Zhe; Ackroyd, Christine; Han, Tingting et al. (2017) Effects from metal ion in tumor endothelial marker 8 and anthrax protective antigen: BioLayer Interferometry experiment and molecular dynamics simulation study. J Comput Chem 38:1183-1190 |
| Cryan, Lorna M; Habeshian, Kaiane A; Caldwell, Thomas P et al. (2013) Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay. J Biomol Screen 18:714-25 |
