Anthrax-toxin-receptor-directed angiogenesis inhibitors

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 1106 Anthrax toxin binds with high affinity to the cellular receptors known as capillary morphogenesis gene protein 2 (CMG2) and tumor endothelial marker 8 (TEM8). We have recently used non-toxic anthrax toxin receptor subunits to demonstrate that interfering with the normal function of anthrax toxin receptors inhibits angiogenesis. Thus, anthrax toxin receptors are useful targets for anti-angiogenic therapy. We have now developed, optimized, and performed pilot screening using homogeneous fluorescence resonance energy transfer assays to identify small molecules that inhibit the high affinity protein-protein interactions between PA and the anthrax toxin receptors, CMG2 or TEM8. Briefly, a PA cysteine mutant (PA E733C) was labeled with a donor fluorophore (Alexa Fluor 488) while soluble receptor binding domain truncation mutants were labeled with an acceptor fluorophore (Alexa Fluor 546). When mixed at equimolar ratios, significant energy transfer is observed. These assays have been optimized for reproducibility, robustness, stability, and sensitivity. Typical Z' values for these assays under screening conditions were >0.90. We have piloted this assay using a library of ~3000 bioactive compounds at Institute of Chemistry and Cell Biology Longwood (ICCB), and have begun more thorough screening of ICCB libraries. Compounds identified in these screens may have utility as anti-angiogenic agents. In addition, because the binding of anthrax protective antigen (PA) is required for anthrax pathogenicity, identification of small molecule inhibitors of PA-receptor binding may lead to anti-anthrax therapeutics for biodefense.