Abstract 3890: Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2.

Abstract

Abstract We have previously shown that the anthrax protective antigen mutant PASSSR can inhibit endothelial cell migration, VEGF and bFGF driven angiogenesis in the corneal micropocket assay, and tumor growth. However anthrax toxin has three reported receptors (CMG2, TEM8, and β1 integrin) that might mediate antiangiogenic effects. Knockdown of β1 integrin in endothelial cells had no effect on PASSSR inhibition of migration and circulating levels of PASSSR in mice are too low for significant integrin occupancy. Using soluble extracellular domain constructs fused to the antibody Fc region to improve pharmacokinetics, we observed that CMG2-Fc inhibits angiogenesis, while TEM8-Fc does not. Similarly, treatment of mice undergoing the corneal micropocket assay with the SR8F7C7 anti-CMG2 antibody resulted in dose-dependent inhibition of bFGF-induced corneal neovascularization, while the L2 anti-TEM8 antibody had no effect at a dose that has potent antitumor activity. Thus, CMG2 blockade, but not TEM8 blockade, can inhibit corneal angiogenesis. To assess the role of CMG2 in angiogenesis in vivo without pharmacologic intervention, we performed the corneal micropocket assay on knockout mice, and observed that knockout mice exhibit a dramatic decrease in angiogenesis vs. wild-type controls. Also, PASSSR does not further inhibit corneal angiogenesis in CMG2−/− mice. This is consistant with the ex vivo observation that CMG2 knockdown reduces the ability of HMVEC to migrate to serum and knockdown cells are no longer inhibited by PASSSR. To allow identification of CMG2 antagonists, we developed a high-throughput FRET-based screening assay. Validation of the assay suggested that tannic acid may inhibit this protein. However, further analysis demonstrated that this activity is attributable not to tannic acid, but instead to a hydrolysis product (PGG) that contaminated our tannic acid sample. We found that PGG inhibits CMG2 with an IC50 of 300 nM, a value consistent with serum levels achieved by antiangiogenic and antitumor dosing. PGG also inhibited endothelial cell migration and angiogenesis in the corneal micropocket assay, demonstrating that this compound can inhibit not only CMG2-PA binding, but also angiogenesis. Further screening of small molecule libraries identified several additional compounds that inhibit endothelial cell migration and angiogenesis in the corneal micropocket assay. In addition, we identified one compound that stimulates both activities. This important result reveals that CMG2 modulators can both stimulate and inhibit angiogenesis, ruling out indirect effects (such as compound toxicity) as a general explanation for the observed effects on angiogenesis. These results demonstrate small molecule modulation of CMG2 and identify potential pharmacoactive leads. Together, these data strongly suggest that small molecule CMG2 antagonists can be used as antiangiogenic therapies. Citation Format: Lorna M. Cryan, P. Christine Ackroyd, Shugeng Cao, Jon Clardy, Kenneth A. Christensen, Michael S. Rogers. Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2013-3890