Abstract 5292: Identification of antiangiogenic small molecule natural products targeting anthrax toxin receptor 2

Abstract

Abstract Bacillus anthracis protective antigen (PA), the B-subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene protein 2 (CMG2) and tumor endothelial marker 8 (TEM8) with high affinity. Both CMG2 and TEM8 are expressed on endothelial cells during angiogenesis. We have shown that wild-type PA inhibits both VEGF-induced and bFGF-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high affinity anthrax receptor ligands, such as PA and PASSSR, are angiogenesis inhibitors, and that anthrax toxin receptors are useful targets for anti-angiogenic therapy. The long-term goal of our research is to determine the extent to which antrax toxin receptor directed therapeutics can control tumor growth and metastasis. Here we show that in addition to inhibition of subcutaneous lung tumors, PASSSR can inhibit the growth of orthotopically implanted breast cancer cells and extend survival in this mouse model. We also sought to identify small molecule natural products that can inhibit the interaction with the anthrax toxin receptor CMG2 and to determine their antiangiogenic and antitumor capacity in vivo. This work was based on the hypothesis that small molecule natural products can inhibit CMG2-ECM interactions required for breast cancer angiogenesis. This work leverages the wide variety of chemical entities generated by endophytic fungi, and the ability of other fungal compounds such as fumagillin to provide interesting insights into angiogenesis inhibitors. Following a high-throughput screen of >10,000 natural product extracts, we identified 48 extracts with significant inhibitory activity, of which ∼0.5% exhibited measurable activity. Of these, 9 exhibited 1:1 binding to CMG2 with IC50 <100 ug/ml total solids. Purification and chemical and in vitro characterization resulting in the identification of six compounds with anti-CMG2 and endothelial cell inhibitory, one of which exhibits antiangiogenic activity. Thus, we have identified several small molecule natural product inhibitors of CMG2 and demonstrated that CMG2 inhibitors can inhibit angiogenesis and breast cancer growth. While none of these molecules is currently suitable as a lead for the development of a breast cancer therapeutic, we believe that these studies confirm that the approach that we have taken is suitable for the development of such a therapeutic and propose to dramatically expand the number of molecules screened for such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5292. doi:1538-7445.AM2012-5292