Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay

TitleIdentification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay
Publication TypeJournal Article
Year of Publication2013
AuthorsCryan, L. M., Habeshian K. A., Caldwell T. P., Morris M. T., Ackroyd P. C., Christensen K. A., and Rogers M. S.
JournalJ Biomol Screen
Date PublishedJul
ISBN Number1552-454X (Electronic)<br/>1087-0571 (Linking)
Accession Number23479355
Keywordsangiogenesis, anthrax, Antigens, Bacterial/*metabolism, Bacillus anthracis/immunology, Biomarkers, Tumor/metabolism, Fluorescence Resonance Energy Transfer, Fret, high-throughput screening, High-Throughput Screening Assays, Humans, Neoplasm Proteins/*antagonists & inhibitors/metabolism, Pilot Projects, Protective Agents/*metabolism, Receptors, Cell Surface/antagonists & inhibitors/*metabolism, Small Molecule Libraries/*pharmacology, tumor endothelial marker 8

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

Short TitleIdentification of Small Molecules That Inhibit the Interaction of TEM8 with Anthrax Protective Antigen Using a FRET Assay