title: Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.
abstract: 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.
authors.affiliation: Boston Children’s Hospital, Harvard Medical School, Vascular Biology Program, Department of Surgery, Karp 11, 300 Longwood Ave, Boston, MA 02115, USA. lorna.cryan@childrens.harvard.edu
authors.forename: Lorna M
authors.initials: LM
authors.lastname: Cryan
authors.forename: Kaiane A
authors.initials: KA
authors.lastname: Habeshian
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Caldwell
authors.forename: Meredith T
authors.initials: MT
authors.lastname: Morris
authors.forename: P Christine
authors.initials: PC
authors.lastname: Ackroyd
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Christensen
authors.forename: Michael S
authors.initials: MS
authors.lastname: Rogers
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism*; D001408:Bacillus anthracis / Q000276:immunology; D014408:Biomarkers, Tumor / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D006801:Humans; D008840:Microfilament Proteins; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010865:Pilot Projects; D020011:Protective Agents / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: C473287:ANTXR1 protein, human; D000942:Antigens, Bacterial; D014408:Biomarkers, Tumor; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D020011:Protective Agents; D011956:Receptors, Cell Surface; D054852:Small Molecule Libraries
keywords: FRET; angiogenesis; anthrax; high-throughput screening; tumor endothelial marker 8