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

J Biomol Screen. 2013 Jul;18(6):714-25. doi: 10.1177/1087057113478655. Epub 2013 Mar 11.

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.

Keywords: FRET; angiogenesis; anthrax; high-throughput screening; tumor endothelial marker 8.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, Bacterial / metabolism*
  • Bacillus anthracis / immunology
  • Biomarkers, Tumor / metabolism
  • Fluorescence Resonance Energy Transfer
  • High-Throughput Screening Assays
  • Humans
  • Microfilament Proteins
  • Neoplasm Proteins / antagonists & inhibitors*
  • Neoplasm Proteins / metabolism
  • Pilot Projects
  • Protective Agents / metabolism*
  • Receptors, Cell Surface / antagonists & inhibitors
  • Receptors, Cell Surface / metabolism*
  • Small Molecule Libraries / pharmacology*

Substances

  • ANTXR1 protein, human
  • Antigens, Bacterial
  • Biomarkers, Tumor
  • Microfilament Proteins
  • Neoplasm Proteins
  • Protective Agents
  • Receptors, Cell Surface
  • Small Molecule Libraries