Identification of small molecules that inhibit the interaction of tem8 with anthrax protective antigen using a FRET assay

Lorna M. Cryan, Kaiane A. Habeshian, Thomas P. Caldwell, Meredith T. Morris, P. Christine Ackroyd, Kenneth A. Christensen, Michael S. Rogers

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)714-725
Number of pages12
JournalJournal of Biomolecular Screening
Volume18
Issue number6
DOIs
StatePublished - Jul 2013

Keywords

  • FRET
  • angiogenesis
  • anthrax
  • high-throughput screening
  • tumor endothelial marker 8

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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