Abstract 1381: A high-throughput assay for tumor endothelial marker-8 (TEM8/ANTXR1) inhibitors
Authors: Cryan Lorna , Habeshian Kaiane , Christensen Kenneth , Rogers Michael S. 
Published: June 2012

TEM8 was first described as a gene upregulated in colorectal tumor endothelium compared to quiescent endothelium. It is also a receptor for anthrax toxin. It is expressed in the endothelium of bladder, esophageal and lung tumors. Mouse knockout studies have shown that TEM8 expression, within the host, promotes tumor growth. Endothelial TEM8 expression can control cell shape, migration, and adhesion to extracellular matrix (ECM) proteins in vitro, supporting a role for TEM8 in tumor angiogenesis. TEM8 interacts with collagen I, collagen VI and gelatin.

Protective antigen is the component of anthrax toxin that binds to its cell surface receptors. We have demonstrated that a mutated form of protective antigen (PASSSR) inhibits endothelial cell migration, corneal angiogenesis, and tumor growth. We hypothesize that PASSSR binds to TEM8 and inhibits its interaction with ECM proteins, thereby inhibiting angiogenesis and tumor growth. As a large immunogenic protein, PASSSR would have significant challenges as a potential therapeutic molecule. Thus our objective is to establish a high-throughput assay to identify small molecule inhibitors of TEM8, which can serve as lead molecules in the identification of novel antiangiogenic agents. Fluorescence resonance energy transfer (FRET) is a sensitive method well suited to high-throughput screening.

We successfully labeled recombinant PAE733C with AlexaFluor546 maleimide. However, identifying a TEM8 labeling strategy proved more challenging. Maleimide or FlAsH labeling of native protein TEM8 extracellular domain, truncations, point mutations or tetracysteine motif extensions resulted in insoluble or inactive protein, as did NHS labeling. We thus investigated GFP labeling, and found that the resulting protein was stable and interacted with PA in the expected manner. When formatted for high-throughput screening, the PA*546 and TEM8-GFP FRET pair gave a Z’ score (a measure of assay performance) of &gt;0.8. We discovered that alterations to the TEM8 molecule, that disrupt either of two pairs of disulfide bonds on cysteine residues, prevent its ability to interact with PASSSR.

The FRET assay was used to identify inhibitors, from a library of over 5,000 known bioactive compounds. Strong hits (&gt;50% inhibition of the PASSSR/TEM8 interaction) included ebselen, and phenylmercuricacetate. Ebselen, a selenium-containing glutathione peroxidase mimic, is known to disrupt disulfide bonds and is likely to have inhibited FRET via binding important cysteine containing residues of TEM8. We will assess anti-angiogenic effects of identified known bioactive molecules. We expect that screening larger small molecule libraries of over 250,000 compounds available at ICCB, Longwood, HMS will result in the identification of a number of candidate TEM8 inhibitors. These molecules are likely to have anti-angiogenic and anti-tumorigenic activities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1381. doi:10.1158/1538-7445.AM2011-1381