A Homogeneous 384-Well High-Throughput Binding Assay for a TNF Receptor Using Alphascreen Technology:

To take advantage of the growing knowledge of cellular signaling pathways, modern-daydrug discoveryfaces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assaytechnologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen™ technologyto develop a high-throughput assayto monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinityconstant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assayin a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assaywas able to detect such peptides and could be used to launch a high- throughput screening campaign for small molecules able to prevent OX40 receptor activation. (Journal of Biomolecular Screening 2003:522-532)