Development of an IgE:Receptor Fluorescence Assay For High Throughput Screening

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DESCRIPTION (provided by applicant): The binding of IgE to its high affinity receptor (Fc5RI) is a key step underlying most allergic responses. We have shown that the IgE antibody can undergo conformational changes affecting the arrangement of the receptor-binding C3 domains, which can adopt a closed, non-receptor binding configuration. We have recently developed a simple mix- and-read fluorescence assay that monitors IgE-Fc binding to the soluble Fc5RI 1- subunit, using purified protein components. In this project, we plan to further optimize this assay for use in high throughput inhibitor screening. We will conduct preliminary screens with test libraries available at Stanford and evaluate secondary biochemical assays for their use in independently verifying preliminary hits obtained in a primary high throughput fluorescence screen. The overall end goal of this project is to identify a set of small molecules that modulate or inhibit IgE binding to its high affinity receptor. These compounds will allow us to probe IgE dynamics and receptor binding in vitro and may form the basis for a novel therapeutic approach to treating the allergic response. PUBLIC HEALTH RELEVANCE: Allergies and asthma have been on a steady rise in the populations of developed countries over the past few decades. IgE antibodies that react with normally benign environmental agents, such as pollen, dust mites or food, can trigger powerful inflammatory responses by activating IgE receptors on mast cells, which then release histamine and other active compounds. In this application, we will optimize and validate a new approach that will allow us to interrogate small molecule libraries to discover potential inhibitors of IgE binding to its receptor. Such inhibitors could act as broad- based treatments for a wide variety of allergies, because they would block a conserved interaction between the antibodies and the cells that are at the heart of the allergic reaction.