HTS Assay for Discovery:Small Molecule Regulators (RMI)

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DESCRIPTION (provided by applicant): The leukocyte specific beta2 integrins are central to the biological function of these cells. These cellular receptors mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell mediated killing. The CD11b/CD18 (alpha-M-beta2) heterodimer is the predominant beta2 integrin receptor in neutrophils and macrophages and mediates the pro-inflammatory functions in these cells. CD11b/CD18 recognizes a wide variety of ligands, including the complement fragment iC3b, fibrinogen, blood clotting factor X and CD54 (ICAM-1). Physiologic binding of CD11b/CD18 receptors to its various ligands is tightly regulated, as defects, its loss or inappropriate activation lead to severe pathological conditions and also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications. Thus, CD11b/CD18 represents an important target for small molecule drugs. Although ligand-mimetic antagonists that block CD11b/CD18 ligand binding, such as anti-CD11b/CD18 monoclonal antibodies (mAbs), have been shown to be effective in certain diseases in animals, small molecule regulators, especially allosteric regulators, are pharmacologically more desirable for human therapeutic applications. There is an inadequate array of selective and potent small molecule beta2 integrin modulators available to the public, and there are no CD11b/CD18 selective small molecule antagonists, especially allosteric inhibitors, currently available. Additionally, no CD11b/CD18 specific agonists are available at this time. A number of established functional assays, including some developed in our laboratory, can be modified and adapted for screening of small molecule regulators of CD11b/CD18. The major goals of our current proposal are to optimize and automate these available assays for a high throughput screening (HTS) environment and use them in a pilot screen to discover small molecule effectors of CD11b/CD18 (Aim 1). Furthermore, we have recently discovered a novel allosteric regulatory site, the beta tail Domain (3TD), in integrins. In this proposal, we would also like to develop HTS assays for identifying small molecules that bind at this site and thus, allosterically regulate integrin activation (Aim 2). We believe that unique molecules discovered in our screen will not only be useful as therapeutic leads against CD11b/CD18, but may also serve as novel probes for structural and mechanistic studies and as integrin conformation sensors. Such studies will undoubtedly shed new light into the mechanism of integrin activation as well. Given the central role of beta2 integrins in regulating leukocyte function, we hope that this research will provide important new tools and further guide future experimentation.