HTS Assay for Discovery:Small Molecule Regulators (RMI)

描述（由申请方提供）：白细胞特异性β 2整联蛋白对这些细胞的生物学功能至关重要。这些细胞受体介导白细胞的二价金属离子依赖性粘附，包括归巢、牢固粘附、迁移、呼吸爆发和通过吞噬作用和细胞介导的杀伤清除病原体。CD 11b/CD 18（α-M-β 2）异二聚体是中性粒细胞和巨噬细胞中主要的β 2整联蛋白受体，并介导这些细胞中的促炎功能。CD 11b/CD 18识别多种配体，包括补体片段iC 3b、纤维蛋白原、凝血因子X和CD 54（ICAM-1）。CD 11b/CD 18受体与其各种配体的生理结合受到严格调节，因为其缺陷、丧失或不适当的活化导致严重的病理状况，并且还导致许多非感染性疾病中的损伤，例如肾衰竭、同种异体移植排斥、心脏病发作、中风和自身免疫性糖尿病并发症。因此，CD 11b/CD 18代表了小分子药物的重要靶点。尽管阻断CD 11b/CD 18配体结合的配体模拟拮抗剂，例如抗CD 11b/CD 18单克隆抗体（mAb），已显示在动物的某些疾病中有效，但小分子调节剂，尤其是变构调节剂，对于人类治疗应用而言是更理想的。公众可获得的选择性和有效的小分子β 2整联蛋白调节剂的数量不足，并且目前没有CD 11b/CD 18选择性小分子拮抗剂，特别是变构抑制剂。此外，目前尚无CD 11b/CD 18特异性激动剂可用。许多已建立的功能测定，包括我们实验室开发的一些，可以进行修改和调整，用于筛选CD 11b/CD 18的小分子调节剂。我们目前的建议的主要目标是优化和自动化这些可用的检测高通量筛选（HTS）的环境，并使用它们在试点筛选，以发现小分子效应的CD 11b/CD 18（目标1）。此外，我们最近发现了一个新的变构调节位点，β尾结构域（3 TD），在整联蛋白。在该提案中，我们还希望开发HTS测定法，用于鉴定在该位点结合并因此变构调节整联蛋白活化的小分子（目的2）。我们相信，在我们的筛选中发现的独特分子不仅可用作抗CD 11b/CD 18的治疗线索，而且还可用作结构和机制研究的新型探针以及整合素构象传感器。这些研究无疑也将为整合素的活化机制提供新的线索。鉴于β 2整合素在调节白细胞功能中的核心作用，我们希望这项研究将提供重要的新工具，并进一步指导未来的实验。

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.