High throughput assay for fibrin-binding probes

描述（由申请人提供）：本申请的目的是开发一种高通量筛选试验来鉴定纤维蛋白特异性化合物。最终目标是开发新的分子探针来成像纤维蛋白、纤维蛋白的形成和溶解。纤维蛋白是血栓（血块）的主要成分，血栓与一系列病理有关，如缺血性中风、心肌梗死、肺栓塞和深静脉血栓形成。除了止血外，已知大多数实体瘤的特征是存在纤维蛋白凝块。最近，纤维蛋白原和纤维蛋白与多发性硬化症有关。利用纤维蛋白特异性分子与光学、磁共振（MR）或放射性报告蛋白结合对纤维蛋白进行成像，可以为所有这些疾病的生物学研究提供深入的见解。纤维蛋白探针可用于鉴别血栓或肿瘤转移并指导患者治疗。我们研究了两个肽类，它们与纤维蛋白上的两个不同位点结合，但不与纤维蛋白原（纤维蛋白的可溶性前体蛋白）结合。我们已经合成了这些肽缀合到光学，磁共振，和/或放射性标签成像。然而，这些肽在体内是不稳定的，会被肝脏和肾脏的肽酶迅速降解。这种快速的体内代谢限制了这种探针的实用性。我们已经证明，这些肽也与可溶性纤维蛋白降解产物DD(E)结合，并且可以实施竞争性结合试验。该试验包括用竞争配体取代DD(E)上的荧光标记肽。利用偏振荧光，蛋白质结合荧光探针的各向异性很高，但当它被移位时，各向异性要低得多。该分析是溶液阶段，可以在一个简单的“混合和测量”格式的微量滴度板格式运行。我们还表明，两类纤维蛋白结合肽可以同时分析，只需在每个肽上使用不同的荧光团。在此应用中，我们建议进一步开发该分析，以翻译到自动化HTS环境。我们还将开发二次筛选，以确认在主筛选中活跃的化合物是否可重复命中，并排除伪影，以及开发反筛选分析，以便为进一步测试确定命中的优先级。这项工作的结果将是一个有效的纤维蛋白结合物的HTS分析，将提交给分子文库探针生产中心网络，通过他们的文库筛选新的有效的和选择性的纤维蛋白结合物。

DESCRIPTION (provided by applicant): The goal of this application is to develop a high throughput screening assay to identify fibrin-specific compounds. The ultimate goal is to develop new molecular probes to image fibrin, its formation, and its dissolution. Fibrin is a major component of thrombus (blood clot), and thrombus is implicated in a range of pathologies, e.g. ischemic stroke, myocardial infarction, pulmonary embolism, and deep vein thrombosis. Beyond hemostasis, it is known that most solid tumors are characterized by the presence of fibrin clots. More recently fibrinogen and fibrin have been implicated in multiple sclerosis. Imaging of fibrin using a fibrin-specific molecule conjugated to an optical, magnetic resonance (MR), or radioactive reporter could provide insights into the biology of all these diseases. Fibrin probes could be used to identify thrombi or tumor metastases and guide patient management. We have worked with two families of peptides that bind to two separate sites on fibrin, but do not bind to fibrinogen, the soluble precursor protein to fibrin. We have synthesized these peptides conjugated to optical, MR, and/or radioactive tags for imaging. However the peptides are unstable in the body and are rapidly degraded by peptidases in the liver and kidneys. This rapid in vivo metabolism limits the utility of such probes. We have shown that these peptides also bind to a soluble fibrin degradation product, DD(E), and that a competitive binding assay can be implemented. The assay involves displacement of a fluorescently labeled peptide from DD(E) by a competitor ligand. Using polarized fluorescence, the anisotropy of the protein-bound fluorescent probe is high, but is much lower when it is displaced. The assay is solution phase and can be run in a microtiter plate format in a simple "mix and measure" format. We have also shown that both classes of fibrin binding peptide can be analyzed simultaneously, simply by using a different fluorophore on each peptide. In this application we propose to further develop this assay for translation to an automated HTS environment. We will also develop secondary screens to confirm compounds active in the primary screen as reproducible hits, and rule out artifacts, as well as develop counter-screening assays to allow prioritization of hits for further testing. The result of this work will be a validated HTS assay for fibrin binders that will be submitted to the Molecular Libraries Probe Production Centers Network for screening with their libraries to identify new potent and selective fibrin binders. PUBLIC HEALTH RELEVANCE: This goal of this project is to develop a high throughput screening assay to identify compounds that bind to the protein fibrin. Fibrin is a marker of blood clots and many solid tumors. The compounds identified in the screening assay may be used to make imaging probes for studying thrombosis and tumor biology, and ultimately to detect blood clots or tumors.