High throughput assay for fibrin-binding probes

描述(由申请人提供)：本申请的目标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、其形成和溶解。纤维蛋白是血栓(血栓)的主要成分，血栓与一系列病理因素有关，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，众所周知，大多数实体肿瘤的特征是纤维蛋白凝块的存在。最近，纤维蛋白原和纤维蛋白被认为与多发性硬化症有关。使用与光学、磁共振(MR)或放射性报告相结合的纤维蛋白特异性分子进行纤维蛋白成像可以为所有这些疾病的生物学提供洞察力。纤维蛋白探针可以用来识别血栓或肿瘤转移，并指导患者的治疗。我们已经研究了两个多肽家族，它们结合到纤维蛋白上的两个不同位置，但不结合纤维蛋白原，纤维蛋白原是纤维蛋白的可溶性前体蛋白。我们已经合成了这些与光学、磁共振和/或放射性标记相结合的多肽，用于成像。然而，这些多肽在体内是不稳定的，并被肝脏和肾脏中的多肽酶迅速降解。这种在体内的快速新陈代谢限制了这种探针的用途。我们已经证明，这些多肽还可以与可溶性纤维蛋白降解产物DD(E)结合，并且可以进行竞争性结合试验。该分析涉及竞争配体取代DD(E)中的荧光标记的多肽。利用偏振荧光，蛋白质结合的荧光探针的各向异性很高，但当它被移位时，各向异性要小得多。该分析为溶液相，可在简单的“混合和测量”形式下以微滴定板形式运行。我们还证明了这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。在这项应用中，我们建议进一步开发该测试，以便将其翻译到自动HTS环境中。我们还将开发二次筛查，以确认在主筛查中活跃的化合物是可重复的命中，并排除伪影，以及开发反筛查分析，以便为进一步测试确定命中的优先顺序。这项工作的结果将是一种经过验证的纤维蛋白结合剂的HTS分析方法，将提交给分子文库探针生产中心网络进行筛选，以确定新的有效和选择性的纤维蛋白结合剂。 与公共健康相关：该项目的目标是开发一种高通量筛选试验，以确定与蛋白质纤维蛋白结合的化合物。纤维蛋白是血栓和许多实体肿瘤的标志。筛选试验中确定的化合物可能用于制造成像探针，用于研究血栓形成和肿瘤生物学，并最终检测血栓或肿瘤。

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.