Specific Inhibition of Ectodomain Shedding of GPIb-alpha

特异性抑制 GPIb-α 的胞外域脱落

• 批准号: 8047809
• 负责人: Renhao Li
• 金额: $ 21.5万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8047809
• 关键词: Address; Affinity; Binding; Biological; Biological Assay; Blood; Blood Platelets; Cell surface; Cells; Cleaved cell; Detection; Environment; Evolution; Excision; FDA approved; Glycoprotein Ib; Goals; Human; Immunoglobulin Fragments; In Vitro; Investigation; Lead; Libraries; Life; Ligands; Mammalian Cell; Measures; Membrane; Metalloproteases; Methods; Molecular Conformation; Monitor; Mutagenesis; N-terminal; Oligonucleotides; Pharmaceutical Preparations; Platelet Glycoproteins; Play; Process; Property; Proteins; RNA; RNA library; Reagent; Recombinant Fusion Proteins; Recombinant Proteins; Recovery; Role; Screening procedure; Site; Stream; Structure; Surface; Testing; Time; Transfusion; Vertebral column; aptamer; base; drug development; glycocalicin; inhibitor/antagonist; interest; novel strategies; receptor; von Willebrand Factor

DESCRIPTION (provided by applicant): The goal of this proposed project is to obtain a macromolecular agent that can block specifically ectodomain shedding of platelet glycoprotein (GP)Ib1. As the primary receptor in platelets for von Willebrand factor (vWF), GPIb1 is proteolyzed or shed by ADAM17 at a juxtamembrane site, resulting in the release of its N-terminal fragment, also known as glycocalicin, from platelet surface. Although the biological significance of ectodomain shedding of GPIb1 and function of glycocalicin remain to be defined, recent evidences suggest that shedding of GPIb1 plays an important role in detection and clearance of pathologically damaged platelets, and that blocking shedding of GPIb1 can hamper clearance of platelets stored in vitro. However, it is not possible at the present time to directly test these hypotheses, because it is not clear whether shedding of GPIb1 is merely an inconsequential indicator for the damaged and to-be-cleared platelets or actually the cause for platelet clearance. Such ambiguity is largely due to the usage of broad-spectrum metalloprotease inhibitors in the investigation and the lack of ability to modulate ectodomain shedding in a substrate-specific manner. Given the compelling need for a shedding inhibitor specific to GPIb1, we propose to screen the scFv phagemid library and the RNA aptamer library for binders that specifically recognize and bind to the juxtamembrane shedding cleavage site in GPIb1. Sufficiently high binding affinity should enable these binders to out-compete ADAM17 for access to, and only to, the GPIb1 shedding cleavage site, and thereby achieving specific inhibition of GPIb1 shedding. Both scFv phagemid and RNA aptamer libraries have been used successfully to produce ligands for various protein targets, some of which have been developed into FDA-approved drugs. Due to their difference in backbone structure, molecule size, folding and amenability to mutagenesis and other related properties, scFv and RNA libraries offer distinct and complementary choices for a shedding inhibitor. For the screening, recombinant proteins that contain the sequence flanking the GPIb1 shedding cleavage site in its native-like conformation will be immobilized as binding targets. Once appropriate binders are identified from the screening, they will be produced in large quantity and tested for their ability to bind the GPIb1 shedding cleavage site and inhibit shedding of GPIb1 in transfected mammalian cells and platelets. With a GPIb1-specific shedding inhibitor, we will be poised to investigate the biological significance of GPIb1 shedding and explore novel strategies to extend the shelf life of stored platelet concentrates. Moreover, our method to develop substrate-specific shedding inhibitors, if successfully developed, can be applied to many other shedding substrates in platelets or cells of interest, which may deliver significantly more membrane receptors as suitable targets for drug development. PUBLIC HEALTH RELEVANCE: Clearance of the stored platelet concentrates may depend on proteolytic removal of GPIb1 from the platelet surface. We plan to produce an agent that specifically inhibits the removal process. This agent is sorely needed to address the mechanism of platelet clearance and it may lead to novel strategies to extend the shelf life of stored platelet concentrates.

描述（由申请人提供）：本拟议项目的目标是获得一种可特异性阻断血小板糖蛋白（GP）Ib 1胞外结构域脱落的大分子药物。GPIb 1作为血小板中血管性血友病因子（vWF）的主要受体，在血小板膜位点被ADAM 17蛋白水解或脱落，导致其N-末端片段（也称为glycocalicin）从血小板表面释放。尽管GPIb 1胞外区脱落的生物学意义和糖钙蛋白的功能仍有待确定，但最近的证据表明，GPIb 1的脱落在检测和清除病理损伤的血小板中起着重要作用，并且阻断GPIb 1的脱落可阻碍体外储存的血小板的清除。然而，目前不可能直接检验这些假设，因为尚不清楚GPIb 1的脱落是否仅仅是受损和待清除血小板的无关紧要的指标，或者实际上是血小板清除的原因。这种模糊性主要是由于在研究中使用广谱金属蛋白酶抑制剂以及缺乏以底物特异性方式调节胞外域脱落的能力。鉴于迫切需要一个脱落抑制剂GPIb 1的特异性，我们建议筛选的scFv噬菌粒库和RNA适体库的粘合剂，特异性识别和结合到GPIb 1的质膜脱落切割位点。足够高的结合亲和力应使这些结合剂能够胜过ADAM 17，以获得且仅获得GPIb 1脱落切割位点，从而实现GPIb 1脱落的特异性抑制。scFv噬菌粒和RNA适体文库都已成功地用于生产各种蛋白质靶标的配体，其中一些已被开发成FDA批准的药物。由于它们在骨架结构、分子大小、折叠和对诱变的顺从性以及其他相关性质方面的差异，scFv和RNA文库为脱落抑制剂提供了不同且互补的选择。为了筛选，将含有天然样构象的GPIb 1脱落切割位点侧翼序列的重组蛋白固定为结合靶标。一旦从筛选中鉴定出合适的结合剂，将大量生产它们，并检测它们结合GPIb 1脱落切割位点和抑制转染的哺乳动物细胞和血小板中GPIb 1脱落的能力。通过GPIb 1特异性脱落抑制剂，我们将准备研究GPIb 1脱落的生物学意义，并探索延长储存血小板浓缩物保质期的新策略。此外，我们开发底物特异性脱落抑制剂的方法，如果成功开发，可以应用于血小板或感兴趣的细胞中的许多其他脱落底物，其可以提供显著更多的膜受体作为药物开发的合适靶点。 公共卫生相关性：储存的浓缩血小板的清除可能取决于血小板表面GPIb 1的蛋白水解清除。我们计划生产一种药剂，专门抑制移除过程。这种药物是迫切需要解决的血小板清除机制，它可能会导致新的策略，以延长储存的血小板浓缩物的保质期。