Roles of fibrin(ogen) in conformational activation of hemostatic proteinase precursors

纤维蛋白（原）在止血蛋白酶前体构象激活中的作用

基本信息

批准号：
10453034
负责人：
INGRID M VERHAMME
金额：
$ 49.57万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-10 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10453034
关键词：
Active Sites Antibiotic Resistance Antibodies Antiplasmin Antithrombins Bacterial Endocarditis Bacterial Proteins Binding Binding Sites Blocking Antibodies Blood Coagulation Disorders C-terminal Catalytic Domain Church Coagulation Process Collaborations Combined Modality Therapy Complex Cryoelectron Microscopy Data Development Environment Enzyme Precursors Epitopes Equilibrium Evaluation Extracellular Matrix Proteins Fibrin Fibrinogen Fluorescence Funding Future Goals Hemostatic Agents Hemostatic function Host Defense Mechanism Human In Vitro Infection Innate Immune System Kinetics Knowledge Mediating Methods Modeling Molecular Conformation Monoclonal Antibodies N-terminal Patients Peptide Hydrolases Physiological Plasmin Plasminogen Play Preclinical Testing Proteins Prothrombin Regulation Resources Risk Role Scanning Serotyping Staphylocoagulase Staphylococcus aureus Streptococcus pyogenes Streptokinase Structure Surface System Terminal Repeat Sequences Testing Therapeutic Embolization Therapeutic antibodies Thrombin Thrombosis Tissues Trypsin Vaccines Variant Virulence Factors Work activation product bacterial resistance base cofactor design expression vector genetic variant humanized monoclonal antibodies in vivo in vivo evaluation in vivo imaging inhibitor mouse model murine monoclonal antibody novel novel therapeutics prevent protein complex research clinical testing side effect

项目摘要

Prothrombin and plasminogen, two central hemostatic zymogens, are activated proteolytically by cleavage of an activation loop. The newly formed N-terminus inserts into a binding pocket and triggers formation of a functional active site. The activation products thrombin and plasmin respectively form and degrade fibrin, but physiological regulation prevents uncontrolled clotting and promiscuous plasmin-mediated tissue degradation. The bacterial virulence factors, staphylocoagulase (SC) and streptokinase (SK), hijack this mechanism by inserting their own N-termini into the host zymogen pockets, and conformationally activating the catalytic site. The SK-plasminogen complex proteolytically activates plasminogen to plasmin. Both the SC and SK complexes with the zymogens and the mature proteases cleave fibrin(ogen) but are impervious to host antithrombin and antiplasmin, and alternative methods are needed to zcontrol their unwanted activity. Our monoclonal antibodies (mAbs) against the SC and SK N-termini block complex formation and activity, counteracting infection-related thrombosis and bacterial spreading in vivo. This illustrates mechanism-based mAb feasibility in an environment of increasing antibiotic resistance. SC and SK have additional, incompletely defined binding sites for fibrin(ogen) independent of substrate recognition, that play a role in localization. Our proposal aims to identify unique SC and SK sequences, and conformational epitopes in their complexes with the zymogens, that promote binding of fibrin(ogen), both in substrate and anchoring modes. Our group has long-standing expertise with SC and SK- mediated zymogen activation, and we recently made good progress identifying fibrin(ogen) fragment D binding to the C-terminal repeats of SC. However, interactions of the SK-plasmin(ogen) complexes with host fibrin(ogen) are still not well understood. Our short-term goals are to define fibrin(ogen) binding, enhancement of cofactor- zymogen reactivity by fibrin(ogen), identify binding epitopes, and develop in vivo effective mAbs that will be added to our existing antibody arsenal. We combine our structure-function and mechanism expertise with that of experts in mAb development (Dr. Bill Church), and in application of mouse models of SC and SK action (Dr. Peter Panizzi). Aim 1 will define dual interaction mechanisms of the SC-prothrombin complex with fibrin(ogen), with the goal of identifying suitable linear and conformational epitopes for blocking fibrin(ogen) binding. Aim 2 will delineate fibrin(ogen)-dependent plasminogen activation mechanisms of S. pyogenes SK variants that to date are not well defined, with the same goal of identifying fibrin(ogen)-binding epitopes on the SK variants. Aim 3 will test our humanized mAbs targeting the N-termini of SC and SK in vivo, and select tight-binding anti- fibrin(ogen) binding site mAbs for in vivo studies. Long-term goals for future funding cycles are the development of mAbs that that cross-react with a wide range of serotypes and allelic variants, and may qualify for pre-clinical and clinical testing. Cocktails of these mAbs would support the patient's hemostatic system by minimizing plasmin-mediated bacterial spreading and unwanted prothrombin activation without causing bacterial resistance.

凝血酶原和纤溶酶原是两种中枢止血酶原，通过裂解激活循环。新形成的 N 末端插入结合口袋并触发功能性结构的形成活跃站点。激活产物凝血酶和纤溶酶分别形成和降解纤维蛋白，但生理上调节可防止不受控制的凝血和混杂的纤溶酶介导的组织降解。细菌毒力因子，葡萄球菌凝固酶（SC）和链激酶（SK），通过插入自己的基因来劫持该机制 N-末端进入宿主酶原口袋，并构象激活催化位点。 SK-纤溶酶原复合物通过蛋白水解将纤溶酶原激活为纤溶酶。 SC 和 SK 均与酶原形成复合物成熟的蛋白酶裂解纤维蛋白（原），但不受宿主抗凝血酶和抗纤溶酶的影响，并且需要替代方法来控制其不需要的活动。我们的单克隆抗体 (mAb) SC 和 SK N 末端阻断复合物的形成和活性，对抗感染相关的血栓形成和细菌在体内传播。这说明了基于机制的 mAb 在日益增加的环境中的可行性抗生素耐药性。 SC 和 SK 具有额外的、不完全定义的独立于纤维蛋白（原）的结合位点底物识别，在定位中发挥作用。我们的提案旨在确定独特的 SC 和 SK 其与酶原的复合物中的序列和构象表位，促进结合纤维蛋白（原），无论是底物模式还是锚定模式。我们的团队在 SC 和 SK 方面拥有长期的专业知识- 介导的酶原激活，我们最近在鉴定纤维蛋白（原）片段 D 结合方面取得了良好进展至 SC 的 C 末端重复。然而，SK-纤溶酶（原）复合物与宿主纤维蛋白（原）的相互作用仍然没有得到很好的理解。我们的短期目标是定义纤维蛋白（原）结合，增强辅因子- 纤维蛋白（原）的酶原反应性，识别结合表位，并开发体内有效的单克隆抗体添加到我们现有的抗体库中。我们将我们的结构功能和机制专业知识与此相结合 mAb 开发专家（Bill Church 博士）以及 SC 和 SK 作用小鼠模型的应用（Dr. 彼得·帕尼兹）。目标 1 将定义 SC-凝血酶原复合物与纤维蛋白（原）的双重相互作用机制，目的是确定合适的线性和构象表位来阻断纤维蛋白（原）结合。目标2 将描述化脓性链球菌 SK 变体的纤维蛋白（原）依赖性纤溶酶原激活机制日期尚未明确定义，其相同目标是识别 SK 变体上的纤维蛋白（原）结合表位。目的 3 将在体内测试我们针对 SC 和 SK N 末端的人源化 mAb，并选择紧密结合的抗用于体内研究的纤维蛋白（原）结合位点单克隆抗体。未来融资周期的长期目标是发展与多种血清型和等位基因变体发生交叉反应的单克隆抗体，可能有资格进行临床前试验和临床测试。这些单克隆抗体的混合物将通过最大限度地减少血液循环来支持患者的止血系统。纤溶酶介导的细菌传播和不需要的凝血酶原激活，而不引起细菌耐药性。