Novel Oxygen Carriers Based on Hexacoordinate Globins to Prevent Renal Toxicity

基于六配位球蛋白的新型氧载体可预防肾毒性

• 批准号: 9257662
• 负责人: Matthew Brian Amdahl
• 金额: $ 4.9万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257662
• 关键词: Address; Adverse effects; Affinity; Anemia; Animal Disease Models; Animal Model; Award; Binding; Biochemistry; Biomedical Engineering; Blood; Blood Pressure; Blood Transfusion; Blood donor; Cells; Characteristics; Chronic; Clinic; Clinical; Clinical Research; Clinical Trials; Cytochromes b5; Data; Development; Doctor of Philosophy; Electron Transport; Encapsulated; Engineering; Exhibits; Extravasation; Failure; Generations; Globin; Heme; Heme Iron; Hemeproteins; Hemoglobin; Hemoglobinuria; Hemorrhage; Histopathology; Human; Hypertension; Immune; In Vitro; Infection; Infusion procedures; Injury; Kidney; Laboratories; Life; Lipids; Liposomes; Liver; Measures; Medicine; Membrane Lipids; Mentors; Methemoglobin; Mus; Mutagenesis; Mutation; National Research Service Awards; Nitric Oxide; Nitric Oxide Donors; Nitrite Reductase; Nitrites; Organ; Oxygen; Pathologic; Pathology; Plasma; Play; Post-Translational Protein Processing; Preparation; Procedures; Process; Property; Protein Engineering; Proteins; Reaction; Recombinants; Renal Blood Flow; Renal function; Research Infrastructure; Resources; Risk; Site; Site-Directed Mutagenesis; Spectrum Analysis; Structure-Activity Relationship; System; Techniques; Testing; Therapeutic; Toxic effect; Training; Transfusion; Translational Research; Universities; Urine; Venous; Whole Blood; base; biomaterial compatibility; career; drug development; experience; in vivo; insight; medical schools; mutant; nephrotoxicity; neuroglobin; novel; novel therapeutics; oxidation; oxygen transport; prevent; protein function; research and development; screening; skills; vasoconstriction

PROJECT SUMMARY/ABSTRACT Blood transfusions are the most common therapy for severe anemia or blood loss, and offer undeniable life- saving benefits in a variety of clinical situations. Nevertheless, chronic shortages of donor blood and continued risks of infectious and immune complications (greatly reduced but not eliminated by screening) post- transfusion have spurred a search for alternatives to whole blood. Purified hemoglobin, and the hemoglobin- based oxygen carriers (HBOCs) that have been derived from it, are one potential substitute for blood transfusions. In clinical studies, however, HBOCs have consistently demonstrated adverse effects, including severe hypertension, renal injury and failure, and other end-organ damage. Extravasation and nitric oxide (NO) scavenging by the protein is thought to underlie these effects. Our lab has developed recombinant mutant proteins based on the hexacoordinate globins neuroglobin (rNgb) and cytoglobin (rCgb) that rapidly generate NO via reduction of nitrite, potentially offsetting any NO scavenging. We, therefore, propose the hypothesis that specific mutations of rNgb and rCgb can be used to engineer a new generation of oxygen carriers that cause reduced hypertension and renal toxicity. To test the hypothesis, we will use UV-Vis spectroscopy to characterize the function of numerous rNgb and rCgb mutants, and identify those mutants with ideal characteristics for oxygen transport and NO generation. We will also encapsulate these proteins within artificial lipid membranes, creating protein-containing liposomes that will be unable to extravasate. Second, we will evaluate cytochrome b5, which reduces methemoglobin in vivo, as a potential reducing system for these mutant proteins, as persistent protein oxidation prevents oxygen binding and nitrite reduction to NO. Finally, we will create liposomes (as in aim 1) containing a promising protein for oxygen delivery as well as the reducing system evaluated in aim 2, and administer these liposomes to mice via venous infusion. Following infusion, we will measure blood pressure, kidney function, and kidney histopathology to determine if any adverse effects are present. The NRSA award will provide me a unique opportunity to leverage the existing research and development infrastructure of my mentor, Dr. Mark Gladwin, to gain advanced training in animal models of disease, protein engineering, and heme biochemistry. The resources and experience of my mentor Dr Gladwin, an expert in NO and globin biochemistry, as well as the translational development of drugs from the laboratory to the clinic, combined with the expansive array of resources available at the University of Pittsburgh, will allow me to develop new translational research skills with a specific focus on development of novel therapeutic strategies. The NRSA will also support my completion of the requirements for a PhD in Bioengineering and defense of my thesis, as well as my return to medical school, where I will begin to develop my clinical skillset in preparation for an eventual career in academic medicine.

项目总结/摘要 输血是严重贫血或失血的最常见疗法，并提供不可否认的生命- 在各种临床情况下节省益处。然而，长期缺乏献血者的血液， 感染和免疫并发症的风险（通过筛查大大降低但不能消除） 输血促使人们寻找全血的替代品。纯化的血红蛋白，而血红蛋白- 由其衍生的氧载体（HBOCs）是一种潜在的血液替代品 输血然而，在临床研究中，HBOCs一直表现出不良反应，包括 严重高血压、肾损伤和衰竭以及其他终末器官损害。外渗和一氧化氮（NO） 被蛋白质清除被认为是这些作用的基础。我们的实验室已经开发出重组突变体 基于六配位球蛋白神经珠蛋白（rNgb）和细胞珠蛋白（rCgb）的蛋白质， NO通过还原亚硝酸盐，可能抵消任何NO清除。因此，我们提出假设： rNgb和rCgb的特定突变可以用来设计新一代的氧载体 从而降低高血压和肾毒性。为了验证假设，我们将使用紫外-可见光谱 光谱来表征许多rNgb和rCgb突变体的功能，并鉴定这些突变体， 氧气输送和NO生成的理想特性。我们还将这些蛋白质包裹在 人工脂质膜，产生含有蛋白质的脂质体，将无法外渗。二是 将评估细胞色素b5，它减少高铁血红蛋白在体内，作为一个潜在的还原系统，这些 突变蛋白质，因为持续的蛋白质氧化阻止氧结合和亚硝酸盐还原为NO。最后，我们 将产生脂质体（如在目标1中），其含有用于氧气输送以及还原的有前景的蛋白质。 系统进行评估，并通过静脉输注将这些脂质体给予小鼠。输注后，我们 将测量血压、肾功能和肾脏组织病理学，以确定是否有任何不良反应 礼物NRSA奖将为我提供一个独特的机会，利用现有的研究， 我的导师Mark Gladwin博士的发展基础设施，以获得动物模型的高级培训， 疾病、蛋白质工程和血红素生物化学。我的导师博士的资源和经验 Gladwin是NO和珠蛋白生物化学方面的专家， 实验室到诊所，结合大学提供的广泛的资源， 匹兹堡，将使我能够发展新的转化研究技能，特别侧重于发展 新颖的治疗策略。NRSA还将支持我完成博士学位的要求， 生物工程和我的论文答辩，以及我回到医学院，在那里我将开始发展 我的临床技能，为最终的学术医学生涯做准备。