Peptide inhibitors of oxidative heme toxicity in acute hemolysis

急性溶血中氧化血红素毒性的肽抑制剂

• 批准号: 9751956
• 负责人: NEEL KUMAR KRISHNA
• 金额: $ 18.75万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751956
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Animal Model; Antioxidants; Autoimmune hemolytic anemia; Bilirubin; Binding; Biological Assay; Blood Chemical Analysis; Cells; Cessation of life; Classical Complement Pathway; Complement; Complement 1q; Complement Activation; Complement Inactivators; Creatinine; Cysteine; Data; Deposition; Development; Disease; Dose; Drug Metabolic Detoxication; Erythrocyte Transfusion; Erythrocytes; Free Radical Formation; Functional disorder; Goals; Heme; Hemoglobin; Hemoglobin concentration result; Hemolysis; Hemolytic Anemia; Histopathology; Human; Immunohistochemistry; In Vitro; Inflammation; Injury; Kidney; Kidney Failure; Laboratories; Lead; Mediating; Medical; Methemoglobin; Modeling; Molecular; Organ; Outcome; Oxidation-Reduction; Oxidative Stress; Oxides; Pathologic; Peptides; Peroxidases; Pharmaceutical Preparations; Phenotype; Play; Process; Property; Publishing; Rattus; Reaction; Reactive Oxygen Species; Renal tubule structure; Research; Research Project Grants; Role; Sickle Cell Anemia; Sulfhydryl Compounds; System; Therapeutic; Tissues; Toxic effect; Transfusion; Weight; Wistar Rats; base; clinically significant; in vitro Assay; in vitro activity; in vivo; inflammatory milieu; inhibiting antibody; inhibitor/antagonist; nephrotoxicity; novel; novel therapeutics; oxidative damage; prevent; prophylactic; renal damage; research clinical testing; transfusion medicine

Acute or severe hemolysis result from the release of significant amounts of toxic free heme leading to organ, tissue and cellular damage manifested in a variety of pathological conditions such as acute hemolytic transfusion reactions (AHTR). In the field of transfusion medicine, AHTR, while rare, is a clinically significant concern. AHTR-mediated RBC hemolysis releases hemoglobin (Hb) into the vasculature which promotes a pro-oxidative and pro-inflammatory environment. Additionally, free heme is exquisitely nephrotoxic leading to renal failure through a number of mechanisms including oxidative damage and heme deposition in the renal tubules. Given the highly destructive properties of cell free heme, development of an inhibitor to detoxify its oxidative properties remains a major unmet medical need. Our laboratory has developed peptide constructs known as Peptide Inhibitors of Complement C1 (PIC1) that inhibit antibody-initiated, classical complement pathway mediated destruction of human RBC in vitro as well as in a rat model of AHTR. Recently, we have discovered that PIC1 can inhibit the peroxidase activity of RBC lysates and purified human Hb in in vitro assays via an antioxidant mechanism mediated by two cysteine residues contained within the PIC1 sequence. Given its dual ability to inhibit complement-mediated RBC destruction and neutralize Hb peroxidase activity, PIC1 provides an exceptional opportunity to mitigate the AHTR disease process. The experimental focus of this project is to assess inhibition of heme peroxidase toxicity of PIC1 in our well-defined rat model as well as define the mechanism by which PIC1 exerts its inhibitory effect on Hb with the long range goal of producing a therapeutic molecule for clinical testing in humans. Our specific hypothesis is that PIC1 will inhibit Hb peroxidase activity in vivo via its reactive cysteine residues, thus limiting toxic free radical formation and inflammation minimizing downstream sequelae, including kidney failure and death. Specific Aim 1 will evaluate the efficacy of PIC1 in preventing heme-mediated tissue and organ injury created by infusing RBC lysates in the AHTR rat model. Following optimization of the amount of RBC lysates required to induce a reliable phenotype, prophylactic and rescue administration of PIC1 in the animal model will be assessed. Readouts will consist of various blood chemistry markers as well as kidney damage evaluated by histopathology. Specific Aim 2 will evaluate the mechanism of PIC1 peroxidase inhibitory activity. PIC1 acts as an antioxidant to inhibit peroxidase activity of Hb via a redox mechanism involving the two cysteine residues. We will evaluate the role each of these cysteine residues play in this activity in vitro and in vivo. First we will utilize derivatives of PIC1 in which one or both cysteine residues are substituted to assess Hb peroxidase inhibitory/antioxidant activity in vitro. Secondly, we will determine the efficacy of selected PIC1 derivatives on inhibition of heme toxicity in the animal model. Successful accomplishment of these aims will establish PIC1 as a novel therapeutic to detoxify free heme in the setting of acute hemolysis such as AHTR, hemolytic anemia and sickle cell disease.

急性或严重的溶血是由于大量无毒无血红素导致器官的释放而引起的， 组织和细胞损伤表现在多种病理条件下，例如急性溶血 输血反应（AHTR）。在输血医学领域，AHTR虽然很少见 忧虑。 AHTR介导的RBC溶血将血红蛋白（HB）释放到促进A的脉管系统中 促氧化和促炎环境。此外，自由血红素是精美的肾毒性，导致 通过多种机制（包括氧化损伤和血红素沉积）的肾衰竭 小管。鉴于无细胞血红素的高度破坏性特性，抑制剂的发展以使其排毒 氧化特性仍然是主要的未满足医疗需求。我们的实验室已经开发了肽构建体 称为补体C1（PIC1）的肽抑制剂，抑制抗体引起的经典补体 途径介导了人类RBC体外以及AHTR大鼠模型的破坏。最近，我们有 发现PIC1可以抑制RBC裂解物的过氧化物酶活性并在体外纯化的人Hb 通过PIC1序列中包含的两个半胱氨酸残基介导的抗氧化剂机制的测定。 鉴于其双重能力抑制补体介导的RBC破坏并中和Hb过氧化物酶活性， PIC1提供了减轻AHTR疾病过程的特殊机会。实验重点 该项目旨在评估我们定义明确的大鼠模型中PIC1的血红素过氧化物酶毒性的抑制 定义PIC1对HB发挥抑制作用的机制，其远距离目标是产生A 用于人类临床测试的治疗分子。我们的具体假设是PIC1将抑制HB 过氧化物酶通过其反应性半胱氨酸残基在体内活性，从而限制了有毒自由基的形成和 炎症最大程度地减少了下游后遗症，包括肾衰竭和死亡。特定目标1将评估 PIC1在防止血红素介导的组织和器官损伤中的功效，通过注入RBC裂解物造成 AHTR大鼠模型。在优化了诱导可靠的RBC裂解物量之后 将评估动物模型中PIC1的表型，预防性和救援给药。读数会 由各种血液化学标记以及通过组织病理学评估的肾脏损害组成。具体的 AIM 2将评估PIC1过氧化物酶抑制活性的机制。 PIC1充当抑制的抗氧化剂 HB的过氧化物酶活性通过涉及两个半胱氨酸残基的氧化还原机制。我们将评估角色 这些半胱氨酸残基中的每一个在体外和体内都在这种活性中发挥作用。首先，我们将在 一个或两个半胱氨酸残基被取代以评估Hb过氧化物酶抑制/抗氧化活性 体外。其次，我们将确定选定的PIC1衍生物对抑制血红素毒性的疗效 动物模型。这些目标的成功实现将确立PIC1作为一种新型的排毒治疗方法 在急性溶血的情况下，例如AHTR，溶血性贫血和镰状细胞病。