Peptide inhibitors of oxidative heme toxicity in acute hemolysis

急性或严重溶血是由于大量有毒的游离血红素释放到器官， 组织和细胞损伤，表现为各种病理情况，如急性溶血 输血反应(AHTR)。在输血医学领域，AHTR虽然罕见，但在临床上具有重要意义 担忧。AHTR介导的RBC溶血释放血红蛋白(Hb)进入血管系统，促进 有利于氧化和促进炎症的环境。此外，游离的血红素具有极强的肾毒性，导致 肾功能衰竭的多种机制，包括肾组织的氧化损伤和血红素沉积 小管。鉴于无细胞血红素的高度破坏性，开发一种抑制剂来解毒其 氧化特性仍然是一个主要的未得到满足的医疗需求。我们的实验室已经开发出多肽结构 已知的补体C1肽抑制物(PIC1)，可抑制抗体启动的经典补体 途径在体外以及在AHTR大鼠模型中介导的人红细胞的破坏。最近，我们有 体外发现PIC1对红细胞裂解物和纯化的人Hb的过氧化物酶活性有抑制作用 通过PIC1序列中包含的两个半胱氨酸残基介导的抗氧化机制进行检测。 鉴于其具有抑制补体介导的红细胞破坏和中和Hb过氧化物酶活性的双重能力， PIC1为缓解AHTR疾病过程提供了一个难得的机会。的实验重点 该项目旨在评估PIC1在我们明确定义的大鼠模型中对血红素过氧化物酶毒性的抑制以及 定义PIC1对Hb发挥抑制作用的机制，长期目标是产生 用于人类临床试验的治疗分子。我们的具体假设是PIC1会抑制Hb 体内过氧化物酶活性通过其活性半胱氨酸残基，从而限制有毒自由基的形成和 炎症可最大限度地减少后续后遗症，包括肾衰竭和死亡。特定目标1将评估 PIC1对输注红细胞裂解物所致组织器官损伤的保护作用 AHTR大鼠模型。在优化了诱导可靠的红细胞裂解物所需的量之后 将在动物模型中评估PIC1的表型、预防和救援给药。读数将 包括各种血液化学标志物以及组织病理学评估的肾脏损害。特定的 目的2探讨PIC1过氧化物酶抑制活性的机制。PIC1作为一种抗氧化剂抑制 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。我们将评估这一角色 在体外和体内，这些半胱氨酸残基中的每一个都参与了这一活动。首先，我们将利用PIC1的导数 哪一个半胱氨酸残基或两个半胱氨酸残基被取代来评估Hb过氧化物酶抑制/抗氧化活性 体外培养。其次，我们将确定选定的PIC1衍生物在抑制血红素毒性方面的有效性。 动物模型。这些目标的成功实现将使PIC1成为一种新的戒毒疗法 急性溶血性贫血、溶血性贫血和镰状细胞病等急性溶血时的游离血红素。

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.