Development of a carbon monoxide scavenging hemoprotein as a novel antidotal therapy to treat inhaled CO poisoning

项目摘要/摘要 在美国，一氧化碳(CO)意外中毒是人类中毒的主要原因， 每年造成约50,000个病例和至少1,500人死亡。没有医疗点解毒治疗 到目前为止，一氧化碳中毒已经存在，常规治疗仅限于吸入100%常压氧 或者高压氧。虽然这些疗法增强了CO的清除，但患者诊断和运输的延误 导致过高的发病率和死亡率。因此，可以部署在 现场、救护车或急诊室可以显著提高患者的存活率和长期预后 病人。鉴于一氧化碳与亚铁血红素紧密结合，我们的实验室试图开发一种基于血红素蛋白的一氧化碳 可结合和清除CO的清除剂作为治疗CO中毒的新方法。基于初步研究 对于重组血红蛋白，我们已经确定了安全有效的基于血红蛋白的四个关键标准。 CO清除剂：(1)高(纳摩尔)CO亲和力，最大限度地从生理血红素部位清除CO，(2) CO选择性，通过氧结合将竞争抑制降至最低，(3)热稳定性和化学稳定性 防止血红素释放和不良反应，以及(4)铁(II)血红素的氧化还原稳定性，以防止自氧化 到不活跃的Fe(III)血红素状态。一氧化碳代谢调节蛋白(RCom)的早期研究 来自土壤微生物的感应转录因子，表明该蛋白具有高CO亲和力和 一氧化碳比氧气具有前所未有的选择性。这项提议的主要目标是将RCom发展成为 安全有效的CO清除剂，将作为CO中毒的改进治疗方法。在……里面 目的1)我们将利用我们实验室开发的体外光谱方法来鉴定1)最小功能 RCom亚基，以及2)关键氨基酸残基，赋予高CO亲和力、选择性和血红素稳定性。在……里面 除了基本的生化特性外，我们还将评估重组表达的能力 RCom变异体在体外清除血红蛋白CO饱和红细胞中的CO。在目标2中，我们将 体内评价两种重组RCom截短体的安全性和有效性。我们将评估系统和器官- RCom静脉给药对健康小鼠体内的特异性影响及其逆转能力的定量研究 先前建立的一氧化碳中毒临床前小鼠模型的血流动力学崩溃和预防死亡 在我们的实验室里。完成所提出的目标将促进我们对血红素蛋白的基本理解 配体选择性，同时也促进了一种治疗吸入性疾病的新解毒疗法的翻译开发 一氧化碳中毒。这些结果，除了职业发展、有指导的培训和授课之外， 最终将为我提供必要的技术专长、背景知识和领导技能 为了实现我长期的学术生涯目标--领导一个研究团队研究CO依赖的信号转导 与人类健康和疾病相关的机制。

PROJECT SUMMARY/ABSTRACT Accidental carbon monoxide (CO) poisoning is the leading cause of human poisoning in the United States, resulting in approximately 50,000 cases and at least 1,500 deaths annually. No point-of-care antidotal therapy exists for CO poisoning to date, and conventional treatments are limited to inhalation of 100% normobaric oxygen or hyperbaric oxygen. While these therapies enhance CO clearance, delays in patient diagnosis and transport contribute to excess morbidity and mortality. Consequently, a fast-acting CO scavenger that can be deployed in the field, ambulance, or emergency room could significantly increase survival and long-term outcomes for patients. Given that CO binds tightly to ferrous heme, our lab seeks to develop a hemoprotein-based CO scavenger that can bind and eliminate CO as a novel therapy for CO poisoning. Based on preliminary studies of recombinant hemoproteins, we have identified four key criteria for a safe and efficacious hemoprotein-based CO scavenger: (1) high (nanomolar) CO affinity to maximize CO scavenging from physiological heme sites, (2) CO selectivity to minimize competitive inhibition by oxygen binding, (3) thermal and chemical stability to prevent heme release and adverse reactivity, and (4) redox stability of the Fe(II) heme to prevent autooxidation to the inactive, Fe(III) heme state. Early investigations of the regulator of CO metabolism (RcoM) protein, a CO- sensing transcription factor from soil microbes, suggest that this protein exhibits high CO affinity and unprecedented selectivity for CO over oxygen. The primary objective of this proposal is to develop RcoM into a safe and efficacious CO scavenger that will serve as an improved therapeutic treatment for CO poisoning. In Aim 1, we will utilize in vitro spectroscopic methods developed in our lab to identify 1) the minimum functional RcoM subunit, and 2) key amino acid residues that confer high CO affinity, selectivity, and heme stability. In addition to characterizing basic biochemical properties, we will assess the ability of recombinantly expressed RcoM variants to scavenge CO from hemoglobin in CO-saturated red blood cells in vitro. In Aim 2, we will evaluate the safety and efficacy of two recombinant RcoM truncates in vivo. We will assess systemic and organ- specific effects of intravenous RcoM delivery in healthy mice in vivo and quantify the ability of RcoM to reverse hemodynamic collapse and prevent death in a preclinical mouse model of CO poisoning previously developed in our laboratory. Completion of the proposed aims will advance our fundamental understanding of hemoprotein ligand selectivity while also advancing the translational development of a novel antidotal therapy to treat inhaled CO poisoning. These outcomes, in addition to career development, mentored training, and didactic coursework, will ultimately provide me with the technical expertise, background knowledge, and leadership skills necessary to accomplish my long-term academic career goal of directing a research team to study CO-dependent signaling mechanisms relevant to human health and disease.