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

项目总结/摘要 意外一氧化碳（CO）中毒是美国人类中毒的主要原因， 导致每年约50，000例病例和至少1，500例死亡。无即时解毒治疗 迄今为止，对于CO中毒，存在一种治疗方法，常规治疗仅限于吸入100%常压氧 或高压氧。虽然这些疗法提高了CO清除率，但患者诊断和运输延迟 导致发病率和死亡率过高。因此，可以在以下环境中部署的快速作用的CO清除剂 现场，救护车或急诊室可以显着提高生存率和长期结果， 患者鉴于CO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的CO 清除剂，可以结合和消除CO作为一种新的治疗CO中毒。根据初步研究， 对于重组血红素蛋白，我们已经确定了安全有效的基于血红素蛋白的 CO清除剂：（1）高（纳摩尔）CO亲和力，以最大限度地从生理血红素位点清除CO，（2） CO选择性，以最小化氧结合的竞争性抑制，（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）Fe（II）血红素的氧化还原稳定性，以防止自氧化 到非活性的Fe（III）血红素状态。CO代谢调节蛋白（RcoM）是一种CO代谢调节蛋白， 从土壤微生物中检测转录因子，表明该蛋白具有高CO亲和力， 对CO的选择性超过对氧气的选择性。本提案的主要目标是将RcoM发展成为 安全有效的一氧化碳清除剂，将作为一氧化碳中毒的改进治疗方法。在 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）最小功能性 RcoM亚基，和2）赋予高CO亲和力、选择性和血红素稳定性的关键氨基酸残基。在 除了表征基本的生化特性，我们还将评估重组表达的能力， 体外CO饱和红细胞中血红蛋白的RcoM变体。在目标2中，我们将 评价两种重组RcoM截短体在体内的安全性和有效性。我们会评估全身和器官- 在健康小鼠体内静脉内RcoM递送的特异性作用，并定量RcoM逆转 先前开发一氧化碳中毒的临床前小鼠模型中的血流动力学崩溃和防止死亡 在我们的实验室里。这些目标的实现将促进我们对血红素蛋白的基本认识 配体选择性，同时也推进了治疗吸入性肺炎的新型解毒剂疗法的转化发展。 CO中毒除了职业发展、辅导培训和教学课程外， 我将最终为我提供必要的技术专长、背景知识和领导技能 为了实现我的长期学术生涯目标，指导一个研究团队研究CO依赖信号传导， 与人类健康和疾病有关的机制。

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.