Optimization of a carbon monoxide (CO) sensing hemoprotein for applications as an antidote for CO poisoning and a biosensor for CO detection in living cells

项目摘要/摘要 一氧化碳(CO)吸入是美国人类中毒的主要原因，导致约 每年50,000例和至少1500人死亡，以及长期的心脏和神经认知后遗症 三分之一的幸存者。不幸的是，到目前为止，还没有针对一氧化碳中毒的解毒疗法。一个领域-- 可部署的不可逆转地清除和隔离CO的试剂可以作为一种改进的治疗方法 提高一氧化碳中毒患者的存活率和长期预后。在这项提案中，我们将 利用一种血红素蛋白开发一氧化碳和亚铁血红素之间独特的强而特异的相互作用 开发一种高亲和力的CO清除剂。我们最近发现了一个值得注意的血球蛋白结构域， 在细菌一氧化碳感应转录因子RCom(一氧化碳代谢调节因子)中发现，它表现出900- 与急性一氧化碳中毒的主要生物靶点--血红蛋白相比，一氧化碳结合亲和力增加了一倍。 这种RCom血蛋白还表现出对CO的精致选择性，而不是氧，这是CO解毒剂的关键特性 它将在有氧的条件下静脉注射到人体内。在目标1中，我们将在体外利用 确定1)最小功能Rcom亚基的光谱方法，以及2)关键氨基酸残基 具有较高的CO亲和力、选择性和血红素稳定性。在目标2中，我们将评估 三个RCom在体内具有最高的CO亲和力和选择性。我们将评估系统性和器官特异性 RCom静脉给药对健康小鼠的影响及输注RCom清除CO的能力 逆转血液动力学崩溃，并防止一氧化碳中毒严重临床前小鼠模型的死亡。这个 这些目标的结果将为血蛋白配体的选择性提供基本的见解，并证明 重组RCom对急性一氧化碳中毒的治疗潜力虽然有毒，但在高 浓度，一氧化碳，作为血红素降解的副产物，在内源产生，起到细胞保护作用 在低浓度时发出信号。临床前和临床研究已经探索了一氧化碳作为一种治疗 条件包括从感染到缺血/再灌注损伤。尽管有潜在的临床益处，但它的作用 CO作为一种信号分子还知之甚少，而CO浓度的变化规律与基础 信号、细胞保护和毒性还没有明确的定义。一种基因编码的共选择性荧光 报告将是梳理CO在生命系统中的生理作用的理想工具。在《目标3》中，我们将 利用RCom的CO敏感功能设计了一种基因编码的荧光报告，表征了CO- 体外依赖反应，并利用CRISPR/Cas9将该报告整合到小鼠基因组中。我们会 量化不同一氧化碳暴露条件下转基因报告鼠的一氧化碳积累并确定机制 在体内产生CO信号、细胞保护和毒性。通过这个目标，我们将开发关键的 生物分子工具，将有助于阐明与人类健康相关的一氧化碳依赖信号机制。

PROJECT SUMMARY/ABSTRACT Carbon monoxide (CO) inhalation is a leading cause of human poisoning in the United States, resulting in about 50,000 cases and at least 1,500 deaths annually, as well as long-term cardiac and neurocognitive sequelae for one-third of survivors. Unfortunately, no point of care antidotal therapy exists for CO poisoning to date. A field- deployable agent that irreversibly scavenges and sequesters CO could serve as an improved therapeutic that increases survival and long-term outcomes for patients suffering from CO poisoning. In this proposal, we will exploit the uniquely strong and specific interaction between CO and ferrous heme by utilizing a hemoprotein scaffold to develop a high-affinity CO scavenger. We recently discovered a remarkable hemoprotein domain, found in the bacterial CO-sensing transcription factor RcoM (regulator of CO metabolism), that exhibits a 900- fold increase in CO binding affinity compared to hemoglobin, the primary biological target in acute CO poisoning. This RcoM hemoprotein also shows exquisite selectivity for CO over oxygen, a critical property for a CO antidote that will be infused intravenously in humans under oxygenated conditions. In Aim 1, we will utilize in vitro spectroscopic methods to identify 1) the minimum functional RcoM subunit, and 2) key amino acid residues that confer high CO affinity, selectivity, and heme stability. In Aim 2, we will evaluate the safety and efficacy of the three RcoM truncates with highest CO affinity and selectivity in vivo. We will assess systemic and organ-specific effects of intravenous RcoM delivery in healthy mice and quantify the ability of infused RcoM to scavenge CO, reverse hemodynamic collapse, and prevent death in a severe preclinical mouse model of CO poisoning. The outcomes of these aims will provide fundamental insight into hemoprotein ligand selectivity and demonstrate the therapeutic potential of recombinant RcoM as a treatment for acute CO poisoning. While toxic at high concentrations, CO, endogenously produced as a by-product of heme degradation, serves as a cytoprotective signal at low concentrations. Preclinical and clinical studies have explored the use of CO as a therapeutic under conditions ranging from infection to ischemia/reperfusion injury. Despite potential clinical benefits, the roles of CO as a signaling molecule are poorly understood, and the CO concentration regimes corresponding to basal signaling, cytoprotection, and toxicity are poorly defined. A genetically encoded, CO-selective fluorescent reporter would be the ideal tool to tease apart physiological roles of CO in living systems. In Aim 3, we will employ the CO-sensing function of RcoM to design a genetically encoded fluorescent reporter, characterize CO- dependent response in vitro, and incorporate this reporter into the mouse genome using CRISPR/Cas9. We will quantify CO accumulation in transgenic reporter mice under different CO exposure conditions and define regimes that give rise to CO signaling, cytoprotection, and toxicity in vivo. Through this aim, we will develop critical biomolecular tools that will enable elucidation of CO-dependent signaling mechanisms relevant to human health.