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

优化一氧化碳 (CO) 传感血红蛋白作为 CO 中毒解毒剂的应用和用于活细胞中 CO 检测的生物传感器

基本信息

批准号：
10643257
负责人：
Matthew Ryan Dent
金额：
$ 16.32万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-10 至 2025-05-31
项目状态：
未结题

项目摘要

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.

项目概要/摘要一氧化碳 (CO) 吸入是美国人类中毒的主要原因，导致约每年有 50,000 例病例和至少 1,500 人死亡，以及长期心脏和神经认知后遗症三分之一的幸存者。不幸的是，迄今为止，还没有针对一氧化碳中毒的即时解毒疗法。一片田野—— 不可逆地清除和隔离二氧化碳的可部署制剂可以作为一种改进的治疗方法增加一氧化碳中毒患者的生存率和长期结果。在本提案中，我们将利用血红素蛋白，利用 CO 和亚铁血红素之间独特的强而特异的相互作用支架开发高亲和力的CO清除剂。我们最近发现了一个显着的血红素蛋白结构域，第 900 章与血红蛋白（急性一氧化碳中毒的主要生物靶标）相比，一氧化碳结合亲和力增加了数倍。这种 RcoM 血红蛋白还表现出对 CO 相对于氧气的精细选择性，这是 CO 解毒剂的关键特性将在含氧条件下静脉注射到人体中。在目标 1 中，我们将利用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。在目标 2 中，我们将评估该药物的安全性和有效性三个 RcoM 截短物在体内具有最高的 CO 亲和力和选择性。我们将评估系统性和器官特异性静脉注射 RcoM 对健康小鼠的影响并量化输注的 RcoM 清除 CO 的能力，在严重的临床前一氧化碳中毒小鼠模型中逆转血流动力学崩溃并防止死亡。这这些目标的结果将为血红蛋白配体选择性提供基本见解，并证明重组 RcoM 作为治疗急性 CO 中毒的治疗潜力。虽然在高浓度下有毒浓度，CO，作为血红素降解的副产品内源产生，作为细胞保护剂低浓度下的信号。临床前和临床研究探索了使用 CO 作为治疗剂从感染到缺血/再灌注损伤的各种情况。尽管有潜在的临床益处，但其作用人们对 CO 作为信号分子知之甚少，并且与基础值相对应的 CO 浓度方案信号传导、细胞保护和毒性的定义不明确。基因编码的共选择性荧光记者将成为梳理二氧化碳在生命系统中生理作用的理想工具。在目标 3 中，我们将利用 RcoM 的 CO 传感功能设计基因编码的荧光报告基因，表征 CO- 体外依赖性反应，并使用 CRISPR/Cas9 将该报告基因整合到小鼠基因组中。我们将量化转基因报告小鼠在不同 CO 暴露条件下的 CO 积累并确定方案产生 CO 信号传导、细胞保护和体内毒性。通过这一目标，我们将开发关键的生物分子工具将能够阐明与人类健康相关的CO依赖性信号传导机制。