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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10643257
• 关键词: Acute; Address; Affinity; Amino Acids; Antidotes; Binding; Biological; Biosensor; CRISPR/Cas technology; Calibration; Carbon Monoxide; Carbon Monoxide Poisoning; Cardiac; Cells; Cessation of life; Chemicals; Clinical; Clinical Research; Cytoprotection; Detection; Development; Diagnosis; Disease; Emergency department visit; Erythrocytes; Exhibits; Fluorescence; Functional disorder; Health; Heme; Hemeproteins; Hemoglobin; Human; In Vitro; Infection; Inflammation; Inhalation; Inorganic Chemistry; Intravenous; Intravenous infusion procedures; Ischemia; Kinetics; Learning; Ligands; Mammalian Cell; Mentorship; Metabolism; Methods; Modeling; Molecular; Molecular Target; Morbidity - disease rate; Mus; Neurocognitive; Neurocognitive Deficit; Organ; Organism; Outcome; Oxygen; Patient-Focused Outcomes; Patients; Pattern; Persons; Physiological; Poisoning; Production; Proliferating; Property; Recombinants; Reperfusion Injury; Reporter; Reporter Genes; Research Personnel; Role; Safety; Signal Pathway; Signal Transduction; Signaling Molecule; Spectrophotometry; Survivors; Testing; Therapeutic; Therapeutic Agents; Tissues; Toxic effect; Toxicology; Transfection; Transgenic Organisms; United States; Variant; biophysical chemistry; career; cell growth; chemical stability; circadian regulation; conventional therapy; design; detector; disability; experience; hemodynamics; improved; in vivo; induced pluripotent stem cell derived cardiomyocytes; innovation; insight; long-term sequelae; microbial; mortality; mouse genome; mouse model; organ injury; organ transplant rejection; point of care; pre-clinical; preclinical study; pressure; prevent; response; safety assessment; scaffold; sensor; skills; standard of care; tool; transcription factor

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.

项目总结/摘要 在美国，一氧化碳（CO）吸入是人类中毒的主要原因， 每年有5万例病例和至少1,500例死亡，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，迄今为止还没有针对CO中毒的即时治疗解毒疗法。一块地- 不可逆地清除和隔离CO的可展开的试剂可以作为改进的治疗剂， 提高了一氧化碳中毒患者的生存率和长期预后。在本提案中，我们将 利用血红素蛋白， 支架开发高亲和力的CO清除剂。我们最近发现了一个显著的血红素蛋白结构域， 发现于细菌CO敏感转录因子RcoM（CO代谢调节因子）中，其表现出900- 与血红蛋白相比，CO结合亲和力增加一倍，血红蛋白是急性CO中毒的主要生物靶点。 这种RcoM血红素蛋白还显示出对CO的精细选择性超过对氧气的选择性，这是CO解毒剂的关键特性 将在充氧条件下通过静脉注入人体。在目标1中，我们将利用体外 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。在目标2中，我们将评估 三种RcoM截短物在体内具有最高的CO亲和力和选择性。我们将评估系统和器官特异性 静脉内RcoM递送在健康小鼠中的作用，并量化输注的RcoM对CO2的能力， 逆转血流动力学崩溃，并防止CO中毒的严重临床前小鼠模型死亡。的 这些目标的结果将提供对血红素蛋白配体选择性的基本认识，并证明 重组RcoM作为急性CO中毒治疗的治疗潜力。虽然毒性高， 浓度，CO，内源性产生的血红素降解的副产品，作为细胞保护作用， 低浓度信号。临床前和临床研究已经探索了CO作为治疗剂的用途， 从感染到缺血/再灌注损伤的病症。尽管有潜在的临床益处， CO作为一种信号分子知之甚少，并且对应于基础代谢的CO浓度机制 信号传导、细胞保护和毒性的定义不明确。一种基因编码的CO选择性荧光 报告者将是梳理CO在生命系统中的生理作用的理想工具。在目标3中，我们 利用RcoM的CO传感功能来设计遗传编码的荧光报告基因，表征CO- 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 在不同的CO暴露条件下量化转基因报告小鼠中的CO积累， 其在体内引起CO信号传导、细胞保护和毒性。通过这一目标，我们将开发关键的 生物分子工具，这将使相关的人类健康的CO依赖性信号机制的说明。