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

开发一氧化碳清除血红蛋白作为治疗吸入性一氧化碳中毒的新型解毒疗法

基本信息

批准号：
10387161
负责人：
Matthew Ryan Dent
金额：
$ 7.68万
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：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387161
关键词：
Accident and Emergency department Aerobic Affinity Ambulances Amino Acids Animal Model Antidotes Area Behavior monitoring Binding Biochemical Biology Biophysics Blood Pressure Carbon Monoxide Carbon Monoxide Poisoning Carboxyhemoglobin Cardiac Catheters Cessation of life Chemistry Data Development Diagnosis Disease Emergency department visit Erythrocytes Escherichia coli Exhibits Functional disorder Goals Health Heme Hemeproteins Hemoglobin Human Hyperbaric Oxygen In Vitro Inhalation Intravenous Investigation Knowledge Laboratories Leadership Ligands Mediating Medicine Mentors Metabolism Methods Microbe Modeling Molecular Cloning Morbidity - disease rate Mus Neurocognitive Organ Outcome Oxidation-Reduction Oxygen Patient-Focused Outcomes Patients Persons Physicians Physiological Poisoning Principal Investigator Property Protein Engineering Proteins Recombinant Proteins Recombinants Research Safety Scientist Signal Transduction Site Site-Directed Mutagenesis Soil Spectrum Analysis Survivors Technical Expertise Testing Therapeutic Toxic effect Training United States Variant Work autooxidation base career career development chemical stability clinically relevant conventional therapy drug development experience heart rate monitor hemodynamics human disease human model improved in vivo long-term sequelae microbial mortality mouse model nanomolar novel novel therapeutics point of care pre-clinical prevent protein metabolism skills transcription factor translational medicine

项目摘要

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.

项目摘要/摘要意外一氧化碳（CO）中毒是美国人类中毒的主要原因，每年导致大约50,000例，至少1,500例死亡。没有护理点解毒疗法迄今为止存在CO中毒，常规治疗仅限于100％正常氧气的吸入或高压氧。尽管这些疗法增强了CO清除率，但在患者诊断和运输方面延迟导致发病率过多和死亡率。因此，可以部署在领域，救护车或急诊室可能会大大提高生存率和长期成果患者。鉴于CO与亚铁血红素紧密结合，我们的实验室试图开发基于血蛋白的CO 可以将CO绑定并消除CO作为CO中毒的新疗法的清道夫。基于初步研究重组血蛋白的重组蛋白，我们已经确定了针对安全有效的血红蛋白的四个关键标准 CO清除剂：（1）高（纳摩尔）CO亲和力，以最大化从生理血红素部位清除的CO，（2） CO选择性以最大程度地减少氧结合的竞争性抑制，（3）（3）热和化学稳定性防止血红素释放和不良反应性，以及（4）Fe（II）血红素的氧化还原稳定性以防止自氧化 Fe（III）血红素状态。对CO代谢（RCOM）蛋白调节剂的早期研究，来自土壤微生物的转录因子，表明该蛋白具有高CO亲和力，并且 CO比氧的前所未有的选择性。该提案的主要目的是将RCOM发展为安全有效的CO清道夫，将作为CO中毒的改进治疗方法。在 AIM 1，我们将利用实验室中开发的体外光谱方法来识别1）最小功能 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 RCOM变体在体外共饱和红细胞中从血红蛋白中清除CO。在AIM 2中，我们将评估两个重组RCOM在体内的安全性和功效。我们将评估系统和器官静脉内RCOM在体内健康小鼠中的特定作用，并量化RCOM反向的能力在先前开发的CO中毒的临床前小鼠模型中，血液动力学崩溃并防止死亡在我们的实验室。拟议目标的完成将提高我们对血蛋白的基本理解配体的选择性，同时还推进了一种新型解毒疗法的翻译发展，以治疗吸入共同中毒。这些成果，除了职业发展外，还指导了培训和教学课程，最终将为我提供必要的技术专长，背景知识和领导能力为了实现我的长期学术职业目标，即指导一个研究团队研究共同依赖的信号与人类健康和疾病有关的机制。