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.

项目概要/摘要意外一氧化碳 (CO) 中毒是美国人类中毒的主要原因，每年导致约 50,000 例病例和至少 1,500 人死亡。无即时抗毒治疗迄今为止存在一氧化碳中毒，常规治疗仅限于吸入100%常压氧或高压氧。虽然这些疗法增强了 CO 清除率，但延迟了患者诊断和转运导致发病率和死亡率过高。因此，一种快速作用的 CO 清除剂可以部署在现场、救护车或急诊室可以显着提高患者的生存率和长期结果患者。鉴于 CO 与亚铁血红素紧密结合，我们的实验室致力于开发基于血红素蛋白的 CO 可以结合并消除 CO 的清除剂，作为 CO 中毒的新型治疗方法。根据初步研究对于重组血红素蛋白，我们确定了安全有效的基于血红素蛋白的四个关键标准 CO 清除剂：(1) 高（纳摩尔）CO 亲和力，可最大限度地从生理血红素位点清除 CO，(2) CO 选择性可最大限度地减少氧结合造成的竞争性抑制，(3) 热稳定性和化学稳定性防止血红素释放和不良反应性，以及 (4) Fe(II) 血红素的氧化还原稳定性，以防止自动氧化到非活性的 Fe(III) 血红素状态。 CO 代谢调节蛋白 (RcoM) 的早期研究，该蛋白是一种 CO- 来自土壤微生物的传感转录因子表明该蛋白质具有高 CO 亲和力对 CO 的选择性超过对氧气的前所未有的选择性。该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。在目标 1，我们将利用我们实验室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。在除了表征基本生化特性外，我们还将评估重组表达的能力 RcoM 变体可在体外从 CO 饱和红细胞的血红蛋白中清除 CO。在目标 2 中，我们将评估两种重组 RcoM 截短体的体内安全性和有效性。我们将评估系统和器官静脉注射 RcoM 递送对健康小鼠体内的具体影响并量化 RcoM 逆转的能力先前开发的一氧化碳中毒临床前小鼠模型中的血流动力学崩溃并防止死亡在我们的实验室。完成所提出的目标将增进我们对血红素蛋白的基本理解配体选择性，同时还促进了治疗吸入性肺炎的新型解毒疗法的转化开发一氧化碳中毒。这些成果，除了职业发展、指导培训和教学课程外，最终将为我提供必要的技术专业知识、背景知识和领导技能实现我指导研究团队研究 CO 依赖性信号传导的长期学术职业目标与人类健康和疾病相关的机制。