Antidote for inhaled CO poisoning based on mutationally engineered neuroglobin

项目总结/摘要 一氧化碳（CO）中毒仍然是死亡和残疾的主要原因，影响50，000人 仅在美国每年就有200万人。从火灾中或暴露于汽车和家中后转移的患者 将发生器排气置于100%氧气上，并转移到具有高压氧输送的设施中 系统尽管大多数大城市都有高压氧治疗中心，但在获得治疗方面存在固有的延误。 和治疗的开始大大限制了疗效。事实上，即使使用高压氧治疗，也有1-2%的患者死亡 并且>25%的存活患者表现出神经认知障碍。目前还没有即时的解毒剂 一氧化碳中毒的治疗方法 在目前的建议中，我们继续研究开发新的一氧化碳中毒解毒疗法， 基于我们的发现，极高亲和力的血红素基分子可以结合并隔离红细胞中的CO， 血细胞和组织线粒体逆转CO中毒的全身缺血。在此前的融资中， 在此期间，我们发现了突变工程化的人的一个令人惊讶的和几乎不可逆的CO结合亲和力。 脑红蛋白（Ngb）。Ngb是一种六配位血红素蛋白，血红素铁与两个组氨酸配位 残基我们将末端组氨酸突变为谷氨酰胺（H64 Q）和三个表面巯基，形成五配位的 血红素蛋白（Ngb-H64 Q-CCC）具有非常高的溶解度（> 10 mM），允许高浓度和 静脉输液这种分子与CO的结合强度比血红蛋白强约500倍。Ngb输注- H64 Q-CCC对CO中毒小鼠红细胞内CO清除的促进作用（25 min ~ 25 min） 秒，恢复心率和血压，将存活率从不到10%提高到85%以上， 随后是CO结合的Ngb-H64 Q-CCC的快速肾消除。 这些发现提供了概念证明，具有非常高CO的血红素基清除剂分子 结合亲和力可被开发为CO中毒的潜在解毒剂。我们的目标是继续发展 我们的Ngb-H64 Q-CCC分子，评估恢复细胞有氧呼吸的功效，安全性， 在临床前模型中对心血管和认知功能以及生存期的急性和长期影响，以及 用于临床开发的重组蛋白的规模化生产。我们还旨在进一步发现新的CO 清除剂小分子，基于我们以前的研究中获得的知识。我们将突变 从微过氧化物酶衍生的血红素结合的、6-12个氨基酸的小肽的工程化，有或没有铁- 钴金属取代以限制氧化还原反应性并增强CO亲和力。 总的来说，这些拟议的研究符合NHLBI和NIH的使命， 具有高度影响力，重要和新颖的研究，具有改善公共卫生的巨大潜力。支持 因为这些拟议的研究有可能改变我们目前治疗CO中毒的范式。

Project Summary/Abstract Carbon monoxide (CO) poisoning remains a major cause of death and disability, affecting 50,000 people per year in the United States alone. Patients removed from fires or following exposure to car and home generator exhaust are placed on 100% oxygen and transferred to a facility with a hyperbaric oxygen delivery system. Despite the availability of hyberbaric therapy centers in most major cities, inherent delays in access to and initiation of therapy greatly limit efficacy. In fact, even with hyberbaric oxygen therapy, 1-2% of patients die and >25% of surviving patients exhibit neurocognitive impairments. There is currently no point-of-care antidote for CO poisoning available clinically. In the present proposal, we continue our studies developing novel antidotal therapies for CO poisoning, based on our findings that extremely high affinity heme-based molecules can bind and sequester CO from red blood cells and tissue mitochondria to reverse the systemic ischemia of CO poisoning. In the previous funding period, we discovered a surprising and near-irreversible CO-binding affinity of mutationally engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and three surface-thiols to form a five-coordinate heme protein (Ngb-H64Q-CCC) that has very high solubility (>10mM), allowing for high concentration and intravenous infusion. This molecule binds CO ~ 500 times more strongly than hemoglobin. Infusions of Ngb- H64Q-CCC in CO-poisoned mice enhanced CO removal from red blood cells in vivo from 25 minutes to 25 seconds, restored heart rate and blood pressure, increased survival from less than 10% to over 85%, and were followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. These findings provide proof-of-concept that heme-based scavenger molecules with very high CO binding affinity can be developed as potential antidotes for CO poisoning. We aim to continue development of our Ngb-H64Q-CCC molecule, evaluating efficacy on the restoration of cellular aerobic respiration, safety, and acute- and long-term effects on cardiovascular and cognitive function and survival in pre-clinical models, and scaling production of recombinant protein for clinical development. We also aim to further discover novel CO scavenger small molecules, based on knowledge derived from our previous studies. We will mutationally engineer heme-bound, small 6-12 amino acid peptides derived from microperoxidase, with and without iron-to- cobalt metal substitutions to limit redox reactivity and enhance CO affinity. Overall, these proposed studies are in keeping with the mission of the NHLBI and NIH to advance highly impactful, significant, and novel studies that have great potential to improve the public health. Support for these proposed studies has the potential to change our current paradigm for therapy of CO poisoning.