Antidote for inhaled CO poisoning based on mutationally engineered neuroglobin

描述（由申请人提供）：一氧化碳（CO）血气中毒仍然是死亡和残疾的主要原因，仅在美国每年就有50，000人受到影响。大于30%的一氧化碳血红蛋白血症患者可能发生脑损伤、长期神经认知缺陷和/或死亡。在本提案中，我们介绍了第一个一氧化碳中毒的解毒疗法，基于突变工程人类脑红蛋白的惊人的和几乎不可逆的CO结合亲和力的发现。与细胞红蛋白和许多植物血红蛋白一样，脑红蛋白是一种六配位血红素蛋白，其血红素铁由近端和远端组氨酸残基配位。为了理解这种双组氨酰结构的功能，我们对近端组氨酸64分子（例如H64 Q）进行了诱变，其打开血红素口袋，形成五配位分子，更类似于Hb或肌红蛋白的血红素口袋。出乎意料的是，该分子对气态配体表现出非常高的亲和力，对氧的P50值（50%的血红素结合氧时的PaO 2值）为0.01 mmHg（正常值为26 mmHg）。这一发现通知我们的主要假设，即H64突变体脑红蛋白将结合CO具有高亲和力，产生一个五坐标“陷阱”的CO。在我们的初步实验中，我们发现，突变工程H64 Q脑红蛋白结合CO的亲和力超过300倍的血红蛋白，可以在体外和体内的CO暴露小鼠在不到2分钟内从完整的红细胞中去除CO。我们建议在一系列体外、临床前生理学和转化研究中将这种分子作为CO中毒的人类生物解毒剂进行精制，并测试其清除CO的能力。 来自红细胞、关键器官（肺、脑和心脏）的CO和线粒体电子传递链的细胞色素C氧化酶。从翻译的角度来看， 这种解毒剂，以改善长期器官功能和生存后，严重的一氧化碳中毒。

DESCRIPTION (provided by applicant): Blood gas poisoning with carbon monoxide (CO) remains a major cause of death and disability, affecting 50,000 persons a year in the U.S. alone. Patients with greater than 30% carboxyhemoglobinemia may develop brain injury, long-term neurocognitive deficits, and/or death. In the present proposal we introduce the first antidotal therapy for carbon monoxide poisoning, based on the finding of a surprising and near-irreversible CO-binding affinity of mutationally engineered human neuroglobin. Neuroglobin, like cytoglobin and many plant Hbs, is a six-coordinate hemoprotein, with the heme iron coordinated by a proximal and distal histidine residues. In an effort to understand the function of this bis-histidyl structure, we performed mutagenesis of the proximal histidine 64 molecule (e.g. H64Q) which opens the heme pocket, forming a five-coordinate molecule, more similar to the heme pocket of Hb or myoglobin. Unexpectedly, this molecule exhibits a remarkably high affinity for gaseous ligands, with a P50 value for oxygen (PaO2 value at which 50% of the heme binds oxygen) of 0.01 mm Hg (normal value is 26 mm Hg). This finding informs our primary hypothesis, that H64 mutant neuroglobin will bind CO with high affinity, producing a five-coordinate "trap" for CO. In our preliminary experiments we find that mutationally engineered H64Q neurglobin binds CO with an affinity more than 300 times that of hemoglobin and can remove CO from intact red cells in vitro and in vivo in CO exposed mice in less than 2 minutes. We propose to refine this molecule in a series of in vitro, pre-clinical physiology, and translational studies as a human biological antidote for CO poisoning and test its ability to clear CO from red blood cells, critical organs (lung, brain, and heart), and cytochrome C oxidase of the mitochondrial electron transport chain. From a translational perspective, will test the ability of this antidote to improve long-term organ function and survival after severe CO poisoning.