Antidote for inhaled CO poisoning based on mutationally engineered neuroglobin

描述（由申请人提供）：一氧化碳（CO）血气中毒仍然是导致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。碳氧血红蛋白血症超过30%的患者可能出现脑损伤、长期神经认知缺陷和/或死亡。在目前的建议中，我们介绍了一氧化碳中毒的第一个解毒剂治疗，基于突变工程人类神经球蛋白惊人的和近乎不可逆的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，神经红蛋白是一种六坐标血红蛋白，其血红素铁由近端和远端组氨酸残基协调。为了了解这种双组氨酸结构的功能，我们对近端组氨酸64分子（例如H64Q）进行了诱变，该分子打开血红素口袋，形成一个五座标分子，更类似于Hb或肌红蛋白的血红素口袋。出乎意料的是，该分子对气体配体表现出非常高的亲和力，氧的P50值（50%血红素结合氧的PaO2值）为0.01 mm Hg（正常值为26 mm Hg）。这一发现证实了我们的初步假设，即H64突变的神经红蛋白将以高亲和力结合CO，产生一个五坐标的CO“陷阱”。在我们的初步实验中，我们发现突变工程的H64Q神经红蛋白结合CO的亲和力超过血红蛋白的300倍，并且可以在不到2分钟的时间内将CO从体外和体内暴露于CO的小鼠的完整红细胞中去除。我们建议在一系列体外、临床前生理学和转化研究中完善该分子，作为人类CO中毒的生物解毒剂，并测试其清除能力

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.