Antidote for inhaled CO poisoning based on mutationally engineered neuroglobin

描述(申请人提供)：一氧化碳(CO)引起的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5万人受到影响。碳氧血红蛋白血症超过30%的患者可能会出现脑损伤、长期神经认知障碍和/或死亡。在目前的提案中，我们介绍了第一种针对一氧化碳中毒的解毒疗法，这是基于发现突变的人脑红蛋白具有令人惊讶的和几乎不可逆的CO结合亲和力。与细胞球蛋白和许多植物HBS一样，脑红蛋白是一种六配位的血红素蛋白，其血红素铁由近端和远端的组氨酸残基协调。为了了解这种双组氨酸结构的功能，我们对近端组氨酸分子(例如H64Q)进行了突变，它打开了血红素口袋，形成了一个五配位的分子，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极高的亲和力，对氧的P50值(氧分压达到50%时，50%的血红素与氧结合)为0.01毫米汞(正常值为26毫米汞)。这一发现提供了我们的初步假设，即H64突变体脑红蛋白将以高亲和力结合CO，产生一个五配位的CO陷阱。在我们的初步实验中，我们发现突变的H64Q神经球蛋白与一氧化碳的亲和力是血红蛋白的300多倍，在体内外CO暴露的小鼠中，可以在不到2分钟的时间内从完整的红细胞中清除一氧化碳。我们建议在一系列体外、临床前生理学和翻译研究中提炼该分子，作为CO中毒的人类生物解毒剂，并测试其清除CO中毒的能力。 来自红细胞、重要器官(肺、脑和心脏)的一氧化碳，以及线粒体电子传输链中的细胞色素C氧化酶。从翻译的角度来看，将测试能力 这种解毒剂可改善严重一氧化碳中毒后的长期器官功能和存活率。

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.