Antidote for inhaled CO poisoning based on mutationally engineered neuroglobin

基于突变工程神经球蛋白的吸入一氧化碳中毒解毒剂

• 批准号: 10660066
• 负责人: Mark T Gladwin
• 金额: $ 70.36万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660066
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Affect; Affinity; Amino Acids; Antidotes; Apoptosis; Binding; Binding Sites; Biochemical; Blood; Blood Pressure; Blood Substitutes; Brain; Brain Edema; Brain Injuries; Carbon Monoxide; Carbon Monoxide Poisoning; Carboxyhemoglobin; Cardiovascular Physiology; Cardiovascular system; Carrying Capacities; Cause of Death; Cell Respiration; Cessation of life; Chemicals; Circulation; Clinical; Cognitive; Development; Diffusion; Distal; Dose; Electron Transport; Emergency department visit; Endotoxins; Engineering; Environment; Erythrocytes; Escherichia coli; Excision; Exhibits; Exposure to; Fire - disasters; Funding; Generations; Glutamine; Half-Life; Heart; Heart Rate; Heme; Heme Iron; Hemeproteins; Hemoglobin; Histidine; Home; Human; Human Engineering; Hydrogen Peroxide; Hyperbaric Oxygen; Hyperbaric Oxygenation; Hyperbaric Therapy; Image; In Vitro; Infusion procedures; Inhalation; Intravenous; Intravenous infusion procedures; Ischemia; Kidney; Knowledge; Lead; Ligands; Long-Term Effects; Magnetic Resonance Imaging; Methods; Mission; Mitochondria; Modeling; Modification; Molecular; Mus; Mutate; Mutation; National Heart, Lung, and Blood Institute; Necrosis; Neurocognitive Deficit; Nitrite Reductase; Nitrogen; Organ; Organoids; Oxidation-Reduction; Oxygen; Paramedical Personnel; Pathologic; Patients; Persons; Poison; Poisoning; Pre-Clinical Model; Precipitation; Production; Property; Protein Engineering; Proteins; Public Health; Reaction; Recombinant Proteins; Recombinants; Regulation; Research; Respiration; Risk; Safety; Site; Solubility; Sulfhydryl Compounds; Surface; System; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; Tubular formation; United States; United States National Institutes of Health; Urea; Work; Yeasts; chemical stability; clinical development; cognitive function; cytochrome c oxidase; design; disability; efficacy evaluation; efficacy testing; exhaust; heme a; improved; in vitro testing; in vivo; induced pluripotent stem cell; innovation; mouse model; nephrotoxicity; neuroglobin; next generation; novel; organ injury; point of care; pre-clinical; prevent; programs; restoration; sensor; water maze

Carbon monoxide (CO) poisoning remains a major cause of death and disability, affecting 50,000 persons each year in the U.S. 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, inherent delays in access to and initiation of therapy greatly limit efficacy. 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 our initial work 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 Hb. 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 provided proof of concept that heme-based scavenger molecules with very high CO binding affinity can be developed as potential antidotes for CO poisoning. In the previous funding period, we continued the 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 showed how infusion of Ngb-H64Q-CCC can restore mitochondrial respiration in tissues and reverse CO-induced effects. We also set out to discover novel CO scavenger molecules which may have improved properties over our lead molecule. Our studies uncovered that RcoM, a bacterial CO sensor, has a high affinity towards CO and presents promising safety profiles in mouse models. In the present proposal we plan to further develop our Ngb-H64Q-CCC molecule, adding modifications that improve its CO affinity and stability for a safer toxicity profile. We also will engineer RcoM to obtain the smallest unit that can scavenge CO with high affinity and present optimal stability and safety properties. Finally, we will leverage all the knowledge on CO and oxygen binding achieved during our research program to develop novel oxygen carrier molecules that can serve as blood substitutes. 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.

一氧化碳（CO）中毒仍然是死亡和残疾的主要原因，每个人影响50,000人 仅在美国一年。病人从火灾中撤离或暴露于汽车和家庭发电机排气管之后 被放置在100％氧气上，并通过高压氧递送系统转移到设施中。尽管有 Hyberbaric疗法中心的可用性，继承了延迟的延迟和疗法的倡议 功效。即使接受河托氧疗法，1-2％的患者和> 25％的生存患者表现出来 神经认知障碍。目前尚无临床可用的CO中毒的护理点解。 在我们的最初工作中，我们发现了突变工程的惊喜和几乎可逆的共同结合亲和力 人类神经白蛋白（NGB）。 NGB是一种六坐标血蛋白，血红素铁由两个组氨酸协调 残留物。我们将远端组氨酸突变为谷氨酰胺（H64Q）和三个表面硫醇，形成了五坐标 血红素蛋白（NGB-H64Q-CCC）具有很高的溶解度（> 10mm），可高浓度和高浓度和 静脉输注。该分子结合CO≈500倍的强度比Hb高500倍。 NGB-H64Q-CCC的输注 在共偶联的小鼠中，从25分钟到25秒就从红细胞中的CO删除，恢复 心率和血压，将生存率从不到10％增加到85％以上，随后迅速 肾脏消除共同绑定的NGB-H64Q-CCC。这些发现提供了基于血红素的概念证明 具有非常高的CO结合亲和力的清道夫分子可以作为CO中毒的潜在解毒剂发展。 在上一个资金期间，我们继续开发NGB-H64Q-CCC分子，评估 对恢复细胞有氧呼吸，安全性以及急性和长期影响的有效性 临床前模型中的心血管和认知功能以及生存，以及重组的缩放产生 临床发育的蛋白质。我们展示了NGB-H64Q-CCC的输注如何恢复线粒体 组织中的呼吸和反向共同诱导的作用。我们还着手发现新颖的清道夫分子 在我们的铅分子上可能具有改善的性能。我们的研究发现了一个细菌CO COM RCOM 传感器对CO具有很高的亲和力，并提出了鼠标模型中的安全性。 在本提案中，我们计划进一步开发我们的NGB-H64Q-CCC分子，并增加了修改 提高其CO亲和力和稳定性，以获得更安全的毒性特征。我们还将设计RCOM以获得最小的 可以清除具有高亲和力的单元，并且目前的稳定性和安全性能。最后，我们会的 利用在我们的研究计划中获得的有关CO和氧结合的所有知识来开发新颖 可以用作血液替代品的氧载体分子。 总体而言，这些拟议的研究与NHLBI和NIH的使命保持一致 有影响力，重要和新颖的研究，具有改善公共卫生的巨大潜力。支持这些 拟议的研究有可能改变我们目前的CO中毒治疗范例。

项目成果

Artificial porphyrin molecules clean up carbon monoxide and cyanide.

• DOI: 10.1073/pnas.2301732120
• 发表时间: 2023-03-14
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Tejero, Jesus

Endogenous Hemoprotein-Dependent Signaling Pathways of Nitric Oxide and Nitrite.

• DOI: 10.1021/acs.inorgchem.1c01048
• 发表时间: 2021-11-01
• 期刊: INORGANIC CHEMISTRY
• 影响因子: 4.6
• 作者: Dent, Matthew R.;DeMartino, Anthony W.;Tejero, Jesus;Gladwin, Mark T.
• 通讯作者: Gladwin, Mark T.

Direct measurement of nitric oxide (NO) production rates from enzymes using ozone-based gas-phase chemiluminescence (CL).

使用基于臭氧的气相化学发光 (CL) 直接测量酶产生一氧化氮 (NO) 的速率。

• DOI: 10.1016/j.niox.2021.10.001
• 发表时间: 2021
• 期刊: Nitric oxide : biology and chemistry
• 作者: Sparacino-Watkins,CourtneyE;LancasterJr,JackR
• 通讯作者: LancasterJr,JackR

Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations.

• DOI: 10.1016/j.niox.2022.06.001
• 发表时间: 2022-08-01
• 期刊: NITRIC OXIDE-BIOLOGY AND CHEMISTRY
• 影响因子: 3.9
• 作者: Kaliszuk, Stefan J.;Morgan, Natasha I.;Ayers, Taylor N.;Sparacino-Watkins, Courtney E.;DeMartino, Anthony W.;Bocian, Kaitlin;Ragireddy, Venkata;Tong, Qin;Tejero, Jests
• 通讯作者: Tejero, Jests