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.

一氧化碳中毒仍然是造成死亡和残疾的主要原因，各有50 000人受到影响 年仅在美国。从火灾中或暴露于汽车和家用发电机废气中的患者 被置于100%氧气中并转移到具有高压氧输送系统的设施中。尽管 高压氧治疗中心的可用性、获得和开始治疗的固有延迟极大地限制了 功效即使使用高压氧治疗，1-2%的患者死亡，并且>25%的存活患者表现出 神经认知障碍目前临床上还没有一氧化碳中毒的即时解毒剂。 在我们最初的工作中，我们发现了一个令人惊讶的和几乎不可逆的CO结合亲和力的突变工程 人脑红蛋白（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CO2的结合强度是Hb的500倍。Ngb-H64 Q-CCC输注液 在CO中毒的小鼠中，从25分钟到25秒增强了体内红细胞中CO的清除， 心率和血压，将存活率从不到10%提高到85%以上，然后快速 CO结合Ngb-H64 Q-CCC的肾消除。这些发现提供了概念证明， 具有非常高的CO结合亲和力的清除剂分子可以被开发为CO中毒的潜在解毒剂。 在上一个资助期，我们继续开发Ngb-H64 Q-CCC分子，评估 恢复细胞有氧呼吸的功效、安全性以及对 临床前模型中的心血管和认知功能和存活率，以及重组的规模化生产 用于临床开发的蛋白质。我们展示了Ngb-H64 Q-CCC的输注如何能够恢复线粒体 呼吸和逆转CO诱导的效应。我们还着手发现新的CO清除剂分子 它可能比我们的铅分子具有更好的性能。我们的研究发现，RcoM，一种细菌CO， 传感器，对CO具有高亲和力，并在小鼠模型中表现出有希望的安全性。 在本提案中，我们计划进一步开发我们的Ngb-H64 Q-CCC分子，添加修饰， 改善其CO亲和力和稳定性，以获得更安全毒性特征。我们还将设计RcoM，以获得最小的 该单元可以以高亲和力吸附CO并呈现最佳的稳定性和安全性。最后我们将 利用我们的研究计划中获得的所有关于CO和氧结合的知识，开发新的 氧载体分子可以作为血液替代品。 总的来说，这些拟议的研究符合NHLBI和NIH的使命， 有影响力的，重要的和新颖的研究，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一氧化碳中毒治疗模式。

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