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

基本信息

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
项目类别：
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

项目摘要

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，每年影响 50,000 人仅在美国一年，就有患者从火灾中救出或暴露于汽车和家用发电机废气中。尽管存在 100% 氧气，但仍被转移到配备高压氧输送系统的设施。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即使采用高压氧治疗，仍有 1-2% 的患者死亡，并且 >25% 的存活患者表现出疗效。目前临床上还没有针对一氧化碳中毒的即时解毒剂。在我们最初的工作中，我们发现了突变工程的令人惊讶且几乎不可逆的共结合亲和力人神经红蛋白 (Ngb) 是一种六配位血红蛋白，其中血红素铁由两个组氨酸配位。我们将远端组氨酸突变为谷氨酰胺（H64Q）和三个表面硫醇以形成五配位。血红素蛋白 (Ngb-H64Q-CCC) 具有非常高的溶解度 (>10mM)，允许高浓度和该分子与 CO 的结合强度比 Ngb-H64Q-CCC 输注强 500 倍。在 CO 中毒小鼠体内，红细胞清除 CO 的能力从 25 分钟增强到 25 秒，恢复心率和血压，将生存率从不到 10% 提高到 85% 以上，随后进行快速肾脏消除 CO 结合的 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 结合亲和力的清除剂分子可以开发作为 CO 中毒的潜在解毒剂。在上一个资助期间，我们继续开发 Ngb-H64Q-CCC 分子，评估恢复细胞有氧呼吸的功效、安全性以及对细胞有氧呼吸的急性和长期影响临床前模型中的心血管和认知功能和生存，以及重组的规模化生产我们展示了 Ngb-H64Q-CCC 的输注如何能够恢复线粒体。我们还着手发现新型二氧化碳清除剂分子。我们的研究发现，RcoM（一种细菌 CO）的特性可能有所改善。传感器，对 CO 具有高亲和力，并在小鼠模型中呈现出有前景的安全性。在目前的提案中，我们计划进一步开发我们的 Ngb-H64Q-CCC 分子，添加修饰提高其 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 以获得最小的毒性。最后，我们将开发一种能够以高亲和力清除 CO 并具有最佳稳定性和安全性的装置。利用我们在研究项目中获得的关于 CO 和氧结合的所有知识来开发新颖的可作为血液替代品的氧载体分子。总体而言，这些拟议的研究符合 NHLBI 和 NIH 的使命，即高度推进有影响力、重要且新颖的研究，具有改善公众健康的巨大潜力。拟议的研究有可能改变我们目前治疗一氧化碳中毒的模式。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
通讯作者：
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
影响因子：
0
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