Bioengineering a Dual Function Protein Construct to Detoxify Heme and Hemoglobin

生物工程双功能蛋白质结构以解毒血红素和血红蛋白

基本信息

批准号：
10663258
负责人：
Paul Werner Buehler
金额：
$ 64.64万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10663258
关键词：
Adverse effects Affinity Apoproteins Binding Biochemical Biological Availability Biomedical Engineering Biophysics Blood Blood Vessels Cardiovascular Diseases Cells Chronic Disease Circulation Complex Disease Dorsal Drug Kinetics Drug Metabolic Detoxication Engineering Evaluation Exercise Test Exposure to Genetic Half-Life Haptoglobins Heme Hemoglobin Hemolysis Hemopexin Hypertension Hypoxia In Vitro Inflammation Lung Lung diseases Macrophage Mediating Methods Microvascular Dysfunction Mus Organ Physiological Plasma Proteins Protocols documentation Reaction Role Stream Temperature Testing Therapeutic Therapeutic Agents Tissues Treatment Efficacy Work apohemoglobin clinically relevant dimer effectiveness evaluation efficacy evaluation haptoglobin-hemoglobin complex in vivo instrument monocyte novel novel therapeutics preclinical evaluation prevent protein complex protein function side effect subcutaneous uptake vascular inflammation vasoconstriction

项目摘要

Abstract During pathophysiological conditions characterized by extensive hemolysis (e.g. acquired and genetic hemolytic diseases), free heme and cell-free hemoglobin (Hb) are released into the blood stream and elicit a variety of adverse effects, namely: vasoconstriction, hypertension, and end organ damage. Thus treatment of hemolytic conditions would benefit from scavengers of free heme and cell-free Hb such as hemopexin (Hpx) and haptoglobin (Hp), respectively. A possible functional alternative to Hpx is apohemoglobin (apoHb). ApoHb is derived by removing heme from Hb, and its vacant heme-binding pockets have a high affinity for heme. Hence, apoHb could serve as a novel in vivo heme scavenger instead of Hpx. However, major potential issues with the use of apoHb as an in vivo heme scavenger are its low thermal stability at physiological temperature, and short circulatory half-life (similar to Hb, 5 min). Fortuitously, previous studies have shown that, similar to Hb, apoHb can bind to Hp forming a highly stable complex. The apoHb-Hp complex retains its ability to bind heme, and is more stable at physiological temperature compared to free apoHb. In addition to being able to bind free heme, the apoHb-Hp complex can scavenge free Hb by exchanging Hp bound apoHb αβ dimers for Hb αβ dimers. Therefore, we hypothesize that the apoHb-Hp complex will have the dual ability to bind and detoxify free heme and Hb that are produced during states of hemolysis. The resulting Hb-Hp complex is much less toxic than free heme or cell-free Hb and is readily cleared from circulation via CD163 mediated monocyte/macrophage uptake. To test this hypothesis, we propose the following specific aims. Specific Aim 1: Biophysical and biochemical characterization of the apoHb-Hp complex and its ability to bind heme and Hb in vitro. Specific Aim 2: In vivo determination of heme and Hb transfer and binding to apoHb-Hp. Specific Aim 3: Systematic pre-clinical evaluation of apoHb-Hp to prevent and/or halt the progression of intravascular hemolysis (Hb/heme)-induced pulmonary cardiovascular disease. Specific Aim 4: Microvascular evaluation of apoHb-Hp to prevent vaso-occlusion and reduce vascular inflammation.

抽象的在以广泛溶血为特征的病理生理条件下（例如获得性和遗传性溶血）溶血性疾病），游离血红素和游离血红蛋白（Hb）被释放到血流中并引起多种不良反应，即：血管收缩、高血压和终末器官损伤。溶血病症将受益于游离血红素和游离血红蛋白的清除剂，例如血红素结合蛋白 (Hpx) 和结合珠蛋白 (Hp) 是 Hpx 的一种可能的功能替代品。通过从 Hb 中去除血红素而获得，其空的血红素结合口袋对血红素具有高亲和力。 apoHb 可以代替 Hpx 作为一种新型的体内血红素清除剂。然而，使用 apoHb 作为体内血红素清除剂的主要潜在问题是其低热生理温度下的稳定性和较短的循环半衰期（类似于 Hb，5 分钟）。研究表明，与Hb类似，apoHb可以与Hp结合，形成高度稳定的复合物apoHb-Hp。复合物保留了结合血红素的能力，并且与游离的 apoHb 相比在生理温度下更稳定。除了能够结合游离血红素外，apoHb-Hp 复合物还可以通过交换 Hp 来清除游离 Hb 因此，我们发现 apoHb-Hp 复合物将具有 Hb αβ 二聚体。结合和解毒溶血状态期间产生的游离血红素和血红蛋白的双重能力。产生的 Hb-Hp 复合物的毒性比游离血红素或无细胞 Hb 低得多，并且很容易从循环中清除通过 CD163 介导的单核细胞/巨噬细胞摄取为了检验这一假设，我们提出以下具体建议。目标。具体目标 1：apoHb-Hp 复合物的生物物理和生化特征及其能力体外结合血红素和血红蛋白。具体目标 2：体内测定血红素和 Hb 转移以及与 apoHb-Hp 的结合。具体目标 3：对 apoHb-Hp 进行系统的临床前评估，以预防和/或阻止疾病进展血管内溶血（Hb/血红素）诱发的肺心血管疾病。具体目标 4：apoHb-Hp 的微血管评估，以防止血管闭塞并减少血管炎。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Increased hemoglobin affinity for oxygen with GBT1118 improves hypoxia tolerance in sickle cell mice.

GBT1118 增加了血红蛋白对氧的亲和力，改善了镰状细胞小鼠的缺氧耐受性。

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Dufu, Kobina;Williams, Alexander T;Muller, Cynthia R;Walser, Cynthia M;Lucas, Alfredo;Eaker, Allyn M;Alt, Carsten;Cathers, Brian E;Oksenberg, Donna;Cabrales, Pedro
通讯作者：
Cabrales, Pedro

Scalable production and complete biophysical characterization of poly(ethylene glycol) surface conjugated liposome encapsulated hemoglobin (PEG-LEH).

聚（乙二醇）表面缀合脂质体封装血红蛋白（PEG-LEH）的可扩展生产和完整的生物物理表征。

DOI：
发表时间：
2022
期刊：
影响因子：
3.7
作者：
Banerjee, Uddyalok;Wolfe, Savannah;O'Boyle, Quintin;Cuddington, Clayton;Palmer, Andre F
通讯作者：
Palmer, Andre F

Hemopexin dosing improves cardiopulmonary dysfunction in murine sickle cell disease.

血红素结合蛋白剂量可改善小鼠镰状细胞病的心肺功能障碍。

DOI：
发表时间：
2021-11-01
期刊：
影响因子：
7.4
作者：
Buehler, Paul W;Swindle, Delaney;Pak, David I;Ferguson, Scott K;Majka, Susan M;Karoor, Vijaya;Moldovan, Radu;Sintas, Chantal;Black, Jennifer;Gentinetta, Thomas;Buzzi, Raphael M;Vallelian, Florence;Wassmer, Andreas;Edler, Monika;Bain, Joseph
通讯作者：
Bain, Joseph

Biophysical Analysis and Preclinical Pharmacokinetics-Pharmacodynamics of Tangential Flow Filtration Fractionated Polymerized Human Hemoglobin as a Red Blood Cell Substitute.

切向流过滤分级聚合人血红蛋白作为红细胞替代品的生物物理分析和临床前药代动力学-药效学。

DOI：
发表时间：
2023-04-10
期刊：
影响因子：
6.2
作者：
Greenfield, Alisyn;Lamb, Derek R;Gu, Xiangming;Thangaraju, Kiruphagaran;Setua, Saini;Yahya, Ahmad;Vahedi, Amid;Khan, Mohd Asim;Wang, Qihong;Buehler, Paul W;Palmer, Andre F
通讯作者：
Palmer, Andre F

Superoxide measurement as a novel probe of red blood cell storage quality.

超氧化物测量作为红细胞储存质量的新型探针。