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

• 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
• 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

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状况中（例如获得和遗传 溶血性疾病），游离血红素和无细胞血红蛋白（HB）被释放到TROOD流中并引起A 多种不良影响，即：血管收缩，高血压和最终器官损害 溶血状况将受益于自由血红素和无细胞HB的清除剂，例如血红素（HPX）和 Haptoglobin（HP），尊敬的人。 通过从HB中取出血红素而得出，其空缺的血红素结合口袋对血红素具有很高的亲和力。 APOHB可以用作无体内血红素清道夫而不是HPX。 但是，使用APOHB用作体内血红素的主要潜力是其低热量 生理温度的稳定性和短循环半衰期（类似于HB，5分钟）。 研究表明，与HB相似，APOHB可以与HP形成高度稳定的复合物 复合物保留是结合血红素的能力，与游离APOHB相比，在生理温度下更稳定。 除了能够结合自由血红素外，APOHB-HP复合物还可以通过交换HP来清除无HB HBαβ二聚体的结合APOHBαβ二聚体。 双重结合排毒的血红素和HB的能力 由此产生的HB-HP复合物的毒性比游离血红素或无细胞HB的毒性要小得多，并且很容易从循环中清除 通过CD163介导的单核细胞/巨噬细胞摄取来检验这种假设 目标。 特定目标1：APOHB-HP复合物的生物物理和生化表征及其能力 在体外结合血红素和HB。 特定目标2：体内测定血红素和HB转移以及与APOHB-HP的结合。 特定目的3：对APOHB-HP进行系统的临时评估，以投影和/或停止该程序 血管内溶血（HB/血红素）诱导的肺心血管疾病。 特定目的4：APOHB-HP的微血管评估以防止血管粘液血管 炎。

项目成果

Metabolic correlates to critical speed in murine models of sickle cell disease.

• DOI: 10.3389/fphys.2023.1151268
• 发表时间: 2023
• 期刊: Frontiers in physiology
• 影响因子: 4

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

• DOI: 10.1371/journal.pone.0269939
• 发表时间: 2022
• 期刊: PloS one
• 影响因子: 3.7

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics.

• DOI: 10.1182/blood.2022015596
• 发表时间: 2022-10-27
• 期刊: BLOOD
• 影响因子: 20.3
• 作者: Vallelian, Florence;Buehler, Paul W.;Schaer, Dominik J.
• 通讯作者: Schaer, Dominik J.

Hemopexin dosing improves cardiopulmonary dysfunction in murine sickle cell disease.

• DOI: 10.1016/j.freeradbiomed.2021.08.238
• 发表时间: 2021-11-01
• 期刊: FREE RADICAL BIOLOGY AND MEDICINE
• 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;Schu, Daniel;Hassell, Kathryn;Nuss, Rachelle;Schaer, Dominik J.;Irwin, David C.
• 通讯作者: Irwin, David C.

Murine models of sickle cell disease and beta-thalassemia demonstrate pulmonary hypertension with distinctive features.

• DOI: 10.1177/20458940211055996
• 发表时间: 2021-10
• 期刊: Pulmonary circulation
• 影响因子: 2.6
• 作者: Buehler PW;Swindle D;Pak DI;Fini MA;Hassell K;Nuss R;Wilkerson RB;D'Alessandro A;Irwin DC
• 通讯作者: Irwin DC