Project 2: Why are mucins so gigantic and is it safe/effective to sever them therapeutically?

项目 2：为什么粘蛋白如此巨大？在治疗上切断它们是否安全/有效？

• 批准号: 10684198
• 负责人: Richard Charles Boucher
• 金额: $ 55.3万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684198
• 关键词: Acetylcysteine; Address; Adhesions; Affect; Alanine; Animal Model; Bicarbonates; Binding; Binding Sites; Biological; Cell surface; Cells; Characteristics; Chelating Agents; Cilia; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coughing; Cysteine; Defect; Deposition; Detergents; Development; Disease; Drug Kinetics; Elasticity; Electrostatics; Failure; Gel; Generations; Genetic; Goals; Health; Host Defense; Host Defense Mechanism; Human; Hydrophobicity; In Vitro; Inhalation; Investigation; Knock-in; Knock-out; Lead; Length; Lung; MUC5B gene; Measures; Mechanics; Mediating; Molecular Weight; Mucins; Mucolytics; Mucous body substance; Mus; Mutagens; Mutant Strains Mice; Neutrons; Obstruction; Osmosis; Patients; Polymers; Production; Property; Radial; Reducing Agents; Reporting; Respiratory Disease; Respiratory System; Rest; Role; Secretory Rate; Side; Structure; Sulfhydryl Compounds; Surface; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Index; Viscosity; airway epithelium; biophysical properties; cohesion; combinatorial; cost; data modeling; dimer; functional restoration; improved; in vivo; light scattering; lung health; molecular size; monomer; mucus clearance; mucus-associated lung diseases; mutant; novel; novel strategies; pathogen; pharmacologic; programs; respiratory; response; sugar; surfactant; viscoelasticity

Project 2, “Why are mucins so gigantic and is it safe/effective to sever them therapeutically?”, Richard C. Boucher, MD, PI, proposes to study fundamental questions with respect to mucus transport in health and how to therapeutically restore mucus clearance in muco-obstructed patients. The project first focuses on a simple question, i.e., why are such gigantic mucin multimers (~ 120 monomers, 300 MDa, 250 nm radius of gyration, Rg) synthesized and secreted by normal airway epithelia? We hypothesize that large multimers are the most economical way to generate the mucus gel-like properties needed for transport. Studies will focus on mucin molecular weight (MW), molecular size (radius of gyration, Rg), concentration (c), overlap concentrations (c*), gel-dependent biophysical properties, and transportability. A corollary goal is to test the hypothesis that mucolytic agents, i.e., agents that decrease multimer length/size, are attractive therapeutic agents for muco-obstructive lung disease. Our approach to investigate both hypotheses rests on the successful generation of a CRISPR knockout mutant mouse (Muc5b D3 cysteine 1128-alanine, cysteine 1170-alanine) that selectively synthesizes and secretes mucin dimers (MW ~ 4 MDa, Rg = 40 nm). SA1 will test the hypothesis that multimeric mucins (large multimers) efficiently occupy space/volume in the mucus layer and, consequently, generate the overlap conditions (c*) needed to produce the gel-like mucus properties required for transport. Structural features of wild- type (WT) vs Muc5b dimeric mucins, utilizing light scattering, neutron scattering, and EM technologies, juxtaposed to biophysical properties, e.g., viscoelastic properties, osmotic moduli, cohesion, and adhesion, will be measured and related to cilial and cough-dependent transport in vitro. SA1 will also test the specific hypothesis that dimeric mucins, because of their small Rg, will not reach the c* conditions under basal conditions (secretory rates) required to generate the elastic properties of a gel needed for cilial-dependent mucus clearance. SA2 will then test the hypothesis that a mucus comprised of small MW mucins is not transportable by cilia in vivo and mucus accumulation in the respiratory tract will result. Responses of WT vs dimeric mice to provocations that perturb mucin concentrations will be tested to explore the requirement of multimeric mucins for host defense. SA3 will test the hypothesis that reducing agents aid mucus clearance in disease but not health. Studies will include comparisons of actions of a novel, efficient thiol-based therapeutic agent (P2165) on WT vs dimeric mucins in vitro and mice in vivo. The goal is to search for potential favorable effects of reducing multimer length on the mucus viscosity required for cough clearance and identify off-target effects mediated by reduction of other intra-mucin cysteines that may produce untoward mucin aggregation/stickiness. The deliverables of the project are to: 1) quantitate the relationships between mucin multimer size, space-occupying characteristics (c*), and mucus function in health; and 2) characterize the mucolytic agent therapeutic index with respect to on- vs off- target effects and provide a roadmap for development of novel mucolytic therapeutics for patients in need.

项目2，“为什么粘蛋白如此巨大，通过治疗方法切断它们是否安全/有效？”，Richard C。 鲍彻，医学博士，PI，建议研究与健康中粘液运输有关的基本问题以及如何 通过治疗恢复粘液阻塞患者的粘液清除。该项目首先关注的是一个简单的 问题，也就是，为什么如此巨大的粘蛋白多聚体(~120个单体，300个丙二醛，250 nm的旋转半径， RG)由正常呼吸道上皮细胞合成和分泌？我们假设大型倍增器是最 以经济的方式产生运输所需的粘液凝胶状特性。研究将集中在粘蛋白上 分子量(MW)、分子大小(回转半径，Rg)、浓度(C)、重叠浓度(c*)、 依赖于凝胶的生物物理性质，以及可运输性。一个必然的目标是检验粘液溶解作用的假设 药物，即减少多聚体长度/大小的药物，是有吸引力的粘膜阻塞性治疗药物 肺部疾病。我们研究这两种假设的方法依赖于CRISPR的成功生成 基因敲除突变小鼠(MUC5B D3半胱氨酸1128-丙氨酸，半胱氨酸1170-丙氨酸)选择性合成 分泌粘蛋白二聚体(Mw~4MDA，Rg=40 nm)。SA1将检验多聚体粘蛋白的假设 (大多聚体)有效地占据粘液层中的空间/体积，从而产生重叠 产生运输所需的胶状粘液特性所需的条件(c*)。野生的结构特征-- 类型(WT)与MUC5B二聚体粘蛋白，利用光散射、中子散射和EM技术， 与生物物理性质并列，例如粘弹性性质、渗透系数、凝聚力和粘附性，将 被测量并与纤毛和咳嗽依赖的体外转运有关。SA1还将测试特定的 假设二聚体粘蛋白由于其相对分子质量较小，在基本条件下不能达到c*条件。 (分泌率)产生依赖纤毛的粘液清除所需的凝胶的弹性性质所需的速率。 然后，SA2将检验由小分子粘蛋白组成的粘液不能被体内纤毛运输的假设 而粘液会积聚在呼吸道中。WT与二聚体小鼠对刺激的反应 将对扰动粘蛋白浓度进行测试，以探索多聚体粘蛋白对宿主防御的需求。 SA3将检验这一假设，即还原剂有助于疾病中的粘液清除，而不是健康。研究将会 包括一种新的、有效的基于硫醇的治疗剂(P2165)对WT和二聚体作用的比较 粘蛋白在体外和小鼠体内。目标是寻找减少多聚体长度的潜在有利影响。 止咳所需黏液黏度的研究及通过减少其他黏液黏度来识别非靶点效应 粘蛋白内的半胱氨酸，可能产生不良的粘蛋白聚集/粘性。项目的可交付成果 是为了：1)量化粘蛋白多聚体大小、空间占据特性(c*)和 粘液在健康中的功能；以及2)表征粘液剂治疗指数的开-关-关- 为有需要的患者开发新的粘液溶解疗法提供了目标效应和路线图。