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，以治疗方式切断它们是否安全/有效？”，Richard C. Boucher，医学博士，PI，建议研究有关健康中粘液运输的基本问题以及如何 治疗性恢复粘液阻塞患者的粘液清除能力 该项目首先关注一个简​​单的问题。 问题，即为什么有如此巨大的粘蛋白多聚体（约 120 个单体，300 MDa，250 nm 回转半径， Rg）由正常气道上皮合成和分泌？我们发现大的多聚体是最多的？ 产生运输所需的粘液凝胶样特性的经济方法 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*)、 凝胶依赖性生物物理特性和可运输性是检验粘液溶解的假设。 药物，即减少多聚体长度/大小的药物，是治疗粘膜阻塞性疾病的有吸引力的治疗剂 我们研究这两种假设的方法依赖于 CRISPR 的成功生成。 选择性合成的敲除突变小鼠（Muc5b D3半胱氨酸1128-丙氨酸、半胱氨酸1170-丙氨酸） 并分泌粘蛋白二聚体（MW ~ 4 MDa，Rg = 40 nm），SA1 将检验多聚粘蛋白的假设。 （大多聚体）有效地占据粘液层中的空间/体积，从而产生重叠 产生运输所需的凝胶状粘液特性所需的条件（c*）。 类型 (WT) 与 Muc5b 二聚粘蛋白，利用光散射、中子散射和 EM 技术， 与生物物理特性（例如粘弹性、渗透模量、内聚力和粘附力）并列，将 被测量并与体外纤毛和咳嗽依赖性运输相关的 SA1 也将测试特定的。 假设二聚体粘蛋白由于其 Rg 小，在基础条件下不会达到 c* 条件 产生纤毛依赖性粘液清除所需的凝胶弹性特性所需的（分泌率）。 然后，SA2 将测试以下假设：由小分子量粘蛋白组成的粘液在体内不能被纤毛运输 WT 与二聚体小鼠对刺激的反应将导致呼吸道中的粘液积聚。 将测试扰乱粘蛋白浓度，以探索多聚粘蛋白对宿主防御的需求。 SA3 将检验还原剂有助于疾病而非健康中粘液清除的假设。 包括新型高效硫醇基治疗剂 (P2165) 对 WT 与二聚体的作用比较 体外粘蛋白和体内小鼠的目标是寻找减少多聚体长度的潜在有利影响。 影响咳嗽清除所需的粘液粘度，并确定通过减少其他物质介导的脱靶效应 粘蛋白内半胱氨酸可能会产生不良的粘蛋白聚集/粘性 该项目的可交付成果。 目的是：1) 量化粘蛋白多聚体大小、占位特征 (c*) 和 健康中的粘液功能；以及 2) 表征粘液溶解剂治疗指数的开启与关闭 目标效果并为有需要的患者开发新型粘液溶解疗法提供路线图。