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？

基本信息

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
项目类别：
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

项目摘要

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，对几个人热有效/有效吗？”，理查德·C（Richard C.）。医学博士Boucher，PI，有关粘液运输的基本问题的提议以及如何在粘液刺发生的患者中热恢复粘液清除率。该项目首先关注一个简单的问题，即为什么如此巨大的粘蛋白多聚体（〜120个单体，300 MDA，250 nm的回旋半径， RG）由正常气道上皮合成并分泌？我们假设大型多培养者是最大的产生运输所需的类似粘液凝胶的特性的经济方法。研究将重点放在粘蛋白上分子量（MW），分子大小（GyRation的半径，RG），浓度（C），重叠浓度（C*），凝胶依赖性生物物理特性和可运输能力。推论的目标是检验粘液溶解的假设代理，即降低多聚体长度/大小的代理，是粘液 - 刺激性的有吸引力的治疗剂肺部病。我们调查这两个假设的方法都取决于成功的CRISPR 敲除突变小鼠（MUC5B D3半胱氨酸1128-丙氨酸，半胱氨酸1170-丙氨酸），可有选择地合成并分泌粘蛋白二聚体（MW〜4 MDA，RG = 40 nm）。 SA1将检验多聚体粘蛋白的假设（大型多单元）有效地占据粘液层中的空间/体积，因此产生重叠产生运输所需的凝胶状粘液特性所需的条件（C*）。野生的结构特征使用光散射，中子散射和EM技术，类型（WT）与MUC5B二聚体粘蛋白，与生物物理特性并置，例如粘弹性特性，渗透模量，内聚和粘合剂，将会在体外进行测量并与纤毛和咳嗽依赖性转运有关。 SA1还将测试特定假设二聚体粘蛋白由于其较小的RG而无法在基本条件下达到C*条件（分泌率）产生依赖纤毛依赖性粘液清除所需的凝胶的弹性特性。然后，SA2将检验以下假设，即cilia在体内积累了小MW粘液的粘液不可传输呼吸道中的粘液积累将导致。 WT与二聚体小鼠对挑衅的反应将测试该敏感性粘蛋白浓度，以探索多聚粘蛋白对宿主防御的需求。 SA3将检验以下假设：减少药物有助于疾病中的粘液清除率，而不是健康。研究将包括在WT与Dimeric上的新型，有效的基于硫醇的治疗剂（P2165）的作用的比较体内体外和小鼠的粘蛋白。目的是寻找减少多聚体长度的潜在有利效果关于咳嗽清除所需的粘液粘度，并识别通过减少其他的介导的脱靶效应可能产生粘蛋白的粘合蛋白半胱氨酸，可能产生不良的粘蛋白聚集/粘性。项目的可交付成果为：1）量化粘蛋白多管尺寸，空间占领特征（C*）和粘液在健康方面的功能； 2）特征相对于ON-OFR-与ON-OFF- 目标效应并为有需要的患者开发新型粘液疗法的路线图。