Force phenotyping of airway smooth muscle cells to develop novel asthma therapies

强制气道平滑肌细胞表型分析以开发新型哮喘疗法

基本信息

批准号：
9452964
负责人：
Dino Di Carlo
金额：
$ 23.48万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9452964
关键词：
Actomyosin Acute Address Adrenal Cortex Hormones Adrenergic Agonists Affect Agonist Animal Model Asthma Biological Assay Bronchoconstriction Bronchodilator Agents Calcium Cardiac Myocytes Cells Chemicals Clinical Cyclic AMP Data Development Disease Drug Screening Drug Targeting Effectiveness Emergency Situation Epigenetic Process Extracellular Matrix FDA approved Fibroblasts Future Generations Genomics Human Immunofluorescence Immunologic Inflammation Inflammation Mediators Inhalation Inhalators Lead Legal patent Libraries Link Lung Measurement Measures Methods Molecular Monitor Muscle Contraction Muscle Tonus Muscle relaxation phase Myosin Light Chain Kinase Natural Products Opsonin Pathway interactions Patients Pattern Pharmaceutical Preparations Pharmacology Phenotype Play Potassium Channel Relaxation Respiratory physiology Role Second Messenger Systems Signal Transduction Slice Smooth Muscle Smooth Muscle Myocytes Specificity Speed Stains Surface System Techniques Testing Time Tissue Model Tissues Vascular Smooth Muscle airway hyperresponsiveness asthma inhaler asthmatic patient base beta-2 Adrenergic Receptors cell type constriction design drug discovery experimental study high throughput screening inhibitor/antagonist macrophage mechanical behavior molecular phenotype mortality new therapeutic target novel novel therapeutics prevent release of sequestered calcium ion into cytoplasm respiratory smooth muscle response screening tool

项目摘要

ABSTRACT Asthma is currently treated with drugs that target inflammation (e.g. corticosteroids) and the subsequent bronchoconstriction (β2 adrenergic receptor agonists) that leads to airway narrowing. Although there are a variety of mechanisms to inhibit cell force generation and contraction, short- and long-acting bronchodilators operate through a single mechanism of action, which has negative consequences, since adaptation to a long-acting beta agonist leads to reduced efficacy of short-acting beta agonist “rescue inhalers.” There is a need for new drugs that target airway smooth muscle contractility through orthogonal pathways to the beta agonists. However, there are no current methods to perform high-throughput screens targeting cell force generation. We have developed a microtechnology-based high-throughput screening approach to characterize cellular force generation at the single-cell level. We hypothesize that new drugs that interfere with airway smooth muscle cell contractility can be found that act through separate pathways and lead to new treatment options for asthma patients. In Aim 1 we will conduct a high-throughput screen to identify compounds that relax contraction in airway smooth muscle cells. We will validate hit compounds in a tissue model - precision cut lung slices. We also anticipate that selective inhibitors of airway smooth muscle contraction can be developed by counter-screening against other contractile cells. Our platform allows for combined measurement of immunofluorescence, calcium levels, and contractile phenotypes for single cells. In Aim 2 we will use this capability to address whether calcium mobilization is increasing and sufficient to evoke HASM cell shortening by contractile agonists. Molecular inputs that modulate smooth muscle actomyosin cross-bridge cycling and the strength of contraction remain less understood given the larger variety of inputs that control smooth muscle tone. Also, we will use this platform to identify new surface markers associated with hyper-responsive contractile phenotypes highlighting potential key ASM subpopulations involved in disease. Such surface markers would also assist in designing cell-targeted anti-contractility drugs for asthma in the future.

抽象的目前治疗哮喘的方法是针对炎症的药物（例如皮质类固醇）和随后的药物导致气道狭窄的支气管收缩（β2 肾上腺素受体激动剂）有多种。抑制细胞力产生和收缩的机制，短效和长效支气管扩张剂起作用通过单一的作用机制，这会产生负面后果，因为适应了长效贝塔激动剂导致短效β激动剂“救援吸入器”的功效降低，因此需要新药。然而，通过与β受体激动剂的正交途径来靶向气道平滑肌收缩力。目前我们还没有开发出针对细胞力生成进行高通量筛选的方法。一种基于微技术的高通量筛选方法，用于表征细胞力的产生我们努力发现干扰气道平滑肌细胞收缩性的新药可以。目标 1：发现它们通过不同的途径发挥作用，并为哮喘患者带来新的治疗选择。我们将进行高通量筛选，以确定能够放松气道平滑肌收缩的化合物我们将在组织模型（精确切割的肺切片）中验证命中化合物。气道平滑肌收缩抑制剂可以通过针对其他收缩性收缩剂的反筛选来开发我们的平台可以结合测量免疫荧光、钙水平和收缩力。在目标 2 中，我们将使用此功能来解决钙动员是否有效的问题。增加并足以通过调节收缩激动剂的分子输入引起 HASM 细胞缩短。鉴于平滑肌肌动球蛋白跨桥循环和收缩强度的了解仍然较少控制平滑肌张力的更多种类的输入此外，我们将使用这个平台来识别新的表面。与高反应收缩表型相关的标记突出了潜在的关键 ASM 亚群这些表面标记物也将有助于设计针对细胞的抗收缩药物。将来会出现哮喘。