Target Validation and Assay Development for Anti-Mucus Therapy

描述(申请人提供)：急性和慢性呼吸道疾病是美国和世界范围内最常见的严重疾病之一，大部分发病率和死亡率是由呼吸道粘液阻塞引起的。反过来，粘液阻塞是粘液产量增加和随后的分泌增加的结果。尽管有医学上的需要，但目前还没有特效的治疗方法来主要阻断粘液的产生。在这里，我们提供了初步数据，人氯通道钙激活1(HCLCA1)提供了一个关键信号(主要通过MAPK13)来调节粘液的产生量。我们进一步假设，hCLCA1水平升高是呼吸道疾病中粘液产生过多的原因。因此，我们认为hCLCAl信号的抑制剂将有益于治疗AIN/VAY粘液的过度产生，并且hCLCA1的水平可以指导治疗。为了进一步发展我们的抗粘液治疗计划，我们提出了两个目标，第一个目标是靶点验证，目标是使用hCLCA1水平作为临床生物标记物，第二个目标是检测开发，目标是使用hCLCA1信号作为高通量筛选(HTS)的目标。目标1将在高保真细胞和小鼠模型以及人类受试者中验证hCLCA1水平和随后的信号与粘液产生耦合。特殊的方法包括一个新的抗hCLCA1单抗的库，一种MAPK13的先导抑制剂，一个新的气雾剂输送系统，以及一个来自具有良好特征的哮喘和COPD受试者的组织样本库。AIM 2将优化和验证可用HTS询问的hCLCA1信号转导分析。特殊的方法包括设计用于诱导hCLCA1表达和随后的Mark激活和粘蛋白基因表达的人上皮细胞系，新的专用HTS系统，该系统完全自动化和自动化，用于在该细胞系和原代培养的ANN/AY上皮细胞中进行基于细胞的分析，以及药物化学，使我们能够对最初的铅和相关化合物进行中试筛选。我们期望这些研究验证hCLCA1作为一种新的诊断和治疗靶点，用于对AINA/AY疾病中粘液分泌过多的患者进行分层和治疗。这种方法利用了一种迄今未知的控制粘液产生的机制。我们预计将开发一种高质量的ELISA来监测hCLCA1水平，以指导患者的分层和治疗，并开发一种基于细胞的分析方法来筛选HTS系统中的hCLCA1抑制剂。多学科方法将由一个在肺部生物学和疾病以及药物开发的所有方面都有专门知识的团队来推动。总之，通过建立抗粘液治疗所需的工具，来自这些目标的信息将对呼吸道疾病的诊断和治疗产生重大影响。相关性(见说明)：从急性呼吸道感染到慢性哮喘和慢性阻塞性肺病的各种呼吸道疾病是最常见的严重疾病类型，许多发病率和死亡率是由于粘液阻塞肺部呼吸道造成的。然而，我们目前还没有特效的抗粘液疗法。这项建议旨在通过验证一种新的机制来解释疾病期间Aina^ay粘液的过度产生，并采取下一步措施来确定监测和治疗这种异常的方法。

DESCRIPTION (provided by applicant): Acute and chronic airway diseases are among the most common forms of serious illness in the U.S. and worldwide, and much of the morbidity and mortality is caused by mucus obstruction of the ainways. In turn, the mucus obstruction is the result of increased mucus production and subsequent secretion. Despite the medical need, there are no specific and effective therapies to primarily block mucus production. Here we present preliminary data that human chloride channel calcium activated 1 (hCLCAl) provides a critical signal (via primarily MAPK13) to regulate the amount of mucus production. We further hypothesize that increased hCLCAl levels are responsible for excess mucus production in airway disease. We therefore propose that an inhibitor of hCLCAl signaling would be beneficial to treat overproduction of ain/vay mucus and that hCLCAl levels can guide therapy. To further develop our plan for anti-mucus therapy, we propose two aims, the first for target validation with the goal of using hCLCAl level as a clinical biomarker, and the second for assay development with the goal of using hCLCAl signaling as a target for high-throughput screening (HTS). Aim 1 will validate that hCLCAl level and consequent signaling is coupled to mucus production in high- fidelity cell and mouse models and in human subjects. Special approaches include a new library of anti- hCLCAl mAb's, a lead inhibitor of MAPK13, a new aerosol delivery system, and a library of tissue samples from well-characterized asthma and COPD subjects. Aim 2 will optimize and verify assays of hCLCAl signal transduction that can be interrogated with HTS. Special approaches include a human epithelial cell line that is engineered for inducible expression of hCLCAl and consequent MARK activation and mucin gene expression, a newly dedicated HTS system that is fully automated and roboticized for cell-based assay in this cell line and primary-culture ainn/ay epithelial cells, and medicinal chemistry to enable a pilot screen of our initial lead and related compounds. We expect these studies to validate hCLCAl as a new diagnostic and therapeutic target for stratifying and treating patients with overproduction of mucus in ainA/ay diseases. The approach takes advantage of a heretofore unknown mechanism for controlling mucus production. We anticipate developing a high-quality ELISA to monitor hCLCAl levels for guiding patient stratification and therapy and a cell-based assay to screen for hCLCAl inhibitors in an HTS system. The multidisciplinary approach will be facilitated by a team with expertise in all aspects of lung biology and disease as well as drug development. Together, the information from these aims will have a significant impact on diagnosis and therapy of airway diseases by establishing the tools needed for anti-mucus therapy. RELEVANCE (See instructions): Airway diseases ranging from acute respiratory infections to chronic asthma and COPD are among the most common types of serious illness, and much of the morbidity and mortality is due to obstruction of the lung airways by mucus. However, we currently have no specific and effective anti-mucus therapies. This proposal aims directly at this issue by validating a new mechanism to explain overproduction of ainA^ay mucus during disease and by taking the next steps to identify the means to monitor and treat this abnormality.