Target Validation and Assay Development for Anti-Mucus Therapy

描述（由申请人提供）：急性和慢性气道疾病是美国和世界范围内最常见的严重疾病形式，大部分发病率和死亡率是由气道粘液阻塞引起的。反过来，粘液阻塞是粘液产生和随后分泌增加的结果。尽管有医疗需求，但没有特异性和有效的治疗方法来主要阻断粘液的产生。在这里，我们提出了初步的数据，人氯离子通道钙激活1（hCLCA 1）提供了一个关键的信号（主要通过MAPK 13），以调节粘液产生的量。我们进一步假设增加的hCLCAl水平是气道疾病中过量粘液产生的原因。因此，我们提出hCLCAl信号传导的抑制剂将有益于治疗ain/vay粘液的过度产生，并且hCLCAl水平可以指导治疗。为了进一步开发我们的抗粘液治疗计划，我们提出了两个目标，第一个目标是目标验证，目标是使用hCLCAl水平作为临床生物标志物，第二个目标是目标开发，目标是使用hCLCAl信号传导作为高通量筛选（HTS）的目标。目的1将验证hCLCAl水平和随后的信号传导与高保真细胞和小鼠模型以及人类受试者中的粘液产生偶联。特殊的方法包括抗hCLCAl mAb的新文库、MAPK 13的主要抑制剂、新的气雾剂递送系统和来自充分表征的哮喘和COPD受试者的组织样品的文库。目的2将优化和验证可以用HTS询问的hCLCAl信号转导的测定。特殊的方法包括人上皮细胞系，其被工程化用于hCLCAl的诱导型表达和随后的MARK活化和粘蛋白基因表达，新的专用HTS系统，其被完全自动化和机器人化用于在该细胞系和原代培养的上皮细胞中进行基于细胞的测定，以及药物化学，以实现我们的初始先导化合物和相关化合物的中试筛选。我们期望这些研究验证hCLCAl作为新的诊断和治疗靶标，用于分层和治疗在免疫缺陷疾病中粘液过度产生的患者。该方法利用了迄今为止未知的控制粘液产生的机制。我们预期开发高质量的ELISA来监测hCLCAl水平以指导患者分层和治疗，以及开发基于细胞的测定来在HTS系统中筛选hCLCAl抑制剂。多学科方法将由一个在肺生物学和疾病以及药物开发的各个方面都具有专业知识的团队来促进。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气道疾病的诊断和治疗产生重大影响。相关性（参见说明）：从急性呼吸道感染到慢性哮喘和COPD的气道疾病是最常见的严重疾病类型，并且大部分发病率和死亡率是由于粘液阻塞肺气道。然而，我们目前没有特异性和有效的抗粘液疗法。该提案直接针对这一问题，通过验证一种新的机制来解释疾病期间AinAy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.