IMAP-Based Fluorescent Polarization Assay for HTS Protein Kinase D Inhibitors

描述（由申请人提供）：蛋白激酶D（PKD）是一个新的丝氨酸/苏氨酸激酶家族，由二酰基甘油靶向。它调节许多基本的细胞功能，包括细胞增殖、存活、分化和蛋白质运输，并且在多器官系统中的病理状况如心脏肥大和癌症中起重要作用。然而，PKD的这些作用的机制尚不清楚，PKD在癌症和其他疾病中的作用尚未完全确定。这部分是由于缺乏在正常细胞过程和病理条件下特异性靶向PKD的有效药理学工具。该提案的直接目标是证明基于IMAP的荧光偏振（FP）测定用于PKD抑制剂的高通量筛选（HTS）的可行性。我们提出的测定是在384孔，小体积格式，并已被改编为高通量筛选。该研究计划旨在实现一个重要的总体目标，特别是发现新的有效和选择性的PKD小分子抑制剂，这将有助于理解PKD的生物学相关性，并具有长期治疗应用的潜力。我们的初步研究为分析优化和质量控制提供了强有力的数据。我们提出的初步分析已经通过了广泛的逻辑分析和变异性评估，并已成功地用于筛选一个小的化合物库在HTS模式。基于这一坚实的基础上的初步数据，我们提出了一个详细的初步筛选试验方案的成本估计和概述的二级和三级筛选试验，以确定和验证新的选择性小分子抑制剂的PKD。这项申请的成功完成将对甘油二酯信号传导研究的进展和治疗由该信号网络失调引起的疾病产生深远的影响。PKD在心脏肥大和多器官系统肿瘤的发病机制中起重要作用。PKD的抑制剂将有助于理解PKD与这些病理条件的相关性，并可作为治疗这些疾病的潜在药物。

DESCRIPTION (provided by applicant): Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based fluorescent polarization (FP) assay for high throughput screening (HTS) of PKD inhibitors. The assay we are proposing is in a 384-well, small volume format and has been adapted for high throughput screening. The research plan has been designed to achieve an important overall objective, specifically the discovery of novel potent and selective small molecule inhibitors of PKD that will be helpful in understanding the biological relevance of PKD and have the potential for long-term therapeutic application. Our preliminary study provided strong data on assay optimization and quality controls. Our proposed primary assay has passed extensive logistical analysis and variability assessment, and has been used successfully to screen a small compound library in HTS mode. Based on this solid foundation of preliminary data, we propose a detailed primary screening assay protocol with a cost estimate and outline the secondary and tertiary screening assays to identify and validate novel selective small molecule inhibitors of PKD. Successful completion of this application will have a profound impact on the advancement of research on diacylglycerol signaling and on the treatment of diseases caused by the deregulation of this signaling network. PKD plays an important role in the pathogenesis of cardiac hypertrophy and cancer in multiple organ systems. Inhibitors of PKD will facilitate the understanding of the relevance of PKD to these pathological conditions and could serve as potential drugs for the treatment of the diseases.