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

描述(申请人提供)：蛋白激酶D(PKD)是一种新的以二酰甘油为靶点的丝氨酸/苏氨酸激酶家族。它调节许多基本的细胞功能，包括细胞的增殖、存活、分化和蛋白质运输，并在多器官系统的心脏肥大和癌症等病理条件下发挥重要作用。然而，PKD的这些作用机制尚不清楚，PKD在癌症和其他疾病中的作用也没有完全确定。这在一定程度上是由于缺乏在正常细胞过程和病理条件下专门针对PKD的有效药理学工具。这项建议的直接目标是证明基于IMAP的荧光偏振(FP)分析用于高通量筛选PKD抑制剂(HTS)的可行性。我们建议的分析是一种384孔、小体积的格式，并已被适应于高通量筛选。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，这将有助于了解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。我们提出的初步实验已经通过了广泛的逻辑分析和变异性评估，并成功地用于HTS模式下的一个小型化合物文库的筛选。基于这一坚实的初步数据基础，我们提出了一种详细的初步筛选方法和成本估算方法，并概述了用于鉴定和验证新型选择性PKD小分子抑制剂的二次和三次筛选方法。这一应用的成功完成将对二酰基甘油信号转导的研究进展以及该信号网络的放松调控引起的疾病的治疗产生深远的影响。PKD在心脏肥厚和多器官系统肿瘤的发病机制中起重要作用。PKD的抑制剂将有助于了解PKD与这些病理状态的相关性，并可能成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.