High-Throughput Screening Platform for Cancer Drug Discovery

项目摘要 靶向治疗在癌症治疗中显示出巨大的前景。高通量筛选(HTS) 竞选活动通常依赖于使用标记配体或酶底物的检测。与以下内容相关联的人工产物 标记导致错误地识别作用于标记底物的化合物，而不是 预定的目标。一种无标记的、基于溶液的HTS方法将有助于识别作用于 特别是针对预定的目标。这一拟议项目的目标是推动发展和 验证这一量热平台是用于高通量ITC分析的可访问方法，展示其 具有研究多种肿瘤治疗靶点的优势。 为了实现这一目标，我们将改进试剂在液滴中的混合，从多路温度读数 单个液滴，自动化流体处理和数据分析，并验证改进后的 研究技术：1)SH2结构域/磷酸肽相互作用；2)脾酪氨酸激酶(Syk)酶 活性，以及3)溴域/乙酰化组蛋白多肽相互作用，进一步应用于小分子 放映。 光谱光学量热技术平台的实现将使全热力学 1536孔板分析中结合反应和酶反应的表征 显著减少了分析开发时间以及更多的重复次数和分析物浓度。这个 增加的吞吐量和减少的样本消耗将改变研究人员的观点 量热法：它不会被视为一种用于有限数量的高价值测量的技术，而将被视为一种 被视为癌症治疗靶点的主要筛查方法。

Project Summary Targeted therapies have shown great promise in the treatment of cancer. High-throughput screening (HTS) campaigns have often relied on assays using labeled ligands or enzyme substrates. Artifacts associated with labeling have led to the erroneous identification of compounds that act in on the labeled substrate rather than the intended target. A label-free, solution-based HTS method will facilitate identification of compounds acting specifically on the intended targets. The goals of this proposed project are to advance development and validate this calorimetry platform as an accessible approach for high throughput ITC analysis, demonstrating its advantages to study a variety of cancer therapeutic targets. To achieve this goal, we will improve mixing of reagents in droplets, multiplex temperature readout from individual droplets, automate fluidic handling and data analysis, and validate the applicability of the improved technology to study: 1) SH2 domain/phosphopeptide interactions, 2) spleen tyrosine kinase (Syk) enzyme activity, and 3) Bromodomain/acetylated histone peptide interactions, further applied in small molecule screening. The realization of the spectro-optical calorimetry technology platform will enable full thermodynamic characterization of binding and enzymatic reactions at the throughput of 1536-well plate assays but with significantly reduced assay development time and larger numbers of repeats and analyte concentrations. The combination of increased throughput and reduced sample consumption will change the way researchers view calorimetry: rather than being seen as a technique for a limited number of high-value measurements, it will be viewed as a primary screening method for cancer therapeutic targets.