Development of the first label-free and high-throughput, cell-based assay that reports on specific enzyme activities

项目摘要/摘要 基于细胞的分析可以量化小分子对细胞行为的影响，是一种强大的工具 癌症研究。评估生理相关环境中的小分子的能力是有用的， 因为检测同时评估化合物的渗透性、毒性和效力。迄今为止，基于单元格的 检测在很大程度上局限于衡量全球变化，如表型、增殖或基因表达。 目前的挑战是开发基于细胞的特定酶活性的分析方法，以增强和 加快药物发现活动。值得注意的是，测量酶的最常见策略 放射性、吸光度和荧光标记等活动通常无法传递到 在蜂窝环境中适当的目标，他们容易受到高假阳性率的影响。这 提案描述了一种新的无标签方法的持续开发和验证，该方法将 在定制的表面化学试剂上进行细胞培养和裂解，以允许对不同的 酶活性。该方法使用呈现细胞的功能化自组装单层(SAM) 黏附配体和相关细胞酶靶标的底物。细胞培养在 在小分子存在的情况下，单层，然后裂解，使裂解物中的酶 可立即将固定化底物转化为产品。在黄金上使用SAMS的一个优势是 它们与基质辅助激光解吸电离(MALDI)质谱学的兼容性，以及 一种名为“SAMDI”的方法，记录裂解物中特定酶的活性。这项技术， 被称为串联培养和裂解(TCAL)的SAMDI提供了一种新的、强大的工具来定量读出 几乎任何细胞酶活性，并代表了高通量和无标记的细胞- 以筛选为基础的药物发现。

Project Summary / Abstract Cell-based assays that quantitate the effect of small molecules on cellular behavior are powerful tools in cancer research. The ability to assess small molecules in a physiologically relevant environment is informative, as the assays simultaneously evaluate compound permeability, toxicity, and potency. To date, cell-based assays are largely limited to measure global changes such as phenotype, proliferation, or gene expression. The current challenge is to develop cell-based assays for specific enzyme activities to augment and accelerate drug discovery campaigns. Notably, the most common strategies for measuring enzyme activities, such as radioactivity, absorbance, and fluorescent labels, often cannot be delivered to the appropriate target within the cellular environment and they are susceptible to high rates of false positives. This proposal describes the continued development and validation of a novel label-free methodology that combines cell culture and lysis on customized surface chemistries to allow a mass spectrometry readout for distinct enzyme activities. The approach uses functionalized self-assembled monolayers (SAMs) that present cell adhesion ligands along with substrates for the relevant cellular enzyme targets. Cells are cultured on the monolayers in the presence of small molecules, and then lysed such that enzymes within the lysate have immediate access to convert the immobilized substrate to a product. An advantage of using SAMs on gold is their compatibility with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), an approach termed “SAMDI”, that records the activity of specific enzymes within the lysate. This technique, termed Tandem Culture and Lysis (TCAL)-SAMDI, offers a novel, powerful tool for a quantitative readout of virtually any cellular enzyme activity and represents new capabilities for high-throughput and label-free, cell- based screens for drug discovery.