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

项目概要/摘要 定量小分子对细胞行为的影响的基于细胞的测定法是测定小分子对细胞行为的影响的有力工具。 癌症研究。在生理相关环境中评估小分子的能力是有益的， 因为该试验同时评价化合物的渗透性、毒性和效力。迄今为止，基于细胞的 测定法很大程度上限于测量整体变化，例如表型、增殖或基因表达。 目前的挑战是开发用于特定酶活性的基于细胞的测定，以增加和 加快药物发现运动。值得注意的是，测量酶的最常见策略 活性，如放射性、吸收和荧光标记，通常不能被递送到细胞。 它们在细胞环境内具有合适的靶点，并且它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， 在定制的表面化学物质上进行细胞培养和裂解，以允许质谱读出不同的 酶活性该方法使用功能化的自组装单分子膜（SAM）， 粘附配体沿着与相关细胞酶靶的底物。将细胞培养在 在存在小分子的情况下，使细胞单层化，然后裂解，使得裂解物内的酶具有 立即接近以将固定化底物转化为产物。在黄金上使用SAM的一个优点是 它们与基质辅助激光解吸电离（MALDI）质谱（MS）的兼容性， 该方法称为“SAMDI”，其记录裂解物内特定酶的活性。这种技术， 称为串联培养和裂解（TCAL）-SAMDI，提供了一个新的，强大的工具，定量读出 几乎任何细胞酶活性，并代表了高通量和无标记，细胞， 基于药物发现的筛选。

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.