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

开发第一个无标记、高通量、基于细胞的检测方法，报告特定的酶活性

基本信息

批准号：
10076910
负责人：
Zachary Gurard-Levin
金额：
$ 107.64万
依托单位：
SAMDI TECH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-10 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10076910
关键词：
Address Advanced Development Benchmarking Biochemical Biological Assay Biotechnology Budgets Businesses Cancer cell line Caspase Cell Adhesion Cell Culture Techniques Cell Line Cells Chemistry Client Cost Savings Cultured Cells Custom Cytolysis Data Development Doctor of Philosophy Drug Screening Environment Enzymes Epigenetic Process Fee-for-Service Plans Fingerprint Funding Gene Expression Generations Genetic Geometry Global Change Goals Gold Immobilization International Label Letters Ligands Liquid substance Malignant Neoplasms Mass Spectrum Analysis Measures Methodology Miniaturization Noise PTPN1 gene Patients Peptides Permeability Pharmacologic Substance Pharmacology Phase Phenotype Phosphoric Monoester Hydrolases Physiological Preparation Protein Tyrosine Phosphatase Radioactivity Reagent Records Reporting Research Research Personnel Robotics Savings Scientist Services Signal Transduction Small Business Innovation Research Grant Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Substrate Specificity Surface Techniques Technology Time Toxic effect Universities Validation anticancer research assay development base biochip cell behavior cost data quality density drug discovery enzyme activity flexibility high throughput screening inhibitor/antagonist interest ionization luminescence monolayer new technology novel novel strategies professor programs prospective research and development screening small molecule symposium therapeutic target tool virtual

项目摘要

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.

项目摘要 /摘要量化小分子对细胞行为影响的基于细胞的测定是强大的工具癌症研究。在身体相关环境中评估小分子的能力是有益的，由于这些测定只是评估了复合渗透性，毒性和效力。迄今为止，基于细胞测定在很大程度上仅限于测量全球变化，例如表型，增殖或基因表达。当前的挑战是开发基于细胞的分析，以增加特定的酶活性，以增加和加速药物发现运动。值得注意的是，测量酶的最常见策略诸如放射性，吸收和荧光标签之类的活动通常无法传递到在细胞环境中的适当目标，它们容易受到高阳性率的影响。这提案描述了一种结合的新型无标签方法的持续开发和验证细胞培养和对定制表面化学的裂解，以允许质谱读数以进行不同酶活性。该方法使用具有官能化的自组装单层（SAM）相关细胞酶靶标的粘附配体以及底物。细胞在单分子在小分子存在下，然后裂解，使裂解物中的酶具有立即访问将固定的基板转换为产品。在黄金上使用sams的优势是它们与基质辅助激光解吸电离（MALDI）质谱法（MS）的兼容性方法称为“ SAMDI”，以记录裂解物中特定酶的活性。这项技术，称为串联文化和裂解（TCAL）-SAMDI，为定量读数提供了一种新颖，有力的工具几乎任何细胞酶活性，代表了高通量和无标签的细胞的新功能基于药物发现的屏幕。