Validation of MALDI-MS-based inhibitor screening technologies for cancer targets

基于 MALDI-MS 的癌症靶标抑制剂筛选技术的验证

• 批准号: 8145493
• 负责人: Kenneth Donald Greis
• 金额: $ 23万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-27 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145493
• 关键词: Accounting; Applied Research; Biological Assay; Categories; Cell physiology; Detection; Development; Disease; Drug Compounding; Effectiveness; Enzyme Inhibition; Enzymes; Evaluation; Fluorescence; Goals; Head; Human Resources; Label; Lead; Libraries; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Mixed Function Oxygenases; Oncogenic; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Preparation; Process; Proteins; Proteomics; Reaction; Reagent; Recording of previous events; Reporting; Sampling; Screening procedure; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Systems Development; Techniques; Technology; Testing; Tumor Suppressor Proteins; Validation; base; biological systems; cancer initiation; cell type; comparative; cost; drug development; drug discovery; enzyme activity; esterase; high throughput screening; inhibitor/antagonist; luminescence; process optimization; protein kinase C iota; protein kinase C zeta; repository; scale up; secondary outcome; success; therapeutic target

DESCRIPTION (provided by applicant): Validation of transformative MALDI-MS-based inhibitor screening technologies for cancer targets. Mass spectrometry (MS) has a long history as a transformative technology. Two specific examples include quantitative MS to measure the fate of drug compounds in biological systems and the development of proteomics techniques for protein identification and characterization. Recently MS-based applications have been demonstrated to be highly effective as the readout for high throughput screening (HTS) assays. The major advantage reported over the commonly used fluorescence and chemi-luminescence readout is the paucity of false positive or false negative readout. Other added benefits include a greatly reduced (>80%) reagent cost by using label-free substrates and the ability to multiplex assays such that multiple therapeutic targets can be screened for inhibitor hits with one pass through the compound repository thus saving millions of dollars in reagent and personnel costs. We have developed MALDI-MS based readout methods for measuring enzyme activity and inhibition for a variety of enzyme classes. However, the limited use of these methods with small test libraries has been insufficient to validate the overall utility of this readout for large screening campaigns. Thus the primary goal of this proposal is to systematically validate the reliability of the MALDI-MS readout compared to a traditional method of HTS using a library of 50,000 compounds. The measurements will include hit rates from the primary screen, the validation rate of the hits in secondary screening and other standard quality assessments common for HTS including signal to background, coefficient of variance (CVs) and Z' values. The target enzymes for these comparative assays will include two related kinases, PKC-zeta and PKC-iota, which appear to regulate two directly opposite effects on cancer initiation and development. Thus as a secondary aim of this proposal, the MALDI-MS readout will be assessed for its effectiveness to distinguish inhibitors from the compound repository that have selectivity for PKC-iota over PKC-zeta. We have shown that MALDI-MS readout is amenable to enzyme assays and inhibitor screening on a small scale with major advantages over existing readout methods. If, the MALDI-MS readout can be scaled to true HTS levels while maintaining all the advantages seen in the proof-of-concept studies, then this MS-based technology would likely transform the way we approach HTS, in much the same way as MS-based technologies have changed bioanalytical and proteomics applications over the past 15-20 years. Furthermore, by targeting kinases (a key class of regulatory enzymes whose dysregulation is often associated with cancer development) to validate the MALDI-MS readout approach, we can be assured that with the success of these studies, it will be clear that this technology can be readily reapplied to other cancer relevant kinases as well as expanded into other enzyme classes and disease categories. PUBLIC HEALTH RELEVANCE: Dysregulation of cellular function is a hallmark of cancer development and progression and can often be traced to the actions of one or a few cellular enzymes, thus these enzymes may act as primary targets to halt cancer development and progression. As such, rapid and accurate methods to evaluated inhibitors of these target enzymes represent an initial phase in the new drug discovery and development process. In this proposal, we investigate a new readout technology, based on mass spectrometry, to rapidly screen for inhibitors of a class of enzymes known to be involved in cancer development and progression.

描述（由申请人提供）：验证用于癌症靶点的基于MALDI-MS的变革性抑制剂筛选技术。质谱（MS）作为一种变革性技术具有悠久的历史。两个具体的例子包括定量MS测量药物化合物在生物系统中的命运和蛋白质组学技术的发展，用于蛋白质鉴定和表征。最近，基于MS的应用已被证明是非常有效的高通量筛选（HTS）测定的读数。与常用的荧光和化学发光读数相比，报告的主要优点是缺乏假阳性或假阴性读数。其他额外的好处包括通过使用无标记底物大大降低（>80%）的试剂成本和多重测定的能力，使得可以通过化合物储存库一次筛选多个治疗靶点的抑制剂命中，从而节省数百万美元的试剂和人员成本。我们已经开发了基于MALDI-MS的读出方法，用于测量各种酶类的酶活性和抑制。然而，这些方法在小测试文库中的有限使用不足以验证该读数在大筛选活动中的整体效用。因此，该提议的主要目标是系统地验证MALDI-MS读出的可靠性，与使用50，000种化合物的库的传统HTS方法相比。测量将包括来自初级筛选的命中率、次级筛选中命中的验证率和HTS常见的其他标准质量评估，包括信号背景、变异系数（CV）和Z'值。用于这些比较测定的靶酶将包括两种相关激酶，PKC-ζ和PKC-1，其似乎调节对癌症起始和发展的两种直接相反的作用。因此，作为该提议的次要目的，将评估MALDI-MS读数区分抑制剂与化合物储存库的有效性，所述化合物储存库对PKC-1的选择性超过PKC-ζ。我们已经表明，MALDI-MS读出是服从酶测定和抑制剂筛选在一个小规模的主要优势，现有的读出方法。如果MALDI-MS读数可以扩展到真正的HTS水平，同时保持概念验证研究中看到的所有优势，那么这种基于MS的技术可能会改变我们接近HTS的方式，就像基于MS的技术在过去15-20年中改变生物分析和蛋白质组学应用一样。此外，通过靶向激酶（其失调通常与癌症发展相关的关键类别的调节酶）来验证MALDI-MS读出方法，我们可以确信，随着这些研究的成功，将清楚的是，该技术可以容易地重新应用于其他癌症相关激酶以及扩展到其他酶类别和疾病类别。 公共卫生关系：细胞功能失调是癌症发展和进展的标志，通常可以追溯到一种或几种细胞酶的作用，因此这些酶可以作为阻止癌症发展和进展的主要靶标。因此，快速和准确的方法来评估这些靶酶的抑制剂代表了新药发现和开发过程的初始阶段。在这项提案中，我们研究了一种基于质谱的新的读出技术，以快速筛选已知参与癌症发展和进展的一类酶的抑制剂。