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

描述（由申请人提供）：基于maldi - ms的癌症靶点变革性抑制剂筛选技术的验证。质谱（MS）作为一项革命性的技术有着悠久的历史。两个具体的例子包括用于测量生物系统中药物化合物命运的定量质谱和用于蛋白质鉴定和表征的蛋白质组学技术的发展。最近，基于ms的应用程序已被证明是非常有效的高通量筛选（HTS）分析的读数。与常用的荧光和化学发光读出相比，报告的主要优点是缺乏假阳性或假阴性读出。其他额外的好处包括，通过使用无标签底物，大大降低了试剂成本（约80%），并且能够进行多重检测，这样就可以通过化合物库一次筛选多个治疗靶点，从而节省数百万美元的试剂和人员成本。我们开发了基于MALDI-MS的读出方法，用于测量各种酶类的酶活性和抑制。然而，这些方法与小型测试库的有限使用不足以验证这种读数对大型筛选活动的总体效用。因此，本提案的主要目标是系统地验证MALDI-MS读数的可靠性，与传统的HTS方法相比，使用5万个化合物的文库。测量将包括主屏幕的命中率、二次筛选中命中的验证率和HTS常见的其他标准质量评估，包括信号与背景、方差系数（cv）和Z值。这些比较分析的靶酶将包括两个相关的激酶，PKC-zeta和PKC-iota，它们似乎在癌症的发生和发展中调节两种直接相反的作用。因此，作为本提案的第二个目标，MALDI-MS读数将评估其区分抑制剂与化合物库中PKC-iota选择性优于PKC-zeta的有效性。我们已经证明MALDI-MS读数适用于酶测定和抑制剂筛选，在小范围内具有比现有读数方法更大的优势。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的所有优势，那么这种基于ms的技术可能会改变我们处理HTS的方式，就像过去15-20年来基于ms的技术改变了生物分析和蛋白质组学的应用一样。此外，通过靶向激酶（一类关键的调节酶，其失调通常与癌症发展相关）来验证MALDI-MS读出方法，我们可以确信，随着这些研究的成功，这项技术可以很容易地重新应用于其他癌症相关的激酶，并扩展到其他酶类和疾病类别。

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.