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

描述(由申请人提供)：针对癌症靶点的基于MALDI-MS的变革性抑制剂筛选技术的验证。作为一种变革性的技术，质谱学有着悠久的历史。两个具体的例子包括用于测量药物化合物在生物系统中命运的定量MS，以及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用于高通量筛选(HTS)分析的高效读数。据报道，与常用的荧光和化学发光读数相比，主要优点是假阳性或假阴性读数较少。其他增加的好处包括通过使用无标签底物大大降低了试剂成本(&gt；80%)，以及多重检测的能力，使得通过化合物储存库一次通过就可以筛选出多个治疗靶点的抑制剂，从而节省了数百万美元的试剂和人员成本。我们开发了基于MALDI-MS的读数方法，用于测量各种酶类别的酶活性和抑制率。然而，这些方法在小型测试库中的有限使用不足以验证这种读数在大型筛查活动中的整体效用。因此，这项提议的主要目标是系统地验证MALDI-MS读数的可靠性，而不是使用50,000个化合物的库来比较传统的高温超导方法。这些测量将包括一次筛查的命中率、二次筛查中命中的确认率以及HTS常见的其他标准质量评估，包括信号与背景、变异系数(CVs)和Z‘值。这些比较分析的目标酶将包括两个相关的激酶，PKC-Zeta和PKC-IOTA，它们似乎调节着癌症发生和发展的两个直接相反的作用。因此，作为这项提议的次要目标，将评估MALDI-MS读数在区分化合物储存库中对PKC-IOTA比PKC-Zeta具有选择性的抑制剂的有效性。我们已经证明，MALDI-MS读数适用于小规模的酶分析和抑制剂筛选，与现有的读数方法相比具有主要优势。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看到的所有优势，那么这种基于MS的技术可能会改变我们处理HTS的方式，就像基于MS的技术在过去15-20年里改变了生物分析和蛋白质组学应用一样。此外，通过针对激酶(其调节失调通常与癌症的发展相关的一类关键调控酶)来验证MALDI-MS读出方法，我们可以确信，随着这些研究的成功，这项技术显然可以很容易地重新应用于其他癌症相关的激酶以及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.