Cell-free assay technologies for the identification of active compounds

该项目导致与Stanley Lipkowitz和Yves Pompersky博士（CCR）合作开发了针对靶点Cbl-b和酪氨酰DNA磷酸二酯酶-2的高通量筛选。PCMBS与Lipkowitz博士（WMB）的实验室合作，正在开发合成化合物和天然产物提取物的高通量筛选（HTS），以发现和表征Cbl-b特异性生化抑制剂。WMB一直处于表征和理解Cbl-b泛素连接酶活性的生物化学的最前沿，并建立了PCMBS能够适应HTS使用的无细胞筛选测定的框架。作为二聚体RING指E3泛素连接酶，Cbl-b协调含泛素的E2酶与待泛素化的最终靶蛋白（底物）之间的相互作用。虽然Cbl-b本身不转移泛素，但它是赋予底物特异性的泛素化级联的单一组分。Cbl-b的结构包含7个不同的基序，包括一个泛素相关（乌巴）结构域，该结构域已被证明对多聚泛素链具有高亲和力和特异性。泛素连接酶反应是在用Cbl-b乌巴预包被的测定板中进行的，以允许多聚泛素化（因此生物素化）的反应产物的结合和保留。与Dr. Yves Pompero（DTB）合作，根据Ogilvie等人开发的测定法重新设计了一种用于发现基于天然产物的Tdp2活性抑制剂的显色测定法。该试验设计用于通过测量底物4-NPPP生成对硝基苯酚的减少来测量Tdp2活性的抑制。基于吸光度的测定将采用透明的、未处理的384孔板，在405 nm处进行检测，这是对硝基苯酚的最大吸收。吸光度信号的降低与推定抑制剂的效力直接相关。在DTB中进行了使用较大酪氨酸化DNA构建体的二级测定。DTB提供了TTRAP/Tdp2酶构建体（由位于FNL，Frederick，MD的NCI蛋白表达实验室生产），并在MTL评价了所有初始酶质量保证。Tdp2对4-NPPP底物的活性被映射到与曼彻斯特组相似的Vmax、Km和Kcat值。我们将4-NPPP的测定浓度设定为低于观察到的Km值（Km = 115 mM），主要是由于检测器在100 mM或以上浓度下饱和，并将测定条件的时间延长至20分钟以上。目前，我们已经筛选了所有可用的MTL纯化合物和合成文库，总计69，793个化合物，初始命中率为0.22%，以及提供命中率为0.82%（命中要求为50%抑制活性）的预分级天然产物文库。HTS已完成，CBL-b和Tdp2抑制化合物的评价正在进行中。与张平博士和凯莉·沃尔特斯博士在DNAJ-PKA和Rpn 2/Rpn 13方面的其他合作也在进行中。这些测定使用多种技术，包括荧光偏振、差示扫描荧光测定和动力学测定。此外，我们最近与丹尼尔·麦克维卡尔博士一起开始了一个项目，使用热熔法鉴定代谢酶Irg 1的潜在抑制剂，并与弗雷迪·埃斯科西亚博士一起鉴定癌症靶点磷脂酰肌醇蛋白聚糖3。我们还与尤金·瓦尔科夫（Eugene Valkov）和伊夫·波米尔（Yves Pommier）启动了与COVID-19相关的项目。

This project resulted in the development high throughput screens for the targets Cbl-b and tyrosyl-DNA phosphodiesterase-2 in collaboration with Drs. Stanley Lipkowitz and Yves Pommier (CCR). The PCMBS, in collaboration with the laboratory of Dr. Lipkowitz (WMB) is developing a high throughput screen (HTS) of both synthetic compounds and natural products extracts for the discovery and characterization of Cbl-b specific biochemical inhibitors. The WMB has been at the forefront of characterizing and understanding the biochemistry of Cbl-b ubiquitin ligase activity and has established the framework for a cell-free screening assay that the PCMBS has been able to adapt for use in an HTS. As a dimeric RING finger E3 ubiquitin ligase, Cbl-b coordinates the interaction between an ubiquitin-containing E2 enzyme and the final target protein to be ubquitinated (the substrate). While Cbl-b itself does not transfer ubiquitin, it is the single component of the ubiquitination cascade that confers substrate specificity. Therefore, Cbl-b is an attractive target for pharmacologic intervention The structure of Cbl-b contains seven different motifs, including an ubiquitin-associated (UBA) domain which has been shown to have a high affinity and specificity for polyubiquitin chains. The ubiqiuitin ligase reaction is performed in assay plates pre-coated with the Cbl-b UBA, to allow for binding and retention of the polyubiquitinated (and hence biotinylated) reaction products.We anticipate that the use of these two agents will reduce the detection of false positive results from aggregating or non-specific protein binding compounds. In collaboration with Dr. Yves Pommier (DTB) a chromogenic assay for discovery of natural products based inhibitors of Tdp2 activity was redesigned from an assay developed by Ogilvie et al.). The assay was designed to measure the inhibition of Tdp2 activity by measuring the decrease in the generation of p-nitrophenol from a substrate, 4-NPPP. The absorbance based assay would employ clear, untreated 384-well plates for detection at 405nm, the absorption maxima of p-nitrophenol. The decrease in absorbance signal would correlate directly with the potency of putative inhibitors. Secondary assays with a larger tyrosinylated DNA construct are taking place in the DTB. DTB provided the TTRAP/Tdp2 enzyme construct (produced by Protein Expression Laboratory, NCI at FNL, Frederick, MD) and all initial enzyme quality assurance was evaluated at the MTL. The Tdp2 activity against the 4-NPPP substrate was mapped to similar values of Vmax, Km, and Kcat as that of the the Manchester group. Our assay concentration of 4-NPPP was set below the observed Km value (Km= 115 mM) primarily due to detector saturation at concentrations 100mM or above and to lengthen the time of the assay conditions beyond 20 minutes. Currently, we have screened all available MTL pure compound and synthetic libraries totaling 69,793 compounds with an initial hit rate of 0.22% and a pre fractionated natural product library which provided a hit rate of 0.82% (hit requirement of 50% inhibition activity). HTS is completed and the evaluation of CBL-b and Tdp2 inhibitory compounds is ongoing. Additional collaborations on DNAJ-PKA and Rpn2/Rpn13 are also underway with Drs. Ping Zhang and Kylie Walters. These assays use a variety of techniques including fluorescence polarization, differential scanning fluorometry and kinetic assays. In addition, we have recently begun a project using a thermal melt assay to identify potential inhibitors of the metabolic enzyme Irg1 with Dr. Daniel McVicar and the cancer target glypican-3 with Dr. Freddy Escorcia. We have also initiated COVID-19 related projects with Eugene Valkov and Yves Pommier.