Cell-free assay technologies for the identification of active compounds

该项目导致与Stanley Lipkowitz博士和Yves Pommier(CCR)博士合作开发了针对Cbl-b和Tyrosyl-DNA磷酸二酯酶-2的高通量筛查。PCMBS与利普科维茨(WMB)博士的实验室合作，正在开发合成化合物和天然产品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一直处于表征和了解Cbl-b泛素连接酶活性的生物化学的前沿，并建立了PCMBS能够用于HTS的无细胞筛选试验的框架。Cbl-b作为一种二聚体环指E3泛素连接酶，协调含有泛素的E2酶与最终被泛素化的目标蛋白(底物)之间的相互作用。虽然Cbl-b本身不转移泛素，但它是泛素化级联的单一成分，赋予底物专一性。因此，Cbl-b是一个有吸引力的药物干预靶点。Cbl-b的结构包含7个不同的基序，其中包括一个泛素相关(UBA)结构域，该结构域已被证明与多泛素链具有很高的亲和力和特异性。泛素连接酶反应是在预涂有Cbl-b UBA的试板上进行的，以允许结合和保留多泛素化(从而生物素化)反应产物。我们预计这两种试剂的使用将减少对聚集或非特异性蛋白质结合化合物的假阳性结果的检测。在与Yves Pommier(DTB)博士的合作下，从Ogilvie等人开发的一种分析中重新设计了一种用于发现基于天然产物的Tdp2活性抑制剂的显色分析方法。该方法旨在通过测量底物4-NPPP生成对硝基苯酚的减少来衡量Tdp2活性的抑制程度。基于吸光度的分析将使用透明的、未经处理的384孔板来在405 nm处检测对硝基苯酚的最大吸收。吸光度信号的降低将与假定的抑制剂的效力直接相关。更大的酪氨酸化DNA结构的二次分析正在DTB进行。DTB提供TTRAP/Tdp2酶结构(由位于马里兰州弗雷德里克的蛋白质表达实验室NCI生产)，所有初始酶质量保证在MTL进行评估。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的检测浓度设置在观察到的Km值(Km=115 mM)以下，主要是因为检测器在浓度为100 mM或更高时饱和，并将检测条件的时间延长到20分钟以上。目前，我们已经筛选了所有可用的MTL纯化合物和合成库，共计69,793个化合物，初始命中率为0.22%，预分提天然产物库的命中率为0.82%(命中要求为50%的抑制活性)。HTS已经完成，CBL-b和Tdp2抑制化合物的评估正在进行中。关于DNAJ-PKA和Rpn2/Rpn13的其他合作也在与张平博士和凯莉·沃尔特斯博士进行中。这些分析使用各种技术，包括荧光偏振法、差示扫描荧光法和动力学分析。此外，我们最近开始了一个项目，与Daniel McVicar博士合作，使用热熔融分析来确定代谢酶IRG1的潜在抑制剂，并与Freddy Ecorcia博士合作，确定癌症靶点Glypcan-3。我们还与尤金·瓦尔科夫、伊夫·庞米尔共同启动了新冠肺炎相关项目。

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.