Cell-free assay technologies for the identification of active compounds

PCMBS与MTP分析开发和筛选科(ADSS/Henrich)密切合作，以自动化、验证完整的筛选，与化学多样性发展科(CDDS/Beutler)密切合作，获取新的化学多样性来源，并与天然产品化学科(NPCS/Grkovic)密切合作，优先选择提取液进行天然产品分离，并对NPCS/Grkovic或CDDS/Beutler分离的纯活性化合物进行生物化学评价。在本审查期间，PCMBS努力的例子包括发表的关于对天然产物进行筛选的论文，以筛选蛋白酶MALT15、泛素连接酶CBLB8、11、13、14和磷酸二酯酶Tdp13的选择性抑制剂。我们对Tdp1天然产物抑制剂的研究也导致了第二篇论文描述了一类新的环肽，这是首次报道的这种磷酸二酯酶的变构抑制剂。我们最近还完成了对磷酸二酯酶TDP2抑制剂的筛选，并正在准备一份手稿。此外，PCMBS继续增加生物物理筛选方法的用途，以确定蛋白质和RNA基序的热稳定性的特定调节因子。最初，这项工作导致了一种新的小分子支架的鉴定，它改变了HIV-1 TAR RNA的稳定性。这项技术现在已得到扩展，允许以高通量格式方便地筛选纯化合物和天然产品样本，以确定RNA和蛋白质稳定性的调节剂。最近发表的一个例子是鉴定了一种天然产物，它选择性地调节了前miR-216的热稳定性。目前，我们正在进行另外两个新的酶筛选，针对嵌合激酶DNAJ/PKAC和肌醇1，3，4-三磷酸5/6激酶(ITPK1)，以及三个新的生物物理筛选；针对免疫相关的代谢酶免疫反应基因1(IRG1)，肝细胞癌GPC-3(GPC-3)和SARS-CoV-2受体结合域(RBD)。PCMBS还评估从MTP筛选中提取的化合物与大分子靶标的特定相互作用。我们使用生物物理和生化技术来评估前miR-21RNA6、Tdp13、9、MALT15和SARS-CoV-2病毒进入的抑制剂。

The PCMBS collaborates closely with the MTP Assay Development and Screening Section (ADSS/Henrich) to automate, validate complete screens, with the Chemical Diversity Development Section (CDDS/Beutler) to access new sources of chemical diversity and with the Natural Products Chemistry Section (NPCS/Grkovic) to prioritize extracts for natural product isolation and biochemically evaluate pure active compounds isolated by NPCS/Grkovic or CDDS/Beutler. Examples of PCMBS efforts during this review period, include published papers on screens of natural product for selective inhibitors of the protease MALT15 , the ubiquitin ligase CBLB8,11,13,14, and the phosphodiesterase Tdp13. Our studies on natural product inhibitors of TDP1 also resulted in a second paper describing a new class of cyclic peptides which are the first reported allosteric inhibitors of this phosphodiesterase. We have recently also completed a screen for inhibitors of the phosphodiesterase TDP2 with a manuscript in preparation. In addition, the PCMBS has continued to increase utility of biophysical screening methods for the identification of specific modulators of the thermal stability of both protein and RNA motifs. Initially, this work resulted in the identification of a new small molecule scaffold that altered the stability of HIV-1 TAR RNA. This technology has now been expanded upon to allow for the facile screening of pure compounds and natural product samples in a high throughput format to identify modulators of RNA and protein stability. One recently published example was the identification of a natural product that selectively modulated the thermal stability of pre-miR-216. We currently have two new other enzymatic screens in progress, against the chimeric kinase DNAJ/PKAC and inositol 1,3,4-triphosphate 5/6 kinase (ITPK1), as well as three new biophysical screens; against the immunologically-pertinent metabolic enzyme Immune Responsive Gene 1 (IRG1), the diagnostic target for hepatocellular carcinoma glypican-3 (GPC-3)and the SARS-CoV-2 receptor binding domain (RBD). The PCMBS also evaluates compounds, derived from MTP screens, for their specific interactions against macromolecular targets. We have used both biophysical and biochemical techniques to evaluate inhibitors of pre-miR-21 RNA6, Tdp13,9, MALT15, and SARS-CoV-2 viral entry.