Development of the HTS Assay:E. Coli RNA Polymerase(RMI)

描述（申请人提供）：RNA聚合酶（RNAP）是基因表达的主要酶，也是遗传调控的靶点。它已被证明是抗菌治疗的靶点，其突变是多药耐药（MDR）结核病问题的核心。这项研究的长期目标是开发基因表达的干预措施，使用小分子化合物特异性干扰RNAP的催化功能或其与细胞调节因子的相互作用。为了寻找RNAP抑制剂，我们设计了用于大化合物库的自动化高通量筛选（HTS）的测定。所提出的测定利用携带荧光基团的核苷酸底物类似物，所述荧光基团在聚合反应中释放时被激活。该测定法已在组分浓度、反应条件方面进行了优化，并适用于HTS。通过从HTS装置中收集的16，000种化合物中检索RNAP抑制剂，证明了该测定的可行性。开发了后续检测试剂盒，以检测初步检测试剂盒中识别的假阳性。实验提出了进一步提高测定性能的小型化和引入额外的后续测定筛选出的化合物，靶向模板DNA，而不是RNAP。该方法将是有用的新的RNAP抑制剂的抗微生物治疗，特别是在克服MDR问题的鉴定。它还将为发现针对特定基因和调控途径的药物开辟新的途径。此外，新的RNAP抑制剂将作为理解RNA合成及其调控的精细机制的有力工具。

DESCRIPTION (provided by applicant): RNA polymerase (RNAP) is the principal enzyme of gene expression and the target for genetic regulation. It has been a proven target for antimicrobial therapy, and its mutations are central to the problem of multi-drug resistant (MDR) TB. The long-term objective of this research is the development of interventions in gene expression using small compounds that specifically interfere with the RNAP's catalytic function or its interaction with cellular regulatory factors. For search of RNAP inhibitors we have designed the assay for automated high throughput screening (HTS) of large libraries of chemical compounds. The proposed assay utilizes nucleotide substrate analogs carrying a fluorescent group, which is activated upon release in the polymerization reaction. The assay has been optimized in respect to concentration of the components, reaction conditions and adapted for HTS. The feasibility of the assay has been demonstrated by retrieving of RNAP inhibitors from the collection of 16,000 compounds in HTS setup. The follow-up assays have been developed to detect false positives identified in the primary assay. Experiments are proposed to further increase the assay performance by miniaturization and introduction of the additional follow-up assay to screen out the compounds that target template DNA rather than RNAP. The methodology would be useful for identification of new inhibitors of RNAP suitable for anti-microbial therapy particularly in overcoming the MDR problem. It will also open new approaches to discovery of drugs targeted to specific genes and regulatory pathways. In addition, new inhibitors of RNAP would serve as a potent tool for understanding of the fine mechanisms of RNA synthesis and its regulation.