Research Project
7056863
[unreadable] 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. [unreadable] [unreadable]
