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High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog
Jyothi Bhat1, Rajendra Rane, Suresh M Solapure
1AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
Abstract:
RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.
Insights
Researchers developed a novel high-throughput screening assay targeting RNA polymerase (RNAP) to find new antibacterial agents. The assay successfully identified compounds active against bacterial RNAP, including those relevant to tuberculosis.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- RNA polymerase (RNAP) is a crucial target for developing antibacterial and antituberculosis drugs.
- Purifying mycobacterial RNAP is challenging, necessitating alternative screening methods.
- Escherichia coli RNAP serves as a surrogate for initial screening.
Purpose of the Study:
- To develop and validate a high-throughput screening (HTS) assay for identifying novel RNAP inhibitors.
- To screen a large compound library for inhibitors of bacterial RNAP.
- To assess the activity of identified hits against relevant mycobacterial RNAP.
Main Methods:
- A novel HTS assay utilizing a fluorescent UTP analog (gamma-AmNS UTP) was developed.
- The assay measures increased fluorescence upon UMP incorporation into RNA, releasing fluorescent gamma-AmNS-PPi.
- The assay was optimized for a 384-well format and used to screen 670,000 compounds at 10 microM.
Main Results:
- Approximately 0.1% of screened compounds exhibited >60% inhibition in the primary HTS.
- All primary active compounds showed dose-response EC(50) values below 100 microM.
- Eighty percent of hits active against E. coli RNAP also showed activity against Mycobacterium smegmatis RNAP.
Conclusions:
- The developed HTS assay is effective for identifying novel inhibitors of bacterial RNA polymerase.
- The assay successfully identified promising hit compounds with activity against mycobacterial RNAP.
- This approach facilitates the discovery of new antibacterial and antituberculosis agents targeting RNAP.
