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Abstract
Grant Number: 1R03NS050811-01 Project Title: LRET-based HT Screening:Inhibitors of Transcription(RMI)
PI Information: Name Title BURGESS, RICHARD R. rburgess@wisc.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): The binding of sigma factors to core RNA polymerase is essential for specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, it is a promising target for drug discovery. A homogeneous assay for sigma-binding to RNA polymerase (E. coli) based on luminescence resonance energy transfer (LRET) has been developed in our lab using an europium-labeled sigma70 and IC5-labeled fragment of the beta-prime subunit of RNA polymerase (amino acid residues 100-309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5-emission.The technical advances offered by LRET resulted in a very robust assay suitable for a high-throughput screening and was successfully used to screen a crude natural product library. We would like to refine this assay, to develop appropriate counter-screens to minimize the number of false positives, and to develop further methods for characterizing the confirmed hits. Then we will use these assays to screen much larger libraries and hopefully to identify and characterize chemical compounds that might represent lead compounds for new antibiotic drug development.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
DNA directed RNA polymerase, antibiotic, bacterial protein, drug discovery /isolation, fluorescence resonance energy transfer, gene induction /repression, genetic transcription, high throughput technology, protein binding, protein protein interaction, technology /technique development, transcription factor
bacterial genetics, microorganism growth
Escherichia coli, biotechnology, chemical registry /resource
Institution: UNIVERSITY OF WISCONSIN MADISON 21 N. Park Street, Suite 6401 MADISON, WI 537151218 Fiscal Year: 2004 Department: ONCOLOGY Project Start: 30-SEP-2004 Project End: 31-AUG-2006 ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE IRG: ZNS1