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Abstract
Grant Number: 1R03NS050782-01 Project Title: Development of a HTS system:topoisomerase targets (RMI)
PI Information: Name Title TSE-DINH, YUK-CHING yuk-ching_tse-dinh@nymc.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): DNA topoisomerases are important targets for therapy against potential viral and bacterial pathogens. The removal of transcription-driven positive and negative supercoils in DNA is a major function of topoisomerases, including poxvirus topoisomerase I. The trapping of covalent intermediates complexed with cleaved DNA formed by topoisomerases during removal of transcription-driven supercoiling can lead to immediate loss of infectivity. However, cell based viral or bacterial count assays cannot distinguish between such topoisomerase-targeting mechanism and other modes of anti-infectivity. In vitro assays with purified enzyme and DNA substrates do not provide twin domains of positive and negative supercoiling that are the sites of action of topoisomerases involved in transcription in vivo. An E. coli based assay would place the target topoisomerase at sites of transcription-driven supercoils, allowing identification of agents that would trap the target topoisomerases during active transcription. The concurrent use of a target topoisomerase active site mutant can immediately confirm if the positive result is due to trapping of covalent topoisomerase complex. Based on our previous work on the effect of vaccinia virus topoisomerase I expression on E. coli DNA supercoiling, we plan to develop a system suitable for high-throughput screening of chemical libraries for potential small pox therapeutic agents that will act by trapping poxvirus topoisomerase complexed with cleaved viral DNA. The screening system should also be applicable for targeting topoisomerases in pathogenic bacteria relevant for biodefense.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
DNA topoisomerase, Escherichia coli, active site, antiviral agent, drug discovery /isolation, genetic transcription, high throughput technology, smallpox virus, technology /technique development
DNA damage, DNA gyrase, Poxviridae disease, camptothecin, chemical cleavage, circular DNA, enzyme complex, gene mutation, genetic strain, microorganism disease chemotherapy, phenylalanine, recombinant protein, reporter gene, vaccinia virus, virus DNA
biotechnology, bioterrorism /chemical warfare, chemical registry /resource
Institution: NEW YORK MEDICAL COLLEGE ADMINISTRATION BUILDING VALHALLA, NY 10595 Fiscal Year: 2004 Department: BIOCHEM AND MOLECULAR BIOLOGY Project Start: 30-SEP-2004 Project End: 31-AUG-2006 ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE IRG: ZNS1