Development of a HTS system:topoisomerase targets (RMI)

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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.