BIOCHEMICAL DISSECTION OF QUINOLONE TARGETS IN MYCOBACTERIUM TUBERCULOSIS

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The recent emergence of multidrug resistant tuberculosis raises the ominous possibility of a high lethal epidemic for a disease that was once considered essentially eliminated from the US population. A combination of factors have brought this problem to the forefront of medical concern. In HIV infected individuals, tuberculosis is a growing epidemic. The strategy that was successful in battling tuberculosis over the last half century in the US has been to give multidrug treatment. However, the world wide explosion in the population of AIDS patients has provided a large and growing reservoir for generating new multidrug resistant forms of several bacterial pathogens. With the appearance of M. tuberculosis strains resistant to all of the standard drugs, there is an urgent need for new approaches and new pharmaceuticals to stop the spread of tuberculosis causing organisms. DNA gyrase is a ubiquitous bacterial enzyme that is essential for maintaining the structure and function of genetic information stored in bacterial cell genome. Gyrase has roles in all aspects of DNA metabolism, including DNA replication, DNA repair, recombination, and gene transcription. Gyrase is a complex enzyme composed of two subunits and it is the target of the quinolone antibiotics. The overall aim of this RFA is to develop new quinolones targeted to kill multidrug resistant forms of M. tuberculosis. There are 4 parts of Project I. Part 1 is focused on gene cloning, sequencing, and obtaining biochemical amounts of the subunits for M. tuberculosis gyrA and gyrB proteins. These aims will be accomplished using PCR cloning and sequencing strategies and by engineering recombinant plasmids that express large amounts of the M. tb gyrase (m- gyrase) in E. coli cells. Part 2 of Project I is developing a quinolone profile of m-gyrase. The quinolone ring has many sites for potential chemical modification and the Parke-Davis Pharmaceutical Research group has synthesized an inventory of 2,300 quinolone drugs. We will screen this collection using gyrase supercoiling and cleavage assays in vitro to find the most potent compounds for testing in cell culture and in animal trials. Part 3 will identify DNA sites that are optimal for M. tb gyrase binding and sites where quinolones most efficiently induce DNA damage by trapping the covalent gyrase-DNA complex. Part 4 will explore the role of supercoil accessory proteins that work with gyrase to form the folded bacterial nucleoid.