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.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Sharp, P M; Bailes, E; Robertson, D L et al. (1999) Origins and evolution of AIDS viruses. Biol Bull 196:338-42
Gao, F; Robertson, D L; Carruthers, C D et al. (1998) A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. J Virol 72:5680-98
Gao, F; Robertson, D L; Carruthers, C D et al. (1998) An isolate of human immunodeficiency virus type 1 originally classified as subtype I represents a complex mosaic comprising three different group M subtypes (A, G, and I). J Virol 72:10234-41
Soares, M A; Robertson, D L; Hui, H et al. (1997) A full-length and replication-competent proviral clone of SIVAGM from tantalus monkeys. Virology 228:394-9
Salminen, M O; Carr, J K; Robertson, D L et al. (1997) Evolution and probable transmission of intersubtype recombinant human immunodeficiency virus type 1 in a Zambian couple. J Virol 71:2647-55
Scheirer, K E; Higgins, N P (1997) The DNA cleavage reaction of DNA gyrase. Comparison of stable ternary complexes formed with enoxacin and CcdB protein. J Biol Chem 272:27202-9
Gao, F; Robertson, D L; Morrison, S G et al. (1996) The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin. J Virol 70:7013-29
Gao, F; Morrison, S G; Robertson, D L et al. (1996) Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J Virol 70:1651-67
Salazar, L; Fsihi, H; de Rossi, E et al. (1996) Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis. Mol Microbiol 20:283-93
Renau, T E; Sanchez, J P; Gage, J W et al. (1996) Structure-activity relationships of the quinolone antibacterials against mycobacteria: effect of structural changes at N-1 and C-7. J Med Chem 39:729-35

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