Tuberculosis is a serious airborne disease for which drug resistance has become a major issue. The long treatment time (6-24 months) is particularly problematic because it creates difficulties in maintaining patient adherence to therapy. Sporadic treatment then fosters the emergence of antimicrobial-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis. Long treatment times are needed, presumably because even active infections contain some non-growing (dormant) bacilli that are not readily killed by most antimicrobials. Our goal is to obtain new fluoroquinolones that rapidly kill non-growing M. tuberculosis and would thereby reduce treatment time radically. As part of their mechanism of action, quinolones trap DNA gyrase as drug-enzyme-DNA complexes in which the DNA is broken but held together by protein. Formation of these complexes, which are thought to block bacterial growth, is reversed by chelation of magnesium ion. Recent work leads us to propose that at lethal quinolone concentrations, which are higher than required to block growth, additional drug binding generates a new complex that is not reversed by chelation of magnesium. We propose that the new complex leads to chromosome fragmentation, which in growing cells is followed by a lethal cascade of reactive oxygen species. Only a quinolone subset kills non-growing cells. These quinolones are proposed to stimulate chromosome fragmentation that causes death directly, without requiring a cascade of reactive oxygen species. The above scenario will be tested by designing, synthesizing, and characterizing new quinolones and quinolone- derived structures for interactions with gyrase and for killing non-growing cells.
In aim 1, tests with cultured cells will examine relationships between experimental compounds and gyrase structure, using gyrase mutants, in-vivo crosslinking, drug structure variation, and molecular modeling. The primary readout will be cell death when protein synthesis is inhibited to eliminate the lethal pathway operating with growing cells.
In aim 2, experiments will focus on biochemical properties of the newly discovered quinolone-gyrase-DNA complex formed at the elevated drug concentrations required to kill bacteria. Attention will focus on drug structures that facilitate release of DNA breaks from gyrase-mediated constraint.
In aim 3, drug structure- lethal activity relationships will be determined with computational analyses (eg. 3-dimensional quantitative structure-activity relationship models) that do not rely on knowledge of drug-gyrase interactions. Superior compounds from work on each of the three aims will be examined for lethality with three mycobacterial growth-arrest systems. The expected outcomes are lead compounds and novel rules for developing new quinolone-type inhibitors that rapidly kill non-growing, persister cells.
New fluoroquinolones will be developed to kill non-growing bacterial subpopulations thought to be responsible for the long treatment times needed to cure tuberculosis. The work will test a new molecular explanation for how fluoroquinolones kill non-growing bacteria. The products of the work will be 1) a molecular understanding of quinolone-mediated killing of non-growing cells, and 2) a new type of quinolone that will help restrict the emergence of quinolone resistance with many bacterial infections.
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