Mechanistic and structural analysis of topo IV and gyrase and their targeting by antibacterial quinolones

Topo IV and gyrase are enzymes that untangle bacterial chromosomes and are important targets of anti-infective drugs. Both enzymes make a DNA break (known as a 'gate') in one DNA molecule and pass a second DNA helix through the gate. Quinolone drugs kill bacteria by stabilizing the enzyme-opened gate. This project aims to understand how topo IV and gyrase mediate gate opening and closure, and how drugs interfere with these fundamental processes. The study builds on insights gained from our recent X-ray crystal structure that revealed how quinolones act at the topo IV-DNA gate. This structure provides a number of intriguing mechanistic hypotheses to be tested and paves the way for the design and evaluation of new inhibitors with novel modes of action that should minimize the emergence of drug resistance.

Topo IV and DNA gyrase regulate DNA supercoiling and chromosome segregation and are important targets of antibacterial quinolones used against Streptococcus pneumoniae and other Gram-positive organisms. Both enzymes act by passing a DNA duplex through a transient double-stranded break in a 'gate' or G-duplex involving formation of a transient covalent enzyme-DNA intermediate known as the 'cleavage complex'. In a very important advance, we recently solved the crystal structure of cleavage complexes formed by the topo IV ParC breakage-reunion- and ParE metal binding (TOPRIM) domains in complex with a G-DNA gate stabilised by moxifloxacin and clinafloxacin, two antipneumococcal fluoroquinolones. The structure suggests how the DNA G-gate may operate in tandem with N- and C- protein gates to facilitate DNA transport, and moreover provides a wealth of drug-enzyme-DNA information to guide drug design. We aim to test a number of hypotheses relating to how ParE interacts with ParC, DNA or ATP to coordinate gate recognition and opening. In addition, a combination of biochemical, structural and chemical approaches will be used to investigate novel topo IV inhibitors engineered to have greater potency, that target topo IV and gyrase equally (dual action to minimise development of resistance) or act irreversibly (to reduce resistance). Completion of the work will provide new insights on the reaction cycle of topo IV and gyrase and their interactions with novel antibacterial drugs.