Using a model Gram (+) organism, Bacillus subtilis, we propose to develop and run robust high throughput screening assays as well as appropriate specificity assays and counterscreens to enable the discovery of small molecule inhibitors that have the potential to be developed into antibacterials and step-specific perturbants of DNA replication pathways. This model organism is closely related to most common Gram (+) human pathogens such as S. aureus, S. pyogenes and the biodefense category A organism, Bacillus anthracis. Over the last three decades only 2 new chemical classes of antibiotics have been approved by the FDA and it is widely recognized that bacterial resistance to exisiting classes of antibiotics is increasing. Presently there are no antibacterials targeting the essential process of DNA replication in bacteria. Bacterial DNA replication is initiated by a specific origin binding protein that recruits helicase assembly proteins and the replicative helicase. The helicase, once assembled on DNA, provides an interaction site for primase, the enzyme that generates RNA primers for DNA synthesis. The helicase also plays a role in recruiting the cellular replicase, DNA polymerase III holoenzyme, which has the processivity to synthesize the entire chromosome without dissociation. In spite of this potential, most replicases encounter damage, resulting in replication fork collapse. This can be counteracted by a special origin-independent replication restart apparatus that can reassemble replication forks. Altogether, these processes employ at least 20 different essential proteins. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterials, and will also serve as an ideal system for developing chemical genetic approaches to perturb the various interactions and reaction stages.
Bacterial pathogens are increasingly becoming resistant to commonly used antibiotics in both community and hospital settings, representing a growing public health problem. This has driven the need for research to discover new antibacterials that affect unexploited targets for which resistance is absent. This work will explore a variety of such unexploited targets, essential for bacterial DNA replication in a model organism closely related to many common human pathogenic bacteria, which will aid the discovery of such new antibacterial compounds.
| Dallmann, H Garry; Fackelmayer, Oliver J; Tomer, Guy et al. (2010) Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential. Biochemistry 49:2551-62 |
