Resistance to antibacterial therapies continues to be an urgent threat to human health, particularly bacteria that are already resistant to multiple antibiotics. To discover and develop new antibacterials, it is important to find and exploit under-utilized antibiotic targets. One attractive candidate is the divisome, the dynamic protein complex that splits bacterial cells in two. The bacterial divisome contains a set of highly conserved and essential proteins that act coordinately to ensure the correct timing and placement of the cell division septum at mid-cell. The septal transpeptidase is already a target of several widely used beta-lactam antibiotics, but no other divisome protein is currently targeted. FtsZ, a highly conserved polymer-forming GTPase that forms a membrane-associated "Z ring" required for organizing the septal transpeptidase and other septum-synthesizing enzymes, is the only other divisome protein that has been studied extensively as a target of small molecules and peptides, and our lab has helped to advance the understanding of FtsZ and its interacting proteins for 30 years. Nevertheless, there is still much to learn about how small molecules perturb FtsZ function at the molecular and cellular level and whether these can lead to potential therapeutics. To address this over-arching theme, this proposal seeks to define, structurally and physiologically, two different sets of promising new small molecule inhibitors of FtsZ. The first is a set of two related benzamide derivatives, synthesized by our medicinal chemistry collaborators, that have high potencies against both Gram-positive bacteria and Gramnegative bacteria with disabled efflux pumps. These derivatives were synthesized to have optimized binding to the interdomain cleft (IDC) of FtsZ, a common target of inhibitors that we have termed FtsZ's "Achilles Heel". Unexpectedly, we found that these two compounds perturb FtsZ by distinct mechanisms in Gram-positive versus Gram-negative bacteria, prompting the hypothesis that they disrupt FtsZ's ability to assemble into the proper condensed polymer architecture needed for cell division to progress further. This model will be tested with our laboratory's unique interdisciplinary array of genetic, cytological, and structural biology methods. The second set of small molecule inhibitors is a pair of bacteriophage peptides that have evolved to target FtsZ as part of their lytic cycle. Each peptide binds directly to FtsZ and blocks Z ring assembly, but also binds to another essential divisome protein that tethers FtsZ to the cytoplasmic membrane. The molecular details of these binding sites are unknown, but we hypothesize that they do not involve the FtsZ IDC and instead perturb Z ring assembly by novel two-pronged mechanisms. Again, we will apply our extensive expertise in genetics, microscopy and structural biology of divisome proteins to elucidate these mechanisms. The insights we will gain from the proposed studies should lay a foundation for the future therapeutic potential of small molecules and peptides, potentially in combination with each other or with other antibiotics, to kill bacteria by disrupting the cell division machinery.

Project Narrative: Pathogenic bacteria are becoming more resistant to most existing antibiotics. As bacterial cells need to divide in order to successfully colonize their host and proliferate, bacterial cell division proteins are an important and relatively under-utilized targets for novel antimicrobials. The design of new and better drugs requires a mechanistic understanding of how small molecule and peptide inhibitors inactivate their targets.