Actions of spiropyrimidinetriones against bacterial type II topoisomerases

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PROJECT SUMMARY Antibacterial resistant infections cause more than 1.2 million deaths around the world each year, and this number is predicted to grow to 10 million by 2050 unless resistance is effectively curbed. A pathogen of particular concern is drug-resistant Neisseria gonorrhoeae, which is listed as one of five “urgent threats” (the highest threat level) by the Centers for Disease Control and Prevention (CDC). Fluoroquinolones (FQs) were recommended as frontline treatment for the 98 million annual cases of gonorrhea since 1993, but their use was discontinued in 2007 due to increasing levels of target-mediated resistance. This resistance occurs when specific mutations in the target for FQs, the bacterial type II topoisomerases, gyrase and topoisomerase IV, arise in response to FQ treatment. One strategy to overcome resistance is to develop new compounds that interact with bacterial type II topoisomerases at amino acid residues distinct from those of FQs. Using this approach, a new class of gyrase/topoisomerase IV-targeted agents with a spiropyrimidinetrione (SPT) pharmacophore was identified. Gyrase and topoisomerase IV regulate the topological state of DNA in bacterial cells. These essential enzymes control levels of DNA supercoiling (over- and underwinding) and remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded DNA break generated in a separate DNA segment. Both FQs and SPTs stabilize the covalent enzyme-cleaved DNA complex (cleavage complex) such that when advancing replication and transcription machinery approach these complexes, they can fragment the genome, triggering the SOS response and other cell death processes. The primary goal of this project is to overcome FQ resistance in N. gonorrhoeae by increasing our understanding of SPT interactions with gyrase and topoisomerase IV and target-mediated resistance development across bacterial species to inform the design of more potent and efficacious antibacterials. This goal will be addressed by three specific aims: In Specific Aim 1, I will assess the actions of SPTs against gyrase and topoisomerase IV from N. gonorrhoeae, Bacillus anthracis, and Escherichia coli to determine how the activities of this class varies across bacterial species. To this end, I will use assays that measure DNA cleavage, cleavage complex stability, and catalytic inhibition with wild-type enzymes. In Specific Aim 2, I will evaluate the ability of novel SPTs to overcome FQ- and SPT-resistance mutations in gyrase and topoisomerase IV and describe the basis by which mutations in these enzymes confer resistance to SPTs. This will require similar enzymological activity assays as described in Specific Aim 1. In addition, I will measure SPT-enzyme binding interactions and conduct competition studies to determine if resistance mutations affect drug binding and/or placement in the gyrase/topoisomerase IV active site. Finally, in Specific Aim 3, I will assess the levels and persistence of SPT-stabilized cleavage complexes generated by gyrase and topoisomerase IV in N. gonorrhoeae cells using an in vivo complex of enzyme bioassay. These studies have the potential to inform the development of new drugs to overcome antibacterial resistance.