We intend to develop new chemical classes of antibacterials by focusing on an under-exploited target in a well- validated pathway. Specifically, we will develop and apply a high-throughput screen for inhibitors of the Escherichia coli DnaA protein, an essential target in the DNA replication pathway. Our overall goal is to identify specific DnaA inhibitors and develop them into novel antibiotics in order to treat resistant bacterial infections. In Phase I, we will optimize a cell-based DnaA assay for a high-throughput screening application. We will use this assay to screen a diverse library of over 100,000 discrete small molecule compounds and purified natural products. The high-throughput screen will measure stimulation of E. coli growth by DnaA inhibitors. Under control conditions, the E. coli strain is unable to grow due to the engineered lethal overinitiation of DNA synthesis by mutant DnaA. This assay will detect inhibitors of any of the multiple essential DnaA functions, including DnaA oligomerization, ATP binding and hydrolysis, unwinding of DNA at the replication origin or recruitment of the DnaB helicase loader. We will confirm hits and eliminate non- specific DNA-binding compounds. Promising compounds from this screen will be tested for antibacterial potency across a panel of Gram-negative and Gram-positive bacterial pathogens. Specificity for DNA replication and cytotoxicity to mammalian cells in culture as well as biochemical (target-based) selectivity will be evaluated to generate a series of validated hits.
The specific aims are to (1) develop a high-throughput screening assay for the essential DnaA protein of E. coli; (2) screen a diverse compound library to identify and confirm inhibitors of E. coli DnaA; and (3) prioritize confirmed screening hits for spectrum, potency, mechanism and selectivity. In Phase II, we will characterize the mechanism of action of the validated hits in more detail and optimize the most promising of these structures utilizing a rational drug design approach to develop antibacterial lead compounds. ? ? Multi-drug resistance is reducing the effectiveness of current antibiotics and very few new antibiotics are in the development pipeline. This proposal describes a new type of screening assay that can identify inhibitors of DnaA, a previously unexploited protein involved in the initiation of DNA synthesis. ? ? ?
