P. aeruginosa is a pathogen known to cause severe illness in humans, particularly patients who are immunocompromised. The bacterium is recognized for its ability to frequently develop multidrug resistance to antibiotics. This project will guide and develop original strategies to find novel antibiotics that are less likely to evolve resistance rapidly. Fragment-Based Drug Discovery is a strategy that screens for small molecules that bind selectively to a target protein; these could then be coupled to reactive warhead inhibitors to covalently inhibit the target protein. Thermal shift assay, using a targeted protein, has provided data for various conditions or fragments simultaneously. Previous aims for this project included finding appropriate dye and protein concentrations, optimizing buffer conditions to enhance protein thermodynamic stability, and screening of a library of small molecules for hits that covalently bind to and further increase protein stability. This assay uses the fluorescence of a hydrophobic dye to monitor the stability of the protein as it is subjected to increasing temperatures. This assay allowed for the screening of various drug fragments that bind the P. aeruginosa glutaredoxin protein, a factor that mitigates reactive oxygen species and is a model for biophysical studies. This factor also has a dithiolate active site that may be inhibited by known warhead chemistries. The use of this thermal shift FBDD assay has provided a fragment screen of hits that can be further analyzed by organic chemists as putative drug leads. Current project goals aim to validate fragment hits that showed a significant shift in the apparent melting temperature during the screening process, as well as attempt to obtain a Kd to help determine binding affinity of the ligand and protein by using nuclear magnetic resonance and isothermal titration calorimetry. Results to obtain these validated hits will be presented.

Thermal- shift assay development for finding novel antibiotics targeting a Cystic Fibrosis pathogen

2019 Spring National Meeting

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