Fragment-Based Drug Discovery via Thermal Shift Assay
Disciplines
Biochemistry | Medicinal-Pharmaceutical Chemistry | Other Chemistry
Abstract (300 words maximum)
The bacterium P. aeruginosa is recognized for its ability to frequently develop multidrug resistance to antibiotics during treatment of infections. My project focuses on strategies to find novel antibiotics that are less likely to evolve resistance rapidly by studying how solution conditions and small fragments can perturb protein binding interfaces. I have established a screen of buffer conditions varying in pH and salt using a thermal shift assay as part of the Fragment- Based Drug Discovery (FBDD) method to optimize thermodynamic stability of the target P. aeruginosa glutaredoxin protein. This method 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. The thermal shift assay uses the fluorescence of a hydrophobic dye to monitor the stability of the protein as it is subjected to increasing temperatures. Progress towards a screen of small fragments from an existing library of lead molecules for hits that non-covalently bind to and increase stability in the target protein will be presented. The optimized buffer conditions will be combined with several previously observed fragment hits that bind to glutaredoxin to observe the thermal shift obtained. Nuclear magnetic resonance (NMR) data will be collected on the newly obtained fragment-protein complexes and compared to existing multidimensional NMR data from previously obtained fragment-protein complexes.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Thomas Leeper
Fragment-Based Drug Discovery via Thermal Shift Assay
The bacterium P. aeruginosa is recognized for its ability to frequently develop multidrug resistance to antibiotics during treatment of infections. My project focuses on strategies to find novel antibiotics that are less likely to evolve resistance rapidly by studying how solution conditions and small fragments can perturb protein binding interfaces. I have established a screen of buffer conditions varying in pH and salt using a thermal shift assay as part of the Fragment- Based Drug Discovery (FBDD) method to optimize thermodynamic stability of the target P. aeruginosa glutaredoxin protein. This method 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. The thermal shift assay uses the fluorescence of a hydrophobic dye to monitor the stability of the protein as it is subjected to increasing temperatures. Progress towards a screen of small fragments from an existing library of lead molecules for hits that non-covalently bind to and increase stability in the target protein will be presented. The optimized buffer conditions will be combined with several previously observed fragment hits that bind to glutaredoxin to observe the thermal shift obtained. Nuclear magnetic resonance (NMR) data will be collected on the newly obtained fragment-protein complexes and compared to existing multidimensional NMR data from previously obtained fragment-protein complexes.