Ellie Nichols, Kaleigh Copenhaver, Josh Beckham (RE), Jon Robertus (PI)
Across the world, medical issues as a result of Staphylococcus aureus infections are a significant public health problem. Exposure to the S. aureus pathogen can lead to a variety of ailments, including cellulitis, pneumonia, meningitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis6. Additionally, antibiotic resistance to current treatments has emerged and continues to create a challenge in not only treating S. aureus infections, but also preventing the spread of the bacteria7. D-alanine:D-alanine ligase is an essential enzyme in peptidoglycan production within S. aureus. Disruption of this process can lead to the death of bacterial cells. The overall objective of this research is to find specific ligands via virtual screening that can bind to this target protein and inhibit its function as validated through wet lab protocols. Several different computational ligand libraries of varying sizes have been screened using GOLD, and the results of these screens compiled into a list of the highest-scoring ligands. Thus far, virtual screening has provided a large list of potential drug compounds, with docking scores from 69.78 to 95.60. These ligands will be further evaluated for their potential to serve as inhibitors of D-alanine:D-alanine ligase of S. aureus. Top ligands found via virtual screening will be tested in biochemical assays, to validate the GOLD predictions. We are working on cloning the coding DNA sequence of the target protein into an expression plasmid. Gene synthesis has successfully been done using primer overlap PCR and cloning will be done via V-PiPE and I-PiPE methods. IPTG induced expression, nickel purification and SDS-PAGE characterization will be conducted to produce and analyze the target protein. Enzyme and inhibition assays will be used to measure enzymatic activity. Any novel compounds found can be used for future drug discovery.
Comments
Thank you for sharing these findings. As a project with a wet lab side and a virtual side, and with several student researchers involved, did you specialize in one side or the other? Or did you get experience on both sides? —Rob Reichle
Thank you for viewing our poster! We each did about 50/50 virtual and wet lab research, so we could both learn the procedures. —Ellie Nichols