Resistance to antibiotics by pathogenic microorganisms poses significant problems to human health. Research and development on novel antibiotics has been slow, particularly in comparison to the rapid development of resistance in microbial populations. The research project described in this narrative is an interdisciplinary effort by Professors Richey, Haile, Toth, and Muzyka to identify novel antibacterial drugs. The goals of the research are to identify antibacterial drug candidates via computer-aided structure-based ligand screening and design (Muzyka and Toth), assay candidate inhibitors for antibacterial activity (Richey), and characterize the biochemical properties (enzyme kinetics) of inhibitors that display antibacterial activity (Haile). This kind of approach has been successful in the development of several drugs that are commercially available, including HIV protease inhibitors.
An antibacterial drug is likely to be most successful if it is directed at enzymes that are necessary to the survival of bacteria but have no human analog. Thus, the Mur family of enzymes (MurA-MurF) has been selected for study as they catalyze the biosynthesis of peptidoglycan in the bacterial cell wall. Without peptidoglycan, the cell wall has less mechanical strength and is unable to resist forces of osmotic pressure. Human cells do not have cell walls, therefore they are not affected by drugs that target the bacterial cell wall. The mechanisms of action for these enzymes have been studied and their X-ray crystal structures are known. Derivatives of phosphinic acid inhibit Mur C through Mur F; however, only one FDA-approved antibiotic (fosfomycin, shown below left in comparison to the native ligand phosophenol pyruvate) targets an enzyme in this family, MurA (shown, above right). Other researchers have reported ligands (molecules that bind to another molecule) which bind to and inhibit these enzymes, but most of these compounds do not exhibit antibacterial activity.
Muzyka and her students are using computational methods to identify potential inhibitors of the MurA enzyme. During the summers of 2009 and 2010, we docked and scored a library of one million compounds that are available commercially. We found over one thousand compounds which have better scores than the natural ligand for this enzyme. We have acquired some of these compounds for testing.
Richey and her students conduct antibacterial assays against a broad range of bacteria for each MurA-binding chemical identified by Dr. Muzyka’s lab. Chemicals that show promising inhibitory activity as detected by the initial antibacterial assays are subjected to MIC (minimal inhibitory concentration) and MLC (minimal lethal concentration) testing against the same panel of bacteria. Throughout these tests, we will directly compare the antibacterial activity of these ligands to fosfomycin, a known MurA inhibitor, as well as to antibiotics that target other bacterial structures.
Haile and her students are isolating and purifying the MurA
enzyme. The purified enzyme will be characterized by measuring
the kinetics for the reaction between UDP-NAG and phosphoenol pyruvate
in the presence of purified MurA enzyme. Potential inhibitors
identified by Muzyka's lab will be studied in the same reaction
conditions, to determine how they influence the reaction.
In 2012, Muzyka and her students began carrying out molecular dynamics calculations with these compounds on the supercomputer at the University of Kentucky. These calculations will allow us to better understand how each compound examined interacts with the amino acids in the protein's active site. We will carry out similar calculations with fosfomycin so that we can compare binding of potential inhibitors to this known inhibitor. The computational results will be analyzed in parallel with the experimental results, hopefully providing insights into the observations.
Joining us in 2014, Dr. Toth and his students carry out virtual screening with AutoDock Vina, enabling us to do consensus scoring by combining the results from AutoDock Vina and DOCK. Dr. Toth and his students use a pair of 64-core servers on campus that are dedicated to drug discovery work. Sometimes he and his students use the XSEDE supercomputers and the Blue Waters supercomputer. One computer science student has created a web-based application and database to enable the group to keep track of all the data generated.
Another aspect of this project is the synthesis of potential inhibitors that are related to high scoring compounds from our docking study. Each compound we synthesize will be analyzed computationally (docking and scoring) as well as molecular dynamics. These computational studies will enable us to determine how they compare to the natural ligands. Samples of the compounds will be tested by our collaborators (Haile and Richey) for antibacterial and enzyme inhibiting abilities.
If you are interested in participating in this research project, download the application and complete it. You may submit it to Dr. Haile, Dr. Muzyka, Dr. Richey, or Dr. Toth in hard copy or by email.
Team MurA, summmer 2015
||Dr. January Haile, Dr. Jennifer Muzyka, Dr. Peggy
Richey, Dr. Dave Toth
|summer 2015||Han He, Rachel Hammer, Stephen Dockins, Matt Silverman, Griffin Cote|
|academic year 2014-15||Blake Martin, Abby Patterson, Grace Anastasio, Matt Silverman, Stephen Dockins, Tanner Lyons|
|summer 2014||Luke Presson, Daniel Graham, Griffin Cote, Bryan Wright, Abby Patterson, Grace Anastasio, Zaid Siddiqui|
|academic year 2013-14||Josh Winner, Leila Samhat, Emily Dennis, Michael Orr, Eric Gantzer, Luke Presson, Daniel Graham|
||Josh Winner, Leila Samhat, Nicki Frost, Emily Dennis, Michael Orr, Ashley Christian|
|academic year 2012-13||Vanessa Song, Ben Boone, Nicki Frost, Calvin Cahall, Travis Carroll, Natalie Orms, Emily Dennis|
||Josh Winner, Vanessa Song, Ben Boone, Nicki Frost, Megan Phelps, Emily Dennis|
|academic year 2011-12
||Natalie Orms, Josh Winner,
Selina Li, Beau Bennett, Vanessa Song, Ben Boone, Marcus Niemann
||Ben Boone, Trinity Hochstetler,
Natalie Orms, Rebecca Juhl, Beau Bennett
|academic year 2010-11
||Zack Sweeney, Emily Hogencamp, Kyle Forte, Katie Lentz, Trinity Hochstetler, Ben Boone
||Zack Sweeney, Trinity Hochstetler, Beau Sauley, DJ Allen
|academic year 2009-10
||Jordan Feigerle, Louesa Akin, Kyle Forte, JB Boone, Max Bishop
||Anita Allison, Louesa Akin, Matt
Williams, Sarah Wilson, Matt Spencer, Max Bishop
|academic year 2008-09