Muzyka, Jennifer L

Project 1:

Structure Based Drug Design:  Attacking COVID

Jennifer Muzyka, Centre College

October 2020

COVID-19 has disrupted many of the routines of everyday life in 2020.  In response to this disruption, my student collaborators and I have shifted our attention from the search for antibiotics to the search for antivirals.  Perhaps we will be able to persuade some collaborators to join us on this project.

The X-ray crystal structure of the main protease for SARS-CoV-2 has been reported and serves as a basis for our structure-based drug design project.  Without the functioning of its protease enzyme, the SARS-CoV-2 virus would be unable to assemble viral proteins and new viral RNA.  Some existing protease inhibitors (lopinavir and ritonavir) have shown to be effective against SARS-CoV-2.

We will begin with a docking study to determine which compounds bind to the active site of the enzyme.  We have previously used DOCK on UK’s supercomputer, when we studied the Mur family of enzymes.  We anticipate again using DOCK for this project, but we are also exploring other docking software at this time.  After completion of the docking study we will follow up with molecular dynamics calculations on high scoring ligands.  For the molecular dynamics calculations, we will use AMBER on the UK supercomputer.

Students:

Anna Bachman, Centre College

Mason Saunders, Centre College

Sam Biggerstaff, Centre College

Chani Akenpaul, Chemistry, Centre College, Added on LCC resources, 11/11/2022 

Student Presentations

November 2014
In November 2014, Luke Presson and Daniel Graham both gave oral presentations on their work at the Kentucky Academy of Science meeting. Daniel won second place in the analytical/physical chemistry section for his presentation. In November 2012, Vanessa (Yang) Song and Josh Winner gave presentations about their work on the MurA project. Vanessa won first place in the chemistry section for her presentation.

Here are the details from these student presentations
“Molecular Dynamics on Potential MurA Inhibitors,” Josh Winner and Jennifer Muzyka, Kentucky Academy of Science annual meeting, Richmond, KY, October 20, 2012.

“Potential MurA Inhibitors,” Yang (Vanessa) Song and Jennifer Muzyka, Kentucky Academy of Science annual meeting, Richmond, KY October 20, 2012.

“A Search for Novel Inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA),” Luke Presson and Jennifer Muzyka, Kentucky Academy of Science annual meeting, Lexington, KY, November 15, 2014.

“Identifying MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) Inhibitors using DOCK 6 Screening,” Daniel Graham and Jennifer Muzyka, Kentucky Academy of Science annual meeting, Lexington, KY November 15, 2014.


August 2013
At this time the only research project we have ongoing involves the study of the Mur enzymes. So far we have only worked with MurA. In the near future we will begin examining MurC and/or MurD. The experimental and computational methods used will be the same, and the goals are the same.

The computational methods used are batch submission of jobs running molecular dynamics calculations (AMBER) and virtual screening calculations (DOCK). AMBER is routinely used by UK scientists. DOCK is not typically used by others at UK. I have installed DOCK in my directory.

All personnel involved in this project are at Centre College. My collaborators are Professor January Haile and Professor Margaret (Peggy) Richey. Students involved in computational work are Joshua Winner, Daniel Graham, and Luke Presson. Students who were formerly involved in this project and whose supercomputer accounts can be disabled include: Calvin Cahall, Natalie Orms, and Trinity Hochstetler.

No publications have resulted from this research project. Some students have given KAS presentations related to their work on this project. Recent presentations from involved students include: Vanessa Song (Muzyka, organic synthesis), Josh Winner (Muzyka, computational chemistry), and Andrea N. Frost (Haile, enzyme inhibition).

No external grant funding has supported this research. Centre College internal grants and access to UK’s supercomputer have been our only support for this interdisciplinary project.

Project 2:

Structure Based Drug Design: Inhibiting Mur Enzymes
Jennifer Muzyka, Centre College
May 2013

Resistance to antibiotics by pathogenic microorganisms poses significant problems to human health. Research and development on novel antibiotics has lagged behind the rise in drug resistance seen in microbial populations. The research project described is part of an interdisciplinary effort with collaborators Peggy Richey and January Haile (both of Centre College) 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), assay candidate inhibitors for antibacterial activity (Richey), and characterize the biochemical properties (enzyme kinetics) of inhibitors that display antibacterial activity (Haile).

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. 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) targets an enzyme in this family, MurA. Recent research has uncovered ligands (molecules that bind to another molecule) that bind to and inhibit these enzymes, but most of these compounds do not exhibit antibacterial activity.

Previously we docked and scored one million commercially available compounds using DOCK on a Teragrid computer at Indiana University. We have purchased a number of the high scoring compounds for experimental testing. Peggy Richey and her students carry out antibacterial testing of the compounds. January Haile and her students are conducting enzyme inhibition studies. We have been carrying out molecular dynamics calculations with these compounds using AMBER on the supercomputer at the University of Kentucky. We have been synthesizing structurally similar compounds for testing.

We have identified three compounds that give promising results in one or more of these screening methods (in vivo, in vitro, and in silico) for MurA. All three of these compounds are things we synthesized at Centre due to their similarities with the high scoring compounds from the 2010 DOCK study. We will be examining each of the compounds via the other two methods before writing up these results for publication. The computational results will be analyzed in parallel with the experimental results, hopefully providing insights into the observations.

In the fall of 2013 we anticipate beginning another round of screening tests with a different member of the Mur family of enzymes. We hope to have access to DOCK on UK’s supercomputer for this round of testing.

Students:

Joshua Winner
Daniel Graham
Luke Presson
Yang Vanessa Song
Yuqian Dai
Griffin Cote - Centre College

Daniel Phillip Thompson - Centre College - Chemistry

Collaborators:

Professor January Haile
Professor Margaret (Peggy) Richey


Software used:

• DOCK is available for free for academic users. We previously used it on Indiana’s Big Red computer, which I understand is now out of commission. As far as I know, this program is not available on UK’s supercomputer. We would probably be interested in using it again in the future if it was available on UK’s supercomputer.
• AMBER is available for a modest license fee to academic users. We have a license to run the program on computers at Centre, but these computers don’t provide adequate computing power. We have been using AMBER on UK’s supercomputer in 2011 and 2012.

Structure Based Drug Discovery: MurA

Collaboration between Dr. Haile, Dr. Muzyka, and Dr. Richey



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 a three-part, interdisciplinary effort by Professors Richey, Haile, 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), 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.

Muzyka and her students recently 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.

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.