Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Room: Exhibit Hall A
Valorie Danielle Chasten - North Carolina Agricultural and Technical State University
Co-Author(s): Dr. Tahl Zimmerman, Department of Family Consumer Science, North Carolina Agricultural and Technical State University, 1601 East Market Street, Greensboro, North Carolina 27411; Dr. Salam A. Ibrahim, Department of Family Consumer Science, North Carolina Agricultural and Technical State University, 1601 East Market Street, Greensboro, North Carolina 27411; Dr. Juan Carlos Lacal, Hospital Universitario, Fuenlabrada, Spain.
Streptococcus pneumoniae and other gram-positive pathogens can be detrimental to both human and animal health. Antibiotic solutions are available; however, resistant strains emerge continuously. Controlling gram-positive pathogen growth requires new strategies. Putative inhibitors of gram-positive isoforms of the enzyme choline kinase have been shown to block the growth of Streptococcus pneumoniae. However, the strength of inhibition and the mechanism of action of these inhibitors on Streptococcus pneumoniae choline kinase (sChok) is unknown. The objective of this project is developing a method for screening sChok inhibitors and categorizing them by inhibition strength and mechanism of action. The plasmid pet28a(+) coding for a His-Tagged form of the sChok enzyme was expressed in Escherichia coli (E. coli) BL21(DE3) cells and purified by affinity chromatography using the His-tag Ni-NTA resin. Lactic Dehydrogenase/Pyruvate Kinase (LDH/PK) reporter assays were used to measure velocities at different concentrations of substrates choline and adenosine triphosphate (ATP). Velocities which were then fit into the Michaelis-Menton equation (vo)=(Vmax [S])/(KM+[S]) to determine the kinetic constants Km and Vmax for both substrates. Km is the concentration of the substrate when the reaction rate or velocity is equal to half of the maximal velocity or Vmax. The half maximal inhibitory concentration (IC50) of choline analogs were keeping the concentration of choline at the Km. Inhibitor strength was ranked by IC50, the concentration of inhibitor required to inhibit the enzyme’s activity by half. A comparative kinetic method was applied to determine inhibitor mechanism of action: competitive, non-competitive, or uncompetitive, or mixed. The Km choline and Km ATP of sChok were 164.247 +/- 59.92 μM and 144.523 +/- 17.8 μM, respectively, while the Vmax choline and Vmax ATP was 103.562 +/- 9.125 absorbance units/min and 67.5896 +/- 2.352 absorbance units/min, respectively. Two promising sChok inhibitors were identified: MN58 and 717, with IC50s of 645 μM and 22 μM respectively. The MN58 and 717 competitive and uncompetitive mechanism of action, respectively. A screening system was developed to screen inhibitors of choline kinase of Streptococcus pneumoniae. sChok inhibitor 717 was the stronger inhibitor due to having the lower IC50 value, but both were the most effective sChok inhibitors described to date. In the future, we plan to elucidate the mechanism of action of other putative inhibitors of Streptococcus pneumoniae such as terpenoids from medicinal plants.
Funder Acknowledgement(s): The 2019 College of Agriculture and Environmental Sciences Undergraduate Research Scholars Program (URSP); United States Department of Agriculture (USDA)
Faculty Advisor: Tahl Zimmerman, firstname.lastname@example.org
Role: Ultimate responsibility for all activities associated with the conduct of a research project. Primary author completed the objectives of the project which includes the following: 1. Express and purify of a choline kinase. 2. Develop a method for screening sChok inhibitors and categorizing them by inhibition strength and mechanism of action. 4. Determine mechanism of action of inhibitors to determine the type of binding: competitive, non-competitive, or uncompetitive inhibition.