Developing a Screening System for Identifying Efficient Inhibitors of the Choline Kinase of Streptococcus Pneumonia
Salam A. Ibrahim, Ph.D.
Department of Family and Consumer Sciences; Food and Nutritional Sciences
Nutrition | Nutritional Epidemiology
Gram-positive pathogens such as Streptococcus pneumoniae can have deleterious effects on both human and animal health. Antibiotics and antimicrobials have been developed to treat infections caused by such pathogens and to prevent food contamination. However, resistant strains emerge continuously. Thus, new strategies for controlling Gram-positive pathogen growth must constantly be developed. Putative inhibitors of Gram-positive isoforms of the enzyme choline kinase have been shown to block the growth of S. pneumoniae. However, the strength of inhibition and the mechanism of action of these inhibitors on S. pneumoniae choline kinase (sChok) is unknown. The aim of this project was to establish a system for screening sChok inhibitors by characterizing their strength of inhibition and determining their mechanisms of action. The sChok enzyme was expressed in BL21(DE3) cells and enriched using IMAC chromatography. The LDH/PK system of quantifying kinase activity was adapted for use with sChok. Inhibitor strength was determined by calculating IC50s. A simple comparative kinetic method was applied to determine inhibitor mechanism of action. The Km choline and Km ATP of sChok were 164.247 +/- 59.92 ÂµM and 144.523 +/- 17.8 ÂµM, respectively, while the Vmax choline and Vmax ATP was 103.562 +/- 9.125 and 67.5896 +/- 2.352, respectively. Two promising sChok inhibitors were identified: MN58 and 717, with IC50s of 500 uM and 0.2 uM respectively. MN58 and 717 had competitive and uncompetitive mechanisms of action, respectively.
Chasten, Valorie, "Developing a Screening System for Identifying Efficient Inhibitors of the Choline Kinase of Streptococcus Pneumonia" (2019). Undergraduate Research and Creative Inquiry Symposia. 172.