A dual choline/phosphocholine colorimetric method for measuring the relative strength of inhibitors of choline kinases of Gram-positive pathogens

Abstract

Using chemical and artificial preservation methods to keep food safe is becoming an issue with consumers. Therefore, it is important to discover antimicrobials from natural sources for use in food safety applications. The objective of the present study was to establish screening system for natural inhibitors of choline kinase (ChoK), a known antimicrobial target. A previously developed dual choline/phosphocholine colorimetric method was used to determine the relative strength of 3 choline kinase inhibitors: Hemocholinium-3, RSM-928A, and MN58 . Whole cell extracts containing the choline kinase of Streptococcus pneumoniae (sChoK) was used as a model. The measured IC50 values of these drugs were >2700 µM, 0.54 µM, and 170-225 µM, respectively. Importantly, not every step of the colorimetric method could be used in the case of every inhibitor, since each had its own particular reactive profile with the colorimetric dyes, which could have lead to confounded measurements. However, in every case, the system was flexible enough to measure choline or phosphocholine, if not both metabolites. We establish here that this dual choline/phosphocholine system is flexible enough to measure the IC50 any possible inhibitor. This colorimetric method is an ideal benchtop method for screening for natural inhibitors of bacterial ChoKs.