Choline Kinase, A Novel Drug Target for the Inhibition of Streptococcus pneumoniae

Abstract

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, these strategies have been increasingly thwarted by the emergence of resistant bacteria strains. Thus, new methods for controlling Gram-positive pathogen growth need to be continuously developed. Choline analogs, such as Hemicholinium-3 (HC-3), have been shown to be useful in blocking cell division in eukaryotic cells through the inhibition of choline kinase, an enzyme which catalyzes the production of phosphocholine from choline and ATP. In some Gram-positive pathogens, choline kinase is an important enzyme in the production of the cell wall element, lipoteichoic acid. However, it is not known if inhibiting this enzyme has any effect on cell division in Gram-positive bacteria. Using the R6 strain as a model, we tested the ability of HC-3 to block the activity of choline kinase in S. pneumoniae and inhibit cell growth. Mass-spectrometry measurements of crude extracts revealed that HC-3 blocked choline kinase activity. Turbidity measurements and population counts showed that HC-3 inhibited cell growth. Competition assays with choline suggested that HC-3 also blocked choline transporters. Western blots showed that lipoteichoic acid production was blocked in the presence of HC-3, and autolytic assays showed that this decrease in lipoteichoic acids caused cells to be more resistant to autolysis. Scanning electron microscopy revealed that HC-3 distorted the cell wall. This study thus establishes choline kinase as a novel drug target for S. pneumoniae.

Identification and validation of novel and more effective choline kinase inhibitors against Streptococcus pneumoniae

“…Both drugs had MICs and MLCs in the low micromolar ranges (see Table 1 ), making them much more powerful inhibitors of S. pneumoniae growth than the previously reported sChoK inhibitor HC-3, which had an MIC of 5,400 µM 3 . To our knowledge, other than HC-3, no other sChoK inhibitors have ever been reported, making MN58b and RSM-932A the most effective sChoK inhibitors thus far reported both on the level of enzyme inhibition and inhibition of cell growth.…”

“…One milliliter (1 mL) of cell samples was centrifuged at 3,000 g , sonicated 3 × 2 min on ices. Western blots were performed as described 3 while total protein on the membrane was measured by fluorescence according to Quickstain instructions.…”

“…This includes establishing novel targets for drug discovery efforts. Recently, choline kinase (ChoK) has been proposed as a drug target for Gram positive species generally 1 after the first ChoK inhibitor capable of inhibiting the growth of S. pneumoniae was discovered 2 , 3 .…”

“…The enzyme of S. pneumoniae has previously been proposed as a drug target 3 . However, the sChoK inhibitor originally tested, Hemicholinium-3 (HC-3), was weak: HC-3 had IC 50 12 and minimum inhibitory concentration (MIC) values in the millimolar range 3 . The aim of this study was to discover stronger inhibitors of sChoK in order to further establish sChoK as a target.…”

Parallel Colorimetric Quantification of Choline and Phosphocholine as a Method for Studying Choline Kinase Activity in Complex Mixtures

“…Experimentally unconfirmed sequence predictions suggest that other Gram-positive pathogens, such as Stapholooccus aureus , Bacillus subtilis , Clostridium perfringens , and Clostridium botulinum also produce Chok isoforms. Importantly, the choline kinase of Streptococcus pneumoniae (sChok) has recently been established as a drug target [ 8 ], and inhibiting sChok was found to effectively slow cell growth and division in this species. However, Chok activity is confirmed in S. pneumoniae alone and has not yet been well characterized in this species of bacteria.…”