Abstract
Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 μM, respectively, and were shown to be 2–4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 μM and 10 μM, respectively, and the minimum lethal concentration (MLC) was 1.6 μM and 20 μM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.
Highlights
Streptococcus pneumoniae choline kinase has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range
The IC50s of each drug against sChoK were measured using two methods: the indirect ldh/pk method that relied on recombinant sChoK and which indirectly measured consumption of ATP14 and a previously reported colorimetric method that relied on BL21 (DE3) extracts expressing recombinant sChoK, which directly measured the consumption of choline and the production of p hosphocholine[16]
Inhibitors were effective in a complex cell extract and in a simplified system relying on a purified enzyme. Both MN58b and RSM-932A had IC50s that fell within the μM range, which was an improvement over the IC50 previously reported for the sChoK inhibitor HC-3 (IC50 of 2.7 mM)[12]
Summary
Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. Both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. New methods for treating S. pneumoniae have to be developed continuously This includes establishing novel targets for drug discovery efforts. The sChoK enzyme is an element of the pathway that mediates the decoration of teichoic acids with PCho via the intermediary CDP-choline. Teichoic acid polymers are cross-linked to peptidoglycan to form WTA by the action of LCP phosphotransferases[11]
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