Inhibition of choline kinase as an antiamoebic approach in Entamoeba histolytica infection
https://doi.org/10.47665/tb.40.4.008
- Author:
Teh, Z.H.
1
;
Lim, B.H.
1
;
See Too, W.C.
1
;
Few, L.L.
1
Author Information
1. School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
- Publication Type:Journal Article
- Keywords:
Entamoeba histolytica;
choline kinase inhibitors;
anti-amoebic approach;
siRNA.
- From:Tropical Biomedicine
2023;40(No.4):430-438
- CountryMalaysia
- Language:English
-
Abstract:
Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or
amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis,
but concerns regarding resistance have emerged due to the identification of resistance pathways in
E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway.
Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK
enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified
EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled
assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors.
Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50
for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene
expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors,
namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth
inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce
cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of
positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive
control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this
study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement
and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific
inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica
by disrupting its phospholipid synthesis pathway.
- Full text:8.2023my1439.pdf
