Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target

Abstract

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Keywords: Amebiasis; Coenzyme A; Drug development; Entamoeba histolytica; Gene silencing; Pantothenate kinase.

Graphical abstract

Fig. 1

Coenzyme A biosynthetic pathway in E. histolytica. ID numbers of individual enzymes in AmoebaDB are also shown. PanK, pantothenate kinase; PPCS-PPCDC, bifunctional phosphopantothenoylcysteine synthetase and phosphopantothenoylcysteine decarboxylase; PPAT, phosphopantetheine adenylyltransferase; and DPCK, dephosphocoenzyme A kinase.

Fig. 2

Effect of CoA, acetyl CoA, and malonyl CoA on the pantothenate kinase activities. Relative activities of EhPanK in the presence of various concentrations of inhibitors to those without inhibitors are shown. The assay was performed in 10 mM MgCl₂, 15 mM HEPES, 20 mM NaCl, 1 mM EGTA, 0.02% Tween-20, 0.1 mg/mL γ−globulins, 0.2 mM pantothenate, and 100 μM ATP with various concentrations of CoA, acetyl CoA, or malonyl CoA at 37 °C at pH 6. The assays were carried out three times independently, and the results are shown as means ± SEM of triplicates.

Fig. 3

Double-reciprocal plots of the recombinant EhPanK in the presence of CoA (A and B), malonyl CoA (C and D), or acetyl CoA (E and F). The enzymatic activities were determined with various concentrations of ATP and 0.2 mM pantothenate (A, C, and E) or various concentrations of pantothenate and 100 μM ATP (B, D, and F), in the presence of three concentrations of inhibitors (0, 0.5, and 2 mM). Data are shown in means ± SEM of triplicate analyses.

Fig. 4

Analyses of EhPanK gene silenced strain. A) RT-PCR analysis of EhPanK gene transcript from E. histolytica transformants. “psAP” indicates control strain transfected with psAP-2-Gunma empty vector, and “PanK gs” indicates EhPanK gene silenced strain. B) Relative levels of gene transcripts encoding all four enzymes involved in CoA biosynthesis by qRT-PCR analysis in psAP and Pan Kgs transformants. qRT-PCR data were normalized against RNA polymerase II, and are shown in percentage relative to the transcript level of each gene in psAP control. C) PanK and DPCK activity in cell lysates of psAP and PanKgs transformants. D) CoA concentration in cell lysates. For B-D, data are shown in mean ± SEM of three biological replicates. Statistical comparison is made by Student's t-test (*P < 0.05, **P < 0.01). E) Growth kinetic of E. histolytica transformants during 96 h incubation in BI-S-33 medium. Data are shown in mean ± SEM of three replicates. Data from one representative experiment of three conducted independently are shown. Statistical comparison is made by Student's t-test (*P < 0.05, **P < 0.01).

Fig. 5

Structures of EhPanK inhibitors identified in this study.