Fig 1.
Prediction of the tertiary structure of the EhAPSK APSK domain.
(A) Schematic illustration for the arrangement of the APS kinase and ATP sulfurylase domains. Red and purple represent APS kinase and ATP sulfurylase domains, respectively. CTP, chloroplast targeting peptides. (B) Sequence alignments of APSK domains from E. histolytica APSK, A. thaliana APSK, and A. aeolicus PAPSS, which were extracted from S1 Fig. The amino acid residues conserved among these three proteins are indicated by *. (C) Homology modeling structures of the EhAPSK APSK domain. Three different homology modeling structures, EhAPSK structure-A, -B, and -C, are shown. PDB IDs shown inside parentheses indicate the template tertiary structure used for generating each structure. The APS binding site of each structure is shown by a dashed circle.
Table 1.
Amino acid sequence identity between the APSK domain of E. histolytica and the APSK domain of nine homologues.
AS, ATP sulfurylase; APSK, APS kinase; PAPSS, PAPS synthase.
Fig 2.
Establishment of the in vitro 96-well plate-based APSK activity assay.
(A) Schematic illustration of the coupling assay to measure APSK activity. APS, adenosine 5′-phosphosulfate; APSK, APS kinase; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; NP1, nuclease P1; PK, pyruvate kinase; PEP, phosphoenolpyruvate; and LDH, lactate dehydrogenase. (B) SDS-PAGE of the affinity-purified recombinant proteins. Lane 1, molecular weight markers; lane 2, rEhAPSK; lane 3, rHsAPSK (recombinant APSK domain of HsPAPSS1); lane 4, recombinant APSK domain of HsPAPSS2; and lane 5, recombinant HsPAPSS1 (full-length HsPAPSS1). (C) Dose-dependent effect of rEhAPSK and rHsAPSK on APSK activity. (D) Validation of screening system for APSK inhibitors.
Fig 3.
Chemical structures of 15 compounds that reproducibly inhibited rEhAPSK activity.
Table 2.
Comparison of results from in vitro activity assay and in silico docking analysis.
Ranking (in vitro) was based on IC50 values, which were determined by more than three independent experiments and expressed as average ± SD. Ranking (in silico) is from S2 Table. A-D-11, A-H-11, and E-H-05 used for IC50 determination were from commercial sources whereas the other 12 compounds were from the Pathogen Box. A, EhAPSK structure-A; B, EhAPSK structure-B; and C, EhAPSK structure-C. PDB IDs shown inside parentheses indicate the template tertiary structures used for generating the homology modeling EhAPSK structures.
Fig 4.
Effects of A-D-11, A-E-11, and E-H-05 (auranofin) on E. invadens cyst formation and of A-D-11 and A-E-11 on E. histolytica trophozoite proliferation.
(A-C) Encystation assay. Encystation-inducing culture was treated with either A-D-11, A-E-11, or E-H-05 (auranofin), immediately after induction (0−72, 0−96, or 0−120 h). Encystation-inducing culture was treated with either A-D-11, A-E-11 or E-H-05 (auranofin) from 48 h after induction (48–72, -96, or -120 h). Flow cytometry results obtained at 72, 96, or 120 h after induction are shown. EB, Evans blue; CF, calcofluor. (D, E) Trophozoite proliferation assay. The total cell number counted is indicated in the upper left corner. PI, propidium iodide; FSC, forward scatter. Representative data are shown from three independent experiments. Results for E-H-05 treatments (0−72 and 48−72 h), which we previously published [18], were confirmed in this study and are shown for ease of comparison.
Table 3.
Summary for IC50 values of A-D-11, A-H-11, and E-H-05 (auranofin) for processes crucial in Entamoeba life cycle and for human cell.
IC50 values for trophozoite proliferation, cyst formation, and human cell were determined by three independent experiments and expressed as average ± SD. IC50 values of E-H-05 for these processes were from [18].
Fig 5.
Cytotoxicity of 15 compounds that inhibited rEhAPSK activity against HFF cells.
(A) Compound treatments at 10 and 100 μM. (B) Dose-dependent effect of E-H-05 on number of viable HFF cells. Data are expressed relative to that from a DMSO-treated control (set as 100%). Data shown are the mean with error bar. Error bars indicate deviation from the mean, obtained from duplicates from three independent experiments.
Fig 6.
Sulfolipid synthesis in E. histolytica treated for 6 h with A-D-11, A-H-11, or E-H-05 (auranofin).
(A) Autoradiography of HPTLC of methanol extracts of [35S]-labeled trophozoites cultivated in the presence of different concentrations of each compound. The autoradiographic images shown are representative of the results from three independent experiments. (B) Quantitation of sulfolipids synthesized in trophozoites cultivated in the presence of different concentrations of each compound. The data were calculated from the autoradiography image and are expressed relative to that in a solvent-treated control (set as 100%) after normalizing to total synthesized sulfolipids and total cell protein. Data shown are the mean with error bar (SD from the mean) from three independent experiments. Samples for A-D-11 and A-H-11 treatments were originally run in a single TLC plate, which included a lane for a control sample, and the obtained image was split into two parts for ease of comparison; therefore, the single control lane was used to calculate the relative values for both A-D-11 and A-H-11.