Fig 1.
Activities of selected kinase inhibitors against E. multilocularis PC.
(A) Phylogenetic tree of human PKs, based on homologies within the kinase domain. Seven groups according to current nomenclature [71] are indicated. (B) Heatmap showing the effects of different kinase inhibitors, covering all 7 groups, on E. multilocularis PC. Colour-code below indicates percentage of luminescence signal (i.e. number of viable cells), normalized to signals from DMSO controls, after 3 d and 7 d of incubation with 10 μM of inhibitor. Inhibitor names, human target proteins, and kinase sub-families are indicated in the table to the left. Black arrow indicates the pan-PIM kinase inhibitor SGI-1776.
Fig 2.
Homologies and structural features of EmPim.
(A) Amino acid sequence alignment of the kinase domains of human Pim-1 (HsPIM1), E. multilocularis Pim (EmPim), human FLT3 kinase (HsFLT3), human haspin kinase (HsHASPIN) and an E. multilocularis haspin kinase ortholog (EmHASPIN1). Residues identical to human Pim-1 are shown in black on grey. Kinase DFG motifs and the hinge regions are marked in red. Black triangles indicate residues known to be involved in the interaction between human Pim-1 and compound CX-6258 (numbered according to human Pim-1). (B) Presence of amino acid residues important for the interaction between human Pim-1 and CX-6258 in different kinases. For each of the 14 known residues of human Pim-1 (HsPIM1), the corresponding residue and position in E. multilocularis Pim (EmPIM), human FLT3 kinase (HsFLT3), human haspin kinase (HsHASPIN), and the E. multilocularis haspin kinase isoform (EmHASPIN1) are shown. Residues identical to those of human Pim-1 are marked in yellow, residues with similar biochemical properties are marked in green. The numbers of identical/similar residues compared to human Pim-1 are listed to the right as well as IC50 values of compounds CX-6258 and SGI-1776 to human enzymes.
Fig 3.
Expression of empim in Echinococcus MV.
(A) WISH on E. multilocularis MV directed against empim. Channels shown are DAPI (blue, nuclear staining), WISH (green, empim+), EdU (red, S-phase stem cells), and merge as indicated. Green arrow indicates example of empim+/EdU- cell, red arrow indicates example of empim-/EdU+ cell, yellow arrow indicates example of empim+/EdU+ cell. Size bar represents 25 μm. A schematic illustration of MV regions where images have been taken is given in S3 Fig. (B) Average numbers of empim+/EdU+ cells per mm2 of germinal layer are shown. Error bar indicates standard deviation.
Fig 4.
Domain structure and homologies of EmCDC25.
(A) Amino acid sequence alignment of the Rhodanese homology domains of EmCDC25 (EmCdc25), two S. mansoni CDC25 orthologs (SmCDC25A, SmCDC25B), and three human CDC25 orthologs (HsCDC25A-C). Conserved Rhodanese domain DCR motifs and the active site are indicated. Residues identical to EmCDC25 are shown in black on grey. (B) Phylogenetic tree based on Rhodanese domains of different CDC25-like phosphatases. Sequences derived from E. multilocularis (EmCDC25), S. mansoni (SmCDC25A/B), H. sapiens (HsCDC25A-C), C. elegans (CeCDC25 1–4), D. melanogaster (TEW, STG), and Saccharomyces cerevisiae (MIH1). Statistical method for the tree was maximum likelihood (ML), substitution model was Jones-Taylor-Thompson, ML heuristic method was Nearest Neighbour Interchange. (C) Domain structures of EmCDC25, two different CDC25 orthologs of S.mansoni (SmCDC25A/B), and three human CDC25 isoforms (HsCDC25A-C). Shown are Rhodanese domains and M-phase inducer phosphatase domains, which are typical for mammalian isoforms.
Fig 5.
Interaction between EmPim and EmCDC25.
(A) Representative pictures of yeast transformant growth on plates selecting for plasmids (-Leu, -Trp) as well as triple dropout (-Leu, -Trp, -His) and quadruple dropout (-Leu, -Trp,—His, -Ade) plates for interaction under medium and high stringency conditions, respectively. Plasmid combinations are indicated to the right, OD600 values for dropout density above. (B) Quantitative assay measuring growth densities of yeast transformants. Plasmid combinations are indicated below the graph. Error bar represents standard deviation. Tukey’s multiple comparison test, followed by one way ANOVA was used to compare all experimental combinations, but only comparisons to the corresponding control are shown. **** indicates p ≤ 0.0001.
Fig 6.
Effects of SGI-1776 and CX-6258 on MV and PC.
(A) Inhibitor effects on mature MV. E. multilocularis MV were incubated for 28 d in the presence of different inhibitor concentrations as indicated below (with medium and inhibitor replacement every 3–4 d), and the number of structurally intact MV was inspected microscopically. One way ANOVA followed by Dunnet’s multiple comparison test was used for comparison to the control DMSO group. ** indicates p ≤ 0.0021. (B) Representative examples of MV incubated with different concentrations of inhibitors as indicated to the left. (C) Inhibitor effectos on the formation of MV from PC. Parasite stem cell cultures were incubated for 21 d in the presence of different inhibitor concentrations as indicated below. Numbers of fully mature MV were subsequently counted. Error bars represent standard deviation. Kruskal-Wallis test followed by Dunn’s multiple comparison test was used for comparisons with control (DMSO) group. * represents p ≤ 0.0332.
Fig 7.
Effects of Pim inhibitors on Echinococcus larvae and human cells.
(A) Heat map showing the effects of 20 in silico screen compounds on MV. Different concentrations (indicated below) of each compound (indicated to the left) were incubated in vitro with MV for 28 d and structural integrity was assessed. Colour-code indicating percentages of surviving vesicles is indicated below. (B) Effects of four in silico screen compounds on MV production from PC. 10 μM of each compound (indicated below) were incubated for 21 d with PC in vitro and the production of MV was assessed. For comparison, SGI-1776 was tested at 10 μM. Error bars represent standard deviation. Kruskal-Wallis test followed by Dunn’s multiple comparison test was used for comparisons with the control (DMSO) group. (C) Effects of Z196138710 and SGI-1776 on MV. Both compounds were tested at different concentrations (shown below) on MV in vitro. Structural integrity was measured after 28 d. Error bar represents standard deviation. One was ANOVA followed by Dunnet’s multiple comparison test was used in comparisons with control (DMSO) group. p values less than 0.0001 are summarized with **** and p values less than 0.0332 are summarized with *. (D) Effects of Z196138710 and SGI-1776 on the in vitro formation of MV from PC. Both inhibitors were incubated at different concentrations (indicated below) for 21 d with PC and the formation of mature MV was measured. Error bar represents standard deviation. Kruskal-Wallis test followed by Dunn’s multiple comparison test was used in comparisons with control (DMSO) group. * represents p ≤ 0.0332. (E) Effects of Z196138710, SGI-1776, and CX-6258 on human HEK293T cells. HEK293T cells were incubated with different concentrations of inhibitors as indicated below. Cell viability was measured after 3 d. (F) Effects of inhibitors on human HepG2 cells. For experimental procedure, see (E). Error bar represents standard deviation. Tukey’s multiple comparison test followed by one way ANOVA was used to compare all experimental settings, only comparisons for equal inhibitor concentrations are shown. P values less than 0.0001 are summarized with **** and p values less than 0.0021 are summarized with **.