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. 2014 Oct 12;4(3):256-66.
doi: 10.1016/j.ijpddr.201409004. eCollection 2014 Dec.

Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

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Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

Marion Morel et al. Int J Parasitol Drugs Drug Resist. .

Abstract

Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.

Keywords: Akt; Chemotherapy; Inhibitor; Protein kinase; Protein kinase B (PKB); Schistosoma mansoni.

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Figures

None
Graphical abstract
Fig. 1
Fig. 1
Protein sequence alignment of SmAkt with Akt proteins from other species. The SmAkt amino acid sequence was aligned using CLUSTAL W algorithm with Akt sequences from Echinococcus multilocularis (EmAkt: CCW28045.1), Drosophila melanogaster (DmAkt1: Q8INB9.3), Anopheles gambiae (AgAkt: XP_003436044.1), Xenopus laevis (XlAkt: AAG59601.1) and Homo sapiens (HsAkt1: AAL55732.1). Pleckstrin Homology (PH) domain is indicated in green, kinase domain (KD) in red and regulatory region in blue. Stars indicate conserved residues (DFG motif of the ATP binding site, threonine and serine phosphorylation sites) implicated in phosphorylating activity. Arrows indicated the PH domain residues (E117 and L150) involved in PH–KD interactions which maintain Akt kinases in inactive state. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Schematic representation of Akt activation. Intramolecular interactions between PH and KD domains maintain Akt in an inactive conformation. Biological activation of Akt occurs by its targeting at the membrane and the binding of PH domain with PIP3 phospholipids which disrupts PH-KD interactions, unmasking the threonine (T) residue and allowing its phosphorylation by PDK1. Activation of Akt is reinforced by the phosphorylation of a second serine residue present in the C-terminal regulatory domain by mTORC2. Mutations of residues in the PH domain (E117 and L150 in the SmAkt protein) disrupt interactions between PH and KD and lead to Akt activation. (PH: Pleckstrin Homology domain, KD: Kinase Domain, RD: Regulatory domain).
Fig. 3
Fig. 3
Inhibition of constitutively active mutants of SmAkt expressed in Xenopus oocytes by Akt inhibitors. (A) Western blot analysis of wild type, E117K and L150R V5-tagged SmAkt proteins expressed in Xenopus oocytes. Anti V5-immunoprecipitates were analyzed in Western blot with anti-V5 or with anti-pT308 active Akt antibodies. SmAkt E117K and L150R active kinases are recognized by anti-pT308 serum and are able to trigger meiosis resumption in oocytes (monitored by GVBD). (B) Oocytes expressing SmAkt E117K or SmAkt L150R were incubated with 10 μM of each Akt inhibitor (A4–8). Anti V5-immunoprecipitates were analyzed as in (A). The five compounds inhibit phosphorylation and activity of SmAkt mutants. (C, D) Oocytes expressing SmAkt L150R (C) or E117K (D) were incubated with variable concentrations of Akt inhibitors (A4–8). For each condition, percentages of oocytes with GVBD were calculated and the results expressed in % of inhibition of maturation using as reference oocytes without inhibitor (mean ± SEM of three independent experiments).
Fig. 4
Fig. 4
Kinetic study of the influence of Akt inhibitors on adult worm pairing. Freshly perfused paired worms (10 couples) were incubated for 72 h with increasing concentrations (10 nM–10 μM) of Akt inhibitor IV (A4), Akt inhibitor X (A7) and PDK1/Akt/Flt Dual Pathway inhibitor (A8). Controls were incubated with the same aliquot of DMSO solvent. The number of paired worms was determined at 24 h, 48 h and 72 h and results expressed in % of total worms (mean ± SEM of three independent experiments).
Fig. 5
Fig. 5
Kinetic study of the influence of Akt inhibitors on schistosomula viability. S. mansoni schistosomula were incubated with different concentrations (50 nM–10 μM) of Akt inhibitor IV (A4), Akt inhibitor X (A7) and PDK1/Akt/Flt Dual Pathway inhibitor (A8). Percentages of alive schistosomula were determined under microscope at 24 h, 48 h and 72 h. Results are expressed as the mean ± SEM of three independent experiments.

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