Vanderstraete M et al. (2013), Dual targeting of insulin and venus kinase Rece...

XB-ART-47158

PLoS Negl Trop Dis 2013 May 16;75:e2226. doi: 10.1371/journal.pntd.0002226.

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Dual targeting of insulin and venus kinase Receptors of Schistosoma mansoni for novel anti-schistosome therapy.

Vanderstraete M , Gouignard N , Cailliau K , Morel M , Lancelot J , Bodart JF , Dissous C .

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Chemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compound has led to emergence of resistant strains of Schistosoma mansoni, therefore pointing out the necessity to find alternative drugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could represent valuable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug.Several commercial RTK inhibitors were tested for their potential to inhibit the kinase activities of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains (ICD) expressed in Xenopus oocytes. We measured the inhibitory effect of chemicals on meiosis resumption induced by the active ICD of the schistosome kinases in oocytes. The IR inhibitor, tyrphostin AG1024, was the most potent inhibitory compound towards SmIR and SmVKR kinases. In vitro studies then allowed us to show that AG1024 affected the viability of both schistosomula and adult worms of S. mansoni. At micromolar doses, AG1024 induced apoptosis and caused schistosomula death in a dose-dependent manner. In adult worms, AG1024 provoked alterations of reproductive organs, as observed by confocal laser scanner microscopy. With 5 µM AG1024, parasites were no more feeding and laying eggs, and they died within 48 h with 10 µM.IRs and VKRs are essential in S. mansoni for key biological processes including glucose uptake, metabolism and reproduction. Our results demonstrate that inhibiting the kinase potential and function of these receptors by a single chemical compound AG1024 at low concentrations, leads to death of schistosomula and adult worms. Thus, AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosome chemotherapy.

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Species referenced: Xenopus
Genes referenced: adm ins myc

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	Figure 2. Sensitivity of SmIR and SmVKR kinase activities to various RTK inhibitors.Sets of 20 oocytes were injected with cRNA encoding constitutively active forms of SmIR and SmVKR cRNA (as described in Figure 1), and incubated with variable concentrations of tyrphostins AG1024 (A), AG538 (B), HNMPA-(AM)3 (C), AG1478 (D), SU11274 (E) and BIBF1120 (F). In each set, the percentage of oocytes that underwent GVBD was calculated. Results are expressed by inhibition in % of control oocytes and represent the mean of three independent experiments.
	Figure 3. Tyrphostin AG1024 inhibits SmIR and SmVKR autophosphorylation.SmIR1YYRE, SmIR2YYHE, SmVKR1YYRE and SmVKR2YYRE proteins were expressed in Xenopus oocytes (as in Figure 1) incubated in ND96 medium containing or not the minimal concentration of AG1024 able to inhibit 100% of their ability to induce GVBD (respectively 100 nM, 1 µM, 100 nM and 100 nM). Immunoprecipitation and Western Blot analyses were performed using anti-Myc and pY-20 antibodies. Inhibition of autophosphorylation correlated with the disability of kinases to induce GVBD (see Figure 2).
	Figure 4. Dose-dependent effect of AG1024 on viability of cultured schistosomula of S.mansoni.(A) Viability of schistosomula cultured for 5 days without or with 1, 10, 20 or 50 µM of AG1024. Results are expressed as % of surviving larvae (mean ±SD, three independent experiments). (B) Morphological examination of schistosomula following 48 h incubation with 10, 20 and 50 µM AG1024. All schistosomula died within 48 h with 50 µM AG1024, characterized by their dark appearance, granular aspect and tegumental abnormalities.
	Figure 5. AG1024 induces apoptosis in S. mansoni schistosomula in culture.Schistosomula were treated for 48 h with DMSO (A, B, C), 10 µM AG1024 (D, E, F) or 50 µM AG1024 (G, H, I), then fixed and stained with DAPI and TUNEL. DAPI (A, C, F), TUNEL (B, D, G) and merged (C, E, H) pictures are shown. TUNEL-positive schistosomula are indicated by arrows. Their number increased in the presence of AG1024, reaching a maximum for the 50 µM treatment (G, H).
	Figure 6. Treatment by AG1024 induces unpairing of adult worms and decrease of egg laying in a dose and time-dependent manner.Freshly perfused paired worms (20 couples) were incubated for 5 days with DMSO solvent, 1 µM or 5 µM of AG1024. (A) Percentages of paired worms were determined every 24 h. Results are expressed as the mean (± SEM) of three independent experiments. (B) In each assay, the total number of eggs laid during the 5 days was determined. Results were expressed as the percentage of eggs laid by AG1024-treated worms compared to controls (mean ± SD, three independent experiments). Statistical analyses were performed using the Student's t-test, and significance is displayed as follows: : p<0,05, : p<0.01, **: p<0.001.
	Figure 7. Alterations of reproductive tissues and organs under treatment by AG1024 of S. mansoni adult worms cultured in vitro.Freshly perfused paired worms (20 couples) were incubated for 5 days with DMSO solvent (A, B), 1 µM AG1024 (C, D) or 5 µM AG1024 (E, F), then fixed and stained with hydrochloric carmine. CLSM examinations showed, particularly at 5 µM, a significant size reduction of female ovary (E), associated with changes in the distribution of immature (io) and mature (mo) oocytes within the ovary lobes (ov), as compared to the control (A). In males treated with AG1024, sperm vesicle (sv, indicated by red arrows) is full of round undifferentiated germinal cells (clearly visible in F), whereas in DMSO-treated control worms it contains elongated spermatozoa. (t) testis.
	Figure 8. Modifications of ootype structures and egg formation in AG1024-treated S. mansoni adult female worms.CLSM examination centered on the female ootype of control parasites (A) or parasites treated with AG1024 1 µM (B) or 5 µM (C, D). Ot (ootype); mg (Mehlis'gland); eg (egg).
	Figure 1. Expression of SmIR and SmVKR intracellular domains in Xenopus oocytes.Sets of 20 oocytes were microinjected with cRNA encoding Myc-tagged versions of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains, in their native (wt) and constitutively active forms (YYXE). Immunoprecipitation and Western Blot analyses were performed using anti-Myc and pY-20 antibodies. Tyrosine phosphorylated active proteins could trigger meiosis resumption in oocytes (monitored by GVBD).

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