Targeting Echinococcus multilocularis PIM kinase for improving anti-parasitic chemotherapy.

BACKGROUND: The potentially lethal zoonosis alveolar echinococcosis (AE) is caused by the metacestode larval stage of the tapeworm Echinococcus multilocularis. Current AE treatment options are limited and rely on surgery as well as on chemotherapy involving benzimidazoles (BZ). BZ treatment, however, is mostly parasitostatic only, must be given for prolonged time periods, and is associated with adverse side effects. Novel treatment options are thus urgently needed. METHODOLOGY/PRINCIPAL FINDINGS: By applying a broad range of kinase inhibitors to E. multilocularis stem cell cultures we identified the proto-oncogene PIM kinase as a promising target for anti-AE chemotherapy. The gene encoding the respective E. multilocularis ortholog, EmPim, was characterized and in situ hybridization assays indicated its expression in parasite stem cells. By yeast two-hybrid assays we demonstrate interaction of EmPim with E. multilocularis CDC25, indicating an involvement of EmPim in parasite cell cycle regulation. Small molecule compounds SGI-1776 and CX-6258, originally found to effectively inhibit human PIM kinases, exhibited detrimental effects on in vitro cultured parasite metacestode vesicles and prevented the formation of mature vesicles from parasite stem cell cultures. To improve compound specificity for EmPim, we applied a high throughput in silico modelling approach, leading to the identification of compound Z196138710. When applied to in vitro cultured metacestode vesicles and parasite cell cultures, Z196138710 proved equally detrimental as SGI-1776 and CX-6258 but displayed significantly reduced toxicity towards human HEK293T and HepG2 cells. CONCLUSIONS/SIGNIFICANCE: Repurposing of kinase inhibitors initially designed to affect mammalian kinases for helminth disease treatment is often hampered by adverse side effects of respective compounds on human cells. Here we demonstrate the utility of high throughput in silico approaches to design small molecule compounds of higher specificity for parasite cells. We propose EmPim as a promising target for respective approaches towards AE treatment.

Activin/Nodal/TGF-ß Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells.

Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-ß pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.