Hedgehog Antagonist Pyrimidine-Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation.

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog-glioma (Hh-Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein-protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine-indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell-based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α-tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α-tubulin from its stabilised form.

Endocrine and paracrine regulation of meiotic cell cycle progression in teleost oocytes: cAMP at the centre of complex intra-oocyte signalling events.

Participation of major endocrine and/or local autocrine/paracrine factors and potential interplay between apparently disparate intra-oocyte signalling events during maintenance and withdrawal of meiotic prophase arrest has been an area of active research in recent years. Studies on oocyte maturation have contributed substantially in the discovery of some of the most important biochemical and cellular events like functional significance of novel membrane-associated steroid receptors, elucidation of maturation promoting factor (MPF), cytostatic factor (CSF) and other signalling cascades that entrain the cell cycle clock to hormonal stimuli. While follicular estrogen has largely been implicated in maintenance of prophase arrest, involvement of maturational steroid and membrane progestin receptor in resumption of meiotic G2-M1 transition in piscine oocytes has been shown earlier. Moreover, detection of ovarian IGF system, maturational gonadotropin stimulation of IGF ligands and potential synergism between maturational steroid and IGF1 in zebrafish oocytes are most recent advancements. Though endocrine/paracrine regulation of cyclic nucleotide-mediated signalling events in meiotic cell cycle progression is well established, involvement of PI3K/Akt signalling cascade has also been reported in fish, amphibian and mammalian oocytes. The major objective of this overview is to describe how fish oocytes maintain high cAMP/PKA activity and how steroid- and/or growth factor-mediated signalling cascade regulate this pathway for the withdrawal of meiotic arrest. Moreover, special emphasis is placed on some recent findings on interaction of PKA with some of the MPF-regulating components (e.g., synthesis of cyclin B or MEK/MAPK signalling cascade) for the maintenance of prophase arrest.

Internal Oligoguanidinium-Based Cellular Transporter Enhances Antisense Efficacy of Morpholinos in In Vitro and Zebrafish Model.

An efficient cellular transporter is highly desirable for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) as Vivo-PMO and PPMO have limitations for in vivo study. We report here a novel internally tetraguanidinium-linked nonpeptidic cellular transporter having a conformationally rigid backbone composed of pharmacologically compatible heterocyclic six-membered rings which internalizes efficiently into cells in full growth medium and ubiquitously distributed into zebrafish embryos. It efficiently transports antisense PMO in vitro and in vivo zebrafish embryos. Comparative study with Gene Tools Vivo-PMO revealed that our cellular-transporter conjugated PMO shows better antisense efficacy.

Piperazic acid derivatives inhibit Gli1 in Hedgehog signaling pathway.

Piperazic acid, a non-proteinogenic amino acid, found in complex secondary metabolites and peptide natural substances, has shown down regulation of Gli1 expression in Hedgehog signaling pathway in cell based assays. Further structure activity relationship study indicated that amide derivatives of piperazic acid are more potent than piperazic acid itself, with little to no toxicity. However, other cellular components involved in the pathway were not affected. To the best of our knowledge, this is the first report on the inhibitory property of piperazic acid in this pathway. Hence, this molecule could serve as a useful tool for studying Hedgehog signaling.

Participation of PI3-kinase/Akt signalling in insulin stimulation of p34cdc2 activation in zebrafish oocyte: phosphodiesterase 3 as a potential downstream target.

Exposure of fully grown oocytes to growth factors (insulin/IGFs) initiates various signalling cascades that culminate to final stages of oocyte maturation. Regulation of signalling pathways during growth factor-induced meiosis resumption in fish is not well characterized. Here we studied the participation of PI3K/Akt signalling pathway during recombinant human insulin (rh-insulin)-induced meiotic maturation in zebrafish (Danio rerio) oocytes. Priming of defolliculated oocytes in vitro with rh-insulin promotes germinal vesicle breakdown (GVBD) in a dose- and time-dependent manner, an effect sensitive to translation but not transcription inhibition. More than 80% of the oocytes underwent GVBD due to 0.8IU/ml rh-insulin within 10h of incubation and the kinetics of p34cdc2 kinase activation corresponded well with GVBD data. PI3K inhibitors, wortmannin and LY294002 blocked insulin, but not 17α, 20ß-DHP-induced GVBD. Immunoblot analyses of oocyte extract revealed that phospho-PI3K (p85α) was up regulated within 30-60 min of insulin stimulation followed by phospho-Akt (Ser473) at 60-120 min. Though PI3K/Akt phosphorylation was largely unaffected, pre-incubation with phosphodiesterase (PDE) inhibitors, IBMX and cilostamide, but not rolipram completely blocked rh-insulin-induced p34cdc2 activation and GVBD. These results suggest that PDE3 may be one potential downstream target to PI3K/Akt signalling necessary for rh-insulin-induced GVBD in zebrafish.