Methanethiosulfonate derivatives as ligands of STAT3-SH2 domain

It is well known that inflammatory conditions in selected organs increase the risk of cancer. Compounds of the inflammatory tumor microenvironment include leukocytes, cytokines, complement components, are orchestrated by transcription factors, such as STAT-3 (Signal Transducer and Activator of Transcription 3) and NF-kB. Therefore drugs able to inhibit one or both transcription factors could be useful tools to treat cancer disease. Two main approaches have been explored to inhibit STAT-3 signalling: \u2022 indirect, inhibiting the upstream tyrosine kinases that are responsible for STAT-3 activation or blocking factors such as JAK, Src, Bcr-Abl, FLT3 and EGFR that are involved in the activation of STAT-3 signalling. This kind of inhibition induces tumour-cell apoptosis but is poor selective. \u2022 direct, by interaction of small molecules with the protein. In this selective approach the starting point is the crystallographic structure of STAT-3 SH2 domain. S-methyl methanethiosulfonate, isolated from cauliflower has been shown to inhibit colon tumor incidence when administered to rats during the post-initiation phase of carcinogenesis [1]. Recently, a new methanethiosulfonate derivative of valproic acid (ACS33) was reported by some of us to show good in vitro antiproliferative activity and to inhibit in vivo the growth of PC3 in subcutaneous xenograft mice models [2]. Fig.1: Structures of the studied thiosulfonate hybrids. Since the influence of methanethiosulfonates on STAT-3 activity has not been yet studied, we decided to synthesize a set of thiosulfonate-drug hybrids (Fig.1) and to submit them and their parent compounds to the AlphaScreen-based assay, to investigate their ability to bind STAT-3 SH2 domain. Moreover, in order to check the selectivity of our molecules on STAT-3, other SH2-containing proteins, such as STAT-1, exhibiting a high degree of sequence homology to STAT-3, have also been tested. Results showed that most of the synthesized thiosulfonate-hybrids are able to strongly and selectively bind STAT-3 SH2 domain, whereas the parent drugs were completely devoid of this ability. Studies are ongoing to better define the profile of our new methanethiosulfonate derivatives as potential dual STAT-3/NFkB inhibitors. References 1. Reddy, B. S.; Kawamori, T.; Lubet, R.; Steele, V.; Kelloff, G.; Rao, C. V. Chemopreventive effect of S-methylmethane thiosulfonate and sulindac administered together during the promotion/progression stages of colon carcinogenesis Carcinogenesis 1999, 20, 1645-8. 2. Wedel S. A.; Sparatore A.; Del Soldato P.; Al-Batran S. E.; Atmaca A.; Juengel E.; Hudak L.; Jonas D.; Blaheta R. A. New histone deacetylase inhibitors as potential therapeutics tools for advanced prostate carcinoma. J. Cell. Mol Med 2008, 12, 2457-66

p66(ShcA) adaptor molecule accelerates ES cell neural induction

SHC genes codify for a family of adaptor molecules comprising four genes. Previous data have implicated the Shc(s) molecules in stem cell division and differentiation. Specifically, the p66(ShcA) isoform has been found to contribute to longevity and resistance from oxidative stress. Here we report that p66(ShcA) is up-regulated during in vitro neural induction in embryonic stem cells. p66(ShcA) over-expression in ES cells reduces GSK-3beta kinase activation and increases beta-catenin stabilization and its transcriptional activity. p66(ShcA) over-expression results in ES cells undergoing an anticipated neural induction and accelerated neuronal differentiation. Similar effects are obtained in human ES cells over-expressing p66(ShcA). This study reveals a role for p66(ShcA) in the modulation of Wnt/beta-catenin pathway and in ES cell neuralization which is consistent between mouse and huma

Wnt5a is a transcriptional target of Dlx homeogenes and promotes differentiation of interneuron progenitors in vitro and in vivo

During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a

Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials

A population of mouse embryonic stem (ES)-derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na(+) currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na(+) channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na(+) currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na(+) current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na(+) channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturatio