Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the ß1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFßR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial-mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376-87. ©2017 AACR.

Development of NMDAR antagonists with reduced neurotoxic side effects: a study on GK11.

The NMDAR glutamate receptor subtype mediates various vital physiological neuronal functions. However, its excessive activation contributes to neuronal damage in a large variety of acute and chronic neurological disorders. NMDAR antagonists thus represent promising therapeutic tools that can counteract NMDARs' overactivation. Channel blockers are of special interest since they are use-dependent, thus being more potent at continuously activated NMDARs, as may be the case in pathological conditions. Nevertheless, it has been established that NMDAR antagonists, such as MK801, also have unacceptable neurotoxic effects. Presently only Memantine is considered a safe NMDAR antagonist and is used clinically. It has recently been speculated that antagonists that preferentially target extrasynaptic NMDARs would be less toxic. We previously demonstrated that the phencyclidine derivative GK11 preferentially inhibits extrasynaptic NMDARs. We thus anticipated that this compound would be safer than other known NMDAR antagonists. In this study we used whole-genome profiling of the rat cingulate cortex, a brain area that is particularly sensitive to NMDAR antagonists, to compare the potential adverse effects of GK11 and MK801. Our results showed that in contrast to GK11, the transcriptional profile of MK801 is characterized by a significant upregulation of inflammatory and stress-response genes, consistent with its high neurotoxicity. In addition, behavioural and immunohistochemical analyses confirmed marked inflammatory reactions (including astrogliosis and microglial activation) in MK801-treated, but not GK11-treated rats. Interestingly, we also showed that GK11 elicited less inflammation and neuronal damage, even when compared to Memantine, which like GK11, preferentially inhibits extrasynaptic NMDAR. As a whole, our study suggests that GK11 may be a more attractive therapeutic alternative in the treatment of CNS disorders characterized by the overactivation of glutamate receptors.

Expression of purinergic P2Y receptor subtypes by INS-1 insulinoma beta-cells: a molecular and binding characterization.

Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35)S]thiotriphosphate) (ATP-alpha-[(35)S]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[(35)S] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[(35)S] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y(1) receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites. Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated.

P2Y receptor mediated modulation of insulin release by a novel generation of 2-substituted-5'-O-(1-boranotriphosphate)-adenosine analogues.

PURPOSE: A series of C2-substituted ATP analogues was previously shown to have potent insulin-secreting properties, yet with poor tissue-selectivity for the pancreatic beta-cell. The present study was designed to evaluate the binding profile on beta-cell membranes and the effects on insulin release and pancreatic vascular resistance of a second generation of P2Y(1) receptor agonists, based on C2-substitution of the adenosine 5'-O-(1-boranotriphosphate) scaffold. MATERIALS AND METHODS: Functional experiments were performed in the rat isolated pancreas model; binding studies with ATP-alpha-[(35)S] were performed in membrane homogenates from the rat insulinoma INS-1 cell line. The diastereoisomers of the compounds are designated by A and B. RESULTS: Under 8.3 mmol l(-1) glucose, 2-methylthio-ATP-alpha-B, A isomer, induced a biphasic and concentration dependent insulin response; its maximal efficacy reaches ninefold the baseline secretion and its EC(50) is 28.1 nmol l(-1). No significant effect of this isomer was observed on vascular resistance, whereas the B isomer, which was a less potent insulin secretagogue, consistently induced a transient vasoconstriction. Interestingly, the insulin response induced by 2-methylthio-ATP-alpha-B, A isomer, was clearly glucose-dependent. This drug competes with ATP-alpha-[(35)S] binding in a complex two sites interaction model, with a K(0.5) value of 17.7 nmol l(-1). 2-Chloro-ATP-alpha-B had a similar insulin-secreting profile as 2-methylthio-ATP-alpha-B, with a lower tissue-selectivity. The non-substituted ATP-alpha-B analog, A isomer, was less potent than the C2-substituted derivatives (A isomers) and had a vasorelaxant effect. CONCLUSIONS: We conclude that 2-methylthio-ATP-alpha-B, A isomer, is a potent and tissue-selective P2Y receptor agonist with high efficacy. Its insulin-releasing action is glucose-dependent, which gives interest to this compound as a drug candidate for treating type 2 diabetes.

Acute exposure to GSM 900-MHz electromagnetic fields induces glial reactivity and biochemical modifications in the rat brain.

The worldwide proliferation of mobile phones raises the question of the effects of 900-MHz electromagnetic fields (EMF) on the brain. Using a head-only exposure device in the rat, we showed that a 15-min exposure to 900-MHz pulsed microwaves at a high brain-averaged power of 6 W/kg induced a strong glial reaction in the brain. This effect, which suggests neuronal damage, was particularly pronounced in the striatum. Moreover, we observed significant and immediate effects on the Kd and Bmax values of N-methyl-D-aspartate (NMDA) and GABA(A) receptors as well as on dopamine transporters. Decrease of the amount of NMDA receptors at the postsynaptic membrane is also reported. Although we showed that the rat general locomotor behavior was not significantly altered on the short term, our results provide the first evidence for rapid cellular and molecular alterations in the rat brain after an acute exposure to high power GSM (Global System for Mobile communication) 900-MHz microwaves.

Re-evaluation of phencyclidine low-affinity or "non-NMDA" binding sites.

TCP and its derivative gacyclidine (+/- GK11) are high-affinity non-competitive antagonists of N-methyl-D-aspartate (NMDA) receptors (NMDARs) and as such exhibit significant neuroprotective properties. These compounds also bind with a low affinity to binding sites whose pharmacological profiles are different from that of NMDARs. With the intention to develop new strategies of neuroprotection, we found it mandatory to investigate whether 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and gacyclidine low-affinity sites are similar. The effects of several drugs selective for either NMDARs or the [(3)H]TCP low-affinity site (or PCP(3) site) on (+), (-)[(3)H]GK11 and [(3)H]TCP specific binding were investigated. Competition experiments on cerebellum homogenates revealed substantial differences between the pharmacological profiles of the PCP(3) site and that of gacyclidine's enantiomers low-affinity sites. Under experimental conditions preventing the interaction of the radioligands with NMDARs, the autoradiographic study showed, however, that the distributions of both [(3)H]TCP and (-)[(3)H]GK11 specific binding were similar. The specific labelling was low and uniform in telencephalic structures, whereas in the cerebellum it was higher in the molecular than in the granular layer. Finally, the analysis of competition experiments performed on tissues slices demonstrated that PCP(3) selective ligands were unable to prevent [(3)H]TCP or (-)[(3)H]GK11 binding to "non-NMDA" binding sites. As a whole, our data suggest that: (1) the different pharmacological profiles of [(3)H]TCP and [(3)H]gacyclidine enantiomers on low-affinity sites are due to their selectivity for specific NMDARs subpopulations; (2) the pharmacological isolation of TCP and gacyclidine "non-NMDA" binding sites is the most appropriate way to further study the low-affinity component of their specific binding. Obtaining reliable and specific pharmacological tools for those binding sites is of particular interest, since it is likely that they play a substantial role in the low neurotoxicity, and therefore tolerability, of gacyclidine, a new neuroprotective drug currently evaluated in clinical trials for the treatment of brain and spinal cord injuries.