In vitro and in vivo pharmacological characterization of SSD114, a novel GABAB positive allosteric modulator.

Positive allosteric modulators (PAMs) of the GABAB receptor have emerged as a novel approach to the pharmacological manipulation of the GABAB receptor, enhancing the effects of receptor agonists with few side effects. Here, we identified N-cyclohexyl-4-methoxy-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine (SSD114) as a new compound with activity as a GABAB PAM in in vitro and in vivo assays. SSD114 potentiated GABA-stimulated [35S]GTPγS binding to native GABAB receptors, whereas it had no effect when used alone. Its effect on GTPγS stimulation was suppressed when GABA-induced activation was blocked with CGP54626, a competitive antagonist of the GABAB receptor. SSD114 failed to potentiate WIN55,212,2-, morphine- and quinpirole-induced [35S]GTPγS binding to cortical and striatal membranes, respectively, indicating that it is a selective GABAB PAM. Increasing SSD114 fixed concentrations induced a leftward shift of the GABA concentration-response curve, enhancing the potency of GABA rather than its efficacy. SSD114 concentration-response curves in the presence of fixed concentrations of GABA (1, 10, and 20µM) revealed a potentiating effect on GABA-stimulated binding of [35S]GTPγS to rat cortical membranes, with EC50 values in the low micromolar range. Bioluminescence resonance energy transfer (BRET) experiments in Chinese Hamster Ovary (CHO)-cells expressing GABAB receptors showed that SSD114 potentiates the GABA inhibition of adenylyl-cyclase mediated by GABAB receptors. Our compound is also effective in vivo potentiating baclofen-induced sedation/hypnosis in mice, with no effect when tested alone. These findings indicate that SSD114, a molecule with a different chemical structure compared to known GABAB PAMs, is a novel GABAB PAM with potential usefulness in the GABAB-receptor research field.

Synthesis and pharmacological characterization of 2-(acylamino)thiophene derivatives as metabolically stable, orally effective, positive allosteric modulators of the GABAB receptor.

Two recently reported hit compounds, COR627 and COR628, underpinned the development of a series of 2-(acylamino)thiophene derivatives. Some of these compounds displayed significant activity in vitro as positive allosteric modulators of the GABAB receptor by potentiating GTPγS stimulation induced by GABA at 2.5 and 25 µM while failing to exhibit intrinsic agonist activity. Compounds were also found to be effective in vivo, potentiating baclofen-induced sedation/hypnosis in DBA mice when administered either intraperitoneally or intragastrically. Although displaying a lower potency in vitro than the reference compound GS39783, the new compounds 6, 10, and 11 exhibited a higher efficacy in vivo: combination of these compounds with a per se nonsedative dose of baclofen resulted in shorter onset and longer duration of the loss of righting reflex in mice. Test compounds showed cytotoxic effects at concentrations comparable to or higher than those of GS39783 or BHF177.

Characterization of COR627 and COR628, two novel positive allosteric modulators of the GABA(B) receptor.

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.

Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria.

Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l(-1) h(-1) of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure L: -lactic acid can be obtained.

Synthesis and characterisation of nonclassical ruthenium hydride complexes containing chelating bidentate and tridentate phosphine ligands.

The synthesis and characterisation of nonclassical ruthenium hydride complexes containing bidentate PP and tridentate PCP and PNP pincer-type ligands are described. The mononuclear and dinuclear ruthenium complexes presented have been synthesised in moderate to high yields by the direct hydrogenation route (one-pot synthesis) or in a two-step procedure. In both cases [Ru(cod)(metallyl)(2)] served as a readily available precursor. The influences of the coordination geometry and the ligand framework on the structure, binding, and chemical properties of the M--H(2) fragments were studied by X-ray crystal structure analysis, spectroscopic methods, and reactivity towards N(2), D(2), and deuterated solvents.