Analysing the effect caused by increasing the molecular volume in M1-AChR receptor agonists and antagonists: a structural and computational study.

M1 muscarinic acetylcholine receptor (M1-AChR), a member of the G protein-coupled receptors (GPCR) family, plays a crucial role in learning and memory, making it an important drug target for Alzheimer's disease (AD) and schizophrenia. M1-AChR activation and deactivation have shown modifying effects in AD and PD preclinical models, respectively. However, understanding the pharmacology associated with M1-AChR activation or deactivation is complex, because of the low selectivity among muscarinic subtypes, hampering their therapeutic applications. In this regard, we constructed two quantitative structure-activity relationship (QSAR) models, one for M1-AChR agonists (total and partial), and the other for the antagonists. The binding mode of 59 structurally different compounds, including agonists and antagonists with experimental binding affinity values (pKi), were analyzed employing computational molecular docking over different structures of M1-AChR. Furthermore, we considered the interaction energy (Einter), the number of rotatable bonds (NRB), and lipophilicity (ilogP) for the construction of the QSAR model for agonists (R2 = 89.64, QLMO2 = 78, and Qext2 = 79.1). For the QSAR model of antagonists (R2 = 88.44, QLMO2 = 82, and Qext2 = 78.1) we considered the Einter, the fraction of sp3 carbons fCsp3, and lipophilicity (MlogP). Our results suggest that the ligand volume is a determinant to establish its biological activity (agonist or antagonist), causing changes in binding energy, and determining the affinity for M1-AChR.

pVHL suppresses Akt/ß-catenin-mediated cell proliferation by inhibiting 14-3-3ζ expression.

The mechanisms controlling degradation of cytosolic ß-catenin are important for regulating ß-catenin co-transcriptional activity. Loss of von Hippel-Lindau protein (pVHL) has been shown to stabilize ß-catenin, increasing ß-catenin transactivation and ß-catenin-mediated cell proliferation. However, the role of phosphoinositide 3-kinase (PI3K)/Akt in the regulation of ß-catenin signaling downstream from pVHL has never been addressed. Here, we report that hyperactivation of PI3K/Akt in cells lacking pVHL contributes to the stabilization and nuclear accumulation of active ß-catenin. PI3K/Akt hyperactivation is facilitated by the up-regulation of 14-3-3ζ and the down-regulation of 14-3-3ε, 14-3-3η and 14-3-3θ. Up-regulation of 14-3-3ζ in response to pVHL is important for the recruitment of PI3K to the cell membrane and for stabilization of soluble ß-catenin. In contrast, 14-3-3ε and 14-3-3η enhanced PI3K/Akt signaling by inhibiting PI3K and PDK1, respectively. Thus, our results demonstrated that 14-3-3 family members enhance PI3K/Akt/ß-catenin signaling in order to increase proliferation. Inhibition of Akt activation and/or 14-3-3 function strongly reduces ß-catenin signaling and decreases cell proliferation. Thus, inhibition of Akt and 14-3-3 function efficiently reduces cell proliferation in 786-0 cells characterized by hyperactivation of ß-catenin signaling due to pVHL loss.

Histamine H3 receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes.

The histamine H3 receptor (H3R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H3Rs couple to Gαi/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca2+ channels. In transfected cells, activation of the human H3R of 445 amino acids (hH3R445) results in phospholipase C (PLC) stimulation and release of Ca2+ from intracellular stores. In this work we have studied whether H3R activation induces Ca2+ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H3R activation induced inositol 1,4,5-trisphosphate (IP3) formation but failed to increase the intracellular calcium concentration ([Ca2+]i). In striatal primary cultures H3R activation resulted in IP3 formation and increased the [Ca2+]i in 18 out of 70 cells that responded with an elevation in the [Ca2+]i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca2+]i induced by H3R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H3R activation stimulates the PLC/IP3/Ca2+ pathway but only in a subpopulation of striatal neurons.

Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 µM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.