Renza Roncarati

Procognitive and Neuroprotective Activity of a Novel α7 Nicotinic Acetylcholine Receptor Agonist for Treatment of Neurodegenerative and Cognitive Disorders

The α7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimers disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of α7 nAChR. SEN12333 shows high affinity for the rat α7 receptor expressed in GH4C1 cells (Ki = 260 nM) and acts as full agonist in functional Ca2+ flux studies (EC50 = 1.6 μM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC50 = 12 μM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at α3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the α7-selective antagonist methyllycaconitine, indicating that it is mediated by α7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel α7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of α7 agonists for treatment of neurodegenerative and cognitive disorders.

Inhibition of Wnt signaling, modulation of Tau phosphorylation and induction of neuronal cell death by DKK1.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimers Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Molecular Cloning and Characterization of the Human Diacylglycerol Kinase β (DGKβ) Gene ALTERNATIVE SPLICING GENERATES DGKβ ISOTYPES WITH DIFFERENT PROPERTIES

Diacylglycerol kinases are key modulators of levels of diacylglycerol, a second messenger involved in a variety of cellular responses to extracellular stimuli. A number of diacylglycerol kinases encoded by separate genes are present in mammalian genomes. We have cloned cDNAs encoding several isoforms of the human homologue of the rat diacylglycerol kinase β gene and characterized two such isoforms that differ at their carboxyl terminus through alternative splicing and the usage of different polyadenylation signals. Quantitative analysis of gene expression in a panel of human tissue cDNAs revealed that transcripts corresponding to both isoforms are co-expressed in central nervous system tissues and in the uterus, with one variant being expressed at relatively higher levels. As green fluorescent protein fusions, the two isoforms displayed localization to different subcellular compartments, with one variant being associated with the plasma membrane, while the other isoform was predominantly localized within the cytoplasm. Differences were also observed in their subcellular localization in response to phorbol ester stimulation. Enzymatic assays demonstrated that the two isoforms display comparable diacylglycerol kinase activities. Therefore, the human diacylglycerol kinase β gene can generate several enzyme isoforms, which can display different expression levels and subcellular localization but similar enzymatic activities in vitro.

Old and New Pharmacology: Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor by the 5-Hydroxytryptamine2B/C Receptor Antagonist SB-206553 (3,5-Dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b′]di pyrrole-1(2H)-carboxamide)

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimers disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b′]-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine2B/2C receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 μM for potentiation of EC20 nicotine responses in GH4C1 cells expressing the α7 receptor. SB-206553 was devoid of direct α7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the α7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 μM) and EC100 (124 μM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 μM), an effect that was entirely blocked by the α7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 μM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of α7 nAChR PAMs in schizophrenia.

Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines.

A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel. The analysis of known blockers of calcium-activated potassium channels revealed the highest potency for apamin (IC(50)=13.2 nM). This result was confirmed by direct recordings of SK3 currents using the whole-cell patch-clamp technique. Tricyclic antidepressants such as desipramine, imipramine and nortriptyline as well as phenothiazines such as fluphenazine, promethazine, chlorpromazine and trifluoperazine blocked the hSK3 channel with micromolar potencies. These compounds also displaced [(125)I]-apamin binding to the hSK3 channel thus suggesting direct and competitive channel blocking activity. Since these compounds share a common three-ring molecular core structure, this feature seems to be important for channel blocking activity. The serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A were able to abolish channel activation with nanomolar potencies, but did not displace [(125)I]-apamin binding. Thus, phosphorylation of hSK3 or an accessory channel subunit seems to be involved in its modulation.

Screening technologies for ion channel drug discovery

For every movement, heartbeat and thought, ion channels need to open and close. It is therefore not surprising that their malfunctioning leads to serious diseases. Currently, only approximately 10% of drugs, with a market value in excess of US

SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist.

10 billion, act on ion channels. The systematic exploitation of this target class has started, enabled by novel assay technologies and fundamental advances of the structural and mechanistic understanding of channel function. The latter, which was rewarded with the Nobel Prize in 2003, has opened up an avenue for rational drug design. In this review we provide an overview of the current repertoire of screening technologies that has evolved to drive ion channel-targeted drug discovery towards new medicines of the future.

Functional Properties of α7 Nicotinic Acetylcholine Receptors Co-expressed with RIC-3 in a Stable Recombinant CHO-K1 Cell Line

Alpha 7 nicotinic acetylcholine receptor (alpha(7) nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimers disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimers disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the alpha(7) nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.

Pharmacological and molecular characterisation of SK3 channels in the TE671 human medulloblastoma cell line.

Heterologous functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs) is difficult to achieve in mammalian cell lines, and the reasons have been associated with a lack of expression of the putative chaperone factor RIC-3. Here, we describe the generation and functional and pharmacological characterization of a Chinese hamster ovary (CHO)-K1 cell line co-expressing the human alpha7 nAChR and RIC-3. Stable recombinant cells expressing alpha7 nAChR on the plasma membrane were selected by binding of fluorochrome-conjugated alpha-bungarotoxin and fluorescence-activated cell sorting. The presence of functional alpha7 channels was demonstrated by whole cell patch clamp recordings. Nicotine and acetylcholine induced rapid desensitizing currents with 50% effective concentration values of 14 and 37 microM, respectively, with agonist-evoked currents detected in approximately 75% of the cell population. Surprisingly, when tested in a FLIPR (Molecular Devices, Sunnyvale, CA) Ca(2+) assay, activation of alpha7 nAChRs was measured only when nicotinic agonists were applied either in the presence of the positive allosteric modulator (PAM) PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein. No Ca(2+) influx was measured upon addition of agonists alone or together with allosteric potentiators such as 5-hydroxyindole that predominantly increase the apparent peak amplitude without robustly affecting the current desensitization rate, as exemplified by PNU-120596. These results show that functional alpha7 nAChRs can stably be expressed in the non-neuronal CHO-K1 cell line. This recombinant cell system is useful for characterization of alpha7 nAChRs and to study the mechanism of action of chemical modulators, in particular the detection of PAMs capable of slowing receptor desensitization kinetics.

Novel Alpha-7 Nicotinic Acetylcholine Receptor Agonists Containing a Urea Moiety: Identification and Characterization of the Potent, Selective, and Orally Efficacious Agonist 1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) Urea (SEN34625/WYE-103914)

The expression of the small conductance calcium-activated potassium channels SK1, SK2 and SK3 was investigated in the TE671 human medulloblastoma cell line using RT-PCR and transcripts were detected only for SK3. Immunodetection experiments confirmed this result, demonstrating the presence of the SK3 protein. This potassium channel was characterised in TE671 cells using whole-cell patch-clamp recordings. Voltage steps to -100 mV from a holding potential of 0 mV in equimolar 140 mM intra- and extracellular K(+) (K(+)(in/out)) elicited an inward current. The reversal potential of this current shifted 56.6 mV per 10-fold increase in K(+)(out) thus suggesting K(+) selectivity. This current was dependent on the concentration of Ca(2+)(in) with an EC(50) of 104.2 nM. A pharmacological characterisation of this current revealed that it was not blocked by 1 microM charybdotoxin (ChTX), 0.3 microM iberiotoxin (IbTX) or 10 microM clotrimazole (CLT) and only modestly inhibited (<50%) by 30 nM scyllatoxin (ScTX), 200 microM dequalinium chloride (Deq) or 300 microM d-tubocurarine (d-TC). The non-selective SK blocker d-TC blocked the current with an IC(50) of 43.2 microM while apamin blocked the current to a much greater extent (87.8% at 1 microM) with an IC(50) of 4.3 nM. Furthermore, the current was significantly increased (132.6+/-5.2%, n=7) by 500 microM 1-ethyl-2-benzimidazolinone (EBIO). Collectively, these data demonstrate the presence of an endogenous SK3 channel in human TE671 cells.