Flora Jow

Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1.

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca2+ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca2+ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca2+-induced cell death by modulating Ca2+ channels and promote neurite outgrowth via FKBP52 binding.

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.

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

Production of GABA by cultured hippocampal glial cells

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.

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)

Alpha-7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha7 nAChR deriving from our continuing efforts in the areas of Alzheimers disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

Discovery of a Novel Alpha-7 Nicotinic Acetylcholine Receptor Agonist Series and Characterization of the Potent, Selective, and Orally Efficacious Agonist 5-(4-Acetyl[1,4]diazepan-1-yl)pentanoic Acid [5-(4-Methoxyphenyl)-1H-pyrazol-3-yl] Amide (SEN15924, WAY-361789)

Alpha-7 nicotinic acetylcholine receptors (α7 nAChR) are implicated in the modulation of many cognitive functions such as attention, working memory, and episodic memory. For this reason, α7 nAChR agonists represent promising therapeutic candidates for the treatment of cognitive impairment associated with Alzheimers disease (AD) and schizophrenia. A medicinal chemistry effort, around our previously reported chemical series, permitted the discovery of a novel class of α7 nAChR agonists with improved selectivity, in particular against the α3 receptor subtype and better ADME profile. The exploration of this series led to the identification of 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (25, SEN15924, WAY-361789), a novel, full agonist of the α7 nAChR that was evaluated in vitro and in vivo. Compound 25 proved to be potent and selective, and it demonstrated a fair pharmacokinetic profile accompanied by efficacy in rodent behavioral cognition models (novel object recognition and auditory sensory gating).

Cloning and functional expression of rKCNQ2 K+ channel from rat brain

By homologue cloning, we have isolated a cDNA encoding a voltage-gated K(+) channel, rKCNQ2, from a rat brain cDNA library using RACE. The open reading frame of the translated protein comprises 852 amino acids with 6 transmembrane segments and a pore motif between S5 and S6. rKCNQ2 shares 96% amino acid identity with human KCNQ2 in which mutations cause a form of epilepsy known as benign familial neonatal convulsions (BFNC). Northern blotting with a rKCNQ2-specific probe revealed a robust single band of 8.6-kb transcript expressed in brain not in other tissues. Functional expression of rKCNQ2 in an HEK 293 cell line by whole-cell current recording and in Xenopus oocytes by two-electrode voltage clamp showed outward K(+) selective currents that displayed delayed rectifier-type kinetics. The G-V curve, fitted with a Boltzmann function, showed voltage dependence of activation with a threshold of activation approximately -60 mV. The rKCNQ2 currents were sensitive to TEA block with a Ki of 0.1 mM. In addition, rKCNQ2 currents were down-regulated upon exposure of cells to either a broad-spectrum tyrosine kinase inhibitor genistein or a Src-like tyrosine kinase inhibitor herbimycin A. Our findings add a rodent member to the KCNQ channel subfamily, providing new information of the channel modulation, and will facilitate generation of rodent models of epilepsy.

Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

1 Cultured human capillary endothelial cells (HCEC) contain a large inward rectifier current, IK(IR), that can be abolished by removing external K+ or by adding 50 μm Ba2+. 2 We show that IK(IR) is responsible for maintaining the hyperpolarized potential (−60.6 ± 0.5 mV, n= 83) of HCEC. Blocking IK(IR) with 50 μm Ba2+ shifts the zero current level and depolarizes HCEC by 36.5 ± 1.3 mV (n= 4). 3 Increasing external Ca2+ concentration ([Ca2+]o) from 0.5 to 7 mm reduces the magnitude of IK(IR) by 36.5 ± 2.3 % (n= 5) and depolarizes the cells by 10.33 ± 2.4 mV (n= 3), whereas decreasing [Ca2+]o from 1.8 to 0.5 mm increases the amplitude of IK(IR) by 6.9 ± 1.9 % (n= 4). The relationship between [Ca2+]o and the percentage block of IK(IR) gives a Kd value of 5.4 ± 0.6 mm at −120 mV. 4 I K(IR) is also blocked by other divalent ions, with Ba2+ >> Sr2+ > Mg2+ > Mn2+= Ca2+, and the block of peak current at −120 mV being 85.3 ± 3.2 % (n= 5) for 50 μm Ba2+, 62.9 ± 2.2 % (n= 5) for 5 mm Sr2+, 40.7 ± 2.5 % (n= 9) for 5 mm Mg2+, 33.4 ± 2.1 % (n= 5) for 5 mm Mn2+ and 32.9 ± 2.1 % (n= 5) for 5 mm Ca2+. 5 The voltage dependence of Sr2+ block of peak IK(IR) occurred with a Kd value of 1.0 ± 0.09 mm for −140 mV, 1.9 ± 0.16 mm for −130 mV, 3.1 ± 0.28 mm for −120 mV, 4.6 ± 0.34 mm for −110 mV and 6.4 ± 0.5 mm for −100 mV (n= 5), with a calculated electrical distance (δ) of 0.44 from the outside.

N-[5-(5-fluoropyridin-3-yl)-1H-pyrazol-3-yl]-4-piperidin-1-ylbutyramide (SEN78702, WYE-308775): a medicinal chemistry effort toward an α7 nicotinic acetylcholine receptor agonist preclinical candidate.

α7 Nicotinic acetylcholine receptors (α7 nAChR) represent promising therapeutic candidates for the treatment of cognitive impairment associated with Alzheimers disease (AD) and schizophrenia. A medicinal chemistry effort around previously reported compound 1 (SEN15924, WAY-361789) led to the identification of 12 (SEN78702, WYE-308775) a potent and selective full agonist of the α7 nAChR that demonstrated improved plasma stability, brain levels, and efficacy in behavioral cognition models.