In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Syntheses and structure-activity relationship (SAR) studies of 2,5-diazabicyclo[2.2.1]heptanes as novel alpha7 neuronal nicotinic receptor (NNR) ligands.

Biaryl substituted 2,5-diazabicyclo[2.2.1]heptanes have been synthesized and tested for their affinity toward alpha7 neuronal nicotinic receptors (NNRs). SAR studies established that 5-N-methyl substituent, heteroaryl linker and the nature of terminal aryl group are critical for the ligand to achieve potent alpha7 NNR agonist activity.

Reduced presynaptic vesicle stores mediate cellular and network plasticity defects in an early-stage mouse model of Alzheimer's disease.

BACKGROUND: Identifying effective strategies to prevent memory loss in AD has eluded researchers to date, and likely reflects insufficient understanding of early pathogenic mechanisms directly affecting memory encoding. As synaptic loss best correlates with memory loss in AD, refocusing efforts to identify factors driving synaptic impairments may provide the critical insight needed to advance the field. In this study, we reveal a previously undescribed cascade of events underlying pre and postsynaptic hippocampal signaling deficits linked to cognitive decline in AD. These profound alterations in synaptic plasticity, intracellular Ca2+ signaling, and network propagation are observed in 3-4 month old 3xTg-AD mice, an age which does not yet show overt histopathology or major behavioral deficits. METHODS: In this study, we examined hippocampal synaptic structure and function from the ultrastructural level to the network level using a range of techniques including electron microscopy (EM), patch clamp and field potential electrophysiology, synaptic immunolabeling, spine morphology analyses, 2-photon Ca2+ imaging, and voltage-sensitive dye-based imaging of hippocampal network function in 3-4 month old 3xTg-AD and age/background strain control mice. RESULTS: In 3xTg-AD mice, short-term plasticity at the CA1-CA3 Schaffer collateral synapse is profoundly impaired; this has broader implications for setting long-term plasticity thresholds. Alterations in spontaneous vesicle release and paired-pulse facilitation implicated presynaptic signaling abnormalities, and EM analysis revealed a reduction in the ready-releasable and reserve pools of presynaptic vesicles in CA3 terminals; this is an entirely new finding in the field. Concurrently, increased synaptically-evoked Ca2+ in CA1 spines triggered by LTP-inducing tetani is further enhanced during PTP and E-LTP epochs, and is accompanied by impaired synaptic structure and spine morphology. Notably, vesicle stores, synaptic structure and short-term plasticity are restored by normalizing intracellular Ca2+ signaling in the AD mice. CONCLUSIONS: These findings suggest the Ca2+ dyshomeostasis within synaptic compartments has an early and fundamental role in driving synaptic pathophysiology in early stages of AD, and may thus reflect a foundational disease feature driving later cognitive impairment. The overall significance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic vesicle stores, synaptic plasticity, and network propagation, which directly impact memory encoding.

Identification and characterization of novel antagonists of the CCR3 receptor.

Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand. Whole cells grown on polylysine-coated plates were used as the receptor source for the screen. Screening of more than 200,000 compounds with this assay yielded a number of screening hits, and of these, 2 active novel antagonists were identified. These compounds showed inhibitory effects on eosinophil chemotaxis in both in vitro and in vivo assays.

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5).

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.

Structure-activity studies of diazabicyclo[3.3.0]octane-substituted pyrazines and pyridines as potent α4ß2 nicotinic acetylcholine receptor ligands.

A series of diazabicyclo[3.3.0]octane substituted pyridines and pyrazines was synthesized and characterized at the α4ß2 neuronal nicotinic acetylcholine receptor (nAChR). The compounds were designed to mimic the profile of ABT-089, high affinity binding ligand for the α4ß2 nAChR, with limited agonist activity. Carboxamide derivatives of 3-(diazabicyclo[3.3.0]octane)-substituted pyridines or 2-(diazabicyclo[3.3.0]octane)-substituted pyrazines were found to have the desired binding and activity profile. The structure-activity relationship of these compounds is presented.

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties.

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

X-ray crystallographic structure of ABT-378 (lopinavir) bound to HIV-1 protease.

The crystal structure of ABT-378 (lopinavir), bound to the active site of HIV-1 protease is described. A comparison with crystal structures of ritonavir, A-78791, and BILA-2450 shows some analogous features with previous reported compounds. A cyclic urea unit in the P(2) position of ABT-378 is novel and makes two bidentate hydrogen bonds with Asp 29 of HIV-1 protease. In addition, a previously unreported shift in the Gly 48 carbonyl position is observed. A discussion of the structural features responsible for its high potency against wild-type HIV protease is given along with an analysis of the effect of active site mutations on potency in in vitro assays.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening microARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the G alpha(q) protein can readily be assessed by fluorescence-based Ca(2+) release measurements, many GPCRs that are coupled to G alpha(s) or G alpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D(4.4) receptor, which normally couples through the G alpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca(2+) release by stably coexpressing this receptor with a chimeric G(alpha qo5) protein in HEK-293 cells. In microARCS format, the cells expressing D(4.4) receptor and G alpha(qo5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable microARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.