Pharmacophore identification and validation for human nAChR α7 agonists

Human nAChR α7 is the potential target for schizophrenia cognitive disorders, and it is meaningful to develop selective human nAChR α7 agonists for the clinical treatment of the disease. Because the crystal structure of α7 receptor has not been resolved, ligand-based drug design strategy was took in this work. A 3D QSAR pharmacophore model was built by HypoGen method, and its quality was evaluated by cost function. Furthermore, the pharmacophore model was validated with activity prediction of test set and was cross-validated based on Fisher’s Randomization Method. By Enrichment Factor and AU-ROC analysis, the final pharmacophore, which is consisted of one HBA, two Hydrophobic and one PosIonizable, was selected and it fitted well with the docking result of α7 homology model and the ligand. The pharmacophore is expected for the following virtual screening and lead optimization of human nAChR α7 agonists, which is important for the development and discovery of novel antipsychotics.     

Sonogashira coupling reaction in the synthesis of novel positive allosteric modulators of α7 nicotinic acetylcholine receptors

A series of 2-arylamino-1,3,5-triazine derivatives (4a–4g), which were designed and synthesized via Sonogashira coupling reaction, were evaluated using two-electrode voltage clamp (TEVC) recordings of human α7 nAChR expressed in Xenopus ooctyes. Compound 4g as a positive allosteric modulator (PAM) showed better efficacy than lead compound 3 (HZZ-A-11) with an EC₅₀ value of 1.23 ± 0.41 μM. Further pharmacological evaluation of compound 4g might lead to the developmental potential for therapy of cognitive deficits commonly shared by neuropsychiatric disorders, such as schizophrenia and Alzheimer’s disease.     

Functional characterization and high-throughput screening of positive allosteric modulators of α7 nicotinic acetylcholine receptors in IMR-32 neuroblastoma cells

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.     

A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia

Background and Purpose

Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596.

Experimental Approach

An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats.

Key Results

Choline (20–200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg⁻¹, s.c. and 1 mg·kg⁻¹, i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg⁻¹, i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments.

Conclusions and Implications

PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting neuroprotective effects of endogenous choline/ACh.     

The research group of Professor Qi Sun has made new progress on α7 nAChR positive allosteric modulator and its improvement in cognitive dysfunction

The research group of Professor Qi Sun has made new progress on α7 nAChR positive allosteric modulator and its improvement in cognitive dysfunction.     

Positive modulation of α7 nAChR responses in rat hippocampal interneurons to full agonists and the α7-selective partial agonists, 4OH-GTS-21 and S 24795

One approach for the identification of therapeutic agents for Alzheimer's disease has focused on the research of α7 nAChR-selective agonists such as the partial agonists 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) and, more recently, 2-[2-(4-bromophenyl)-2-oxoethyl]-1-methyl pyridinium (S 24795). An alternative approach for targeting α7 nAChR has been the development of positive modulators for this receptor. In this study we examined the interactions between full or partial agonists and positive modulators of α7 nAChRs in situ in brain tissue. Three positive modulators were used, 5-hydroxyindole (5-HI), 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), and genistein. Whole-cell recordings were performed in stratum radiatum interneurons from rat brain slices. Hippocampal interneurons were stimulated by ACh, choline, S 24795, or 4OH-GTS-21, before and after bath perfusion with the positive modulators. 5-HI was not effective at potentiating 200 μM 4OH-GTS-21-evoked responses, however 5-HI induced a sustained potentiation of responses evoked by 30 μM 4OH-GTS-21. When 1 mM ACh and 200 μM 4OH-GTS-21 were applied alternately α7-mediated responses to both agonists were reduced, suggesting that high concentration of 4OH-GTS-21 produces residual inhibition or desensitization and that 5-HI is not effective at overcoming receptor desensitization. Similar results were obtained with α7 receptors expressed in Xenopus oocytes. Interestingly, responses evoked by S 24795 were potentiated by 5-HI but not by genistein. Additionally, PNU-120596 was able to potentiate α7-mediated responses, regardless of the nature of the agonist. We demonstrated that the potentiation of α7 nAChR response would depend on the nature and the effective concentration of the agonist involved and its particular interaction with the positive modulator.     

Solution-phase parallel synthesis and SAR of homopiperazinyl analogs as positive allosteric modulators of mGlu₄

Using a functional high-throughput screening (HTS) and subsequent solution-phase parallel synthesis approach, we have discovered a novel series of positive allosteric modulators for mGlu?, a G-protein coupled receptor. This series is comprised of a homopiperazine central core. The solution-phase parallel synthesis and SAR of analogs derived from this series will be presented. This series of positive allosteric modulators of mGlu? provide critical research tools to further probe the mGlu?-mediated effects in Parkinson's disease.     

The quest for the treatment of cognitive impairment: α7 nicotinic and α5 GABAA receptor modulators

Age, age-related pathologies, certain psychiatric disorders, head traumas and other conditions are characterised by an impairment of cognitive functions. Cognition is a complex process involving a large number of neurotransmitters that can modulate, positively or negatively, learning and memory; therefore, their receptors may represent suitable targets to develop cognition-enhancing drugs. Among others, the α₇ nicotinic cholinergic receptor and the α₅ GABA_A receptor are emerging as attractive targets for developing therapeutics in this field. The important role of α₇ nicotinic receptors has been proven thanks to the discovery of α₇-selective agonists, such as GTS-21 and AR-R17779, which has stimulated the synthesis of a large number of new compounds, some of which are in clinical trials. The observation that the classical tranquilliser benzodiazepines (agonists that potentiate GABA_A receptor functions), are amnesic, while inverse agonists (that attenuate the functions of the same receptor) improve cognitive tasks, stimulated the search for modulators mainly directed toward the α₅-containing GABA_A receptor, which seems at present the most important GABA_A receptor subtype involved in cognitive processes. This article reviews the patents on modulators of α₇ nicotinic acetylcholine and GABA_A receptors disclosed during the period 2000 – 2006.     

Regulatory role of the α7nAChR in cancer

Exposure to tobacco products is responsible for the majority of all human cancers. Nicotinic acetylcholine receptors (nAChRs) were identified as early as 1989 as important regulators of cancer cells. In analogy to its function in the brain, the homomeric α7nAChR has "accelerator function" on the most common human cancers by stimulating the synthesis and release of excitatory neurotransmitters (serotonin in small cell lung cancer, noradrenaline/adrenaline in most other cancers) that drive cell proliferation, migration, angiogenesis, neurogenesis and metastasis while inhibiting apoptosis. These effects are not only caused by α7nAChRs expressed in cancer cells but also by α7nAChRs in ganglia and nerves of the sympathetic part of the autonomic nervous system that release noradrenaline/adrenaline into the tumor environment. In the nervous system, α7nAChR protein undergoes paradoxical upregulation without concomitant desensitization upon chronic exposure to nicotine. The same phenomenon has been reported for α7nAChR expressed in cancer cells of the lungs and pancreas where chronic nicotine or nicotine-derived nitrosamines upregulated the receptor protein, resulting in hyperactivity of its effectors. Strategies that target the α7nAChR for cancer intervention are highly promising but should aim to reduce signaling downstream of the receptor rather than blocking the receptor because of its numerous vital functions in the mammalian organism.     

In vivo pharmacological interactions between a type II positive allosteric modulator of α7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model

Background and Purpose

The α7 nicotinic ACh receptor subtype is abundantly expressed in the CNS and in the periphery. Recent evidence suggests that α7 nicotinic ACh receptor (nAChR) subtypes, which can be activated by an endogenous cholinergic tone comprising ACh and the α7 agonist choline, play an important role in chronic pain and inflammation. In this study, we evaluated whether type II α7 positive allosteric modulator PNU-120596 induces antinociception on its own and in combination with choline in the formalin pain model.

Experimental Approach

We assessed the effects of PNU-120596 and choline and the nature of their interactions in the formalin test using an isobolographic analysis. In addition, we evaluated the interaction of PNU-120596 with PHA-54613, an exogenous selective α7 nAChR agonist, in the formalin test. Finally, we assessed the interaction between PNU-120596 and nicotine using acute thermal pain, locomotor activity, body temperature and convulsing activity tests in mice.

Key Results

We found that PNU-120596 dose-dependently attenuated nociceptive behaviour in the formalin test after systemic administration in mice. In addition, mixtures of PNU-120596 and choline synergistically reduced formalin-induced pain. PNU-120596 enhanced the effects of nicotine and α7 agonist PHA-543613 in the same test. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, hypomotility and antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via activation of α7 nAChRs.

Conclusions and Implications

Our results demonstrate that type II α7 positive allosteric modulators produce antinociceptive effects in the formalin test through a synergistic interaction with the endogenous α7 agonist choline.     

The α7 nicotinic acetylcholine receptor positive allosteric modulator prevents lipopolysaccharide-induced allodynia,hyperalgesia and TNF-α in the hippocampus in mice

BackgroundPrevious studies have shown that α7 nicotinic acetylcholine receptor (nAChR) has a critical role in the regulation of pain sensitivity and neuroinflammation. However, pharmacological effects of α7 nAChR activation in the hippocampus on neuroinflammatory mechanisms associated with allodynia and hyperalgesia remain unknown. We have determined the effects of 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an α7 nAChR positive allosteric modulator, on lipopolysaccharide (LPS)-induced allodynia and hyperalgesia in mice. We also evaluated the effects of TQS on immunoreactivity of microglial marker ionized-calcium binding adaptor molecule 1 (Iba-1), phospho-nuclear factor-κB (p-NF-κB p⁶⁵), tumor necrosis factor-alpha (TNF-α), and norepinephrine (NE) level.MethodsMice were treated with (0.25, 1 or 4 mg/kg, ip) followed by LPS (1 mg/kg, ip) administration. Allodynia and hyperalgesia were determined using von Frey filaments and hot plate respectively. Immunoreactivity of Iba-1, p-NF-κB p⁶⁵, and TNF-α, were measured in the hippocampus using immunofluorescence assay. Hippocampal NE level was evaluated using high performance liquid chromatography.ResultsLPS administration resulted in allodynia and hyperalgesia in mice after six h. Systemic administration of TQS prevented LPS-induced allodynia and hyperalgesia. TQS pretreatment significantly decreased the immunoreactivity of Iba-1, p-NF-κB, and TNF-α in CA1 and DG regions of the hippocampus. In addition, TQS reversed LPS-induced NE reduction in the hippocampus.ConclusionsTaken together, our results suggest that TQS prevented LPS-induced allodynia and hyperalgesia, upregulation of TNF-α expression and NE level reduction involving microglial α7 nAChR in part in the hippocampus. Therefore, these findings highlight the important effects of α7 nAChR allosteric modulator against symptoms of inflammatory pain.     

Competitive binding at a nicotinic receptor transmembrane site of two α7-selective positive allosteric modulators with differing effects on agonist-evoked desensitization

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the ‘M2–M3 segment’ in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site.