Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ～12 ? from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.     

Identification of novel α7 nicotinic receptor ligands by in silico screening against the crystal structure of a chimeric α7 receptor ligand binding domain

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK_i ?5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [³H]epibatidine on chimeric α7/5-HT₃ receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.     

SAR of α7 nicotinic receptor agonists derived from tilorone: exploration of a novel nicotinic pharmacophore

The well-known interferon-inducer tilorone was found to possess potent affinity for the agonist site of the α7 neuronal nicotinic receptor (K(i)=56 nM). SAR investigations determined that both basic sidechains are essential for potent activity, however active monosubstituted derivatives can also be prepared if the flexible sidechains are replaced with conformationally rigidified cyclic amines. Analogs in which the fluorenone core is replaced with either dibenzothiophene-5,5-dioxide or xanthenone also retain potent activity.     

Induction of antibodies to particular sites of the α7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the α7 subunit of the human nicotinic acetylcholine receptor (α7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2^d, H-2^b, and H-2^k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2^d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (α7 nAChRα. The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the α7 subunit: 1–23, 98–106, 159–168, and 173–188 (or 179–188).     

Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the?medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.     

Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

We introduce the term ‘silent agonists’ to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5′-phenylanabaseine) was synthesized and identified as a new silent agonist for the α7 nAChR; it binds to the receptor but does not activate α7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the α7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of α7 nAChR ligands may constitute a new alternative for the development of α7 nAChR therapeutics.     

Structure–activity relationships of N-substituted ligands for the α7 nicotinic acetylcholine receptor

A series of α7 neuronal nicotinic acetylcholine receptor ligands were designed based on a structural combination of a potent, but non-selective ligand, epibatidine, with a selective lead structure, 2. Three series of compounds in which aryl moieties were attached via a linker to different positions on the core structure were studied. A potent and functionally efficacious analog, (3aR,6aS)-2-(6-phenylpyridazin-3-yl)-5-(pyridin-3-ylmethyl)octahydropyrrolo[3,4-c]pyrrole (3a), was identified.     

Estrogen receptor α and β are involved in the activation of lordosis behavior in estradiol-primed rats

The present study was designed to assess the participation of estrogen receptors alpha (ERα) and beta (ERβ) in the short-term facilitation of lordosis behavior in ovariectomized (ovx), estradiol (E₂) primed rats. In experiment 1, dose response curves for PPT and DPN (ERα and ERβ agonists, respectively) facilitation of lordosis behavior (lordosis quotient and lordosis score) were established by infusing these agonists into the right lateral ventricle (icv) in female rats injected 40 h previously with 5 μg of E₂ benzoate. PPT doses of 0.08 and 0.4 ng produced high lordosis quotients starting at 30 min and continuing at 120 and 240 min post-injection. DPN induced high levels of lordosis behavior at all times tested. However, the intensity of lordosis induced by both agonists was weak. In experiment 2, we tested the involvement of each ER in facilitation of lordosis by icv infusion of MPP (ERα-selective antagonist) or PHTPP (ERβ-selective antagonist) prior to infusion of 2 ng of free E₂. Icv infusion of either MPP or PHTPP 30 min before free E2 significantly depressed E₂ facilitation of lordosis. The results suggest that both forms of ER are involved in the short-latency facilitation of lordosis behavior in E₂-primed rats.     

Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 μg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (～ 50%), NADPH oxidase 4 (NOX4; ～100%), and transforming growth factor-β expression (～200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.     

Multiple nicotinic acetylcholine receptor α-subunits are expressed in the arterial system of the rat

Neuronal nicotinic acetylcholine receptors (nAChRs) are hetero- and homopentamers built up by nine different alpha-subunits and three different beta-subunits. The subtype composition within the receptor determines ligand specificity, affinity and cation permeability. In this study we focused on the distribution of the ligand binding alpha-subunits in the rat arterial system by means of RT-PCR and immunohistochemistry. Subtypes alpha3, alpha5, alpha7 and alpha10 were found to be expressed by endothelial cells, suggesting that they are equipped both with calcium-preferring (alpha7 homopentamers) and monovalent cation-preferring (heteropentamers containing alpha3- and alpha5-subunits) nAChR channels. All alpha-subtypes except alpha9 were expressed by vascular smooth muscle cells with a highly specific distribution pattern along the vascular tree. While every alpha-subunit except alpha9 was detected in the thoracic aorta, intrapulmonary arterial branches contained only alpha7 immunoreactivity, and other vascular beds held intermediate positions with respect to the extent of alpha-subunit expression. Current knowledge does not allow to correlate these distribution patterns to specific functions, but it can be anticipated that at least some components of nAChR-mediated signalling in the arterial wall are highly specific for individual arteries.     

Phosphorylation of Akt/GSK-3β/eNOS amplifies 5-HT2B receptor blockade mediated anti-hypertrophic effect in rats

Herein, we studied the cross talk between 5-HT(2B) receptor blocker (SB-204741) and GSK-3β inhibitor (SB-216763) in isoproterenol-induced cardiac hypertrophy for 28 days. SB-204741 treatment significantly ameliorated (P<0.05) myocardial dysfunction, myocyte area, fibrosis and myocardial architecture in isoproterenol insulted myocardium. Moreover, this improvement in functional and morphological changes was associated with suppression of hypertrophic (BNP and CK-MB), inflammatory (IKK-β/NF-κB/TNF-α and CRP), and apoptotic markers (TUNEL positivity and Bax expression) along with phosphorylation of Akt/GSK-3β/β-catenin/eNOS. Intriguingly, co-treatment with GSK-3β inhibitor (P<0.01) further amplified the anti-hypertrophic effect of SB-204741 (P<0.05) such that the effect was indistinguishable from that of vehicle treated rats. Thus, 5-HT(2B) receptor blockade mediated anti-hypertrophic effect is atleast in part is governed through phosphorylation of Akt/GSK-3β/β-catenin/eNOS via attenuating inflammatory and apoptotic pathways.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.     

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg₃ (Rg₃) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg₃. We further characterized Rg₃ regulation of L247T receptors. We found that Rg₃ inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg₃ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg₃ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg₃ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg₃ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg₃ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg₃ at the channel pore.     

Toxicity study in juvenile rats with the α4β2 nicotinic acetylcholine receptor partial agonist CP-601,927

BACKGROUND: CP‐601927 is a selective α₄β₂ nicotinic acetylcholine receptor (nAChR) partial agonist. The objective of this study was to assess the potential effects persisting into adulthood when CP‐601,927 was administered to neonatal/juvenile rats. Since the juvenile toxicity study was being performed early in the development program and this study would represent the longest dosing period yet evaluated, the study design incorporated standard endpoints typically evaluated in a general toxicity screening study. METHODS: CP‐601,927 was administered to Sprague‐Dawley rats from postnatal day (PND) 7–70 by oral gavage at doses of 0.3, 1, or 3 mg/kg. During treatment animals were evaluated for growth, development, and sexual maturation. At the end of the treatment period general toxicity screening endpoints were collected (e.g., organ weights, histology, clinical chemistry). Following a 2‐week latency period, animals were evaluated for CNS function in a comprehensive behavioral training battery consisting of a functional observational battery, motor activity, acoustic startle response, and learning and memory evaluations. Reproductive competency was evaluated by mating treated rats and allowing pregnant dams to deliver and rear their litters until PND 10. RESULTS AND CONCLUSIONS: Treatment‐related findings included the death of 2 males receiving 3 mg/kg CP‐601,927, and transient reductions in body weight for both males and females during the third week of dosing which quickly recovered to control levels. The only treatment‐related alteration in behavior was decreased motor activity, which occurred only in females at the highest dose tested. CP‐601,927 had no effect on acoustic startle response, learning and memory, sexual maturation, reproductive capacity, or general toxicity endpoints. Birth Defects Res (Part B) 92:323–332, 2011. © 2011 Wiley‐Liss, Inc.