Synthesis of desformylflustrabromine and its evaluation as an α4β2 and α7 nACh receptor modulator

doi:10.1016/j.bmcl.2007.06.047

Bioorganic & Medicinal Chemistry Letters

Volume 17, Issue 17, 1 September 2007, Pages 4855-4860

https://doi.org/10.1016/j.bmcl.2007.06.047 Get rights and content

Abstract

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of α4β2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.

Graphical abstract

The present investigation reports the synthesis of desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) as their water-soluble hydrochloride salts and examines their action as positive allosteric modulators of α4β2 nicotinic acetylcholine receptors using two-electrode voltage clamp recordings.

Section snippets

Note added in proof

While our manuscript was being reviewed, Lindel et al., Organic Lett., 2007, 9, 283, published an alternative synthesis of desformylflustrabromine.

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Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI
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The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.
Desformylflustrabromine (dFBr), a positive allosteric modulator of the α<inf>4</inf>β<inf>2</inf> nicotinic receptor modulates the hypnotic response to ethanol
2021, Alcohol
Citation Excerpt :
It is also important to consider the possibility of channel block. dFBr does result in channel block of α4β2 nAChR at higher concentrations (Weltzin & Schulte, 2010), with an IC50 value of ~150 μM (Kim et al., 2007). Liu et al. (2013) showed that dFBr reaches a concentration of ~100 ng/mL (i.e., ~79.5 nM) in cerebrospinal fluid 30 and 90 minutes post s.c. injection of 3 mg/kg in Sprague–Dawley rats.
Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α₄β₂ nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α₄β₂ nAChR in vitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol.
: Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The β₂ nAChR selective antagonist dihydro-β-erythroidine (DHβE) was used to study the involvement of the β₂ subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α₄ nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α₄β₂ nAChR.
Pretreatment with 6 mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHβE suggest that dFBr reduced ethanol-induced LORR duration via the β₂ nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α₄ nAChR subunit protein levels. This ethanol-induced α₄ nAChR upregulation was significantly reduced in rats pretreated with 6 mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α₄β₂ nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents.
Our results suggest that thalamic nAChRs containing the α₄ subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α₄β₂ nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on β₂ nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.
Advances in smoking cessation pharmacotherapy: Non-nicotinic approaches in animal models
2020, Neuropharmacology
The landscape of worldwide tobacco use is changing, with a decrease in traditional smoking and an exponential rise in electronic cigarette use. No new nicotine cessation pharmacotherapies have come to market in the last 10 years. The current therapies that have been approved by the United States Food and Drug Administration for nicotine cessation include nicotine replacement therapy, varenicline, a nicotinic acetylcholine receptor partial agonist, and the atypical antidepressant bupropion. Nicotine replacement therapy and varenicline both act on nicotinic acetylcholine receptors. Bupropion inhibits the dopamine transporter, the norepinephrine transporter, and the nicotinic acetylcholine receptors to inhibit smoking behavior. Notwithstanding these treatments, rates of successful nicotine cessation in clinical trials remain low. Recent pharmacological approaches to improve nicotine cessation rates in animal models have turned their focus away from activating nicotinic acetylcholine receptors. The present review focuses on such pharmacological approaches, including nicotine vaccines, anti-nicotine antibodies, nicotine-degrading enzymes, cannabinoids, and metformin. Both immunopharmacological and enzymatic approaches rely on restricting and degrading nicotine within the periphery, thus preventing psychoactive effects of nicotine on the central nervous system. In contrast, pharmacologic inhibition of the enzymes which degrade nicotine could affect smoking behavior. Cannabinoid receptor agonists and antagonists interact with the dopamine reward pathway and show efficacy in reducing nicotine addiction-like behaviors in preclinical studies. Metformin is currently approved by the Food and Drug Administration for the treatment of diabetes. It activates specific intracellular kinases that may protect against the lower metabolism, higher oxidation, and inflammation that are associated with nicotine withdrawal. Further studies are needed to investigate non-nicotinic targets to improve the treatment of tobacco use disorder.
This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.
Advances in the In vitro and In vivo pharmacology of Alpha4beta2 nicotinic receptor positive allosteric modulators
2020, Neuropharmacology
Citation Excerpt :
dFBr by itself did not elicit nAChR-mediated current (i.e. did not activate nAChR). However, it enhanced peak ACh-induced current responses of α4β2 nAChR and α2β2 at submicromolar concentrations (Sala et al., 2005; Kim et al., 2007; Weltzin and Schulte, 2010; Pandya and Yakel, 2011). Further, it was found that dFBr potentiates both (α4)2(β2)3 and (α4)3(β2)2 nAChR isoforms with potentiation EC50s of ~0.4 and ~1.6 μM and potentiation Imax, of ~400 and ~300%, respectively, suggesting a stronger potentiation of (α4)3(β2)2 than (α4)2(β2)3 nAChR (Hamouda et al., 2016).
Receptors containing α4 and β2 subunits are a major neuronal nicotinic acetylcholine receptor (nAChR) subtype in the brain. This receptor plays a critical role in nicotine addiction, with potential smoking cessation therapeutics producing modulation of α4β2 nAChR. In addition, compounds that act as agonists at α4β2 nAChR may be useful for the treatment of pathological pain. Further, as the α4β2 nAChR has been implicated in cognition, therapeutics that act as α4β2 nAChR agonists are also being examined as treatments for cognitive disorders and neurological diseases that impact cognitive function, such as Alzheimer's disease and schizophrenia. This review will cover the molecular in vitro evidence that allosteric modulators of the α4β2 neuronal nAChR provide several advantages over traditional α4β2 nAChR orthosteric ligands. Specifically, we explore the concept that nAChR allosteric modulators allow for greater pharmacological selectivity, while minimizing potential deleterious off-target effects. Further, here we discuss the development and preclinical in vivo behavioral assessment of allosteric modulators at the α4β2 neuronal nAChR as therapeutics for smoking cessation, pathological pain, as well as cognitive disorders and neurological diseases that impact cognitive function.
This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.
Positive allosteric modulators of α7* or β2* nicotinic acetylcholine receptors trigger different kinase pathways in mitochondria
2018, International Journal of Biochemistry and Cell Biology
Mitochondrial nicotinic acetylcholine receptors (nAChRs) regulate the early stage of mitochondria-driven apoptosis, including cytochrome c release. Mitochondrial nAChR signaling is mainly mediated by intra-mitochondrial kinases, in an ion-independent manner. To determine the relationship between specific nAChR subtypes and mitochondrial kinases, the effects of a set of nAChR subtype-selective positive allosteric modulators (PAMs) on cytochrome c release from mouse liver mitochondria stimulated by 0.9 μM Ca²⁺, 0.5 mM H₂O₂ or 1.0 μM wortmanin is studied. The results indicate that Ca²⁺-stimulated cytochrome c release from wild-type, but not α7-/-, mice mitochondria is attenuated by the potent agonist PNU-282987 or type II PAMs (PNU-120596, 4BP-TQS, and PAM-2-4), but not by NS-1738, a type I PAM. In contrast, wortmannin-stimulated cytochrome c release from wild-type and, to a lesser extent, α7-/- mice mitochondria is efficiently attenuated by the β2-selective PAM desformylfrustrabromine. In conclusion, the ligand-evoked α7* nAChR conformational changes required to induce intra-mitochondrial signaling can be triggered through orthosteric (agonists) and transmembrane (type II PAMs) sites, but not by the interaction with type I PAMs. The α7 and β2 nAChR subunits are responsible for the engagement of distinct kinase pathways, supporting the concept that multiple heteromeric nAChR subtypes ensure mitochondria resistance to various exogenous and endogenous apoptogenic agents.

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Synthesis of desformylflustrabromine and its evaluation as an α4β2 and α7 nACh receptor modulator

Abstract

Graphical abstract

Section snippets

Note added in proof

Neurochem. Int.

Int. J. Biochem. Cell Biol.

Eur. J. Pharmacol.

Neurosci. Lett.

Tetrahedron

J. Neurosci. Methods

J. Neurobiol.

J. Neurosci. Res.

Alzheimer Dis. Assoc. Disord.