Acute Administration of Desformylflustrabromine Relieves Chemically Induced Pain in CD-1 Mice.

Neuronal nicotinic acetylcholine receptors are cell membrane-bound ion channels that are widely distributed in the central nervous system. The α4ß2 subtype of neuronal nicotinic acetylcholine receptor plays an important role in modulating the signaling pathways for pain. Previous studies have shown that agonists, partial agonists, and positive allosteric modulators for the α4ß2 receptors are effective in relieving pain. Desformylflustrabromine is a compound that acts as an allosteric modulator of α4ß2 receptors. The aim of this study was to assess the effects of desformylflustrabromine on chemically induced pain. For this purpose, the formalin-induced pain test and the acetic acid-induced writhing response test were carried out in CD-1 mice. Both tests represent chemical assays for nociception. The results show that desformylflustrabromine is effective in producing an analgesic effect in both tests used for assessing nociception. These results suggest that desformylflustrabromine has the potential to become a clinically used drug for pain relief.

Absolute Configuration and Pharmacology of the Poison Frog Alkaloid Phantasmidine.

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.

Levamisole: A Positive Allosteric Modulator for the α3ß4 Nicotinic Acetylcholine Receptors Prevents Weight Gain in the CD-1 Mice on a High Fat Diet.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the function of multiple neurotransmitter pathways throughout the central nervous system. This includes nAChRs found on the proopiomelanocortin neurons in the hypothalamus. Activation of these nAChRs by nicotine causes a decrease in the consumption of food in rodents. This study tested the effect of subtype selective allosteric modulators for nAChRs on the body weight of CD-1 mice. Levamisole, an allosteric modulator for the α3ß4 subtype of nAChRs, prevented weight gain in mice that were fed a high fat diet. PNU-120596 and desformylflustrabromine were observed to be selective PAMs for the α7 and α4ß2 nAChR, respectively. Both of these compounds failed to prevent weight gain in the CD-1 mice. These results suggest that the modulation of hypothalamic α3ß4 nAChRs is an important factor in regulating food intake, and the PAMs for these receptors need further investigation as potential therapeutic agents for controlling weight gain.

Desformylflustrabromine: A Novel Positive Allosteric Modulator for beta2 Subunit Containing Nicotinic Receptor Sub-Types.

Nicotinic acetylcholine receptors are ligand-gated transmembrane ion channels that are present at the neuromuscular junction and in different locations in the nervous system. The different subtypes of neuronal nicotinic acetylcholine receptors that are found in the brain are thought to be involved in many neurological processes such as pain, cognitive function and depression, as well as in the pathophysiology of numerous neurological diseases and conditions. While the neurotransmitter acetylcholine is an endogenous agonist for all nicotinic receptors subtypes, many drugs that act as agonists and antagonists have also been identified or developed for these receptors. In addition, a novel class of compounds described as allosteric modulators have also been identified or developed for nicotinic acetylcholine receptors. Allosteric modulators are ligands that bind to nicotinic receptors at sites other than the orthosteric site where acetylcholine binds. One such allosteric modulator is desformylflustrabromine. Five chemical analogs along with desformylflustrabromine act as positive allosteric modulator for nAChRs that contain the beta2 subunit in their pentameric structure. Here the discovery and development, medicinal chemistry and pharmacological actions of desformylflustrabromine have been discussed. Desformylflustrabromine and its chemical analogs have the potential to develop into clinically used drugs for neurological diseases and conditions where nicotinic acetylcholine receptors are involved.

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4ß2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer's disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain.

Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4ß2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs.

Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist.

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors.

Deconstruction of the α4ß2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine.

Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4ß2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4ß2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4ß2 nACh receptors by 1 were identified.

Allosteric modulators of the α4ß2 subtype of neuronal nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The α4ß2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for α4ß2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the α4ß2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the α4ß2 receptors. Here we discuss the concept of allosterism as it pertains to the α4ß2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

Allosteric modulator Desformylflustrabromine relieves the inhibition of α2ß2 and α4ß2 nicotinic acetylcholine receptors by ß-amyloid(1-42) peptide.

Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins that belong to the cys-loop ligand-gated ion channel family. These receptors are widely expressed in the brain and implicated in the pathophysiology of many neurological conditions, including Alzheimer's disease (AD), where typical symptoms include the loss of cognitive function and dementia. The presence of extracellular neuritic plaques composed of ß amyloid (Aß(1-42)) peptide is a characteristic feature of AD. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) for α4ß2 nAChRs since it increases peak ACh responses without inducing a response on its own. Previously, the effect of dFBr on the α2ß2 nAChR subtype was not known. The action of dFBr was tested on α2ß2 receptors expressed in Xenopus oocytes. It was found that dFBr is also a PAM for the α2ß2 receptor. Next we tested whether dFBr had any effect on the previously known block of both the α4ß2 and α2ß2 receptors by Aß(1-42). We found that the functional blockade of ACh-induced currents in oocytes expressing α4ß2 and α2ß2 receptors by Aß(1-42) was prevented by dFBr. We conclude that dFBr is a positive allosteric modulator for both α4ß2 and α2ß2 subtypes of nAChRs and that it also relieves the blockade of these receptors by Aß(1-42). This study demonstrates that PAMs for the non-α7 nAChRs have the potential to develop into clinically applicable drugs for AD and other disorders.