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Protean agonism at the muscarinic M2 receptor
No full textProtean agonism at the muscarinic M2 receptor
De Min A. (1), Matera C. (2), Messerer R. (3), Dallanoce C. (2), Holzgrabe U. (3), Mohr K. (1)
(1) University of Bonn Pharmacology and Toxicology - Bonn, Deutschland
(2) University of Milan Pharmaceutical Sciences - Milan, Italien
(3) University of Würzburg Pharmaceutical and Medicinal Chemistry - Würzburg, Deutschland
Muscarinic acetylcholine receptors, with their five different subtypes, belong to the class A of GPCRs and have been extensively studied with the purpose of finding selective ligands for their modulation. In this respect, in the last years a new strategy was developed, i.e. the synthesis of so-called dualsteric ligands that bind simultaneously to both the highly conserved orthosteric site and the less conserved allosteric site of the M2 receptor subtype [1].
Recently, we found out that one dualsteric ligand, a hybrid derived from the orthosteric superagonist iperoxo and the negative allosteric modulator naphmethonium, showed a peculiar behavior known as “protean agonism”. Given that protean agonism has not been described so far for muscarinic receptors, additional studies were performed, in order to gain better insight into structure/activity-relationships. Therefore, a series of compounds of different middle chain length and iperoxo-related orthosteric agonist moieties were chosen for testing.
[35S]GTPγS binding assays were carried out in order to study the effect of the hybrids in the Gi signaling pathway. Experiments were performed in Tris buffer either with low sodium concentration, that assured a stable spontaneously active M2 receptor system, or in Tris buffer supplemented with 200 mM NaCl, that abolished M2 constitutive activity.
The results revealed a great variety of activities of the structurally related hybrids, including inverse agonism, partial agonism and protean agonism. The tested compounds had similar potencies (pEC50) in this assay, ranging between 7.5-8.5, except for two protean agonists and one inverse agonist. The former two revealed significantly lower potencies and efficacies in Tris buffer with “low sodium” concentration. These results might suggest that in this buffer the ligands adopt a purely allosteric pose rather than a dualsteric pose. Concerning the inverse agonist, it had a low pEC50 in both buffers. This result might indicate that the hybrid adopts a purely allosteric binding pose in both conditions, thus inactivating the receptor through the negative allosteric fragment.
Taken together, linker length and nature of orthosteric moiety are both relevant in determining the intensity and the direction of the effect. Two protean agonists were identified for the muscarinic M2 receptor.
Antony, J. et al.: FASEB J. 2009, 23: 442-50
In silico design of agonists targeting the alpha7 nicotinic acetylcholine receptors, their synthesis and preliminary pharmacological evaluation
No full textAs an extension of previous molecular modeling studies on nicotinic acetylcholine receptor (nAChR) ligands,[1] we now adopted the LIR (Linear Interaction Response) approach to reach a realistic compromise between accuracy and calculation rate, during the delta(G)bind value estimation of a training set of known agonists for the alpha7 nAChR subtype.
The same strategy was applied to a text set of potential alpha7 nAChR agonists, which allowed identification of general structures A and B as the most promising ligands in the series. Thus, some of these derivatives were synthesized along with procedures previously utilized by us,[2] and were then assayed for binding affinity at alpha7 and alpha4beta2 nAChR subtypes.
[1] Grazioso, G.; Cavalli, A.; De Amici, M.; Recanatini, M.; De Micheli, C. J. Comput. Chem. 2008, 29, 2593-2602. [2] Rizzi, L.; Dallanoce, C.; Matera, C.; Magrone, P.; Pucci, L.; Gotti, C.; Clementi, F.; De Amici, M. Bioorg. Med. Chem. Lett. 2008, 18, 4651-4654
Allosteric ligands for G protein-coupled receptors : a novel strategy with attractive therapeutic opportunities
No full textAllosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter-bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein-coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (Class A, B, and C receptors). In addition, the behavior of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated
Selective nicotinic agonists to treat pathologies of the central nervous system
No full textI recettori nicotinici neuronali costituiscono una famiglia di recettori canale a struttura pentamerica attivati da un ligando e costituiti sia da svariate combinazioni eteromeriche di subunità alfa e beta sia da assemblaggi omomerici delle subunità alpha7, alpha8 e alpha9.[1,2] I due sottotipi maggiormente espressi nel sistema nervoso centrale (SNC) sono la combinazione eteromerica alpha4beta2 e quella omomerica alpha7.[3] Studi di tipo neuroanatomico ed elettrofisiologico, convalidati da test comportamentali, hanno evidenziato l'importanza di questi due sottotipi recettoriali nei processi di apprendimento e di memoria.[4] Da queste premesse, i due recettori nicotinici sopra menzionati sono stati individuati come bersagli di farmaci utilizzabili in importanti patologie del SNC quali il morbo di Alzheimer, il morbo di Parkinson, la sindrome di Tourette, l'ansia, stati depressivi, l'epilessia, il dolore neuropatico, la schizofrenia e la dipendenza da nicotina.[1] La progettazione di ligandi selettivi per un solo sottotipo recettoriale è essenziale per valutarne il coinvolgimento in stati patologici e successivamente per sviluppare farmaci caratterizzati da limitati effetti collaterali. Pertanto molte delle ricerche attuali in questo settore sono rivolte alla progettazione di agonisti alpha4beta2 selettivi da impiegare nel trattamento del dolore neuropatico, nel trattamento sintomatico di patologie neurodegenerative del SNC e nei programmi di disuassefazione da nicotina. Per quest'ultima indicazione terapeutica, nel 2006 la FDA ha approvato la Vareniclina. Intensi studi sono anche rivolti alla progettazione di agonisti alpha 7 selettivi avendo come obiettivo lo sviluppo di farmaci in grado di trattare affezioni neurologiche e psichiatriche, quali il morbo di Alzheimer e la schizofrenia, e processi infiammatori.[5,6] In questa relazione verranno illustrate le strategie utilizzate per progettare, da un lato, agonisti selettivi per il sottotipo recettoriale alpha4beta2 e, dall'altro, di generare ligandi capaci di interagire selettivamente con il sottotipo alpha7. Questi studi sono stati effettuati utilizzando modelli recettoriali sviluppati utilizzando le più moderne tecniche computazionali e validati con dati di letteratura. Bibliografia [1] A. A. Jensen et al., J. Med. Chem. 2005, 48, 4705-4745. [2] P. Séguél et al., J. Neurosci. 1993, 13, 596-604. [3] S. Weiland et al.., Behav. Brain Res. 2000, 113, 43-56. [4] E. D. Levin, B. B. Simon Psycopharmacology 1998, 138, 217-230. [5] L. Ulloa. Nature Reviews - Drug Discovery 2005, 673-68
The enantiomers of epiboxidine and of two related analogs: Synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors
No full textEpiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes. Chirality 24:543-551, 2012
Novel ligands for neuronal nicotinic receptor subtypes: synthetic, pharmacological and molecular docking studies
No full textNeuronal nicotinic acetylcholine receptors (nAChRs) are pentameric proteins made up
of homomeric or heteromeric combinations of α and α/β subunits, whose differential
association confers specific structural and functional properties to the resulting
subtypes. The α4β2 and the α7 nAChRs are by far the most expressed in the central
nervous system (CNS), whereas the α3β4 subtype is predominant in the sensory and
autonomic ganglia and in a subpopulation of neurons in the medial habenula (MHN)
and interpeduncular nucleus (IPN) in the CNS [1]. Recently, behavioral and functional
studies have further indicated the crucial role of nAChRs in nicotine reward, addiction
and expression of withdrawal. The α4 and β2 subunits appear to be crucial for nicotine
dependence and the α3β4 nAChR subtype seems to be implicated in addiction to
nicotine and other drugs of abuse [2]. As an extension of our research in this field [3],
we aimed at deepening the investigation on the involvement of nAChR subtypes in
each of the aspects of tobacco addiction. To such an end, we designed and
synthesized the set of novel compounds 4-9, which may be related to Nicotine 1,
Epibatidine 2 and Anabaseine 3.
In the new derivatives, the pyridine ring featuring the parent ligands was replaced by a
3-hydroxybenzene or a 3-hydroxymethylbenzene moiety. The synthetic approach to the
target compounds, the data of their pharmacological screening as well as the results of
molecular modeling investigations will be presented and discussed
Synthesis of novel epibatidine-related derivatives through 1,3-dipolar cycloaddition of pyridinenitrile oxides
No full textThe Delta(2)-isoxazoline derivatives 3a- c and 4a- c, structurally related to epibatidine, and the simplified analogues 5a-c were synthesized by means of a 1,3-dipolar cycloaddition of regioisomeric pyridinenitrile oxides to suitable dipolarophiles. Target compounds were assayed at alpha 4 beta 2 and alpha 7 neuronal acetylcholine receptor (nAChR) subtypes. Competition binding experiments at alpha 4 beta 2 nAChRs showed an overall significant reduction in affinity for the compounds under study in comparison to the reference radioligand [H-3]-epibatidine. On the other hand, compounds 3b, 3c, and 4b exhibited a noticeable affinity for the alpha 7 receptors and 3c showed also a slight degree of alpha 7 over alpha 4 beta 2 selectivity
Epiboxidine and Novel-Related Analogues: A Convenient Synthetic Approach and Estimation of Their at Neuronal Nicotinic Acetylcholine Receptor Subtypes
No full textSynthesis of epibatidine-related Delta(2)-isoxazoline derivatives and evaluation of their binding affinity at neuronal nicotinic acetylcholine receptors
No full textThe group of Δ2-isoxazoline derivatives 5a–c and 6a–c, structurally related to epibatidine, and the simplified analogues
7a–c were synthesized by means of a 1,3-dipolar cycloaddition-based strategy and tested at α4β2 and α7 neuronal acetylcholine receptor (nAChR) subtypes. Competition binding experiments at α4β2 nAChR subtypes showed an overall significant reduction in affinity for the compounds under study in comparison to the reference radioligand [3H]-epibatidine. These outcomes have been rationalized by taking into account the ligand-based pharmacophore models reported in
the literature and the recently proposed molecular model of the α4β2 receptor subtype. Conversely, compounds 5b, 5c,
and 6b exhibited a noticeable affinity for the α7 receptors and, in the case of 5c, also some subtype selectivit
Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-Coupled Receptor
Full text linkG protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to GPCRs induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist-receptor complexes on a molecular level. Employing all-atom molecular dynamics (MD) simulations, dynophores (i.e. a combination of static 3D-pharmacophores and MD-based conformational sampling), ligand design and receptor mutagenesis, we show that inactive agonist-receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) vs. dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist-receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists, where binding modes will be only subtly different and confined to only one binding site
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