Design, Synthesis, and Action of Oxotremorine-Related Hybrid-Type Allosteric Modulators of Muscarinic Acetylcholine Receptors

Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; Amici, Marco De; Holzgrabe, Ulrike

doi:10.1021/jm050769s

Cited by 71 publications

(80 citation statements)

References 40 publications

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“…Consequently, potent and selective muscarinic receptor ligands, developed using a multivalent approach, might prove to be valuable therapeutics. Others have used a similar strategy and have reported the advantages of a series of agonist-allostere bivalent ligands (Disingrini et al, 2006). Here, we provide evidence for a multivalent interaction between the antagonist THRX-160209 and the muscarinic M 2 receptor.…”

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confidence: 52%

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

et al. 2007

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THRX-160209 is a potent antagonist at the M 2 muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M 2 receptor subtype (apparent pK I ϭ 9.51 Ϯ 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK I values Ͻ 6.0), 2) specificity of THRX-160209 for the M 2 receptor subtype compared with the closely related M 4 (apparent pK I ϭ 8.78 Ϯ 0.24) and M 1 , M 3 , and M 5 receptors (apparent pK I values Յ 8.0), and 3) acceleration (Ͼ10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M 2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3 H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [ 3 H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M 2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.The muscarinic receptor family consists of five subtypes that are found in smooth and cardiac muscle, epithelial and endothelial cells, secretory cells, neurons, and inflammatory cells (Caulfield, 1993;Racke and Matthiesen, 2004). These receptors represent attractive targets for drug design in a variety of therapeutic areas, including overactive bladder, chronic obstructive pulmonary disease, and Parkinson's disease (Eglen et al., 2001;Katzenschlager et al., 2003;Barnes, 2004;Hegde et al., 2004). However, most currently marketed, muscarinic receptor-targeted therapies exhibit significant side effects, some of which are due to potent drug interactions at undesirable muscarinic receptor subtypes outside the intended organ system. High sequence homology within the orthosteric site, across the five receptor subtypes, makes the synthesis of tissue-specific or subtype-specific drugs challenging (Hulme et al., 1990). A drug with specificity for a given subtype or tissue is predicted to have increased efficacy but decreased side effects relative to a drug with equal affinity for all subtypes (Gainetdinov and Caron, 1999).Muscarinic receptors are known to be subject to modulation by ligands that do not bind to the ACh binding pocket (the "orthosteric site") (Ellis et al., 1991). Targeting these "secondary" or "allosteri...

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confidence: 52%

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

et al. 2007

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“…ACh, atropine, and NMS were obtained from Sigma-Aldrich. Iperoxo (56), Isox (57), 6-naph (25), iper-6-naph (25), isox-6-naph (58), and iper-8-naph (26) have been synthesized exactly as described previously. The synthesis of iper-rigid-naph is described in the supplemental information.…”

Section: Methodsmentioning

confidence: 99%

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Böck

Bermudez

Krebs

et al. 2016

Journal of Biological Chemistry

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G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors 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 M 2 receptors to demonstrate the existence and function of such inactive agonist⅐receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist⅐receptor 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) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist⅐receptor 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.Specific and coordinated cell-to-cell communication regulates the flow of information between cells, and proper information processing ensures physiological functions of biological systems. G protein-coupled receptors (GPCRs), 8 constituting the largest class of membrane proteins in mammals, are essential mediators of chemically and light-encoded information (1-4). GPCRs sense a great variety of extracellular stimuli, e.g. neurotransmitters and hormones, and subsequently translate this information into an intracellular response via G proteins, ␤-arrestins, and possibly GPCR-interacting proteins (2-5). Because of their abundance and relevance in regulating the majority of (patho-)physiological processes in humans, GPCRs have for a long time represented the most important drug targets being addressed by at least a third of all currently marketed drugs (6, 7).Agonist binding leads to receptor activation, which is followed by intracellular G protein recruitment and subsequent cell signaling. Breakthroughs in GPCR crystallography have led to inactive and active crystal structures of the same receptor protein. Among these are rhodopsin (8 -10) and more recently the ␤ 2 -adrenergic (11-14), M 2 muscarinic (15, 16), and -opioid receptors (17, 18). These structures most likely represent energetically favored, relatively stable inactive and active receptor⅐ligand complexes. Despite the diversity of crystallized receptors, a common...

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“…For example, although the M 1 mAChR-selective agonist N-desmethylclozapine was originally suggested to be allosteric, definitive evidence for an allosteric interaction with the receptor is lacking (10 -12). Moreover, novel bitopic ligands have recently been described, which concomitantly associate with both the orthosteric binding site and an allosteric binding site (13)(14)(15)(16)(17)(18)(19); it is possible that previous studies of selective agonists have classified such bitopic ligands as purely allosteric.…”

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confidence: 99%

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

Keov

López

Devine

et al. 2014

Journal of Biological Chemistry

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Background: Bitopic ligands bind concomitantly to orthosteric and allosteric receptor sites. Results: Residues affecting binding and biased signaling of the selective agonists TBPB and 77-LH-28-1 were identified at the M 1 muscarinic receptor. Conclusion: Novel bitopic ligand binding poses and mechanisms of receptor activation were identified. Significance: Understanding the basis of bitopic ligand mechanisms can enable the design of selective ligands.

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Design, Synthesis, and Action of Oxotremorine-Related Hybrid-Type Allosteric Modulators of Muscarinic Acetylcholine Receptors

Cited by 71 publications

References 40 publications

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

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Design, Synthesis, and Action of Oxotremorine-Related Hybrid-Type Allosteric Modulators of Muscarinic Acetylcholine Receptors

Cited by 71 publications

References 40 publications

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

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A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor