Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

Lazareno, Sebastian; Popham, Angela; Birdsall, N. J. M.

doi:10.1124/mol.62.6.1492

Cited by 92 publications

(96 citation statements)

References 23 publications

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“…This finding is in general agreement with our prior mutagenesis study (Chan et al, 2008), which suggested an engagement of the modulator with extracellular loop regions of the M 4 mAChR and an important function of D 432 in the third extracellular loop; this residue has been implicated in the binding of the prototypical modulator, gallamine, to the M 4 mAChR (Gnagey et al, 1999). In contrast, the interaction between LY2033298 and atropine was negatively cooperative, whereas that between LY2033298 and WIN51708 was neutrally cooperative; both findings clearly indicate that LY2033298 does not interact with either the orthosteric site or a second allosteric site on the M 4 mAChR recognized by staurosporine, KT5720, and various WIN compounds (Lanzafame et al, 2006;Lazareno et al, 2000Lazareno et al, , 2002.…”

Section: Discussionmentioning

confidence: 54%

“…Preliminary mutagenesis experiments with LY2033298 also implicated extracellular loop regions in the actions of the modulator (Chan et al, 2008). However, there is another class of 'atypical' modulators, such as WIN51708, WIN62577, staurosporine, and KT5720, that bind to a second, currently unresolved, allosteric site on mAChRs (Lanzafame et al, 2006;Lazareno et al, 2000Lazareno et al, , 2002. Thus, to gain more definitive insight into which allosteric site is involved in the binding of LY2033298, we exploited the ability of the modulator to also act as an agonist and performed functional interaction studies between this compound and either the classic orthosteric antagonist, atropine, the 'prototypical-site' allosteric modulator, C 7 /3-phth, or the 'second-site' allosteric modulator, WIN51708.…”

Section: Ly2033298 Potentiates Both the Affinity And The Efficacy Of mentioning

confidence: 99%

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Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Leach

Loiacono

Felder

et al. 2009

Neuropsychopharmacol

146

169

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We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M 4 muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligandbinding studies revealed that LY2033298 displayed a preference for the active state of the M 4 mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [ 35 S]GTPgS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3b phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M 4 mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [ 3 H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M 4 mAChR knockout mice, validating the M 4 mAChR as a key target of action of this novel allosteric ligand.

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Section: Discussionmentioning

confidence: 54%

Section: Ly2033298 Potentiates Both the Affinity And The Efficacy Of mentioning

confidence: 99%

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Leach

Loiacono

Felder

et al. 2009

Neuropsychopharmacol

146

169

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“…In radioligand binding assays: (a) LY2033298 exhibited a lack of significant affinity modulation of the muscarinic orthosteric antagonists NMS and QNB (3-quinuclidinyl benzilate), indicating no overlap with the orthosteric binding site In [ 35 S]GTPγS binding assays: (b) coaddition of the mAChR antagonist atropine caused a substantial reduction in the maximal effect of LY2033298 with a minimal change in potency, characteristic of noncompetitive negative cooperativity (c) LY2033298 demonstrated a competitive interaction with the mAChR negative allosteric modulator C 7 /3-phth, known to bind the "common" muscarinic allosteric site 79 (d) LY2033298 exhibited neutral cooperativity upon coaddition with WIN51708, a known "second allosteric site" mAChR ligand, 73 confirming the allosteric agonist's site of action to be the "common" allosteric site on the M 4 mAChR The Prospect of Bitopic Interactions. While the M 1 mAChR-selective ligand literature reviewed herein provides substantial evidence to suggest purely allosteric modes of action, there exist several examples where this evidence is inconclusive.…”

Section: ■ Important Considerations In Allosteric Ligand Classificatimentioning

confidence: 99%

Development of M₁ mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

Davie

Christopoulos

Scammells

2013

ACS Chem. Neurosci.

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Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer's disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M 1 muscarinic acetylcholine receptor (M 1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M 1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M 1 mAChR, comprehensively summarize and critically evaluate the M 1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues. KEYWORDS: M 1 mAChR, allosteric ligands, Alzheimer's disease, schizophrenia, cognitive deficits, memory T he muscarinic acetylcholine receptor (mAChR) family is a group of rhodopsin-like (Family A) G protein-coupled receptors (GPCRs) consisting of five distinct subtypes M 1 −M 5 . Activation of the M 1 , M 3 , and M 5 receptor subtypes primarily results in coupling to the G q/11 family of G proteins, activation of phospholipase C (PLC), release of inositol-1,4,5-trisphosphate (IP 3 ), and subsequent mobilization of intracellular calcium Ca 2+ . Activation of the M 2 and M 4 receptor subtypes primarily results in coupling to the G i/o family of G proteins, inhibition of adenylate cyclase (AC), reduction in cyclic AMP (cAMP), and a decrease in neurotransmitter release via the blockage of voltage-gated calcium channels (Figure 1). These pathways represent a generalized view of each receptor's coupling capacity, as all five subtypes couple to a broader range of G protein-and non-G protein-mediated signaling and regulatory pathways, 1 ultimately leading to the regulation of enzymes and neurotransmitters critical for intercellular chemical communication and biological function. In a ...

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“…Theoretically, the EC 50 or IC 50 of the affinity ratio plot corresponds to the K d of the test agent at the free receptor, and the asymptotic level corresponds to the cooperativity constant for the test agent and primary ligand (Lazareno and Birdsall, 1995). Affinity ratios were calculated from the specific binding data as follows (Lazareno and Birdsall, 2000;Lazareno et al, 2002):…”

Section: Methodsmentioning

confidence: 99%

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M₄Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

Lazareno

Doležal²,

Popham³

et al. 2004

Mol Pharmacol

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Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of L-[ 3 3 H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M 4 receptors, but not from hippocampal slices, which contain presynaptic M 2 receptors. We conclude that thiochrome is a selective M 4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M 1 to M 3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.Most receptor-active ligands bind to the same site as the endogenous ligand, the so-called orthosteric site. Agonists mimic the actions of the endogenous ligand, whereas antagonists physically prevent the endogenous ligand from binding but lack its actions. The property of a ligand that determines its effect when bound to a receptor is called its efficacy. Different degrees of efficacy lead to so-called full agonists, partial agonists with a smaller maximal effect than full agonists; neutral antagonists, which occupy the active site without exerting any effect; and inverse agonists, which reduce the activity of constitutively active receptors (Kenakin, 2002). The selectivity of an orthosteric ligand for one receptor or receptor subtype is determined by its affinity for the receptor and its efficacy at that receptor. The difference in affinity between the target receptor and other receptors must be large for an orthosteric ligand to have useful selectivity. This can be difficult to achieve for receptors that show close homology at the orthosteric binding region such as the five subtypes of muscarinic receptor (M 1 -M 5 ) (Hulme et al., 1990). Selectivity derived from efficacy can also be hard to achieve, because the effect of a partial agonist depends on properties of the tissue, such as receptor density and downstream amplification mechanisms, which vary between cells and tissues, so a ligand with no apparent functional effect on tissues in vitro may nevertheless activate tissues in vivo, leading to unacceptable side effects (Terry et al., 2002).An alternative approach for developing selective ligands is to look for allosteric ligands that bind to a site on the receptor which is different from the site to which the endogenous ligand binds. This allows both types of ligand to bind simultaneously. If the affinity (or efficacy) of the endogenous ligand is different when it is bound to the allosteric liganded receptor compared with when it is bound to the free receptor, then the allosteric ligand exhibits positive or negative coop-

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Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

Cited by 92 publications

References 23 publications

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Development of M₁ mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M₄Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

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Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

Cited by 92 publications

References 23 publications

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M4Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

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Product

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Development of M₁ mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M₄Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity