Allosteric modulation
Dualsteric GPCR targeting and functional selectivity: the paradigmatic M2 muscarinic acetylcholine receptor

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Muscarinic acetylcholine receptors belong to Class A seven transmembrane helical receptors and serve as important drug targets in the treatment of various diseases such as chronic obstructive pulmonary disease, overactive bladder, bronchial asthma and glaucoma. Despite intensive research the discovery of experimental ligands which activate or block specific muscarinic receptor subtypes has only been successful for the M1 and M4 subtypes but remains a challenging task at the other subtypes. In recent years, ligands have been introduced which bind simultaneously to the acetylcholine binding site, that is, the orthosteric site, and to an allosteric binding site. These so-called dualsteric ligands display M2 subtype preference due to the addressing of the allosteric binding site. As proven recently, dualsteric receptor activation goes along with a pronounced signaling bias which follows clear structure–bias-relationships. Dualsteric receptor targeting might represent a common strategy to generate functional selectivity.

Introduction

Muscarinic acetylcholine receptors (mAChRs) belong to the Class A of G protein-coupled receptors (GPCRs, also referred to as 7 transmembrane helical receptors: 7TMRs) which constitute the largest family of cell surface receptors in humans. 7TMRs are able to modulate almost every physiological process making them to important targets for drug discovery [1]. In fact, about one third of current marketed drugs are directed at this protein family [2]. 7TMRs translate extracellular stimuli of broad chemical diversity into a cellular response via a variety of intracellular adaptor proteins, in particular G proteins and β-arrestins [3].

Muscarinic AChRs are divided into five subtypes (M1–M5) which share a common architecture of their helical bundle regions harboring the acetylcholine (ACh) binding site, that is, the orthosteric site [4, 5, 6, 7]. Drugs which act at specific mAChR subtypes with high selectivity are needed to fine-tune pathophysiological processes modulated by single subtypes. This therapeutic goal has yet not been achieved. However, subtype-selective experimental compounds have been described, that is, allosteric agonists and positive allosteric modulators for the M1 and M4 mAChR subtypes [8, 9]. For the other members of the muscarinic receptor family including the M2 subtype, however, subtype-selectivity remains a challenging task [10].

Very recently, novel muscarinic ligands have been introduced which display M2 subtype preference by taking advantage of the structural heterogeneity between the allosteric domains of the receptor subtypes. In principle, there are three modes of allosteric targeting: (a) binary complex formation: the allosteric ligand binds to the free receptor and modulates receptor activity via the allosteric site, either displaying allosteric agonism, neutral antagonism or inverse agonism itself, (b) ternary complex formation: the allosteric ligand co-binds with ACh to the receptor and modulates ACh binding and/or the ACh-elicited response in a positive, neutral or negative manner and (c) a bitopic allosteric/orthosteric ligand binds simultaneously to both sites: dualsteric targeting.

With respect to nomenclature, we regard dualsteric binding as a special case of bitopic binding. Literally, bitopic means ‘two locations’ without further specification thereof. In fact, heterobivalent ligands – molecules encompassing two structurally distinct pharmacophores – may undergo different modes of bitopic binding. First, a bitopic ligand may be engaged in monovalent interactions with each of two possible targets (Fig. 1a, left panel). Second, a bitopic ligand may bridge two neighboring target proteins such as a receptor-heterodimer (Fig. 1a, middle panel). Addressing multiple target proteins, the aforementioned compounds have also been named ‘designed multiple ligands’ [11]. Third, and most important in the present context, a bitopic ligand bind simultaneously to the orthosteric and the allosteric site of one receptor protein: dualsteric binding (=bitopic orthosteric/allosteric binding; Fig. 1a, right panel).

Simplistically, the orthosteric moiety of a dualsteric ligand may encode the message for receptor activation while the allosteric moiety may serve as the address for a receptor subtype thus providing subtype-selectivity [12]. If the allosteric moiety conveys or modulates activity on its own, the situation will likely be more complex. Beyond that, dualsteric agonists may tie receptor signaling to a subset of possible pathways thus inducing a signaling bias.

Dualsteric receptor targeting has first been described for mAChRs, in particular the M2 subtype which is archetypal for the study of allosteric/orthosteric interactions [13, 14]. This review will focus on the discovery of dualsteric ligands for the M2 receptor as a paradigm and the goals achieved in the past few years. We present and discuss the experimental challenges which have to be faced in the process of dualsteric drug discovery and characterization.

Section snippets

Heterobivalent ligands for the M2 receptor

The following chapter briefly presents heterobivalent ligands known to interact with the M2 receptor. A detailed report has been provided elsewhere [15].

McN-A-343 (Fig. 1b and c) is an atypical partial agonist at all mAChR subtypes [16]. It displays highest efficacy at the M1 and M4 subtypes and lowest efficacy at the M2 subtype [17]. McN-A-343 was shown by Christopoulos and coworkers to adopt a bitopic orthosteric/allosteric binding mode at the M2 receptor [13]. Intriguingly, efficacy of

Dualsteric binding: verification and orientation

Principally, dualsteric ligands can bind in two different orientations, first the dualsteric binding pose, that is, both moieties bind simultaneously to the orthosteric and the allosteric binding site, and, second, the pure allosteric mode [15, 29]. Because of this complex binding topography, dualsteric binding is inferred from a set of different experimental approaches rather than from a single assay system. In the following section we review strategies which have been successfully co-applied

Quantification of ligand bias – a case study

7TMRs are considered as fluctuating macromolecules that may adopt various conformations and couple to multiple adaptor proteins such as different G protein subclasses and β-arrestins [35]. Often an endogenous ligand activates several adaptor proteins which is referred to as promiscuous signaling. In recent years, ligands have been described which for example favor coupling to G proteins over β-arrestins or even distinguish between G protein subtypes [35, 36, 37].

The functional selectivity of

Conclusions

The M2 muscarinic receptor subtype is archetypal for the study of allosterism as sensitivity of 7TMRs to allosteric modulation was first discovered with this receptor subtype [22, 23, 43]. Moreover, the topography of the M2 receptor's allosteric binding site, including the exact location of essential amino acids, has been elucidated [32, 33, 44]. Years ago, site-directed mutagenesis together with docking simulations already suggested the allosteric binding site to be located in the

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgements

A.B. is member of the graduate school THEME (Theoretical and Experimental Medicine), University of Bonn. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) by grants to K.M. (MO 821/2-1).

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