Current Advances in Allosteric Modulation of Muscarinic Receptors
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biomolecules Review Current Advances in Allosteric Modulation of Muscarinic Receptors Jan Jakubik 1,* and Esam E. El-Fakahany 2,* 1 Department of Neurochemistry, Institute of Physiology CAS, 142 20 Prague, Czech Republic 2 Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, MN 55455, USA * Correspondence: [email protected] (J.J.); [email protected] (E.E.E.-F.) Received: 17 January 2020; Accepted: 16 February 2020; Published: 18 February 2020 Abstract: Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer’s disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents. Keywords: acetylcholine; muscarinic receptors; allosteric modulation 1. Introduction Slow metabotropic responses to acetylcholine are mediated by muscarinic receptors. Five distinct subtypes of muscarinic acetylcholine receptors (M1–M5) have been identified in the human genome [1]. The structure of all five receptor subtypes was resolved by X-ray crystallography [2–6]. Muscarinic receptors are members of class A of the G-protein-coupled receptor (GPCR). M1,M3, and M5 subtypes preferentially activate phospholipase C and calcium mobilization through Gq/11, whereas M2 and M4 receptors inhibit the activity of adenylyl cyclase by activation of the α-subunit of the Gi/o family of G-proteins. The latter two receptors also modulate the conductance of ion channels (e.g., inward rectifying potassium ion channels) by βγ-dimers of the Gi/o G-proteins [7]. Muscarinic receptors mediate a wide range of physiological functions in the central and peripheral nervous system and innervated tissues. Muscarinic receptors thus represent a potential therapeutic target for the treatment of psychiatric and neurologic conditions (e.g., schizophrenia, Alzheimer’s disease, Huntington disease) [8,9] as well as internal diseases (e.g., type 2 diabetes, asthma, chronic pulmonary obstruction, incontinence) [10–12]. The concept of allosterism was formally introduced into the field of enzymology by Monod et al. [13] and Koshland et al. [14] in 1965 and 1966, respectively. The former model was termed concerted, the latter one sequential. Allosteric modulation of GPCR is much simpler than that of enzymes. GPCR allosteric modulators bind to a site on the receptor that is spatially distinct from that of the endogenous transmitter, acetylcholine, in the case of muscarinic receptors. Consequently, binding of an allosteric modulator and an orthosteric ligand is not mutually exclusive, i.e., both ligands may bind to the receptor simultaneously to form a ternary complex (Figure1). Binding of allosteric Biomolecules 2020, 10, 325; doi:10.3390/biom10020325 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 325 2 of 16 Biomolecules 2020, 10, x 2 of 15 modulatorsligands may induces bind to athe change receptor in the simultaneously conformation to of form the receptor a ternary that complex results (Figure in changes 1). Binding in affinity of (eventuallyallosteric modulators potency and induces efficacy) a change of the orthostericin the conformation ligand [15 of]. the receptor that results in changes in affinity (eventually potency and efficacy) of the orthosteric ligand [15]. FigureFigure 1.1. AnAn orthostericorthosteric ligandligand LL bindsbinds toto thethe receptorreceptor RR withwith equilibriumequilibrium dissociationdissociation constantconstant KKDD, andand anan allostericallosteric modulatormodulator AA bindsbinds toto thethe receptorreceptor RR withwith anan equilibriumequilibrium dissociationdissociation constantconstant KKAA. TheThe orthostericorthosteric ligand ligand L L and and the the allosteric allosteric modulator modulato Ar canA can bind bind concurrently concurrently to the to receptor the receptor R to formR to aform ternary a ternary complex complex LRA. Binding LRA. Binding of one ligand of one to theligand receptor to the changes receptor the changes equilibrium the dissociationequilibrium constantdissociation of the constant other ligandof the byother a factor ligand of by cooperativity a factor of cooperativityα [15]. α [15]. BasedBased onon thethe eeffectsffects ofof anan allostericallosteric modulatormodulator onon thethe aaffinityffinity ofof anan orthostericorthosteric ligand,ligand, allostericallosteric modulatorsmodulators maymay bebe classifiedclassified intointo threethree categories:categories: 1.1. Positive allosteric modulators (PAM) thatthat increaseincrease thethe aaffinityffinity ofof orthostericorthosteric ligands;ligands; 2.2. NegativeNegative allosteric modulators (NAM) that decrease the aaffinityffinity ofof orthostericorthosteric ligands; ligands; and and 3. 3. Neutral Neutral allosteric allosteric modulators modulators that that do do not not aff ectaffect the the affi nityaffinity of the of orthostericthe orthosteric ligand. ligand. When When the intrinsicthe intrinsic efficacy efficacy of allosteric of allosteric modulator modulator is taken is taken into account, into account, these threethese categoriesthree categories expand expand to six: 1.to Pure six: PAMs;1. Pure 2. PAM-agonistsPAMs; 2. PAM-agonists that possess that intrinsic possess agonistic intrinsic propensity agonistic in thepropensity absence ofin the orthostericabsence of agoniststhe orthosteric they modulate; agonists 3. they PAM-antagonists modulate; 3. PAM-antagonists that lower the effi cacythat oflower the agoniststhe efficacy they of modulate the agonists [16]; they 4. Pure modulate NAMs; [16]; 5. NAM-agonists 4. Pure NAMs; that 5. possessNAM-agonists own agonistic that possess propensity own inagonistic the absence propensity of the agonistsin the absence and activate of the theagonists receptor, and while activate they the negatively receptor,modulate while they endogenous negatively agonistmodulate [17 endogenous]; 6. Silent allosteric agonist [17]; modulators 6. Silent (SAMs)allosteric that, modulators although (SAMs) they bind that, to although the receptor, they bind do not to atheffect receptor, the affinity, do not potency, affect orthe effi affinity,cacy of potency, the orthosteric or efficacy ligand of and the doorthosteric not have ligand agonistic and propensity do not have on theiragonistic own. propensity In interaction on their with own. agonist, In interaction an allosteric with modulator agonist, an may allosteric affect modulator both agonist may affi affectnity both and eagonistfficacy. affinity Thus, each and efficacy. of the six Thus, abovementioned each of the six categories abovementioned has three categories sub-categories has three based sub-categories on positive, negative,based on orpositive, neutral negative, effects (cooperativity) or neutral effects of the (cooperativity) allosteric modulator of the onallosteric agonist modulator efficacy. However, on agonist six basicefficacy. categories However, are six suffi basiccient categories for the general are sufficient classification. for the general classification. AsAs earlyearly as in 1969, Lüllmann et al. showed in their pioneering work that alkane-bis-ammonium compoundscompounds inhibited inhibited the the functional functional response response to tothe the conventional conventional muscarinic muscarinic agonist agonist carbachol carbachol non- non-competitivelycompetitively [18]. [ 18Later,]. Later, Clark Clark and and Mitchelson Mitchelson discovered discovered that that gallamine gallamine similarly similarly inhibited inhibited thethe actionaction ofof acetylcholine and carbachol on the functionfunction of heart atria in a non-competitive manner [19]. [19]. TheThe concentration-response curves curves to to the the agonists agonists we werere shifted shifted to tothe the right, right, but but the the magnitude magnitude of the of theprogressive progressive shifts shifts diminished diminished with with increasing increasing co concentrationsncentrations of of gallamine. gallamine. When When the the action ofof acetylcholineacetylcholine on the heartheart waswas evaluatedevaluated inin thethe combinedcombined presencepresence ofof gallaminegallamine andand thethe antagonistantagonist atropine,atropine, thethe inhibitioninhibition of of functional functional response response to to carbachol carbachol was was smaller smaller than than expected expected for for the the eff ectseffects of twoof two competitive competitive antagonists. antagonists. These These observations observations led led to to the the conclusion conclusion that that thethe actionaction ofof gallaminegallamine takestakes placeplace atat anan allostericallosteric site site on on the the receptor, receptor, resulting resulting in in negative negative cooperativity