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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 , 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 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 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 . 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 and an orthosteric 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- 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 [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 agonist 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, 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 cooperativity with with the the binding binding of bothof both orthosteric orthosteric agonists agonists and and antagonists. antagonists. Since Since then, then, a wide a wide variety variety of allosteric of allosteric modulators modulators has been has discovered.been discovered. These These include include inhibitors inhibitors of , of acetylcholinesterase, ion blockers,channel variousblockers, alkaloids, various smallalkaloids, , small etc.peptides, For review, etc. For see review, [20]. Thanks see [20]. to Thanks early intensive to early research,intensive muscarinicresearch, muscarinic receptors becamereceptors a usefulbecame prototype a useful prototype of allosterically-modulated of allosterically-modulated GPCRs. GPCRs. 2. Advantages of Allosteric Modulators as Therapeutics 2. Advantages of Allosteric Modulators as Therapeutics 2.1. Selectivity by Targeting Less Conserved Domains on the Receptor 2.1. Selectivity by Targeting Less Conserved Domains on the Receptor Muscarinic receptor subtypes share high structural homology in the transmembrane domains Muscarinic receptor subtypes share high structural homology in the transmembrane domains where the orthosteric binding site is located. On the other hand, domains out of the membrane are where the orthosteric binding site is located. On the other hand, domains out of the membrane are Biomolecules 2020, 10, x 3 of 15 Biomolecules 2020, 10, 325 3 of 16 less conserved. Targeting allosteric domains allows achieving binding selectivity for certain receptor lesssubtypes conserved. to the Targetingextent which allosteric is not domainspossible with allows orthosteric achieving ligands. binding selectivity for certain receptor subtypes to the extent which is not possible with orthosteric ligands. 2.2. Conservation of Space and Time Pattern of Signaling 2.2. Conservation of Space and Time Pattern of Signaling Theoretically, a pure PAM of acetylcholine would only induce an action when endogenous acetylcholineTheoretically, is released. a pure Consequently, PAM of acetylcholine its action would would only be restricted induce an in action space whenand time endogenous to those acetylcholinesynapses where is released. signaling Consequently, is currently itshappening. action would Thus, be restrictedspace and in time space patterns and time of to signaling those synapses could wherebe restored signaling under is currentlydiminished happening. acetylcholine Thus, release space andthat timeis typical patterns in neurodegenerative of signaling could be disorders, restored undere.g., Alzheimer’s diminished acetylcholinedisease. Successful release action that is typicalof PAMs in neurodegenerativerequires functional disorders, post-synaptic e.g., Alzheimer’s receptors. disease.Encouragingly, Successful post-synaptic action of PAMsreceptors requires are relatively functional intact post-synaptic in neurodegenerative receptors. Encouragingly, diseases like post-synapticAlzheimer’s disease receptors or senile are relatively dementia intact of Lewy in neurodegenerative body type [21,22]. diseases like Alzheimer’s disease or senile dementia of Lewy body type [21,22]. 2.3. Absolute Selectivity 2.3. Absolute Selectivity In practice, absolute selectivity due to exclusive binding to a single receptor subtype is hard to achieve.In practice, Alternatively, absolute the selectivity absolute dueselectivity to exclusive of an bindingallosteric to agent a single can receptor be achieved subtype by ishaving hard toa achieve.ligand with Alternatively, the desired the cooperativity absolute selectivity at a given of anrecept allostericor subtype agent and can neutral be achieved (silent) by cooperativity having a ligand at withthe rest the of desired the receptor cooperativity subtypes. at a givenSelectivity receptor may subtype be derived and neutralfrom binding (silent) cooperativity cooperativity as at thewell rest as offrom the effects receptor on subtypes. potency and Selectivity efficacy. may be derived from binding cooperativity as well as from effects on potency and efficacy. 2.4. Selective Blocking of Activated Receptors 2.4. Selective Blocking of Activated Receptors PAM-antagonists that exhibit partial or absolute selectivity for a given receptor subtype may be usedPAM-antagonists to selectively target that receptors exhibit partialactivated or absoluteby the endogenous selectivity forneurotransmitter a given receptor located subtype in specific may be usedtissues to or selectively organs [16]. target This receptors featureactivated can be used bythe to endogenousselectively reverse neurotransmitter persistent excessive located in agonism specific tissuesunder orcertain organs pathological [16]. This feature conditions can be (e.g., used tobronchospasm selectively reverse in asthma persistent [23]) excessive or overstimulation agonism under of certainsalivary pathological and lacrimal conditions glands (e.g.,after bronchospasm in asthma poisoning [23]) or in overstimulation case of M3-selective of salivary PAM- and lacrimalantagonists. glands after organophosphate poisoning in case of M3-selective PAM-antagonists. 3. Location of the Allosteric Binding Sites on Muscarinic Receptors 3. Location of the Allosteric Binding Sites on Muscarinic Receptors The binding site of classical allosteric modulators like gallamine, alcuronium, or The binding site of classical allosteric modulators like gallamine, alcuronium, or alkane-bis- alkane-bis-ammonium compounds has been located between the second (o2) and third extracellular ammonium compounds has been located between the second (o2) and third extracellular (o3) loops (o3) loops (Figure2, green) [ 24–26]. The charged EDGE motif in the o2 loop plays a critical role in the (Figure 2, green) [24–26]. The charged EDGE motif in the o2 loop plays a critical role in the binding binding of these ligands [27]. In the crystal structure of the M2 receptor [4], the allosteric modulator of these ligands [27]. In the crystal structure of the M2 receptor [4], the allosteric modulator LY2119620 LY2119620 (that binds to the same site between o2 and o3) does not form a hydrogen bond to E172 or (that binds to the same site between o2 and o3) does not form a hydrogen bond to E172 or E175 of the E175 of the EDGE motif but makes π–π interactions to the adjacent Y177 in the o2 and Y426 in the EDGE motif but makes π–π interactions to the adjacent Y177 in the o2 and Y426 in the extracellular extracellular edge of TM7. edge of TM7.

Figure 2. Side view with TM6 front (left), and extracellular view (right) of the orthosteric (yellow), Figure 2. Side view with TM6 front (left), and extracellular view (right) of the orthosteric (yellow), common allosteric (green), and (cyan) binding sites at the M2 receptor (4MQT) (colored in common allosteric (green), and cholesterol (cyan) binding sites at the M2 receptor (4MQT) (colored in red-white-blue gradient). red-white-blue gradient). Biomolecules 2020, 10, 325 4 of 16

Biomolecules 2020, 10, x 4 of 15 Biomolecules 2020, 10, x 4 of 15 Computer modeling studies of allosteric ligand binding to M2 receptors have revealed two centers for bindingComputer of the modeling electropositive studies partof allosteric of allosteric ligand ligands binding (Figure to 3M)[2 28receptors]. The first have center revealed consists two of centers Computerfor binding modeling of the electropositivestudies of allosteric part ofligand allosteric binding ligands to M 2(Figure receptors 3) [28].have Therevealed first centertwo Y177,centers N410, for N419, binding and of W422, the electropositive and the second part consists of allosteric of Y80, ligands Y83, T84, (Figure and 3) T423. [28]. Further, The first this center study consists of Y177, N410, N419, and W422, and the second consists of Y80, Y83, T84, and T423. Further, showedconsists that of Y177, alkane-bis-ammonium N410, N419, and W422, compounds, and the second gallamine, consists alcuronium, of Y80, Y83, and T84, and T423. bind Further, to the this study showed that alkane-bis-ammonium compounds, gallamine, alcuronium, and strychnine fistthis center. study M showed1-selective that PAM alkane-bis-ammonium benzyl quinolone compounds, carboxylic acid gallamine, (BQCA, alcuronium, Figure4) binds and tostrychnine Y179 and bind to the fist center. M1-selective PAM benzyl quinolone carboxylic acid (BQCA, Figure 4) binds to F182bind in to the the o2 fist loop, center. and M E3971-selective and W400 PAM in benzyl TM7 [29 quinolone]. Thus, thecarboxylic binding acid site (BQCA, of BQCA Figure overlaps 4) binds with to the Y179 and F182 in the o2 loop, and E397 and W400 in TM7 [29]. Thus, the binding site of BQCA commonY179 and allosteric F182 in binding the o2 loop, site. and In contrast, E397 and M W4004-selective in TM7 PAM [29]. LY2033298 Thus, the (Figurebinding4 )site interacts of BQCA with overlaps with the common allosteric binding site. In contrast, M4-selective PAM LY2033298 (Figure theoverlaps o1, o2, andwith o3 the loops common [30]. allosteric Key binding binding residues site. In of contrast, LY2033298 M4-selective are K95 PAM in the LY2033298 o1, F186 in(Figure o2, and 4) interacts with the o1, o2, and o3 loops [30]. Key binding residues of LY2033298 are K95 in the o1, D4324) interacts in o3 (M with4 numbering). the o1, o2, and A cryptic o3 loops allosteric [30]. Key binding binding pocket residues in of the LY2033298 extracellular are K95 domain in the that o1, is F186 in o2, and D432 in o3 (M4 numbering). A cryptic allosteric binding pocket in the extracellular absentF186 inin existingo2, and D432 crystal in o3 structures (M4 numbering). has been A predicted cryptic allosteric by simulation binding ofpocket molecular in the dynamicsextracellular and domainconfirmeddomain that that by is mutagenesis isabsent absent in in existing experimentsexisting crystal crystal [31 structurstructur]. This cryptices has been pocket predictedpredicted is dynamically by by simulation simulation formed of inof molecular themolecular vicinity dynamicsof thedynamics common and and confirmed allosteric confirmed siteby by mutagenesis centermutagenesis 1 by rearrangement experiments experiments [31]. ofconserved ThisThis cryptic cryptic E 7.36pocket pocketin TM7, is is dynamically dynamically Y2.64 in the formed formed o2 loop 7.36 2.64 inclose thein the tovicinity TM2,vicinity andof of the the conserved common common Callosteric allosteric45.50 in the site site middle centercenter 1 of1 by the rearrangement o3 loop (general of of conserved conserved GPCR numbering E 7.36E in in TM7, TM7, [32 Y2.64 ,Y33 ]). in inthe the o2 o2 loop loop close close to to TM2, TM2, and and conservedconserved CC45.5045.50 in the middlemiddle ofof thethe o3 o3 loop loop (general (general GPCR GPCR The site is preferentially formed at the M1 receptor and has been identified as a binding site of highly numberingnumbering [32,33]). [32,33]). The The site site is is preferentially preferentially formedformed at the MM11 receptorreceptor and and has has been been identified identified as asa a M1-selective PAM BQZ12 (Figure4)[34,35]. bindingbinding site site of ofhighly highly M M1-selective1-selective PAM PAM BQZ12 BQZ12 (Figure(Figure 4) [34,35].[34,35].

FigureFigure 3. 3.Extracellular Extracellular view view of of two two binding binding centers centers (green) (green) in in the the common common allosteric allosteric binding binding site site at at the Figurethe M 3.2 receptorExtracellular (4MQT) view (colored of two in binding red-white-blue centers gradient). (green) in the common allosteric binding site at M2 receptor (4MQT) (colored in red-white-blue gradient). the M2 receptor (4MQT) (colored in red-white-blue gradient).

Figure 4. Structures of M -selective benzyl quinolone carboxylic acid (BQCA), BQZ12, M -selective Figure 4. Structures of M1 1-selective benzyl quinolone carboxylic acid (BQCA), BQZ12, M2-selective2 LY2119620,LY2119620, and and M M4-selective4-selective LY2033298 LY2033298 allosteric allosteric modulator.modulator. Figure 4. Structures of M1-selective benzyl quinolone carboxylic acid (BQCA), BQZ12, M2-selective

LY2119620, and M4-selective LY2033298 allosteric modulator. Biomolecules 2020, 10, 325 5 of 16

While the majority of known muscarinic allosteric ligands bind to the site between the o2 and o3 loops, sterol-based WIN-compounds have been found to interact with gallamine and strychnine in a non-competitive manner [36]. Thus, the binding site for WIN-compounds is not between the o2 and o3 loops but somewhere else. However, the precise location of the WIN-compound binding site has not been determined yet. Interestingly, cholesterol also allosterically modulates the binding and function of muscarinic receptors [37–40]. Using site-directed mutagenesis, the binding site for membrane cholesterol has been located to the groove between TM6 and TM7 in the intracellular leaflet of the membrane [40]. Membrane cholesterol allosterically modulates many GPCRs [41]. Cholesterol has co-crystallized with GPCRs at various sites, both in the extracellular and intracellular leaflet of the membrane [41]. Computer modeling of the interaction between membrane cholesterol and GPCR suggests the possibility of several cholesterol binding sites per one molecule of GPCR [42]. How many cholesterol binding sites muscarinic receptors do have and whether sterol-based WIN-compounds bind to the cholesterol-binding site remains to be elucidated.

4. Molecular Mechanisms of Action of Allosteric Modulators The maximum magnitude of the effects of an allosteric modulator at a given receptor (binding cooperativity) varies from one orthosteric ligand to another. Comparison of the binding cooperativity of a given allosteric ligand with several orthosteric ligands has suggested that cooperativity is dependent on the distance between the electropositive and electronegative part of the orthosteric ligand [43]. Thus, data has suggested that allosteric modulators change the distance between TM3 interacting with electropositive and TM6 interacting with an electronegative part of the orthosteric ligand. This hypothesis was confirmed later by crystal structures and molecular modeling [4,44]. Numerous studies have identified differential key amino acids to govern allosteric action of various allosteric modulators, suggesting the existence of multiple allosteric switches on muscarinic receptors. For example, M4-selective PAM LY2033298 gains its efficacy from interaction with K95 in the o1 loop, and its binding cooperativity results from interaction with F186 in the o2 loop (M4 numbering) [30]. 7.35 In contrast, in the o2 loop (Y179 in M1, Y177 in M2) and in TM7 (W , Ballesteros-Weinstein numbering [45]) have been identified as key residues, defining the magnitude of binding cooperativity of M1-selective PAM of acetylcholine (BQCA) as well as M2-selective PAM of iperoxo (LY2119620, Figure4)[ 4,29,34]. The common feature found for all PAMs is shrinkage of the vestibule to the orthosteric binding site that is accompanied by the closure of the binding pocket [46]. Divergent mechanisms underlying how shrinkage of the vestibule is achieved probably represent the molecular basis of PAM receptor subtype selectivity. Many GPCRs, including muscarinic receptors, activate several signaling pathways upon activation by agonists [47–49]. It is generally accepted that structurally different agonists induce specific changes in the conformation of GPCRs that can lead to non-uniform modulation of signaling pathways. This preferential orientation of the signaling of a GPCR towards a subset of its signal transducers is termed signaling bias [50]. In principle, the structure of the receptor in a ternary complex with agonist and the allosteric modulator is distinct from the one in a binary complex with agonist only and may result in biased signaling too. Attaining signaling bias via allosteric modulation could bring a new plethora of possibilities to modulate receptor function. It has been shown at M1 receptors that the allosteric modulator VU0029767 (Figure5) acts as a strong PAM of acetylcholine-induced intracellular calcium mobilization while its effects on acetylcholine-induced activation of phospholipase D were marginal [51]. Based on this observation, it has been suggested that differential modulation of receptor coupling to downstream signaling pathways by allosteric modulators may result in signaling bias. However, structurally divergent M1 PAMs BQCA (Figure4) and MIPS1674 (Figure5) have shown only small differences in the modulation of the phosphate, β-arrestin, and ERK1/2 pathways [52]. Residues near the common allosteric binding site have been implicated in mediating biased signaling even of orthosteric ligands at several GPCRs [53]. Thus, divergent mechanisms of shrinkage of the Biomolecules 2020, 10, 325 6 of 16 vestibule to the binding pocket may not only stand behind subtype-selective effects of allosteric Biomolecules 2020, 10, x 6 of 15 modulators but also drive ligand bias that is common to the entire A class of GPCRs [54].

Figure 5. Structures of M1 allosteric modulators MIPS1674 and VU0029767. Figure 5. Structures of M1 allosteric modulators MIPS1674 and VU0029767. 5. Role of the Common Allosteric Binding Site in the Binding of Orthosteric Ligands 5. Role of the Common Allosteric Binding Site in the Binding of Orthosteric Ligands The common allosteric binding site located between the o2 and o3 loops represents the vestibule to the orthostericThe common binding allosteric site. Thebinding model site in located which anbetween orthosteric the o2 ligand and o3 binds loops transiently represents to the a secondary vestibule siteto the before orthosteric moving binding to the orthosteric site. The model binding in site whic (so-calledh an orthosteric tandem two-siteligand binds model transiently [55]) has beento a proposedsecondary tosite explain before apparent moving to receptor the orthosteric isomerization binding upon site antagonist(so-called tandem binding two-site [56,57]. model Interaction [55]) ofhas orthosteric been proposed ligands to with explain the allostericapparent domainreceptor between isomerization the o2 andupon o3 antagonist loops has beenbinding confirmed [56,57]. andInteraction studied of in orthosteric detail by computer ligands with modeling the alloster [3,58,59ic]. domain These studies between showed the o2 that and two-step o3 loops binding has been is commonconfirmed for and all studied orthosteric in detail ligands by computer studied and modelin that orthostericg [3,58,59]. ligands These studie interacts showed primarily that with two-step Y177 (commonbinding is allostericcommon centerfor all orthosteric 1), N410, N419, ligands and studied W422 (commonand that orthosteric allosteric centerligands 2) interact (M2 numbering). primarily Subtypewith Y177 variations (common of aallostericffinity and center kinetics 1), ofN410, orthosteric N419, ligandsand W422 may (common be attributed allosteric to variation center in2) their(M2 interactionnumbering). with Subtype the allosteric variations site of [6 ].affinity and kinetics of orthosteric ligands may be attributed to variationThe strongin their interaction interaction of with tiotropium the allosteric with the site allosteric [6]. binding site also explains the discrepancy betweenThe extremelystrong interaction slow binding of tiotropium kinetics with and immediatethe allosteric inhibitory binding actionsite also of explains this bronchodilator the discrepancy [60]. Tiotropiumbetween extremely binding slow to the binding allosteric kinetics site is and fast andimmediat resultse inhibitory in antagonism action of theof this functional bronchodilator effects of [60]. the Tiotropium binding to the allosteric site is fast and results in antagonism of the functional effects of M3 receptor. the M3 receptor. The M2-selective antagonist competitively inhibits the binding of orthosteric ligands.The HighM2-selective concentrations antagonist slow methoctramine down the dissociation competitively of [3 H]N-methylscopolamine.inhibits the binding of orthosteric Detailed analysisligands. ofHigh methoctramine concentrations binding slow has down revealed the dissociation two modes of of interaction [3H]N-methylscopolamine. with the receptor. TransientDetailed bindinganalysis of to methoctramine the common allosteric binding has site revealed center 1 two (namely modes E175 of interaction in the o2 loop)with the is followedreceptor. Transient by stable binding thatto the occurs common concurrently allosteric to bothsite center the allosteric 1 (namely and orthostericE175 in the sites o2 loop) [61]. Methoctramineis followed by isstable thus abinding bitopic that dualsteric occurs antagonist. concurrently to both the allosteric and orthosteric sites [61]. Methoctramine is thus a bitopic dualsteric antagonist. 6. Bitopic Ligands 6. Bitopic Ligands The idea of utilizing high efficacy of orthosteric ligands and subtype diversity of the allosteric bindingThe sites idea has of utilizing led to the high development efficacy of of orthosteri bitopic ligandsc ligands that and span subtype both sites diversity and interact of the with allosteric them concurrently.binding sites has In principle, led to the adevelopment bitopic ligand of consists bitopic oflig twoands molecules, that span both one targetingsites and theinteract orthosteric with them site, andconcurrently. the other interactingIn principle, with a bitopic the allosteric ligand site.consis Thets of two two parts molecules, are connected one targeting by a linker the oforthosteric a proper length.site, and A the combination other interacting of the nonselective with the allosteric muscarinic site. agonist The two iperoxo parts and are itsconnected M1-selective by a PAM linker BQCA of a hasproper led tolength. a set ofA M combination1-selective agonists of the withnonselective graded e ffimuscariniccacy that isagonist dependent iperoxo on the and chemical its M1-selective structure ofPAM the BQCA linker [62has]. led The to orientation a set of M of1-selective the orthosteric agonists moiety with within graded the ef orthostericficacy that bindingis dependent site is crucialon the forchemical subtype structure selectivity of the [63 linker]. Therefore, [62]. The the orientation structure of of the the orthosteric linker affects moiety not onlywithin effi thecacy orthosteric but also thebinding selectivity site is crucial of bitopic for ligands.subtype Anotherselectivity example [63]. Therefore, of a bitopic the structure agonist that of the gains linker its selectivityaffects not fromonly interactionefficacy but with also the the common selectivity allosteric of bitopic binding ligands. site isAnother TBPB (Figure example6)[ of64 ,a65 bitopic]. Mutations agonist of that the allostericgains its selectivity from interaction with the common allosteric binding site is TBPB (Figure 6) [64,65]. Mutations of the allosteric and orthosteric binding sites have revealed that NDMC (N- ) that is considered an allosteric agonist and McN-A-343 (4-(m-chlorophenyl- carbamoyloxy)-2-butynyltri-methylammonium) that is considered an orthosteric agonist are in fact Biomolecules 2020, 10, 325 7 of 16 andBiomolecules orthosteric 2020, 10 binding, x sites have revealed that NDMC (N-desmethylclozapine) that is considered7 of an 15 allostericBiomolecules agonist 2020, 10, andx McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltri-methylammonium)7 of 15 thatbitopic is consideredligands [66,67]. an orthosteric Allosteric agonist effects areof the in fact bitopic bitopic agonists ligands McN-A-343 [66,67]. Allosteric and NDMC effects and of thethe bitopic ligands [66,67]. Allosteric effects of the bitopic agonists McN-A-343 and NDMC and the2 bitopicallosteric agonists agonists McN-A-343 AC-42 and and 77-LH-28-1 NDMC and (Figure the allosteric 6) are mediated agonists via AC-42 Y177 and in 77-LH-28-1the o2 loop (Figureof the M6) allostericreceptor [66].agonists Taken AC-42 together, and 77-LH-28-1these bitopic (Figure ligands 6) alareso mediated interact with via Y177the center in the 1 o2 (Figure loop of 3) the of theM2 are mediated via Y177 in the o2 loop of the M2 receptor [66]. Taken together, these bitopic ligands also receptor [66]. Taken together, these bitopic ligands also interact with the center 1 (Figure 3) of the interactcommon with allosteric the center binding 1 (Figure site (Figure3) of the 2). common allosteric binding site (Figure2). common allosteric binding site (Figure 2).

Figure 6. Structures of bitopic ligands AC-42, 77-LH-28-1, and TBPB. Figure 6. Structures of bitopic ligands AC-42, 77-LH-28-1, and TBPB. Figure 6. Structures of bitopic ligands AC-42, 77-LH-28-1, and TBPB. Further developmentdevelopment of of bitopic bitopic ligands ligands has has led led to theto the discovery discovery of the of photo-switchablethe photo-switchable ligands ligands [68]. In[68]. the FurtherIn case the ofcase development a photo-switchableof a photo-switchable of bitopic ligand, li gandsligand, the has conformationthe led conformation to the discovery of the of the linker of linker the is photo-switchable sensitive is sensitive to exposureto exposure ligands to light[68].to light In of atheof specific acase specific of wavelength, a photo-switchablewavelength, which which leads ligand, leads to the the to isomerization conformationthe isomerization of of the the of linker linker the andlinker is sensitive change and change in to the exposure mutual in the orientationtomutual light orientationof a of specific orthosteric of wavelength, orthosteric and allosteric andwhich allosteric moieties leads to (Figuremoieties the isomerization7). (Figure In one linker7). ofIn conformation,theone linkerlinker andconformation, the change orthosteric in the andmutualorthosteric allosteric orientation and moieties allosteric of orthosteric are moieties in an orientation and are inallosteric an that orientation doesmoieties not that allow (Figure does simultaneous not7). In allow one simultaneouslinker interaction conformation, of interaction individual the moietiesorthostericof individual with and moieties their allosteric respective with moieties their binding respectiveare in sites an thatorientation bind makesing sites athat ligand that does makes inactive. not allow a ligand After simultaneous inactive. linker isomerization, After interaction linker theofisomerization, individual orientation moieties ofthe individual orientation with moietiestheir of individualrespective allows simultaneous bindmoietiesing sitesallows interaction that simultaneous makes of a individual ligand interaction inactive. moieties of After individual with linker their respectiveisomerization,moieties with binding their the orientation sites,respective and the bindingof ligandindividual sites, becomes and moieties the active. ligand allows Photo-switchable becomes simultaneous active. ligandsPhoto-switchableinteraction are anof invaluableindividual ligands toolmoietiesare an in invaluable basic with research their tool respective since in basic they researchbinding allow an sites,since experimenter andthey the allow ligand to an apply experimenter becomes ligand active. at theto apply Photo-switchable desired ligand place at and the desired ligands timeareplace an in and invaluable the desired blink oftool time a laser.in in basic the researchblink of a since laser. they allow an experimenter to apply ligand at the desired place and desired time in the blink of a laser.

Figure 7.7. Scheme of isomerizationisomerization of photo-switchablephoto-switchable ligand, consisting of the orthostericorthosteric (O) andand allostericFigure 7. (A)(A)Scheme moietymoiety of connectedisomerizationconnected byby the theof photo-switchablelinker.linker. ligand, consisting of the orthosteric (O) and allosteric (A) moiety connected by the linker. Besides beingbeing an an interesting interesting research research tool, bitopictool, bitopic muscarinic muscarinic ligands, ligands, consisting consisting of the orthosteric of the andorthosteric allostericBesides andbeing moiety, allosterican have interesting also moiety, a translationalresearch have tool,also potential. bitopic a translational Formuscarinic example, ligands,potential. bis(ammonio)alkane-type consisting For example, of the compoundsorthostericbis(ammonio)alkane-type (hybridand allosteric molecules compounds moiety, of the orthosteric (hybridhave alsomolecules agonist a iperoxotranslational of the and orthosteric allosteric potential. moietyagonist For derivediperoxo example, fromand bis(ammonio)alkane-typeallosteric moiety derived fromcompounds naphmethonium (hybrid molecules or W84) canof thebe usedorthosteric as non- agonist non-steroidaliperoxo and allostericanalgesics moiety [69] or derivedinhibitors from of cell naphmethonium growth for glioblastoma or W84) can therapy be used [70]. as non-opioid non-steroidal analgesics [69] or inhibitors of cell growth for glioblastoma therapy [70]. 7. Novel Allosteric Modulators 7. Novel Allosteric Modulators Biomolecules 2020, 10, 325 8 of 16 naphmethonium or W84) can be used as non-opioid non-steroidal analgesics [69] or inhibitors of cell growth for glioblastoma therapy [70].

7. Novel Allosteric Modulators Biomolecules 2020, 10, x 8 of 15 Although positive allosteric modulation of acetylcholine was presented as proof of concept in the 1990sAlthough [71,72], it positive took more allosteric than 10 modulation years to develop of acet firstylcholine PAMs withwas physiologicallypresented as proof active of propertiesconcept in suitablethe 1990s for [71,72], further it drugtook developmentmore than 10 [ 73years–76]. to However, develop thefirst progress PAMs with of research physiologically on muscarinic active allostericproperties modulators suitable for has further accelerated, development and selective [73–76]. PAMs forHowever, each subtype the progress have been of research discovered on duringmuscarinic the lastallosteric decade. modulators has accelerated, and selective PAMs for each subtype have been discoveredNamely, during two new the classeslast decade. of M1 -selective PAMs have been developed. Derivatives of heterocyclic carboxamidesNamely, displaytwo new various classes degrees of M1-selective of intrinsic PAMs activity have and been various developed. modulatory Derivatives profiles of of heterocyclic affinity and ecarboxamidesfficacy of acetylcholine display various [77]. Similarly, degrees derivatives of intrinsic of ac 4-phenylpyridin-2-onetivity and various modulatory (Figure8 )profiles display of a diverseaffinity rangeand efficacy of activities, of acetylcholine ranging from [77]. pure Similarly, PAMs to derivat pure allostericives of 4-phenylpyridin- agonists [78]. All2-one 4-phenylpyridin-2-one (Figure 8) display derivativesa diverse range have retainedof activities, exquisite ranging selectivity from forpure the PAMs M1 receptor. to pure allosteric agonists [78]. All 4- phenylpyridin-2-one derivatives have retained exquisite selectivity for the M1 receptor.

Figure 8.8. The core structure of 4-phenylpyridin-2-one derivatives.derivatives.

The availability of the M receptor crystal structure in an inactive [2], as well as active The availability of the M22 receptor crystal structure in an inactive [2], as well as active conformationconformation [4] [4] and and advances advances in computer in computer modeling, modeling, has allowed has allowed for the acceleration for the acceleration of structure- of structure-basedbased design of design chemically of chemically diverse diverseallosteric allosteric modulators modulators of this ofreceptor this receptor [79]. The [79]. combination The combination of in ofsilico in silicosimulation simulation of molecular of molecular dynamics dynamics and virtual and virtualscreening screening has led to has the led identification to the identification of allosteric of allosteric modulators of the M receptor of chemically novel structures that have been verified as modulators of the M2 receptor 2of chemically novel structures that have been verified as NAMs and NAMsPAMs of and superagonist PAMs of superagonist iperoxo in binding iperoxo experiments. in binding experiments. PositivePositive allostericallosteric modulatorsmodulators of antagonistsantagonists maymay turnturn otherwiseotherwise aa non-selectivenon-selective orthostericorthosteric antagonistantagonist to the subtype-selective antagonist antagonist with with therapeutic therapeutic potential. potential. Recently, Recently, as asa proof a proof of of concept, M -selective positive modulators of orthosteric antagonists have been developed [80]. concept, M2-selective2 positive modulators of orthosteric antagonists have been developed [80]. In this In this study, triazolo-quinazolinone analogs were identified as M selective positive modulators of study, triazolo-quinazolinone analogs were identified as M2 selective2 positive modulators of N-methylscopolamineN-methylscopolamine (NMS)(NMS) byby dockingdocking aa largelarge librarylibrary ofof moleculesmolecules toto thethe allostericallosteric bindingbinding sitesite ofof the M receptor in an inactive conformation. Resulting allosteric modulators increased NMS affinity the M22 receptor in an inactive conformation. Resulting allosteric modulators increased NMS affinity about 5-times and slowed NMS dissociation from the M receptor about 50-fold while having no such about 5-times and slowed NMS dissociation from the M22 receptor about 50-fold while having no such eeffectsffects atat thethe otherother subtypessubtypes ofof muscarinicmuscarinic receptors.receptors. TestingTesting ofof novel novel muscarinic muscarinic allosteric allosteric modulators modulators in animal in animal models models of human of human diseases diseases has shown has theirshown good their effi goodcacy. efficacy. Cholinergic neurons neurons regulate regu glutamatergiclate neurons neurons in the striatum in the striatum that regulate that selection and decision-making behavior [81]. The M -selective PAM of acetylcholine BQCA has regulate selection and decision-making behavior [81]. The1 M1-selective PAM of acetylcholine BQCA improvedhas improved efficacy efficacy of the antipsychoticsof the , haloperidol, , andclozapine, and inaripiprazole the glutamatergic in the deficit mouse model of behavior [82]. M receptors play a critical role in cognitive processes [7]. Thus, glutamatergic deficit mouse model of 1behavior [82]. M1 receptors play a critical role in cognitive positive allosteric modulation of M receptors appears to be the way to treat cognitive deficits in processes [7]. Thus, positive allosteric1 modulation of M1 receptors appears to be the way to treat Alzheimer’scognitive deficits disease in orAlzheimer’s schizophrenia disease [83]. or However, schizophrenia cortex, [83]. hippocampus, However, andcortex, striatum hippocampus, post mortem and samplesstriatum frompost somemortem patients samples with from schizophrenia some patients have with shown schizophrenia a profound have loss ofshown muscarinic a profound receptors loss and decreased responsiveness to M -selective PAM of acetylcholine BQCA. These findings may explain of muscarinic receptors and decreased1 responsiveness to M1-selective PAM of acetylcholine BQCA. whyThese some findings individuals may explain with schizophreniawhy some individuals may not wi respondth schizophrenia to such [84 ].may not respond to such [84]. The striatal M receptors attenuate and glutamatergic neurotransmission [85]. Thus, The striatal M4 4 receptors attenuate dopaminergic and glutamatergic neurotransmission [85]. selective positive modulation of M receptors may provide a novel treatment strategy of psychotic Thus, selective positive modulation4 of M4 receptors may provide a novel treatment strategy of symptomspsychotic symptoms in schizophrenia in schizophrenia that are considered that are toconsidered result from to dopaminergic result from dopaminergic hyperactivity hyperactivity [83]. A series [83]. A series of M4 PAMs have been found to be centrally active and efficient in an animal model of schizophrenia [86]. Huntington’s disease results from increased transmission at glutamatergic corticostriatal synapses that may be attenuated by the cholinergic system. In accordance, M4 PAMs have improved behavioral symptoms in the mouse model of Huntington disease [76]. M5 muscarinic receptors are expressed solely in the substantia nigra and ventral tegmental area (VTA) [7]. Cholinergic neurons in the nucleus accumbens (NAcc) project to VTA, where they stimulate dopaminergic neurons. The VTA dopaminergic neurons project back to NAcc, where they Biomolecules 2020, 10, 325 9 of 16

of M4 PAMs have been found to be centrally active and efficient in an animal model of schizophrenia [86]. Huntington’s disease results from increased transmission at glutamatergic corticostriatal synapses that may be attenuated by the cholinergic system. In accordance, M4 PAMs have improved behavioral symptoms in the mouse model of Huntington disease [76]. M5 muscarinic receptors are expressed solely in the substantia nigra and ventral tegmental areaBiomolecules (VTA) 2020 [7]., 10 Cholinergic, x neurons in the nucleus accumbens (NAcc) project to VTA, where9 they of 15 stimulateBiomolecules dopaminergic2020, 10, x neurons. The VTA dopaminergic neurons project back to NAcc, where9 they of 15 stimulatestimulate cholinergiccholinergic neurons.neurons. ThisThis positivepositive feedbackfeedback worksworks asas thethe rewardreward circuitcircuit [[87].87]. AA blockadeblockade oror stimulate cholinergic neurons. This positive feedback works as the reward circuit [87]. A blockade or negativenegative allostericallosteric modulationmodulation ofof MM55 receptors could break the reward cycle and prevent addiction. negative allosteric modulation of M5 receptors could break the reward cycle and prevent addiction. TheThe M5-selective-selective NAM NAM of of acetylcholine acetylcholine ML375 ML375 (Figure (Figure 9)9 [88])[ 88 has] has effectively effectively prevented prevented cocaine self- The M5-selective NAM of acetylcholine ML375 (Figure 9) [88] has effectively prevented cocaine self- self-administrationadministration and andthe development the development of addiction of addiction in rats inrats [89]. [89 ]. administration and the development of addiction in rats [89].

Figure 9. Structure of M5-selective negative allosteric modulators (NAM) of acetylcholine ML375. FigureFigure 9. 9. StructureStructure of of M M5-selective5-selective negative negative allosteric allosteric modu modulatorslators (NAM) (NAM) of of acetylcholine acetylcholine ML375. ML375. In type 2 diabetes, pancreatic β cells are unable to release sufficient amounts of to InIn typetype 2 2 diabetes, diabetes, pancreatic pancreaticβ cells β cells are unableare unable to release to release sufficient sufficient amounts amounts of insulin of to insulin maintain to maintain physiological blood glucose levels. Parasympathetic activation of pancreatic β cells by physiologicalmaintain physiological blood glucose blood levels. glucose Parasympathetic levels. Parasympathetic activation of activation pancreatic ofβ pancreaticcells by acetylcholine β cells by acetylcholine stimulates the release of insulin [90]. Effects of acetylcholine on insulin secretion are stimulatesacetylcholine the stimulates release of insulinthe release [90]. of Eff insulinects of acetylcholine[90]. Effects of on acetylcholine insulin secretion on insulin are mediated secretion by theare mediated by the M3 receptor [91]. In accordance, M3 PAM of acetylcholine VU0119498 (Figure 10) has Mmediated3 receptor by [the91]. M In3 receptor accordance, [91]. M In3 PAMaccordance, of acetylcholine M3 PAM of VU0119498 acetylcholine (Figure VU0119498 10) has been(Figure shown 10) has to been shown to enhance glucose-stimulated insulin secretion and greatly improve glucose tolerance enhancebeen shown glucose-stimulated to enhance glucose-stimulated insulin secretion insulin and greatly secretion improve and glucosegreatly toleranceimprove glucose in and tolerance obese in lean and obese glucose-intolerant mice [92]. These effects were absent from mice lackingβ M3 glucose-intolerantin lean and obese mice glucose-intolerant [92]. These effects mice were [92]. absent These from effects mice were lacking absent M3 receptors from mice in theirlackingcells. M3 receptors in their β cells. receptors in their β cells.

FigureFigure 10.10. Structure of M33-selective positive allosteric modu modulatorslators (PAM) ofof acetylcholineacetylcholine VU0119498.VU0119498. Figure 10. Structure of M3-selective positive allosteric modulators (PAM) of acetylcholine VU0119498. 8.8. Perspectives 8. Perspectives RecentRecent findingsfindings of of the the diversity diversity of actions of actions of allosteric of allosteric modulators modulators and bitopic and ligands bitopic of muscarinicligands of Recent findings of the diversity of actions of allosteric modulators and bitopic ligands of receptorsmuscarinic have receptors greatly influencedhave greatly the influenced development the of development new therapies of for new a variety therapies of disorders. for a variety Recent of muscarinic receptors have greatly influenced the development of new therapies for a variety of discoveriesdisorders. ofRecent selective discoveries positive allostericof selective modulators positive of allosteric acetylcholine modulators with therapeutic of acetylcholine potential inwith the disorders. Recent discoveries of selective positive allosteric modulators of acetylcholine with treatmenttherapeutic of psychiatricpotential in and the neurologic treatment disordersof psychiat likeric Alzheimer’s and neurologic or schizophrenia, disorders like as wellAlzheimer’s as internal or therapeutic potential in the treatment of psychiatric and neurologic disorders like Alzheimer’s or schizophrenia, as well as internal disorders like type 2 diabetes, are encouraging. Research on several schizophrenia, as well as internal disorders like type 2 diabetes, are encouraging. Research on several muscarinic allosteric modulators has surpassed the stage of optimization of selectivity, efficacy, and muscarinic allosteric modulators has surpassed the stage of optimization of selectivity, efficacy, and bioavailability and advanced to a translational stage of medicinal research. In the field of basic bioavailability and advanced to a translational stage of medicinal research. In the field of basic research, further progress is mainly achieved by the introduction of novel concepts and techniques. research, further progress is mainly achieved by the introduction of novel concepts and techniques. Novel photo-switchable ligands may greatly increase the accuracy of timely delivery as well as allow Novel photo-switchable ligands may greatly increase the accuracy of timely delivery as well as allow varied permutations of experimental design. Thus, focus on the design of novel photo-switchable varied permutations of experimental design. Thus, focus on the design of novel photo-switchable ligands is currently gaining momentum. ligands is currently gaining momentum. Biomolecules 2020, 10, 325 10 of 16 disorders like type 2 diabetes, are encouraging. Research on several muscarinic allosteric modulators has surpassed the stage of optimization of selectivity, efficacy, and bioavailability and advanced to a translational stage of medicinal research. In the field of basic research, further progress is mainly achieved by the introduction of novel concepts and techniques. Novel photo-switchable ligands may greatly increase the accuracy of timely delivery as well as allow varied permutations of experimental design. Thus, focus on the design of novel photo-switchable ligands is currently gaining momentum.

Author Contributions: J.J. and E.E.E.-F. contributed to the review equally. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Czech Academy of Sciences institutional support [RVO:67985823] and the Grant Agency of the Czech Republic grant [19-05318S]. Conflicts of Interest: The authors declare no conflict of interest.

Nomenclature

Compounds Chemical Names 77-LH-28-1 1-[3-(4-Butylpiperidino)propyl]-3,4-dihydroquinoline-2(1H)-one AC-42 4-(4-Butylpiperidin-1-yl)-1-o-tolylbutan-1-one Alcuronium 2-[(1S,9Z,11S,13S,17S,25Z,27S,33S,35S,36S)-38-(2-hydroxyethylidene)-14,30-bis(prop-2-enyl)- 8,24-diaza-14,30-diazoniaundecacyclo [2 5.5.2.211,14.11,26.110,17.02,7.013,17.018,23.030,33.08,35.024,36] octatriaconta-2,4,6,9,18,20,22,25-octaen-28-ylidene]ethanol BQCA 1-(4-Methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid BQZ12 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo [h]quinazolin-4(3H)-one Gallamine 2-[2,3-bis [2-(triethylazaniumyl)ethoxy]phenoxy]ethyl-triethylazanium LY2119620 3-Amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno [2,3-b]pyridine-2-carboxamide LY2033298 3-Amino-5-Chloro-N-Cyclopropyl-6-Methoxy-4-Methylthieno [2,3-B]Pyridine-2-Carboxamide McN-A-343 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltri-methylammonium Methoctramine N,N0-bis [6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane]diamine MIPS1674 1-[(1S,2S)-2-hydroxycyclohexyl]-4-[2-[[4-(1H-pyrazol-4-yl)phenyl]methoxy]phenyl]pyridin- 2-one ML375 (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3- dihydro-1H-imidazo [2,1-a]isoindol-5(9bH)-one Naphmethonium [3-(1,3-dioxobenzo [de]isoquinolin-2-yl)-2,2-dimethylpropyl]-[6-[3-(1,3-dioxoisoindol-2-yl) propyl-dimethylazaniumyl]hexyl]-dimethylazanium NDMC N-desmethylclozapine; 3-chloro-6-piperazin-1-yl-11H-benzo [b] [1,4]benzodiazepine NMS N-methylscopolamine; [(1S,2S,4R,5R)-9,9-dimethyl-3-oxa-9-azoniatricyclo [3.3.1.02,4]nonan-7-yl] (2S)-3-hydroxy-2-phenylpropanoate Strychnine (4aR,5aS,8aR,13aS,15aS,15bR)-4a,5,5a,7,8,13a,15,15a,15b,16-decahydro-2H-4,6-methanoindolo [3,2,1-ij]oxepino [2,3,4-de]pyrrolo [2,3-h]quinolin-14-one TBPB 1-(10-(2-methylbenzyl)-1,40-bipiperidin-4-yl)-1H-benzo [d]imidazol-2(3H)-one Tiotropium [(1S,2S,4R,5R)-9,9-dimethyl-3-oxa-9-azoniatricyclo [3.3.1.02,4]nonan-7-yl]2-hydroxy-2,2-dithiophen-2-ylacetate VU0119498 1-(4-bromobenzyl)indole-2,3-dione VU0029767 (E)-2-(4-ethoxyphenylamino)-N0-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide W84 6-[dimethyl-[3-(4-methyl-1,3-dioxoisoindol-2-yl)propyl]azaniumyl]hexyl-dimethyl-[3- (4-methyl-1,3-dioxoisoindol-2-yl)propyl]azanium Biomolecules 2020, 10, 325 11 of 16

References

1. Bonner, T.I.; Buckley, N.J.; Young, A.C.; Brann, M.R. Identification of a family of muscarinic genes. Science 1987, 237, 527–532. [CrossRef][PubMed] 2. Haga, K.; Kruse, A.C.; Asada, H.; Yurugi-Kobayashi, T.; Shiroishi, M.; Zhang, C.; Weis, W.I.; Okada, T.; Kobilka, B.K.; Haga, T.; et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 2012, 482, 547–551. [CrossRef][PubMed] 3. Kruse, A.C.; Hu, J.; Pan, A.C.; Arlow, D.H.; Rosenbaum, D.M.; Rosemond, E.; Green, H.F.; Liu, T.; Chae, P.S.; Dror, R.O.; et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 2012, 482, 552–556. [CrossRef][PubMed] 4. Kruse, A.C.; Ring, A.M.; Manglik, A.; Hu, J.; Hu, K.; Eitel, K.; Hübner, H.; Pardon, E.; Valant, C.; Sexton, P.M.; et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 2013, 504, 101–106. [CrossRef][PubMed] 5. Thal, D.M.; Sun, B.; Feng, D.; Nawaratne, V.; Leach, K.; Felder, C.C.; Bures, M.G.; Evans, D.A.; Weis, W.I.; Bachhawat, P.; et al. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 2016, 531, 335–340. [CrossRef][PubMed] 6. Vuckovic, Z.; Gentry, P.R.; Berizzi, A.E.; Hirata, K.; Varghese, S.; Thompson, G.; van der Westhuizen, E.T.; Burger, W.A.C.; Rahmani, R.; Valant, C.; et al. Crystal structure of the M5 muscarinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 2019, 116, 26001–26007. [CrossRef] 7. Eglen, R.M. Overview of muscarinic receptor subtypes. In Handb Exp Pharmacol; Springer: Heidelberg, Germany, 2012; pp. 3–28. ISBN 0171-2004. 8. Conn, P.J.; Jones, C.K.; Lindsley, C.W. Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders. Trends Pharmacol Sci 2009, 30, 148–155. [CrossRef] 9. Bock, A.; Schrage, R.; Mohr, K. Allosteric modulators targeting CNS muscarinic receptors. Neuropharmacology 2018, 136, 427–437. [CrossRef] 10. Gautam, D.; Han, S.-J.; Duttaroy, A.; Mears, D.; Hamdan, F.F.; Li, J.H.; Cui, Y.; Jeon, J.; Wess, J. Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis. Diabetes Obes. Metab. 2007, 9, 158–169. [CrossRef] 11. Gosens, R.; Zaagsma, J.; Meurs, H.; Halayko, A.J. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir. Res. 2006, 7, 73. [CrossRef] 12. Hegde, S.S. Muscarinic receptors in the bladder: From basic research to therapeutics. Br. J. Pharmacol. 2006, 147, S80–S87. [CrossRef][PubMed] 13. Monod, J.; Wyman, J.; Changeux, J.P. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 1965, 12, 88–118. [CrossRef] 14. Koshland, D.E.; Némethy, G.; Filmer, D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 1966, 5, 365–385. [CrossRef] 15. Jakubík, J.; El-Fakahany, E.E. Allosteric Modulation of Muscarinic Receptors. In Muscarinic Receptor: From Structure to Animal Models; Myslivecek, J., Jakubik, J., Eds.; Neuromethods; Humana Press; Springer Science + Business Media LLC: New York, NY, USA, 2016; Volume 107, pp. 95–130. ISBN 978-1-4939-287-6. 16. Kenakin, T.; Strachan, R.T. PAM-Antagonists: A Better Way to Block Pathological Receptor Signaling? Trends Pharmacol. Sci. 2018, 39, 748–765. [CrossRef][PubMed] 17. Jakubík, J.; Bacáková, L.; Lisá, V.; El-Fakahany, E.E.; Tucek, S. Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc. Natl. Acad. Sci. USA 1996, 93, 8705–8709. [CrossRef] 18. Lüllmann, H.; Ohnesorge, F.K.; Schauwecker, G.C.; Wassermann, O. Inhibition of the actions of carbachol and DFP on guinea pig isolated atria by alkane-bis-ammonium compounds. Eur. J. Pharmacol. 1969, 6, 241–247. [CrossRef] 19. Clark, A.L.; Mitchelson, F. The inhibitory effect of gallamine on muscarinic receptors. Br. J. Pharmacol. 1976, 58, 323–331. [CrossRef] 20. Jakubík, J.; El-Fakahany, E.E. Allosteric Modulation of Muscarinic Acetylcholine Receptors. Pharmaceuticals 2010, 3, 2838–2860. [CrossRef] 21. Mash, D.C.; Flynn, D.D.; Potter, L.T. Loss of M2 receptors in the cerebral cortex in Alzheimer’s disease and experimental cholinergic denervation. Science 1985, 228, 1115–1117. [CrossRef] Biomolecules 2020, 10, 325 12 of 16

22. Perry, E.K.; Smith, C.J.; Court, J.A.; Perry, R.H. Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types. J. Neural Transm. Park. Dis. Dement. Sect. 1990, 2, 149–158. [CrossRef] 23. Buels, K.S.; Fryer, A.D. Muscarinic receptor antagonists: Effects on pulmonary function. Handb. Exp. Pharmacol. 2012, 208, 317–341. 24. Krejˇcí, A.; Tuˇcek,S. Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors. Mol. Pharmacol. 2001, 60, 761–767. 25. Huang, X.-P.; Prilla, S.; Mohr, K.; Ellis, J. Critical residues of the common allosteric site on the M2 muscarinic acetylcholine receptor: more similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide. Mol. Pharmacol. 2005, 68, 769–778. [PubMed] 26. Jakubík, J.; Krejcí, A.; Dolezal, V. Asparagine, valine, and in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators. J. Pharmacol. Exp. Ther. 2005, 313, 688–696. [CrossRef][PubMed] 27. Leppik, R.A.; Miller, R.C.; Eck, M.; Paquet, J.L. Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor. Mol. Pharmacol. 1994, 45, 983–990. 28. Dror, R.O.; Green, H.F.; Valant, C.; Borhani, D.W.; Valcourt, J.R.; Pan, A.C.; Arlow, D.H.; Canals, M.; Lane, J.R.; Rahmani, R.; et al. Structural basis for modulation of a G-protein-coupled receptor by allosteric . Nature 2013, 503, 295–299. [CrossRef][PubMed] 29. Abdul-Ridha, A.; López, L.; Keov, P.; Thal, D.M.; Mistry, S.N.; Sexton, P.M.; Lane, J.R.; Canals, M.; Christopoulos, A. Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor. J. Biol. Chem. 2014, 289, 6067–6079. [CrossRef][PubMed] 30. Nawaratne, V.; Leach, K.; Felder, C.C.; Sexton, P.M.; Christopoulos, A. Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: Identification of ligand-specific and global activation mechanisms. J. Biol. Chem. 2010, 285, 19012–19021. [CrossRef] 31. Hollingsworth, S.A.; Kelly, B.; Valant, C.; Michaelis, J.A.; Mastromihalis, O.; Thompson, G.; Venkatakrishnan, A.J.; Hertig, S.; Scammells, P.J.; Sexton, P.M.; et al. Cryptic pocket formation underlies allosteric modulator selectivity at muscarinic GPCRs. Nat. Commun. 2019, 10, 3289. [CrossRef] 32. Ballesteros, J.A.; Weinstein, H. Integrated methods for the construction of three dimensional models and computational probing of structure function relations in G protein-coupled receptors. In Methods in Neurosciences; Sealfon, S.C., Conn, P.M., Eds.; Academic Press: San Diego, CA, USA, 1995; Volume 25, pp. 366–428. ISBN 9780121852955. 33. Isberg, V.; De Graaf, C.; Bortolato, A.; Cherezov, V.; Katritch, V.; Marshall, F.H.; Mordalski, S.; Pin, J.-P.P.; Stevens, R.C.; Vriend, G.; et al. Generic GPCR residue numbers—Aligning topology maps while minding the gaps. Trends Pharmacol. Sci. 2015, 36, 22–31. [CrossRef] 34. Abdul-Ridha, A.; Lane, J.R.; Mistry, S.N.; López, L.; Sexton, P.M.; Scammells, P.J.; Christopoulos, A.; Canals, M. Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor. J. Biol. Chem. 2014, 289, 33701–33711. [CrossRef][PubMed] 35. Bradley, S.J.; Bourgognon, J.M.; Sanger, H.E.; Verity, N.; Mogg, A.J.; White, D.J.; Butcher, A.J.; Moreno, J.A.; Molloy, C.; Macedo-Hatch, T.; et al. M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss. J. Clin. Investig. 2016, 127, 487–499. [CrossRef][PubMed] 36. Lazareno, S.; Popham, A.; Birdsall, N.J.M. Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors. Mol. Pharmacol. 2002, 62, 1492–1505. [CrossRef][PubMed] 37. Michal, P.; Rudajev, V.; El-Fakahany, E.E.; Doležal, V. Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways. Eur. J. Pharmacol. 2009, 606, 50–60. [CrossRef][PubMed] 38. Michal, P.; El-Fakahany, E.E.; Doležal, V. Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors. Neurochem. Res. 2014, 40, 2068–2077. [CrossRef] Biomolecules 2020, 10, 325 13 of 16

39. Janickova, H.; Rudajev, V.; Dolejsi, E.; Koivisto, H.; Jakubik, J.; Tanila, H.; El-Fakahany, E.E.; Dolezal, V. -Based Diets Improve Muscarinic Neurotransmission in the Hippocampus of Transgenic APPswe/PS1dE9 Mice. Curr. Alzheimer Res. 2015, 12, 923–931. [CrossRef] 40. Randáková, A.; Dolejší, E.; Rudajev, V.; Zimˇcík, P.; Doležal, V.; El-Fakahany, E.E.; Jakubík, J. Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound . Neuropharmacology 2018, 133, 129–144. [CrossRef] 41. Gimpl, G. Interaction of G protein coupled receptors and cholesterol. Chem. Phys. 2016, 199, 61–73. [CrossRef] 42. Lee, A.G. Interfacial Binding Sites for Cholesterol on G Protein-Coupled Receptors. Biophys. J. 2019, 116, 1586–1597. [CrossRef] 43. Zlotos, D.P.; Buller, S.; Stiefl, N.; Baumann, K.; Mohr, K. Probing the pharmacophore for allosteric ligands of muscarinic M2 receptors: SAR and QSAR studies in a series of bisquaternary salts of caracurine V and related ring systems. J. Med. Chem. 2004, 47, 3561–3571. [CrossRef] 44. Bermudez, M.; Wolber, G. Structure versus function—The impact of computational methods on the discovery of specific GPCR-ligands. Bioorganic Med. Chem. 2015, 23, 3907–3912. [CrossRef][PubMed] 45. Ballesteros, J.A.; Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 1995, 25, 366–428. 46. Burger, W.A.C.; Sexton, P.M.; Christopoulos, A.; Thal, D.M. Toward an understanding of the structural basis of allostery in muscarinic acetylcholine receptors. J. Gen. Physiol. 2018, 150, 1360–1372. [CrossRef][PubMed] 47. Laugwitz, K.L.; Allgeier, A.; Offermanns, S.; Spicher, K.; Van Sande, J.; Dumont, J.E.; Schultz, G. The human thyrotropin receptor: A heptahelical receptor capable of stimulating members of all four G protein families. Proc. Natl. Acad. Sci. USA 1996, 93, 116–120. [CrossRef] 48. Hermans, E. Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors. Pharmacol. Ther. 2003, 99, 25–44. [CrossRef] 49. Jakubik, J.; El-Fakahany, E.E.; Doležal, V. Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors. Mol. Pharmacol. 2006, 70, 656–666. [CrossRef][PubMed] 50. Kenakin, T.; Christopoulos, A. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 2013, 12, 205–216. [CrossRef] 51. Marlo, J.E.; Niswender, C.M.; Days, E.L.; Bridges, T.M.; Xiang, Y.; Rodriguez, A.L.; Shirey, J.K.; Brady, A.E.; Nalywajko, T.; Luo, Q.; et al. Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity. Mol. Pharmacol. 2009, 75, 577–588. [CrossRef] 52. Van der Westhuizen, E.T.; Spathis, A.; Khajehali, E.; Jörg, M.; Mistry, S.N.; Capuano, B.; Tobin, A.B.; Sexton, P.M.; Scammells, P.J.; Valant, C.; et al. Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors. Mol. Pharmacol. 2018, 94, 770–783. [CrossRef] 53. Wacker, D.; Stevens, R.C.; Roth, B.L. How Ligands Illuminate GPCR Molecular Pharmacology. Cell 2017, 170, 414–427. [CrossRef] 54. Bermudez, M.; Bock, A. Does Divergent Binding Pocket Closure Drive Ligand Bias for Class A GPCRs? Trends Pharmacol. Sci. 2019, 40, 236–239. [CrossRef][PubMed] 55. Jakubik, J.; El-Fakahany, E.E.; Tucek, S. Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors. J. Biol. Chem. 2000, 275, 18836–18844. [CrossRef][PubMed] 56. Järv, J.; Hedlund, B.; Bartfai, T. Isomerization of the muscarinic receptor. antagonist complex. J. Biol. Chem. 1979, 254, 5595–5598. [PubMed] 57. Järv, J.; Hedlund, B.; Bartfai, T. Kinetic studies on -agonist competition. J. Biol. Chem. 1980, 255, 2649–2651. 58. Redka, D.S.; Pisterzi, L.F.; Wells, J.W. Binding of orthosteric ligands to the allosteric site of the M(2) muscarinic cholinergic receptor. Mol. Pharmacol. 2008, 74, 834–843. [CrossRef] 59. Jakubík, J.; Randáková, A.; Zimˇcík, P.; El-Fakahany, E.E.; Doležal, V. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors. Sci. Rep. 2017, 7, 40381. [CrossRef] Biomolecules 2020, 10, 325 14 of 16

60. Kistemaker, L.E.M.; Elzinga, C.R.S.; Tautermann, C.S.; Pieper, M.P.; Seeliger, D.; Alikhil, S.; Schmidt, M.; Meurs, H.; Gosens, R. Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium. Br. J. Pharmacol. 2019, 176, 2864–2876. [CrossRef] 61. Jakubík, J.; Zimˇcík, P.; Randáková, A.; Fuksová, K.; El-Fakahany, E.E.; Doležal, V. Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors. Mol. Pharmacol. 2014, 86, 180–192. [CrossRef] 62. Bock, A.; Bermudez, M.; Krebs, F.; Matera, C.; Chirinda, B.; Sydow, D.; Dallanoce, C.; Holzgrabe, U.; De Amici, M.; Lohse, M.J.; et al. Ligand binding ensembles determine graded agonist efficacies at a G protein-coupled receptor. J. Biol. Chem. 2016, 291, 16375–16389. [CrossRef] 63. Schmitz, J.; Van Der Mey, D.; Bermudez, M.; Klöckner, J.; Schrage, R.; Kostenis, E.; Tränkle, C.; Wolber, G.; Mohr, K.; Holzgrabe, U. Dualsteric muscarinic antagonists-orthosteric binding pose controls allosteric subtype selectivity. J. Med. Chem. 2014, 57, 6739–6750. [CrossRef] 64. Avlani, V.A.; Langmead, C.J.; Guida, E.; Wood, M.D.; Tehan, B.G.; Herdon, H.J.; Watson, J.M.; Sexton, P.M.; Christopoulos, A. Orthosteric and allosteric modes of interaction of novel selective agonists of the M1 muscarinic acetylcholine receptor. Mol. Pharmacol. 2010, 78, 94–104. [CrossRef][PubMed] 65. Keov, P.; Valant, C.; Devine, S.M.; Lane, J.R.; Scammells, P.J.; Sexton, P.M.; Christopoulos, A. Reverse

engineering of the selective agonist TBPB unveils both orthosteric and allosteric modes of action at the M1 muscarinic acetylcholine receptor. Mol. Pharmacol 2013, 84, 425–437. [CrossRef][PubMed] 66. Gregory, K.J.; Hall, N.E.; Tobin, A.B.; Sexton, P.M.; Christopoulos, A. Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias. J. Biol. Chem. 2010, 285, 7459–7474. [CrossRef][PubMed] 67. Valant, C.; Gregory, K.J.; Hall, N.E.; Scammells, P.J.; Lew, M.J.; Sexton, P.M.; Christopoulos, A. A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic McN-A-343 as a bitopic orthosteric/allosteric ligand. J. Biol Chem 2008, 283, 29312–29321. [CrossRef] 68. Agnetta, L.; Kauk, M.; Canizal, M.C.A.; Messerer, R.; Holzgrabe, U.; Hoffmann, C.; Decker, M. A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy. Angew. Chem. Int. Ed. 2017, 56, 7282–7287. [CrossRef] 69. Matera, C.; Flammini, L.; Quadri, M.; Vivo, V.; Ballabeni, V.; Holzgrabe, U.; Mohr, K.; De Amici, M.; Barocelli, E.; Bertoni, S.; et al. Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity. Eur. J. Med. Chem. 2014, 75, 222–232. [CrossRef] 70. Cristofaro, I.; Spinello, Z.; Matera, C.; Fiore, M.; Conti, L.; De Amici, M.; Dallanoce, C.; Tata, A.M. Activation of M2 muscarinic acetylcholine receptors by a hybrid agonist enhances cytotoxic effects in GB7 glioblastoma cancer stem cells. Neurochem. Int. 2018, 118, 52–60. [CrossRef] 71. Jakubík, J.; Bacáková, L.; El-Fakahany, E.E.; Tucek, S. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol. Pharmacol. 1997, 52, 172–179. [CrossRef] 72. Birdsall, N.J.; Farries, T.; Gharagozloo, P.; Kobayashi, S.; Kuonen, D.; Lazareno, S.; Popham, A.; Sugimoto, M. Selective allosteric enhancement of the binding and actions of acetylcholine at muscarinic receptor subtypes. Life Sci. 1997, 60, 1047–1052. [CrossRef] 73. Bridges, T.M.; Marlo, J.E.; Niswender, C.M.; Jones, C.K.; Jadhav, S.B.; Gentry, P.R.; Plumley, H.C.; Weaver, C.D.; Conn, P.J.; Lindsley, C.W. Discovery of the first highly M5-preferring muscarinic acetylcholine receptor ligand, an M5 positive allosteric modulator derived from a series of 5-trifluoromethoxy N-benzyl isatins. J. Med. Chem. 2009, 52, 3445–3448. [CrossRef] 74. Kuduk, S.D.; Chang, R.K.; Di Marco, C.N.; Pitts, D.R.; Greshock, T.J.; Ma, L.; Wittmann, M.; Seager, M.A.; Koeplinger, K.A.; Thompson, C.D.; et al. Discovery of a selective allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold. J. Med. Chem. 2011, 54, 4773–4780. [CrossRef] 75. Davie, B.J.; Valant, C.; White, J.M.; Sexton, P.M.; Capuano, B.; Christopoulos, A.; Scammells, P.J. Synthesis and pharmacological evaluation of analogues of benzyl quinolone carboxylic acid (BQCA) designed to

bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor. J. Med. Chem. 2014, 57, 5405–5418. [CrossRef][PubMed] Biomolecules 2020, 10, 325 15 of 16

76. Pancani, T.; Foster, D.J.; Moehle, M.S.; Bichell, T.J.; Bradley, E.; Bridges, T.M.; Klar, R.; Poslusney, M.; Rook, J.M.; Daniels, J.S.; et al. Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice. Proc. Natl. Acad. Sci. USA 2015, 112, 14078–14083. [CrossRef] [PubMed] 77. Dallagnol, J.C.C.; Khajehali, E.; Van Der Westhuizen, E.T.; Jörg, M.; Valant, C.; Gonçalves, A.G.; Capuano, B.; Christopoulos, A.; Scammells, P.J. Synthesis and Pharmacological Evaluation of Heterocyclic Carboxamides: Positive Allosteric Modulators of the M1Muscarinic Acetylcholine Receptor with Weak Agonist Activity and Diverse Modulatory Profiles. J. Med. Chem. 2018, 61, 2875–2894. [CrossRef][PubMed] 78. Mistry, S.N.; Jörg, M.; Lim, H.; Vinh, N.B.; Sexton, P.M.; Capuano, B.; Christopoulos, A.; Lane, J.R.; Scammells, P.J. 4-Phenylpyridin-2-one Derivatives: A Novel Class of Positive Allosteric Modulator of the M1 Muscarinic Acetylcholine Receptor. J. Med. Chem. 2016, 59, 388–409. [CrossRef][PubMed] 79. Miao, Y.; Goldfeld, D.A.; VonMoo, E.; Sexton, P.M.;Christopoulos, A.; McCammon, J.A.; Valant, C. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor. Proc. Natl. Acad. Sci. USA 2016, 113, E5675–E5684. [CrossRef][PubMed] 80. Korczynska, M.; Clark, M.J.; Valant, C.; Xu, J.; Von Moo, E.; Albold, S.; Weiss, D.R.; Torosyan, H.; Huang, W.; Kruse, A.C.; et al. Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 2018, 115, E2419–E2428. [CrossRef] [PubMed] 81. Goldberg, J.A.; Ding, J.B.; Surmeier, D.J. Muscarinic modulation of striatal function and circuitry. Handb. Exp. Pharmacol. 2012, 208, 223–241. 82. Choy, K.H.C.; Shackleford, D.M.; Malone, D.T.; Mistry, S.N.; Patil, R.T.; Scammells, P.J.; Langmead, C.J.; Pantelis, C.; Sexton, P.M.; Lane, J.R.; et al. Positive Allosteric Modulation of the Muscarinic M1 Receptor Improves Efficacy of Antipsychotics in Mouse Glutamatergic Deficit Models of Behavior. J. Pharmacol. Exp. Ther. 2016, 359, 354–365. [CrossRef] 83. Nickols, H.H.; Conn, J.P. Development of allosteric modulators of GPCRs for treatment of CNS disorders. Neurobiol. Dis. 2014, 61, 55–71. [CrossRef] 84. Hopper, S.; Pavey, G.M.; Gogos, A.; Dean, B. Widespread Changes in Positive Allosteric Modulation of the Muscarinic M1 Receptor in Some Participants With Schizophrenia. Int. J. Neuropsychopharmacol. 2019, 22, 640–650. [CrossRef][PubMed] 85. Tzavara, E.T.; Bymaster, F.P.; Davis, R.J.; Wade, M.R.; Perry, K.W.; Wess, J.; McKinzie, D.L.; Felder, C.; Nomikos, G.G. M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies. FASEB J. 2004, 18, 1410–1412. [CrossRef][PubMed] 86. Brady, A.E.; Jones, C.K.; Bridges, T.M.; Kennedy, J.P.; Thompson, A.D.; Heiman, J.U.; Breininger, M.L.; Gentry, P.R.; Yin, H.; Jadhav, S.B.; et al. Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats. J. Pharmacol. Exp. Ther. 2008, 327, 941–953. [CrossRef][PubMed] 87. Yeomans, J.S. Muscarinic receptors in brain stem and mesopontine cholinergic arousal functions. Handb. Exp. Pharmacol. 2012, 208, 243–259. 88. Gentry, P.R.; Kokubo, M.; Bridges, T.M.; Kett, N.R.; Harp, J.M.; Cho, H.P.; Smith, E.; Chase, P.; Hodder, P.S.; Niswender, C.M.; et al. Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo [2,1-a]isoindol-5(9bH)-one (ML375). J. Med. Chem. 2013, 56, 9351–9355. [CrossRef] 89. Gunter, B.W.; Gould, R.W.; Bubser, M.; McGowan, K.M.; Lindsley, C.W.; Jones, C.K. Selective inhibition of M5 muscarinic acetylcholine receptors attenuates cocaine self-administration in rats. Addict. Biol. 2018, 23, 1106–1116. [CrossRef] 90. Gilon, P.; Henquin, J.C. Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr. Rev. 2001, 22, 565–604. Biomolecules 2020, 10, 325 16 of 16

91. Duttaroy, A.; Zimliki, C.L.; Gautam, D.; Cui, Y.; Mears, D.; Wess, J. Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in M3 muscarinic acetylcholine receptor-deficient mice. Diabetes 2004, 53, 1714–1720. [CrossRef] 92. Zhu, L.; Rossi, M.; Cohen, A.; Pham, J.; Zheng, H.; Dattaroy, D.; Mukaibo, T.; Melvin, J.E.; Langel, J.L.; Hattar, S.; et al. Allosteric modulation of β-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice. Proc. Natl. Acad. Sci. USA 2019, 116, 18684–18690. [CrossRef]

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