Designing Hybrids Targeting the Cholinergic System by Modulating the Muscarinic and Nicotinic Receptors: a Concept to Treat Alzheimer’S Disease

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Review Designing Hybrids Targeting the Cholinergic System by Modulating the Muscarinic and Nicotinic Receptors: A Concept to Treat Alzheimer’s Disease

Daniela Volpato and Ulrike Holzgrabe *

Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Am Hubland, 97074 Würzburg, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-931-31-85460

Academic Editors: Diego Muñoz-Torrero and Michael Decker Received: 22 November 2018; Accepted: 5 December 2018; Published: 7 December 2018

Abstract: The cholinergic hypothesis has been reported ﬁrst being the cause of memory dysfunction in the Alzheimer’s disease. Researchers around the globe have focused their attention on understanding the mechanisms of how this complicated system contributes to processes such as learning, memory, disorientation, linguistic problems, and behavioral issues in the indicated chronic neurodegenerative disease. The present review reports recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer’s disease (AD) and the study of their therapeutic relevance. The rationale and the design of the bifunctional compounds will be discussed in order to understand their potential as tools to investigate the role of the cholinergic system in AD.

Keywords: AChE inhibitor; Alzheimer’s disease; bitopic ligand; cholinergic system; hybrid molecules; muscarinic receptors; nicotinic receptors

1. Introduction Alzheimer’s disease (AD) is the most common form of dementia among geriatric people since the beginning of the 21st century. In 2016, over 47.5 million people around the world suffered from dementia. The World Health Organization hypothesizes that the number may rise to 75.6 million by 2030. The clinical condition of AD is characterized by a progressive loss of memory and other intellectual abilities up to serious levels where the daily life is severely affected [1]. Of note, no definitive cause and no known cure have been found yet. The characteristic features of the neurodegenerative decline include a decrease in levels of acetylcholine (ACh) followed by dysfunction and eventual death of cholinergic neurons. Furthermore, soluble β-amyloid oligomers (sAβ), β-amyloid fibrils (fAβ) misfolding and aggregation as well as formation of tau protein tangles in the nervous tissue of brain occur as a result of tau-protein hyperphosphorylation [1–3]. Water-soluble Aβ aggregates, detected in human brain lysates from AD patients, were found to be more toxic than their insoluble fibrillar counterparts by Li et al. [4]. Several other co-factors were hypothesized, such as oxidative stress and heavy metal dyshomeostasis [5]. Etiologic evidence exists between AD, cholinergic, and glutamatergic neurochemical systems. ACh is a crucial neurotransmitter responsible for mental and learning abilities. In AD patients, a dramatic decrease in cholinergic innervation in the cortex and the hippocampus is related to the loss of neurons in the basal forebrain [6]. Presynaptic cholinergic dysfunctions, loss of the cholinergic neural network, and overactivation of acetylcholinesterase (AChE), which leads to weakened neurotransmission, support the cholinergic hypothesis of the disease. This assumption

Molecules 2018, 23, 3230; doi:10.3390/molecules23123230 www.mdpi.com/journal/molecules Molecules 2018, 23, 3230 2 of 28 Molecules 2018, 23, x FOR PEER REVIEW 2 of 29 suggestscholinergic that restoration cholinergic therapy restoration could therapy be useful could for be alleviating useful for alleviatingthe cognitive the symptoms cognitive symptoms [7]. Another [7]. Anotherneurochemical neurochemical hypothesis hypothesis is mediated is mediatedby N-methyl- by ND-methyl--aspartateD -aspartate(NMDA). Glutamate (NMDA). Glutamate is an excitatory is an excitatoryneurotransmitter neurotransmitter for NMDA for receptors NMDA receptors which are which essential are essential in cognitive in cognitive processes. processes. However, However, in inaddition addition to tothe the physiological physiological stimulation, stimulation, glutamate glutamate receptors receptors cause cause neuronal neuronal damage damage due due to excitotoxicity under certaincertain circumstances [[8].8]. The conventional pharmacotherapy of AD make makess use of compounds which inhibit the enzyme AChE (donepezil,(donepezil, rivastigmine,rivastigmine, and and galantamine) galantamine) in in order order to to increase increase the the levels levels of acetylcholineof acetylcholine in thein nervousthe nervous tissue tissue of the of brain the ((1–3),brain ((1–3), Figure 1Figure). However, 1). However, these compounds these compounds can improve can theimprove cognitive the deﬁcitscognitive of thedeficits disease of the only disease for a couple only for ofmonths a couple before of months losing activity.before losing The shortness activity. ofThe the shortness effect could of bethe due effect to could a decrease be due of theto a AChEdecrease itself of inthe the AChE relevant itself regions in the relevant of the brain regions [9]. Laterof the inbrain time, [9]. another Later drugin time, was another introduced drug was for the introduc treatmented for of the AD, treatment i.e., memantine of AD, i.e. ((4), memantine Figure1) being ((4), Figure a glutamatergic 1) being a antagonistglutamatergic which antagonist protects nervewhich tissue protects against nerve glutamate-mediated tissue against glutamate-mediated excitotoxicity. Unfortunately, excitotoxicity. both classesUnfortunately, of drugs both only provideclasses of a symptomaticdrugs only prov reliefide without a symptomatic preventing relief the progression without preventing of the disease, the aprogression fact which of is notthe surprisingdisease, a fact given which the intricacyis not surp ofrising the pathology. given the Thus,intricacy there of isthe a desperatepathology. need Thus, to ﬁndthere selective is a desperate and powerful need to drugsfind selective to improve and thepowerful treatment drugs of ADto improve [1]. the treatment of AD [1].

Figure 1. StructuresStructures of donepezil, rivastigmine, galantamine and memantine.

So far, developingdeveloping new leading compounds with a lower risk-to-benefit risk-to-benefit ratio where benefits benefits clearly overcomeovercome the the side side effects effects has nothas beennot accomplishedbeen accomplished yet due yet to thedue complexity to the complexity and incomplete and understandingincomplete understanding of the pathophysiological of the pathophysiological processes in processes AD. Progress in AD. through Progress clinical through trials clinical is limited trials by pooris limited selectivity by poor of candidate selectivity molecules of candidate anda molecules lack of knowledge and a lack of theof exactknowledge role of of proteins the exact considered role of asproteins crucial considered targets. To as combat crucial this targets. complex To combat multifactorial this complex disease, multifactorial the challenge disease, is to discover the challenge which andis to howdiscover many which biological and how targets many have biological to be modulated targets have by developingto be modulated powerful by developing and selective powerful drugs. Hence,and selective the idea drugs. of creating Hence, hybrid the idea molecules of creating containing hybrid molecules two discrete containing recognition two unitsdiscrete linked recognition through aunits spacer, linked resulting through in thea spacer, so-called resulting hybridization in the approach,so-called hybridization is followed. Aapproach, supplemental is followed. molecular A segmentsupplemental next tomolecular the first standardsegment pharmacophorenext to the first couldstandard serve pharmacophore to address different could molecularserve to address targets ordifferent distinct molecular domains oftargets the same or dist protein.inct domains In addition of the to traditional same protein. methods, In addition various to modern traditional drug discoverymethods, approachesvarious modern must bedrug investigated discovery in approaches parallel and must complementarily be investigated to bring in newparallel hopes and in thiscomplementarily therapeutic field. to bring The resultingnew hopes hybrid in this molecules therapeutic can field. serve The as tools resulting for opening hybrid new molecules interesting can perspectivesserve as tools infor medicinal opening new chemistry interesting with perspect more promisingives in medicinal therapeutic chemistry opportunities with more to promising fight AD (fortherapeutic discussions opportunities on the further to fight potential AD (for of discussions the multifunctional on the further approach potential see ref. of [ 10the]). multifunctional approach see ref. 10). [10]

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2. The2. The Cholinergic Cholinergic Hypothesis Hypothesis of of Memory Memory DysfunctionDysfunction TheThe substantial substantial loss loss of presynapticof presynaptic cholinergic choliner neuronsgic neurons in human in human postmortem postmortem Alzheimer’s-diseased Alzheimer’s- brains,diseased the dysfunction brains, the ofdysfunction cholinergic of markers cholinergic such ma as ACh,rkers choline,such as andACh, choline choline, acetyl and transferase choline acetyl (ChAT, antransferase enzyme responsible (ChAT, an for enzyme ACh production)responsible asfor well ACh as production) the consequent as well loss as of the cognitive consequent skills loss and of the mentalcognitive improvement skills and observed the mental in patients improvement upon artificial observ restorationed in patients of cholinergic upon artificial activity restoration unequivocally of confirmcholinergic the validity activity of theunequivocally cholinergic confirm hypothesis the in validity AD [2]. of Acetylcholine the cholinergic receptors hypothesis (AChRs) in AD propagate [2]. theAcetylcholine cognitive ability receptors and consist (AChRs) of propagate two primary the memberscognitive ability namely and muscarinic consist of (mAChRs)two primary and members nicotinic receptorsnamely (nAChRs).muscarinic While(mAChRs) mAChRs and nicotinic are G-protein receptors coupled (nAChRs). receptors While (GPCRs), mAChRs nAChRs are G-protein are ligand coupled receptors (GPCRs), nAChRs are ligand gated ion channels. After release, ACh is hydrolyzed gated ion channels. After release, ACh is hydrolyzed to choline and acetate in the synaptic cleft by two to choline and acetate in the synaptic cleft by two cholinesterases, i.e. the AChE and the cholinesterases, i.e., the AChE and the butyrylcholinesterase (BChE) (see Figure2). Over the course of butyrylcholinesterase (BChE) (see Figure 2). Over the course of the disease, the concentration of the disease, the concentration of AChE decreases by 85%, whereas high levels of BChE were found to AChE decreases by 85%, whereas high levels of BChE were found to influence the aggregation of Aβ, influence the aggregation of Aβ, the main component of the amyloid plaques found in the brains of the main component of the amyloid plaques found in the brains of Alzheimer patients [11,12]. Alzheimer patients [11,12]. Furthermore, synaptic dysfunction and neuronal cell death in vitro [13–16] as Furthermore, synaptic dysfunction and neuronal cell death in vitro [13–16] as well as impaired wellbehavioral as impaired performance behavioral in performanceanimal models in [17–19] animal seem models to be [17 directly–19] seem correlated to be directly with sA correlatedβ aggregates with sA[20].β aggregates For this reason, [20]. For mAChRs, this reason, nAChRs, mAChRs, as well nAChRs, as the ashydrolases well as the AChE hydrolases and BChE AChE are and important BChE are importanttargets in targets the treatment in the treatment of AD [21–23]. of AD [ 21–23].

FigureFigure 2. 2.In In the the red red circles circles some some hallmarkshallmarks ofof Alzheimer’sAlzheimer's disease disease are are reported, reported, the the cholinergic cholinergic hypothesishypothesis is is emphasized emphasized through through thethe schematicschematic re representationpresentation of of a apre pre and and postsynaptic postsynaptic neuron. neuron. ZoomZoom in onin on the the cholinergic cholinergic synapse synapse in particularin particular biosynthesis, biosynthesis, storage, storage, release, release, hydrolysis hydrolysis of of ACh ACh and AChRand locationsAChR locations are illustrated are illustrated (modiﬁed (modified from from Auld Auld et al. et [24 al.]). [24]).

3. Hybrid3. Hybrid Compounds Compounds as as Drug Drug Discovery Discovery ToolsTools inin ADAD DueDue to to the the absolute absolute relevancerelevance of of the the cholinergic cholinergic system system in AD in AD it may it may be desirable be desirable to address to address the theinvolved involved cholinergic cholinergic receptors receptors and and enzymes enzymes simultaneously simultaneously by bymeans means of ofa multi-target a multi-target strategy strategy and/orand/or selectively selectively through through a dualsterica dualsteric approach. approach. This This can becan realized be realized by addressing by addressing different different binding pocketsbinding of pockets the same of protein’sthe same protein' surfaces withsurface one with hybrid one hybrid molecule molecule or different or different proteins proteins involved involved in the samein the pathology. same pathology. “Bivalent “Bivalent ligand” andligand” “hybrid” and “hybrid” are the general are the termsgeneral deﬁning terms defining compounds compounds which are composedwhich are of composed two functional of two pharmacophores functional pharmacophores linked by alinked spacer by [25 a spacer]. [25].

3.1.3.1. One One Single Single Molecule—Different Molecule—Different Targets Targets BecauseBecause of of the the multifactorial multifactorial dimensiondimension of AD and and the the difficulty difﬁculty to to apply apply the the classical classical “one “one target—onetarget—one disease” disease” concept concept [ 26[26],], the the multitargetmultitarget compounds approach approach has has often often been been considered considered in recent years [27]. In this review, only hybrids intended to activate the cholinergic system will be in recent years [27]. In this review, only hybrids intended to activate the cholinergic system will described. The advantages of the multipotent strategy are obvious, but it remains absolutely be described. The advantages of the multipotent strategy are obvious, but it remains absolutely

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Molecules 2018, 23, x FOR PEER REVIEW 4 of 29 challenging. In fact, molecular hybridization involves integration of the structure-activity relationships challenging. In fact, molecular hybridization involves integration of the structure-activity of two individual chemical species into a single molecular entity which should result in a balance of relationships of two individual chemical species into a single molecular entity which should result potency, efﬁcacy, and selectivity for all the targets. The new hybrids can be designed with different in a balance of potency, efficacy, and selectivity for all the targets. The new hybrids can be designed degrees of pharmacophore overlapping, with linking or merging techniques, and using distinct with different degrees of pharmacophore overlapping, with linking or merging techniques, and connectionusing distinct positions connection of the activepositions moieties. of the Furthermore,active moieties. the Furthermore, combination the of combination several pharmacophoric of several unitspharmacophoric usually results units in large usually molecules results whichin large have molecules limits inwhich terms have of molecular limits in terms weight, of lipophilicity,molecular permeabilityweight, lipophilicity, of membranes, permeability and hydrogen of membranes, bonds acceptance and hydrogen and donation bonds acceptance [27]. The techniqueand donation lends itself[27]. well The to technique address the lends cholinergic itself well system to address in AD the by cholinergic concomitant system inhibition in AD of by AChE concomitant and direct muscarinicinhibition and of AChE nicotinic and activation. direct muscarinic and nicotinic activation.

3.2.3.2. One One Single Single Molecule—Different Molecule—Different Binding Binding Pockets Pockets ofof aa Protein’sProtein's SurfaceSurface AsAs already already mentioned, mentioned, the the hybridization hybridization strategy strategy can can be be used used to to obtain obtain molecules molecules containing containing two pharmacophorictwo pharmacophoric entities entities able to able bind to topographically bind topographically different different binding binding sites of sites the of same the targetsame target protein, e.g.,protein, the endogenous e.g. the endogenous orthosteric orthosteric site and the site allosteric and the one. allosteric Allosterism, one. Allosterism, first defined first to defined describe to the modificationdescribe the of modification any enzyme of activityany enzyme by the activity binding by the of binding ligands of to ligands sites that to sites were that different were different from the substrate-bindingfrom the substrate-binding site itself [ 28site], itself is widely [28], is explored widely explored for GPCRs, for GPCRs, representing representing the largest the largest class of class drug targetsof drug in the targets human in the genome human [25 genome]. It provides [25]. It theprov greatides opportunitythe great opportunity of selectively of selectively affecting affecting one GPCR subtypeone GPCR and simultaneouslysubtype and simultaneous avoiding thely avoiding problem the of problem targeting of the targ structurallyeting the structurally conserved conserved orthosteric bindingorthosteric site [29 binding]. Novel site drugs [29]. targeting Novel drugs GPCRs targetin do notg GPCRs enter the do marketnot enter as frequentthe market as as expected frequent due as to unwantedexpected off-target due to unwanted effects resulting off-target from effects the lackresulting of selectivity from the withinlack of aselectivity distinct receptor within a family distinct [30 ]. receptor family [30]. 3.2.1. Allosteric Modulation 3.2.1. Allosteric Modulation GPCRs constitute a superfamily of integral membrane proteins characterized by seven transmembraneGPCRs constitute domains beinga superfamily connected of by integral three extracellular membrane (EL)proteins and intracellularcharacterized (IL) by loops seven [31 ]. Allosterictransmembrane modulators domains do not being bind connected to the orthostericby three extr bindingacellular site(EL) but and act intracellular at a distinct (IL) bindingloops [31]. site Allosteric modulators do not bind to the orthosteric binding site but act at a distinct binding site (i.e., (i.e., an allosteric site) to either potentiate or inhibit activation of the receptor by its endogenous an allosteric site) to either potentiate or inhibit activation of the receptor by its endogenous ligand ligand [32]. An innovative perspective in drug development takes advantage of allosteric binding sites [32]. An innovative perspective in drug development takes advantage of allosteric binding sites which usually altered between the different receptor subtype throughout the evolutionary processes. which usually altered between the different receptor subtype throughout the evolutionary processes. Conformational changes of the receptor upon allosteric ligand binding can modify the activity of Conformational changes of the receptor upon allosteric ligand binding can modify the activity of an anorthosteric agent agent in in terms terms of of the the binding affinity affinity and/or and/or the the downstream downstream efficacy. efficacy. This This means means emphasizingemphasizing signals signals to to some some pathways pathways over over othersothers amongamong the various various possible possible combinations combinations which which areare associated associated with with receptor receptor activation, activation, a a phenomenon phenomenon knownknown as bias signaling signaling (cf. (cf. Figure Figure 3)3 )[[33,34].33,34].

FigureFigure 3. 3.Schematic Schematic ofof GPCRGPCR signalsignal transduction:transduction: a allostericllosteric modulators modulators (spheres) (spheres) bind bind to toa a topographicallytopographically different different site site (yellow (yellow sphere) sphere) than than the the orthosteric orthosteric site site (yellow (yellow triangle) triangle) which which hosts hosts an orthosterican orthosteric ligand ligand (triangles). (triangles). Reproduced Reproduced with with permission permission from from [35 [35].].

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Besides subtype selectivity, allosteric modulators offer the advantage of saturable effects. Since most of them lack agonistic activity, they can perfectly “fine-tune” any orthosteric ligand activity to a ceiling level above which no further modulation occurs; this means they can be administered with higher doses and lower propensity to toxicity compared to classic agonists or antagonists [25]. Since the discovery of allosterism, several theories were postulated in order to identify and quantify this phenomenon: Ehlert’s ternary complex model is one of the most significant methods describing how two distinct ligands, orthosteric and allosteric, may influence the binding and functioning of each other [36]. The cooperativity level, indicated by the cooperativity factor α, distinguishes between positive and negative allosteric modulators (PAMs and NAMs) and neutral allosteric ligands (NALs). It is important to keep in mind that the obtained cooperativity is always referred to a single allosteric modulator for a specific orthosteric probe and that an accurate interpretation of a combination of functional and binding assays is necessary to understand the actual kind of modulation. Probe dependency and cooperativity reveal a new level of subtype selectivity rather than affinity, a phenomenon which was termed “absolute subtype selectivity”. For example, the binding of ACh is selectively enhanced at the M4 muscarinic receptor by thiochrome binding to the allosteric site, even though this allosteric modulator shows almost equal affinity for all muscarinic subtypes [37]. A particular beneficial therapeutic meaning may be associated with allosteric agonist compounds which differ from pure allosteric modulators. They are able to directly activate a receptor by binding to an allosteric binding site even in the absence of an orthosteric ligand. These allosteric agonists might be more suitable for the treatment of AD, either because of the reduced endogenous tone of neurotransmitters or even their complete lack [38].

3.2.2. Bitopic Ligand Principle The structural diversity among the extracellular vestibules allowed an innovative medicinal chemistry strategy for the design of novel ligands, i.e., hybrid compounds. They are characterized by chemical fragments which allow an interaction with both orthosteric and allosteric recognition sites. Merging the best of both allosteric and orthosteric worlds in one ligand launches the bitopic or dualsteric compounds [39,40], a strategy derived from the “message-address” concept by Schwyzer [41] which was first applied to GPCRs by Portoghese et al. [42]. This concept explicitly underlines the orthosteric/allosteric combination, in opposite to the more general umbrella term bivalent [25]. The orthosteric interaction provides high affinity binding and activation/inhibition of a receptor (message), whereas the allosteric interaction yields receptor subtype-selectivity (address) and may modulate both efficacy and intracellular signaling pathway activation [43]. In fact, the transduction pathway of GPCRs include a large set of intracellular signaling proteins like different G proteins, GRKs (protein coupled receptor kinase), and arrestins [44]. Such hybrid molecules would allow to selectively identify therapeutically relevant signaling pathways, attenuate harmful signals, or selectively block the ability of the agonist to produce unselective signals resulting in side effects [45]. The recent functional screening of biased ligands using multiple assay formats suggest that the phenomenon represents an existing and relevant therapeutic opportunity for the treatment of different disorders [43]. The dualsteric principle was validated through numerous bitopic ligands examples: Iper-6-phth ((5a-C6), Figure4) and iper-6-naph (( 5b-C6), Figure4) are derived from the M2 allosteric hexamethonium compounds W84 and naphmethonium (NAM regarding orthosteric agonists) linked with the non-selective orthosteric superagonist iperoxo. The dualsteric compounds possess relatively good selectivity for the M2 receptor regarding potency and affinity. Agonistic activation and unexpected selectivity in the intracellular G protein pathway were observed: activation of Gi signaling at the expense of the Gs. Molecules 2018, 23, 3230 6 of 28

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O O Br - - X X Br N Ar Ar N N N N N X n n X - O O Br Br - O O n =n 6-10 = 6-10 N N O O HybridHybrid 1 5a-C6 1 5a-C6 HybridHybrid 2 5b-C6 2 5b-C6

Ar Ar= = Ar Ar= =

a groupa group b groupb group X=H X=CH X=H X=CH3 3 FigureFigureFigure 4. 4.Structures 4.StructuresStructures of ofbitopic ofbitopic bitopic hybrids. hybrids. hybrids. 5a-C6, 5a-C6,5a-C6, 5b-C6 5b-C6 5b-C6 M2 M2 muscarinicM2 muscarinic muscarinic hybrid. hybrid. hybrid. Green: Green: Green: orthosteric orthosteric orthosteric buildingbuildingbuilding block, block, block, red: red: allosteric red: allosteric allosteric building building building block. block. block.

StartingStartingStarting from from from the the thestructure structure structure of ofofthe thethe M2 M2M2 preferring preferring preferring antagonist antagonist DIBADIBA DIBA ((((6 6),((), Figure6 Figure), Figure5), 5), the 5), the homo-dimeric the homo- homo- dimericdimericdibenzodiazepinone dibenzodiazepinone dibenzodiazepinone derivatives derivatives derivatives UNSW-MK250 UNSW-MK250 UNSW-MK250 ((7), Figure (( 7((),57 )),Figure and Figure UNSW-MK262 5) 5)and and UNSW-MK262 UNSW-MK262 (( 8), Figure 5(() as8((), well8 ), FigureFigureas the5) 5)as heterodimeric aswell well as asthe the UR-SK75 heterodimeric heterodimeric ((9), Figure UR-SK75 UR-SK755) compounds (( 9((),9 ),Figure Figure were 5) prepared. 5)compounds compounds All moleculeswere were prepared. prepared. exhibited All All high moleculesmoleculesM2 receptor exhibited exhibited afﬁnities high high M2 and M2 recept werereceptor capable oraffinities affinities of and retarding and were were capable [3H]NMS capable of ofretarding dissociation, retarding [3H]NMS [3H]NMS indicating dissociation, dissociation, an allosteric indicatingindicatinginteraction an an allosteric [ 46allosteric]. Saturation interaction interaction binding [46]. [46]. experiments Saturation Saturation in binding the binding presence experiments experiments of M2 allosteric in inthe the modulatorspresence presence of and ofM2 M2 some allostericallostericradiolabeled modulators modulators dibenzodiazepinone and and some some radiolabeled radiolabeled compounds dibenzodiazepinone highlighteddibenzodiazepinone a competitive compounds compounds mechanism highlighted highlighted suggesting a a a competitivecompetitivedualsteric mechanism bindingmechanism mode suggesting suggesting [47,48]. a dualsteric a dualsteric binding binding mode mode [47,48]. [47,48].

N N R R

DIBADIBA (6) (6)

R R R= N N R= N O O R N R HNHN

UNSW-MK250UNSW-MK250 (7) (7) N N O N R O H N R H N O O N N N O N N O H H N N N H H O N N O N N N H H R R N N O O UNSW-MK262UNSW-MK262 (8) (8)

S N N S N N N N O O N N R R N UR-SK75 (9) N UR-SK75 (9) FigureFigureFigure 5. Structures5. Structures 5. Structures of homo-heteroof ofhomo-hetero homo-hetero dimeric dimeric dimeric dibenzodiazepinone dibenzodiazepinone dibenzodiazepinone M2 M2 derivatives. M2 derivatives. derivatives.

ExtendingExtendingExtending the the approach the approach approach to toother other to GPCRs otherGPCRs includes GPCRs includes the includes the adenosine adenosine the A1 adenosine A1 receptor receptor with A1 with receptor the the dualsteric dualsteric with the ligandsligandsdualsteric LUF6258, LUF6258, ligands VCP746 VCP746 LUF6258, and and VCP171 VCP171 VCP746 (( 10(( and,10 11, ),11 VCP171 Figure), Figure 6; (( 106;VCP171,, VCP171,11), Figure structure structure6; VCP171, not not disclosed). disclosed). structure An An not orthostericorthostericdisclosed). adenosine adenosine An orthostericagonist agonist was was adenosine connected connected agonistthrough through wasan an alkyl connectedalkyl chain chain linker throughlinker to toa PAM ana PAM alkyl of ofthe chainthe PD81,723 PD81,723 linker to type.type. The The resulting resulting bitopic bitopic ligands ligands were were evaluat evaluateded pharmacologically pharmacologically in inradioligand radioligand displacement displacement studiesstudies suggesting suggesting a dualsterica dualsteric receptor receptor interact interactionion of ofLUF6258 LUF6258 [49]. [49]. VCP746 VCP746 was was subsequently subsequently

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Moleculesa PAM 2018, of23, thex FOR PD81,723 PEER REVIEW type. The resulting bitopic ligands were evaluated pharmacologically7 of 29 in radioligand displacement studies suggesting a dualsteric receptor interaction of LUF6258 [49]. developedMolecules 2018by, 23, Valantx FOR PEER et REVIEW al. [50] by inclusion of the PAM (2-amino-4-(2-7 of 29 VCP746 was subsequently developed by Valant et al. [50] by inclusion of the PAM (2-amino-4-(2- (trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)-methanone) in the dualsteric structure. The bitopic (triﬂuoromethyl)phenyl)thiophen-3-yl)(phenyl)-methanone)developed by Valant et al. [50] by inclusion in theof dualsteric the PAM structure. (2-amino-4-(2- The bitopic ligand 11 was found to induce significant biased cellular signaling in favor of cytoprotection and to ligand(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)-methanone)11 was found to induce signiﬁcant biased cellular signaling in the in dualsteric favor of cytoprotectionstructure. The andbitopic to the detriment of bradycardia side effects. the detriment of bradycardia side effects. ligand 11 was found to induce significant biasedR cellular signaling in favor of cytoprotection and to the detriment of bradycardia side effects. HN N N R HN

HO N NN N O HO N N HO OHO

HO OH n=6 HN O n=6 HN

n=8O N S Cl n=8 CF3 N Cl O Cl S H2N S CF3 Cl H N O O S 2

LUF6258 (10)O VCP746 (11)

Figure 6. Structures of bitopic ligands 10LUF6258, 11 at (10)A1 adenosineVCP746 receptor.(11) Green: orthosteric building block, red: allosteric building block. FigureFigure 6. 6.Structures Structures of of bitopic bitopic ligands ligands10 10, ,11 11at at A1 A1 adenosine adenosine receptor. receptor. Green: Green: orthosteric orthosteric building building block,block, red: red: allosteric allosteric building building block. block. The compound THR-160209 ((12), Figure 7) was obtained by Steinfeld et al. [51] by connection of an orthostericTheThe compound compound 3-benzylhydrylpyrrolinyl THR-160209 THR-160209 (( 12((12),), Figure buildingFigure7) was7) block was obtained obtainedto an allosteric by by Steinfeld Steinfeld 4-aminobenzylpiperidine et al. et [al.51 [51]] by connectionby connection of an orthosteric 3-benzylhydrylpyrrolinyl building block to an allosteric 4-aminobenzylpiperidine motif motifof using an orthosteric a heptane 3-benzylhydrylpyrrolinyl chain. A significant increase building in affinity block was to an noted allosteric for the 4-aminobenzylpiperidine bitopic ligand (12), using a heptane chain. A signiﬁcant increase in afﬁnity was noted for the bitopic ligand (12), exhibiting exhibiting a certain preference for the M2 AChR compared with its orthosteric or allosteric fragments amotif certain using preference a heptane for thechain. M2 A AChR significant compared increase with in its affinity orthosteric was ornoted allosteric for the fragments bitopic ligand [25,31 ].(12), [25,31].exhibiting a certain preference for the M2 AChR compared with its orthosteric or allosteric fragments [25,31].

Figure 7. Figure 7. StructuresStructures of bitopic of bitopic hybrids. hybrids. 12 M2 12 M2acetylcholine acetylcholine receptor receptor antagonist. antagonist. Green: Green: orthosteric orthosteric buildingbuilding block, block, red: red:allosteric allosteric building building block. block. Figure 7. Structures of bitopic hybrids. 12 M2 acetylcholine receptor antagonist. Green: orthosteric TheThe buildingdopamine dopamine block, D2-like D2-likered: allosteric receptor receptor building is is objectobject block. ofof research research as drugas drug target target for neuropsychiatric for neuropsychiatric disorders. disorders.The bitopic The modebitopic of actionmode withof action D2 and with D3 receptorsD2 and wasD3 demonstratedreceptors was fordemonstrated the dopaminergic for the ligand The dopamine D2-like receptor is object of research as drug target for neuropsychiatric dopaminergicSB269652 ((ligand13), Figure SB2696528). Only ((13), a Figure few years 8). Only after a its few publication years after by its Stemppublication et al. by [ 52 Stemp,53], SB269652 et al. disorders. The bitopic mode of action with D2 and D3 receptors was demonstrated for the [52,53],was SB269652 described was as andescribed atypical as bitopic an atypical ligand. bitopic It behaves ligand. like It behaves a competitive like a competitive antagonist antagonist with receptor dopaminergic ligand SB269652 ((13), Figure 8). Only a few years after its publication by Stemp et al. withmonomers receptor monomers and allosterically and allosterically across receptor across dimersreceptor by dimers changing by changing the ability the of ability ligands of ligands to bind the [52,53], SB269652 was described as an atypical bitopic ligand. It behaves like a competitive antagonist to bindorthosteric the orthosteric binding binding pocket onpocket the other on the protomer other protomer of the dimer of the [54 dimer,55]. [54,55]. with receptor monomers and allosterically across receptor dimers by changing the ability of ligands A new and potent bitopic sphingosine-1-phosphate subtype 3 receptor (S1P3) antagonist, SPM- to bind the orthosteric binding pocket on the other protomer of the dimer [54,55]. 354 ((15), Figure 8), with in vivo activity was a useful tool to define the spatial and functional A new and potent bitopic sphingosine-1-phosphate subtype 3 receptor (S1P3) antagonist, SPM- properties of S1P3 in regulating cardiac conduction. SPM-354 can compete with both the allosteric 354 ((15), Figure 8), with in vivo activity was a useful tool to define the spatial and functional S1P3 selective agonist CYM-5541 as well as the orthosteric agonist S1P, showing bitopic properties. properties of S1P3 in regulating cardiac conduction. SPM-354 can compete with both the allosteric SPM-354, a 2-propyl derivative of the previously described SPM242, was chosen for in vivo studies S1P3 selective agonist CYM-5541 as well as the orthosteric agonist S1P, showing bitopic properties. ((14) Figure 8) because of its improved potency and in vivo efficacy [56,57]. SPM-354, a 2-propyl derivative of the previously described SPM242, was chosen for in vivo studies

((14) Figure 8) because of its improved potency and in vivo efficacy [56,57].

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A new and potent bitopic sphingosine-1-phosphate subtype 3 receptor (S1P3) antagonist, SPM-354 ((15), Figure8), with in vivo activity was a useful tool to deﬁne the spatial and functional properties of S1P3 in regulating cardiac conduction. SPM-354 can compete with both the allosteric S1P3 selective agonist CYM-5541 as well as the orthosteric agonist S1P, showing bitopic properties. SPM-354, a 2-propyl derivative of the previously described SPM242, was chosen for in vivo studies ((14) Figure8) becauseMolecules of2018 its, 23 improved, x FOR PEER potency REVIEW and in vivo efﬁcacy [56,57]. 8 of 29

Figure 8. Structures of bitopic hybrids. 13 D3 dopaminergic ligand SB269652; 14, 15 bitopic S1P3 Figure 8. Structures of bitopic hybrids. 13 D3 dopaminergic ligand SB269652; 14, 15 bitopic S1P3 antagonist. Orthosteric building block in green allosteric building block in red. antagonist. Orthosteric building block in green allosteric building block in red. All these examples illustrate that this principle is applicable across the GPCR landscape. Additionally,All these thanks examples to the illustrate progress that in understandingthis principle is dualsteric applicable ligands, across severalthe GPCR ligands landscape which. wereAdditionally, thought to thanks be allosteric to the progress modulators in understanding were re-classified dualsteric later as ligands, dualsteric several ligands ligands (e.g., which 77-LH-28-1 were andthought AC-42 to atbe M1allosteric receptor modulators described were below re-classified see par. 4.1.4). later as Hence, dualsteric one canligands be optimistic (e.g. 77-LH-28-1 that in and the future,AC-42 moreat M1 dualstericreceptor described ligands atbelow different see par. GPCRs 4.1.4). will Hence, be discovered one can be and optimistic designed that using in the rational future, structure-basedmore dualsteric drugligands discovery. at different GPCRs will be discovered and designed using rational structure- based drug discovery. 3.2.3. Hybrid Strategies Design 3.2.3. Hybrid Strategies Design Both orthosteric and allosteric fragments and the linker play an important role in the interaction with theBoth target orthosteric protein, and thus allosteric an optimization fragments and process the linker considering play an theseimportant moieties role isin indispensable.the interaction Bothwith pharmacophoresthe target protein, have thus to an be enhancedoptimization separately process in considering a kind of fragment-based these moieties designis indispensable. approach, investigatingBoth pharmacophores the chemical have environment to be enhanced of the separately two binding in a pockets.kind of fragment-based However, one shoulddesign rememberapproach, thatinvestigating the binding the of chemical one pharmacophore environment isof influencingthe two binding the binding pockets. of However, the other one moiety. should For remember example, itthat is possiblethe binding that of upon one pharmacophore binding of the ligandis influenc theing receptor the binding goes throughof the other conformational moiety. For example, changes causingit is possible negative that cooperativity upon binding for of the the complementary ligand the receptor moiety. goes Therefore, through it isconformational difficult to predict changes the pharmacologicalcausing negative outcome cooperativity of such for bitopic the complementary molecules. Furthermore, moiety. Therefore, the linker mustit is difficult fit perfectly, to predict allowing the thepharmacological allosteric and orthostericoutcome of fragments such bitopic to reach molecules. their target Furthermore, pockets. Length,the linker flexibility, must fit connection perfectly, position,allowing and the chemicalallosteric characteristics and orthosteric of thefragments linker play to reach a fundamental their target role pockets. in the bitopicLength, interaction. flexibility, connectionBitopic position, ligands range and chemical from the characteristics first combination of the of linker agonist-NAM play a fundamental (e.g., iperoxo-6-phth) role in the bitopic [58] to agonist-PAMinteraction. (e.g., iperoxo-BQCAd) [49,50,59,60] and antagonist-NAM (e.g., atr-6-naph) [61] for the M2 and M1Bitopic muscarinic ligands receptors. range from All the possible first combination combinations of easily agonist-NAM widen the (e.g. spectrum iperoxo-6-phth) of opportunities. [58] to agonist-PAMAlternative (e.g. approaches iperoxo-BQCAd) may also [49,50,59,60] include the an used antagonist-NAM of bitopic ligands (e.g. to atr-6-naph) study the low-affinity [61] for the andM2 transientand M1 bindingmuscarinic sites whichreceptors. are definedAll possible as metastable combinations sites [62 easily]. Several widen research the groupsspectrum have of hypothesizedopportunities. the existence of such a binding site at the entrance of the receptor to which ligands bind transientlyAlternative [63–68 approaches]. If an energetically may also favorableinclude the conformation use of bitopic change ligands of to the study complex the low-affinity ligand-receptor and occurs,transient the binding ligand sites continues which its are path defined down as to metastable the orthosteric sites site[62]. whereas Several otherwise,research groups its route have is preventedhypothesized through the existence dissociation. of such This a binding metastable site siteat the acts entrance as a selectivity of the receptor filter: to access which is ligands denied tobind molecules transiently that [63–68]. do not If have an energetically the required favorable physico-chemical conformation characteristics change of forthe thecomplex binding ligand- site. receptor occurs, the ligand continues its path down to the orthosteric site whereas otherwise, its route is prevented through dissociation. This metastable site acts as a selectivity filter: access is denied to molecules that do not have the required physico-chemical characteristics for the binding site. Metastable binding sites offer new receptor sections that can be explored, opening new strategic opportunities in the field of bitopic ligands. Since several GPCRs are organized in dimeric or oligomeric complexes, respectively, bivalent ligands intended to target an allosteric and orthosteric binding sites or even two orthosteric sites of two different protomers in a receptor dimer. This will constitute a new design prospective. Rather

Molecules 2018, 23, 3230 9 of 28

Metastable binding sites offer new receptor sections that can be explored, opening new strategic opportunities in the field of bitopic ligands. Since several GPCRs are organized in dimeric or oligomeric complexes, respectively, bivalent ligands intended to target an allosteric and orthosteric binding sites or even two orthosteric sites of two different protomers in a receptor dimer. This will constitute a new design prospective. Rather than promoting a ligand-induced dimerization, the bivalent ligands tend to stabilize the pre-existing dimers. Unique pharmacological properties including receptor selectivity could be achieved as bivalent ligands are able to bridge different receptor units. Heterodimers can be highly selective compounds targeting only those tissues in which both receptors are co-expressed. Apart from the opioid receptor field, the potential of these ligands has been poorly explored up to now [69]. The bitopic approach used for the first time in 2006 by De Amici et al. [70] on the M2 muscarinic receptor was recently extended in the research field of AD on M1 receptor.

4. Muscarinic Receptor The muscarinic receptors are involved in diverse functions throughout the body with particular focus on the bladder, gastrointestinal tract, eye, heart, brain, and salivary glands [71]. Physiologically, these functions are activated and deactivated selectively thanks to the specific tissue distribution of receptors and the existence of five different receptor subtypes (M1–M5). The M1-type muscarinic AChRs, predominantly expressed in the hippocampus and cerebral cortex, play a central role in cognitive processing, memory, and learning. Since structure and density of these AChRs remain almost unchanged in the disease state, the M1 receptor is considered a very promising target to increase the cholinergic tone [72]. Highly subtype selective drugs are needed in order to avoid side effects originating from unwanted receptor activation or inhibition [38]. A number of M1 muscarinic agonists was developed to treat AD. These are promising agents as they display significant neurotrophic effect, decrease β-amyloid plaque deposition, and improve oxidative stress-induced damage. Several compounds have shown an efficacy on the M1 as a therapeutic target for the AD as they not only enhance the cholinergic functions but can also shift the APP (amyloid precursor protein) processing towards the nonamyloidogenic pathway through activation of protein kinase C, the metalloproteinase domain 17 (ADAM17), and a disintegrin [3]. Activation of M1 muscarinic receptors attenuates tau hyperphosphorylation via glycogen synthase kinase-3b (GSK-3b) inhibition, reduces β amyloid peptide levels, and increases ERK activation and potentiation of NMDA receptors [38,73,74]. Considerable efforts were made to obtain agonists for M1 but their off-target effects preclude the therapeutic use. Various agonists for M1 muscarinic receptor including arecoline ((16), Figure9) CI-1017 ((17), Figure9), xanomeline (( 18), Figure9), tazomeline (( 19), Figure9), talsaclidine (( 20), Figure9) and milameline (( 21), Figure9) do not show sufficient selectivity for the M1 subtype. The most prominent candidates in the past few years were the M1 muscarinic agonists AF102B ((22), Figure9) (cevimeline; approved in the USA and Japan for Sjogren’s Syndrome and having excellent pharmacokinetic properties), AF150(S) ((23), Figure9) and AF267B (( 24), Figure9) which additionally show a significant neurotrophic effect, inhibit Aβ and oxidative stress induced cell death as well as apoptosis in PC12 cells transfected with the M1 muscarinic receptor [5,75]. Molecules 20182018,, 2323,, 3230x FOR PEER REVIEW 1010 of of 29 28 Molecules 2018, 23, x FOR PEER REVIEW 10 of 29

Figure 9. Structures of moderately active and selective orthosteric M1 agonists. Figure 9. StructuresStructures of moderately active an andd selective orthosteric M1 agonists. Furthermore, the M1 selective agonist lead candidates VU0364572 ((25), Figure 10) and VU0357017Furthermore, ((26), theFigurethe M1 M1 selective10) selective were agonist tested agonist lead on candidatesleadcell linescandidates as VU0364572 well VU0364572 as ((animal25), Figure models((25 ),10 )Figure andand VU0357017 exhibited10) and ((VU0357017encouraging26), Figure ((10 as26) werewell), Figure as tested effective 10) on were cell properties lines tested as wellon on cellvarious as animallines parameters. as models well andas Theanimal exhibited compound models encouraging VU0357017and exhibited as wellnot asencouragingonly effective showed properties asexcellent well as onM1 effective various subtype properties parameters. selectivity on Thebut various also compound a parameters. good VU0357017blood Thebrain compound notbarrier only (BBB) showed VU0357017 penetration excellent not M1onlyand subtype inshowed vivo selectivityefficacy excellent [38]. butM1 EVP-6124 alsosubtype a good selectivity (enceniclin) blood brain but (( also barrier27), aFigure good (BBB) blood10) penetration was brain the barrierlast and cholinergicin (BBB) vivo efﬁcacypenetration receptor [38]. EVP-6124andagonist in vivowhich (enceniclin) efficacy entered [38]. (( phase27 ),EVP-6124 Figure 3 trials 10 (enceniclin) )but was it the was last wi((27 cholinergicthdrawn), Figure due receptor10) towas severe the agonist lastgastrointestinal whichcholinergic entered receptoradverse phase 3agonisteffects trials [3]. butwhich it was entered withdrawn phase due3 trials to severe but it gastrointestinal was withdrawn adverse due to effects severe [ 3gastrointestinal]. adverse effects [3].

Figure 10. Structures of M1 lead candidates. Figure 10. Structures of M1 lead candidates. Some highly M1-selective allosteric agonists must also be mentioned here which do not require an orthosterorthosterSome highly bound M1-selective to thethe receptorreceptor allosteric forfor activation.agonistsactivation. must For For also example, example, be mentioned the the TBPB TBPB here allosteric allosteric which do agonist agonist not require (( ((2828),), Figurean orthoster 1111)) interacts bound with to the slightly receptor different for activati bindingon. domains For example, of the M1 the receptor TBPB allosteric which are agonist all adjacent ((28), toFigureto the orthosteric 11) interacts site. with LuAE51090 slightly different (( 29), Figure binding 11)11) displayeddomains of a thehigh M1 de degree receptorgree of selectivity which are whenall adjacent tested intoin the aa broadbroad orthosteric panelpanel site. ofof GPCRs,GPCRs, LuAE51090 ionion channels,channels,((29), Figure transporters,transporters, 11) displayed and a high enzymes, degree and of selectivity showed an when acceptable tested pharmacokineticinpharmacokinetic a broad panel proﬁleprofileof GPCRs, and BBBion channels, penetration transporters, properties inand vivo enzymes, [[76].76]. Compound and showed 30 (Figure an acceptable 11)11) was identiﬁedpharmacokineticidentified byby highhigh profile throughputthroughput and BBB screeningscreening penetration (HTS) (HTS) properties and and virtual virtual in screening screening vivo [76]. at at Compound GlaxoSmithKline, GlaxoSmithKline, 30 (Figure unraveling unraveling 11) was a moleculeidentifieda molecule with by with high improved improvedthroughput binding binding screening and pharmacokineticand (HTS) pharmacokinetic and virtual parameters. screening parameters. For at GlaxoSmithKline,30 ,For good 30, M1 good agonist M1 unraveling potency,agonist intrinsicapotency, molecule activity,intrinsic with and activity,improved subtype and binding selectivity subtype and selectivity for pharmacokinetic M1 are for reported M1 are [parameters.reported77,78]. [77,78]. For 30, good M1 agonist potency, intrinsic activity, and subtype selectivity for M1 are reported [77,78].

Molecules 2018, 23, x FOR PEER REVIEW 11 of 29 Molecules 2018, 23, 3230 11 of 28

Molecules 2018, 23, x FOR PEER REVIEW 11 of 29

FigureFigure 11. 11. StructuresStructures Structures of of M1 allosteric allosteric agonists. agonists.

Both thetheBoth comparisoncomparison the comparison of of the theof allosteric the allosteric allosteric agonists agonists agonists among amongamong each each each other other other and and even andeven with evenwith M1-PAMs M1-PAMswith M1-PAMs reveals reveals structural similarities. They all consist of an heterobicycle system with at least one nitrogen structural similarities. They all consist of an heterobicycle system with at least one nitrogen atom and reveals atomstructural and a similarities.carbonyl group They close all to consist the nitrogen of an. heterobicycleFurthermore, the system heterocycle with isat connectedleast one tonitrogen aatom carbonyl andpiperidine groupa carbonyl rings close which group to the are nitrogen.close linked to to anotherthe Furthermore, nitrogen cyclic moiety.. theFurthermore, heterocycle the is connectedheterocycle to piperidineis connected rings to whichpiperidine are linked rings which to another are linked cyclic moiety.to another cyclic moiety. 4.1. Design of Hybrid Compounds to Investigate the Muscarinic Receptor 4.1. Design of Hybrid Compounds to Investigate the Muscarinic Receptor 4.1. Design ofThe Hybrid allosterically Compounds less conserved to Investigate site of thethe mAChR Muscarinic which Receptor is located in the extracellular vestibule (ECV) and separated from the orthosteric pocket by a tyrosine lid [79] lends itself well as binding site The allosterically less conserved site of the mAChR which is located located in in the the extracellular extracellular vestibule vestibule (ECV) andfor new separated dualsteric from ligands. the orthosteric The close proximity pocket byof the a tyrosine orthosteric lid and [79 ]the lends allosteric itself subdomains, well as binding a site (ECV) andtypical separated feature of from the Classthe orthosteric A GPCRs, allows pocket the by dualsteric a tyrosine compounds lid [79] lendsto simultaneously itself well asinteract binding site for newwith dualstericdualsteric the two binding ligands. sites. The The close close proximity of of the orthosteric and the allosteric subdomains, a a typical featurefeatureThe of ofchemical the the Class Class space A A GPCRs,between GPCRs, allowsthe allows orthosteric the the dualsteric dualsteric and allosteric compounds compounds portions to of simultaneously tothe simultaneously M2 receptor interact was interact with thewith two theinvestigated binding two binding sites. thanks sites. to the pharmacological analysis of Iper-8-naph ((5b-C8), Figure 4) [80,81], McN- TheA-343 chemical ((31), Figure space 12), between and its fragments the orthosteric and analogues and [82].allosteric portions of of the M2 receptor was investigated thanks thanks to to the the pharmacological pharmacological analysis analysis of Iper-8-naph of Iper-8-naph ((5b-C8 ((5b-C8), Figure), Figure 4) [80,81],4)[ 80McN-,81], McN-A-343A-343 ((31), Figure ((31), Figure 12), and 12), its and fragments its fragments and analogues and analogues [82]. [82].

Figure 12. Structures of McN-A-343.

The long-known partial agonist McN-A-343 interacts with the allosteric EL2 vestibule through its 3-chlorphenylcarbamate. The binding to the orthosteric sites is guaranteed by the tetramethylammonium cation. The two parts are linked via a rigid butynyl chain. However, this Figure 12. Structures of McN-A-343. molecule is probably too small to entirely occupy the allosteric pocket and thereby achieves no subtype selectivity. It binds to both M2 and M4 receptors [31]. The long-known partial agonist McN-A-343 interacts with the allosteric EL2 vestibule The long-knownIper-8-naph partialis a partial agonist agonist McN-A-343 at the M2 receptor interacts. The partialwith theagonism allosteric of most EL2 of the vestibule dualsteric through throughits 3-chlorphenylcarbamate.agonists its 3-chlorphenylcarbamate. depends on both theThe dualsteric binding The binding binding to themode to orthostericitself the orthostericand on thesites dynamic sites is isguaranteed equilibrium guaranteed ofby by the the tetramethylammoniummultiple inactive and cation. cation. active The Thestates, two two i.e. parts parts the ligandare are lin linked bindingked via ensemble. a rigid In butynyl the “dualsteric chain. However, However,binding this moleculemode”, isis probablyprobably iperoxo too istoo smallbound small toto entirelyto the entirely orthosteric occupy occupy pocket the allostericthe while allosteric the pocket allosteric pocket and therebynaphthalimide and thereby achieves moiety achieves no subtype no selectivity.subtypeprotrudes selectivity. It binds towards toIt bothbinds the M2extrac to both andellular M4M2 part receptorsand of M4 the receptorsreceptor. [31]. This [31]. po se is responsible for a functionally selective activation of the G proteins and cellular signaling [81,83]. In the alternative pose, which is a Iper-8-naph is a partial agonist at the M2 receptor receptor.. The partial agonism of most most of of the the dualsteric dualsteric “purely allosteric binding mode”, the entire bitopic ligand (both the orthosteric and the allosteric agonists depends on both the dualsteric binding mode itself and on the dynamic equilibrium of agonistsmoieties) depends is inon the both allosteric the vestibule.dualsteric In thisbinding case, themode inactive itself receptor and onconformation the dynamic is stabilized. equilibrium It of multiplewould inactive be difficult and activeactiveto pharmacologically states,states, i.e.,i.e. the distinguish ligand the binding “flip-flop” ensemble.ensemble. mechanism In In fromthe the a“dualsteric ligand with abinding mode”, purely iperoxoiperoxo bitopic is is bound boundbinding to themodeto orthostericthe. In orthostericcase ofpocket the iperoxo-dualsteric pocket while thewhile allosteric derivativesthe allosteric naphthalimide at the naphthalimide M2R, moietythe bitopic protrudes moiety towardsprotrudesmode the towards extracellularof interaction the extrac was part validatedellular of the part receptor.by theof thefollowing Thisreceptor. poseexperiments: This is responsible po radioligandse is responsible for binding a functionally foranalysis, a functionally in selective activationselective activation of the G proteinsof the G andproteins cellular andsignaling cellular signaling [81,83]. In [81,83]. the alternative In the alternative pose, which pose, is which a “purely is a allosteric“purely allosteric binding mode”,binding themode”, entire the bitopic entire ligand bitopi (bothc ligand the (both orthosteric the orthosteric and the allosteric and the allosteric moieties) ismoieties) in the allosteric is in the allosteric vestibule. vestibule. In this case, In this the case, inactive the inactive receptor receptor conformation conformation is stabilized. is stabilized. It would It bewould difficult be difficult to pharmacologically to pharmacologically distinguish distinguish the “flip-flop” the “flip-flop” mechanism mechanism from a from ligand a ligand with a with purely a bitopicpurely bitopic binding binding mode. Inmode case. ofIn thecase iperoxo-dualsteric of the iperoxo-dualsteric derivatives derivatives at the M2R, at the the M2R, bitopic the mode bitopic of interactionmode of interaction was validated was byvalidated the following by the experiments: following experiments: radioligand bindingradioligand analysis, binding in wild-type analysis, and in

Molecules 2018, 23, 3230 12 of 28 mutant receptors, and downstream signaling assays in addition to receptor docking simulations [34]. Partial agonism is the direct consequence of this dynamic balance. Measuring the partial agonism depends on the afﬁnity of the two pharmacophores and of the dualsteric ligand itself for the respective binding sites [30].

4.1.1. BQCAd Hybrid Derivatives The same approach was applied for exploring the M1 receptor binding pockets. Among several selective M1 allosteric modulators known, the promising group of N-benzyl quinolone carboxylic acids (BQCAs) was selected as positive allosteric moieties of these dualsteric M1 hybrids [59,60]. The BQCA allosteric modulator ((32a), Figure 13) was ﬁrst described by the Merck laboratory. The compound is a positive allosteric modulator, an allosteric agonist, and exhibits pure subtype selectivity for the M1 receptor: no agonism, antagonism, nor potentiation activity was observed at other mAChRs up to 100 µM. The concentration of ACh which needed to activate the receptor decreased up to 129-fold in the presence of BQCA and an inﬂection point of 845 nM is reported in the presence of 3 nM ACh. Its allosteric modulation is mediated by the interaction with the residues Y179 and W400, suggesting the binding of BQCA to the "common" allosteric site [84,85]. As an orthosteric fragment, the non-selective superagonist iperoxo ((33), Figure 13)[59] and the endogenous ligand ACh were linked to the BQCA derivative (BQCAd ((32b), Figure 13)) [60]. The two parts were connected by alkylene chains of differentMolecules 2018 lengths, 23, x FOR (C4, PEER C6, C8,REVIEW C10) ((34a, b, c, d), Figure 13). 13 of 29

Figure 13. Structures of BQCAd/Iperoxo-AChBQCAd/Iperoxo-ACh hybrids. hybrids.

The compounds showed The compounds showed

1. subtype1. subtype selectivity: selectivity: receptor receptor activation activation and signaling and signaling pathway pathway studies performedstudies performed with (dynamic with mass redistribution) DMR in the presence and absence of specific Gi and Gq signaling blockers (dynamic mass redistribution) DMR in the presence and absence of specific Gi and Gq signaling indicate a preferentially Gq mediated signal of the hybrids. 2. partialblockers agonism indicate which a preferentially may provide Gq controlled mediated activation signal of withoutthe hybrids. overdriving the M1 signaling and2. withpartial possible agonism improved which sidemay effectprovide profile controlled over the activation control of cognitionwithout overdriving [86]. the M1 3. potencysignaling and and efficacy with possible dependency improved on the side length effect of profile the chain over andthe control on the of substitution cognition [86]. pattern of3. the potency allosteric and fragment. efficacy dependency Elongating on the the chain length from of C4the to chain C6 switches and on the the substitution receptor fractional pattern occupancy from the inactive, purely allosteric pose to the active binding pose which defines the of the allosteric fragment. Elongating the chain from C4 to C6 switches the receptor fractional efficacy of the dualsteric ligands [87]. occupancy from the inactive, purely allosteric pose to the active binding pose which defines Thethe interaction efficacy of ofthe these dualsteric compounds ligands with [87]. the M1 muscarinic receptor was further investigated by Fluorescence Resonance Energy Transfer (FRET) measurements in a living cell system for a closer understandingThe interaction of the of receptorthese compounds movement. with Here, the M1 some muscarinic other ligands receptor were was added further to investigated the hybrid by Fluorescence Resonance Energy Transfer (FRET) measurements in a living cell system for a closer understanding of the receptor movement. Here, some other ligands were added to the hybrid series including longer chained (C8, C10) compounds and ACh hybrids ((35a,b,c,d), Figure 13) [60]. The optimal linker length for the activation of the receptor was confirmed to be a C6 chain, e.g. in the case of iperoxo-C6-BQCA with a maximal detected signal of about 24% compared to 100 µM of the iperoxo reference. Surprisingly, a new receptor conformation was observed: a FRET signal with opposite direction to the typical agonist signal occurred for the longer chain hybrids. How this unknown receptor activation process at a molecular level is connected with the bitopic compound interaction mode, the different G-protein activation, β-arrestin recruitment, or further downstream signaling must be investigated to completely understand this unexpected M1 receptor arrangement. The distinct receptor movement that occurs upon binding of dualster ligands may help to illuminate the interplay between G proteins and β-arrestins preferentially coupling to different receptor conformations [88]. Particularly within the M1 pharmacology, functionally selective properties can be exploited to characterize which signaling outcomes would address neuropsychiatric illnesses [89]. Of note, replacing the flexible alkyl chain with a rigid alkynylbenzene linker ((36), Figure 14) leads to a delayed FRET signal, indicating slower receptor kinetics. The compound with a flexible chain of corresponding length afforded a sharper FRET signal, indicating the responsibility of the rigidification in the kinetic modification [60,90].

Molecules 2018, 23, 3230 13 of 28 series including longer chained (C8, C10) compounds and ACh hybrids ((35a,b,c,d), Figure 13)[60]. The optimal linker length for the activation of the receptor was confirmed to be a C6 chain, e.g., in the case of iperoxo-C6-BQCA with a maximal detected signal of about 24% compared to 100 µM of the iperoxo reference. Surprisingly, a new receptor conformation was observed: a FRET signal with opposite direction to the typical agonist signal occurred for the longer chain hybrids. How this unknown receptor activation process at a molecular level is connected with the bitopic compound interaction mode, the different G-protein activation, β-arrestin recruitment, or further downstream signaling must be investigated to completely understand this unexpected M1 receptor arrangement. The distinct receptor movement that occurs upon binding of dualster ligands may help to illuminate the interplay between G proteins and β-arrestins preferentially coupling to different receptor conformations [88]. Particularly within the M1 pharmacology, functionally selective properties can be exploited to characterize which signaling outcomes would address neuropsychiatric illnesses [89]. Of note, replacing the flexible alkyl chain with a rigid alkynylbenzene linker ((36), Figure 14) leads to a delayed FRET signal, indicating slower receptor kinetics. The compound with a flexible chain of Molecules 2018, 23, x FOR PEER REVIEW 14 of 29 corresponding length afforded a sharper FRET signal, indicating the responsibility of the rigidification in theMolecules kinetic 2018 modification, 23, x FOR PEER [REVIEW60,90]. 14 of 29

Figure 14. Structures of BQCAd/Iperoxo hybrids connected by a rigid linker and a photo-switchable Figuremoiety.Figure 14. Structures 14. Structures of BQCAd/Iperoxo of BQCAd/Iperoxo hybrids hybrids connected connected by aby rigid a rigid linker linker and and a photo-switchablea photo-switchable moiety. moiety. π 3.33 6.51 7.39 Probably,Probably, a cation-a cation-interactionπ interaction between between the the three three tyrosines tyrosines Y104 Y104,3.33 Y403, Y4036.51and and Y426 Y4267.39of of the the 3.33 6.51 7.39 so-calledso-calledProbably, tyrosine tyrosine a lidcation- lid with withπ the interaction the rigid rigid linker betweenlinker contributes contri the threebutes to tyrosines the to delayedthe delayed Y104 FRET , FRETY403 signal. signal. and (Remodeling Y426 (Remodelingof the of the of so-called tyrosine lid with the rigid linker contributes to the delayed FRET signal. (Remodeling of tyrosinethe tyrosine lid. in lid. Figure in Figure 15). 15). the tyrosine lid. in Figure 15).

FigureFigureFigure 15. 15.15.Left: Left: top toptop view viewview snapshots snapshotssnapshots fromfrom from a a molecular molecular dynamics dynamics dynamics simulation simulation simulation of of theof the theiperoxo-bound iperoxo-bound iperoxo-bound crystalcrystalcrystal structure structurestructure unveil unveilunveil the thethe conformational conformational conformational ﬂexibilityflexibility flexibility of ofthe the tyrosine tyrosine tyrosine lid, lid, lid, especially especially especially Tyr-426 Tyr-426 Tyr-4267.397.39. Right:7.39. Right:. Right: basedbasedbased on onon the the sampling sampling of ofof side side side chain chain chain conformations,conformations, conformations, the the tyrosine tyrosine tyrosine lid lid lid was was was re remodeledmodeled remodeled for for bindingfor binding binding mode mode mode investigationsinvestigationsinvestigations on on dualsteric dualstericdualsteric agonist. agonist. agonist. Reproduced Reproduced Reproduced withwith with permission permission from from from [83]. [83 [83].].

AA furtherfurther step in thethe studystudy of of M1 M1 activation activation processes processes was was performed performed with with the thesynthesis synthesis and and pharmacologicalpharmacological investigation of of the the first first new new photo-switchable photo-switchable dualsteric dualsteric ligand. ligand. Agnetta Agnetta et al. et [90] al. [90] designeddesigned aa pharmacologicalpharmacological tooltool in in which which the the phot photochromicochromic azobenzene azobenzene moie moiety wasty was inserted inserted as a as a spacer in the hybrid compound. Regarding the time course of M1 activation, compound 37 was found spacer in the hybrid compound. Regarding the time course of M1 activation, compound 37 was found to be as slow as with the rigid compound, underlining the fundamental role of the chain structure of to be as slow as with the rigid compound, underlining the fundamental role of the chain structure of the dualsteric hybrids in the M1 receptor kinetic. More interestingly, with the BQCAAI ((37), Figure the dualsteric hybrids in the M1 receptor kinetic. More interestingly, with the BQCAAI ((37), Figure 14) compound, the drastic influence of the iperoxo-BQCA hybrid geometry on the activity of the 14) compound, the drastic influence of the iperoxo-BQCA hybrid geometry on the activity of the compound was demonstrated. A simple light-induced isomerization from the cis to the less stable compoundtrans BQCAAI was isomer demonstrated. turns the antagonistA simple light-inducedcompound into isomerization an agonist. from the cis to the less stable trans BQCAAI isomer turns the antagonist compound into an agonist. 4.1.2. LY593093 4.1.2. LY593093 Another allo/ortho partial agonist hybrid was designed at Eli Lilly: LY593093 ((38), Figure 16). The Anothermolecule allo/orthodisplaced thepartial [3H] agonistNMS antagonist hybrid was from designed M1 muscarinic at Eli Lilly: receptor LY593093 (pKi of 6.21)((38), without Figure 16). Theany moleculeeffect on displaced [3H] NMS the binding [3H] NMS at theantagonist M3-M5AChR from M1 and muscarinic stimulating receptor β-arrestin (pK i recruitment.of 6.21) without anyAdditionally, effect on LY593093[3H] NMS had binding no modulatory at the M3-M5AChRrole in potentiating and stimulatingACh functional β-arrestin activity recruitment.and the Additionally,binding was notLY593093 affected had by anno increasingmodulatory conc roleentration in potentiating of the PAM ACh BQCA, functional indicating activity that andthe the binding was not affected by an increasing concentration of the PAM BQCA, indicating that the

Molecules 2018, 23, 3230 14 of 28

A further step in the study of M1 activation processes was performed with the synthesis and pharmacological investigation of the ﬁrst new photo-switchable dualsteric ligand. Agnetta et al. [90] designed a pharmacological tool in which the photochromic azobenzene moiety was inserted as a spacer in the hybrid compound. Regarding the time course of M1 activation, compound 37 was found to be as slow as with the rigid compound, underlining the fundamental role of the chain structure of the dualsteric hybrids in the M1 receptor kinetic. More interestingly, with the BQCAAI ((37), Figure 14) compound, the drastic inﬂuence of the iperoxo-BQCA hybrid geometry on the activity of the compound was demonstrated. A simple light-induced isomerization from the cis to the less stable trans BQCAAI isomer turns the antagonist compound into an agonist.

4.1.2. LY593093 Another allo/ortho partial agonist hybrid was designed at Eli Lilly: LY593093 ((38), Figure 16). 3 The molecule displaced the [ H] NMS antagonist from M1 muscarinic receptor (pKi of 6.21) without any effect on [3H] NMS binding at the M3-M5AChR and stimulating β-arrestin recruitment. Additionally, LY593093Molecules 2018 had, 23, nox FOR modulatory PEER REVIEW role in potentiating ACh functional activity and the binding was15 of not 29 affected by an increasing concentration of the PAM BQCA, indicating that the interaction between LY593093interaction and between M1AChR LY593093 is different and M1AChR from the is interaction different from of the the receptor interaction with of the the endogenous receptor with ligand the ACh.endogenous High subtypeligand ACh. selectivity High issubtype achieved selectivity through is multiple achieved points through of bindingmultiple pocket points interactions of binding whichpocket wasinteractions shown inwhich the dockingwas shown model in the [86 ].docking Binding model was [86] also. observedBinding was for also the M2observed receptor, for butthe inM2 terms receptor, of functional but in terms activity of functional the Eli Lilly activity compound the Eli Lilly is 5 co andmpound 120 times is 5 moreand 120 potent times on more M1 potent AChR thanon M1 on AChR other muscarinicthan on other receptor muscarinic subtypes receptor and shows subtypes an E maxandat shows least 2-foldan Emax higher at least [91 ].2- Progressionfold higher through[91]. Progression clinical trials through was clinical however trial interrupteds was however due tointerrupted its modest due brain to penetration.its modest brain penetration.

Figure 16. StructureStructure of LY593093 LY593093..

4.1.3.4.1.3. AC-42AC-42 and AC-260584AC-260584 The allostericallosteric andand orthostericorthosteric mode mode of of action action was was also also demonstrated demonstrated by by means means of of a combinationa combination of functionalof functional and and binding binding assays assays for thefor AC-42the AC compound-42 compound ((39), (( Figure39), Fig 17ure). The 17). compound The compound slows slows down 3 thedown dissociation the dissocia of [tionH]NMS of [3H] fromNMS the from M1 muscarinic the M1 muscarinic receptor, indicating receptor, its indicating allosteric its capabilities; allosteric moreover,capabilities; in moreover, equilibrium in equilibrium binding studies, binding AC-42 studies almost, AC-42 completely almost completely inhibits inhibits the binding the binding of the 3 [ofH]NMS the [3H]NMS orthosteric orthosteric probe probe and, inand, the in functional the functional test IP1,test IP1, the antagonistthe antagonist atropine atropine decreases decreases the potencythe potency of the of ligand the ligand in a concentration-dependent in a concentration-dependent manner. manner. Later findings Later indicated findings an indicated additional an orthostericadditional orthosteric binding pose binding of the pose ligand of [ the2,92 , ligand93]. AC-42, [2,92,93 unlike]. AC oxo-M,-42, unlike arecoline, oxo-M, and arecoline pilocarpine,, and is unable to promote M1-Gα protein coupling, proving the ability of bitopic ligands to activate pilocarpine, is unable to promotei1/2 M1-Gαi1/2 protein coupling, proving the ability of bitopic ligands to differentactivate different down streaming down streaming effectors effectors [94]. AC-260584 [94]. AC-260584 ((40), Figure((40), Figure 17), the 17), orally the orally bioavailable bioavailable and structurallyand structurally related related compound compound with greater with greater potency potency and efficacy and than efficacy AC-42, than was AC initially-42, was defined initiall asy andefined allosteric as an agonist. allosteric Mutation agonist of. Mutation amino acids of amino in the acids TM3 in of the the TM3 M1 receptor of the M1 (ACh receptor binding (ACh site) binding affects thesite) binding affects the of the binding compound of the andcompound hence supports and hence the supports hypothesis the of hypothesis the bitopic of behavior the bitopic [95 ].behavior [95]. 4.1.4. 77-LH-28-1

4.1.4.77-LH-28-1 77-LH-28-1 ((41), Figure 17), a close structural analogue of AC-42, was initially described as an allosteric agonist. However, it might have a bitopic binding mode because the antagonist 77-LH-28-1 ((41), Figure 17), a close structural analogue of AC-42, was initially described as an scopolamine is able to shift its dose-response curve rightward indicating an orthosteric binding mode. allosteric agonist. However, it might have a bitopic binding mode because the antagonist Further evidence supporting this theory is the competition at the NMS-occupied receptor with a scopolamine is able to shift its dose-response curve rightward indicating an orthosteric binding mode. Further evidence supporting this theory is the competition at the NMS-occupied receptor with a prototypical muscarinic negative allosteric bis-quaternary phthalimidopropyl modulator (heptane- 1,7-bis(dimethyl-3'-phthalimidopropylammonium bromide, abbreviated as C7/3-phth) [92]. 77-LH-28-1 was, however, used as an allosteric fragment of the bitopic hybrids synthesized by Piergentili et al. [89] The compounds containing xanomeline as orthosteric fragment were pharmacologically investigated as described below (See par. 5.1.4).

Molecules 2018, 23, 3230 15 of 28 prototypical muscarinic negative allosteric bis-quaternary phthalimidopropyl modulator (heptane- 1,7-bis(dimethyl-30-phthalimidopropylammonium bromide, abbreviated as C7/3-phth) [92]. 77-LH-28-1 was, however, used as an allosteric fragment of the bitopic hybrids synthesized by Piergentili et al. [89] The compounds containing xanomeline as orthosteric fragment were pharmacologicallyMolecules 2018,, 23,, xx FORFOR investigated PEERPEER REVIEWREVIEW as described below (See par. 5.1.4). 16 of 29

Figure 17. StructuresStructures of of AC-42, AC-42, AC-260584 AC-260584 and 77-LH-28-1.

5. Acetylcholinesterase (AChE) 5. Acetylcholinesterase (AChE) The decreasing cholinergic tone is one of the physiological reasons of the debilitating AD. To switch The decreasing cholinergic tone is one of the physiological reasons of the debilitating AD. To off a cholinergic signal, ACh is catabolized by the acetylcholinesterase (AChE) which hydrolyzes the switch off a cholinergic signal, ACh is catabolized by the acetylcholinesterase (AChE) which ester of the neurotransmitter to acetic acid and choline. The enzyme also appears to be involved in the hydrolyzes the ester of the neurotransmitter to aceticic acidacid andand choline.choline. TheThe enzymeenzyme alsoalso appearsappears toto aggregation of Aβ by a peripheral anionic site (PAS) in addition to its classical enzymatic hydrolysis be involved in the aggregation of Aβ byby aa peripheralperipheral anionicanionic sitesite (PAS)(PAS) inin additionaddition toto itsits classicalclassical of ACh [96,97]. AChE inhibitors compensate the cholinergic tone in the synaptic transmission and enzymatic hydrolysis of ACh [96,97]. AChE inhibitors compensate the cholinergic tone in the thus, because of an augmenting ACh concentration, usually relieve the symptoms and can decrease synaptic transmission and thus, because of an augmenting ACh concentration, usually relieve the the rate of cognitive decline for some months. However, they are not able to arrest disease progression symptoms and can decrease the rate of cognitive decline for some months. However, they are not and inexorable neurodegeneration [27]. Already in 1991, Sussman et al. described the enzyme as a able to arrest disease progression and inexorable neurodegeneration [27]. Already in 1991, Sussman narrowet al. described cavity that the enzyme drives the as a substrate narrow cavity through that a peripheraldrivesdrives thethe substratesubstrate anionic site throthro (PAS)ugh a to peripheral the catalytic anionic site (CAS),site (PAS) where to the the catalytic catalytic site triad (CAS), Glu-His-Ser where the is located catalyticlytic [98 triadtriad]. (Figure Glu-His-SerGlu-His-Ser 18). isis locatedlocated [98].[98]. (Figure(Figure 18).18).

FigureFigure 18.18. 3D3D structure structure of of native native torpedotorpedo californicacalifornica AChE.AChE. The The catalytic catalytic triad triad in in red,red, red, Trp84Trp84 Trp84 inin thethe the CAS,CAS, CAS, Trp279Trp279 atat thethe PAS,PAS, andand thethe bottleneckbottleneck residue Phe330 inin blue.blue. Reproduc Reproduceded with permission from [99]. [99].

TheThe knowledgeknowledge of of the the active active site site and and of of the the exact exac functiontt functionfunction ofeach ofof eacheach amino aminoamino acid acidacid of the ofofcatalytic thethe catalyticcatalytic triad allowedtriadtriad allowedallowed the discovery thethe discoverydiscovery of enzymatic ofof enzymaticenzymatic inhibitors. inhibitoinhibito The AChErs. The inhibitors AChE (AChEIs)inhibitors donepezil, (AChEIs) galantamine, donepezil, galantamine, rivastigmine ((1–3), Figure 1), and tacrine ((42), Figure 19) were successfully developed and approved by international authorities. Later, tacrinetacrine waswas withdrawnwithdrawn fromfrom thethe marketmarket duedue toto aa significant hepatic toxicity.

Molecules 2018, 23, 3230 16 of 28 Molecules 2018, 23, x FOR PEER REVIEW 17 of 29

rivastigmine ((1–3), Figure1), and tacrine (( 42), Figure 19) were successfully developed and approved by international authorities. Later, tacrine was withdrawn from the market due to a signiﬁcant hepaticMolecules toxicity.2018, 23, x FOR PEER REVIEW 17 of 29

Figure 19. Structure of tacrine.

Figure 19. Structure of tacrine. These compounds inhibit the enzymeFigure differently: 19. Structure ofseveral tacrine. ligands, e.g. galantamine, covalently bind to the serineThese residue compounds and inhibit form thethea bond enzymeenzyme that differently:differently: is stable severalseveral for about ligands,ligands, 15–20 e.g.,e.g. galantamine, min; during covalently this time, the enzyme is inhibited.bind to the serineTacrine residue blocks and formthe pass a bondage that of is the stable neurotransmitter for about 15–20 min; due during to athis strong time, theπ-stacking enzyme is inhibited. Tacrine blocks the passage of the neurotransmitter due to a strong π-stacking interaction enzymewith Trp84 is inhibited. (Figure Tacrine 20) [100].blocks the passage of the neurotransmitter due to a strong π-stacking interaction withwith Trp84Trp84 (Figure(Figure 2020))[ [100].100].

Figure 20. Overlay of the trigonal crystal structure of tacrine (black) and Tc AChE (gray) complex: showing the catalytic anionic site, aromatic gorge, and peripheral anionic site. Reproduced with permission from [100]. Figure 20. Overlay of the trigonal crystal structure of tacrine (black) and Tc AChE (gray) complex: Figure 20. Overlay of the trigonal crystal structure of tacrine (black) and Tc AChE (gray) complex: showing the catalytic anionic site, aromatic gorge, and peripheral anionic site. Reproduced with Rivastigmine is a pseudo-irreversible arylcarbamate inhibitor of both AChE and BChE. Clinical showing thepermission catalytic from anionic [100]. site, aromatic gorge, and peripheral anionic site. Reproduced with studies comparing the effect of the two approved drugs donepezil (only AChE inhibition) and permission from [100]. rivastigmineRivastigmine (AChE is aand pseudo-irreversible BChE inhibition) arylcarbamate revealed an inhibitoradditional of benefit both AChE in daily and BChE.living, Clinical global studiesfunction, comparing and behavior the effectfor rivastigmine of the two in approved patients drugswith wild-type donepezil BuChE (only AChEallele due inhibition) to the dual and Rivastigminerivastigmineinhibition is of a (AChE AChEpseudo-irreversible and and BChE BChE inhibition) [101–103]. arylcarbamate revealed Anyway an additional, the inhibitoruse of beneﬁt AChEIs of in both daily for living,AChEAD is global consideredand function, BChE. a Clinical studies comparingandsuboptimal behavior the therapy for effect rivastigmine and of is stillthe in undertwo patients discussionapprov with wild-typeed [104]. drugs This BuChE donepezilreview allele will due not(only to give the dual AChEdetails inhibition aboutinhibition) all of and rivastigmineAChEAChEIs (AChE and because BChE and they [ 101BChE –were103]. alreadyinhibition) Anyway, summarized the userevealed of AChEIsexcellently an for additional in AD ref. is 105–108 considered benefit [105–108]. a suboptimal in daily therapy living, global and is still under discussion [104]. This review will not give details about all AChEIs because they function, and5.1. Designbehavior of Hybrid for Compoundrivastigmines Inhibiting in patienthe Cholinesterasets with wild-type BuChE allele due to the dual were already summarized excellently in ref. [105–108]. inhibition of AChE and BChE [101–103]. Anyway, the use of AChEIs for AD is considered a suboptimal5.1.5.1.1. therapy Design Tacrine ofand HybridHybrids is Compoundsstill under Inhibiting discussion the Cholinesterase [104]. This review will not give details about all In order to investigate the potency of tacrine for the inhibition of AChE with simultaneously AChEIs because5.1.1. Tacrine they were Hybrids already summarized excellently in ref. 105–108 [105–108]. reduced hepatotoxicity, tacrine was intensively used for the development of hybrid molecules. Pang et al.In found order a to bis-C7-tacrine investigate the homodimer potency of (( tacrine43-C7), forFigure the inhibition21) exhibiting of AChE a 1000-fold with simultaneously higher AChE 5.1. Design of Hybrid Compounds Inhibiting the Cholinesterase reducedinhibitory hepatotoxicity, potency in rats tacrine than the was monomer, intensively indica usedting for the absence development of steric of hybridinteraction molecules. problems Pang in etthe al. narrow found cavity a bis-C7-tacrine preceding the homodimer catalytic site ((43-C7 of th),e enzyme. Figure 21 The) exhibiting tacrine dimers a 1000-fold are able higher to establish AChE 5.1.1. Tacrineinhibitoryinteractions Hybrids potency with both in rats the than catalytic the monomer, and the periph indicatingeral thesite. absence A shorter of steric tether interaction (<5 methylene problems units) in hampers the simultaneous binding to both interaction sites of AChE (dual-site binding). In addition, In orderthe tolipophilicity investigate of the the hybrid potency was found of tacrine to be increased for the compared inhibition to tacrine of AChE itself [109,110]. with simultaneously reduced hepatotoxicity, tacrine was intensively used for the development of hybrid molecules. Pang et al. found a bis-C7-tacrine homodimer ((43-C7), Figure 21) exhibiting a 1000-fold higher AChE inhibitory potency in rats than the monomer, indicating the absence of steric interaction problems in the narrow cavity preceding the catalytic site of the enzyme. The tacrine dimers are able to establish interactions with both the catalytic and the peripheral site. A shorter tether (<5 methylene units) hampers the simultaneous binding to both interaction sites of AChE (dual-site binding). In addition, the lipophilicity of the hybrid was found to be increased compared to tacrine itself [109,110].

Molecules 2018, 23, 3230 17 of 28 the narrow cavity preceding the catalytic site of the enzyme. The tacrine dimers are able to establish interactions with both the catalytic and the peripheral site. A shorter tether (<5 methylene units) hampers the simultaneous binding to both interaction sites of AChE (dual-site binding). In addition, theMolecules lipophilicity 2018,, 23,, xx ofFORFOR the PEERPEER hybrid REVIEWREVIEW was found to be increased compared to tacrine itself [109,110]. 18 of 29

Figure 21. Bis tacrine homodimers.

5.1.2. Tacrine/Iperoxo hybrids hybrids Some bitopic bitopic ligands ligands combining combining the the M2 M2 allosteric allosteric structure structure of the of theallosteric allosteric modulator modulator W84 W84and andnaphmethonium naphmethonium and the and non-selective the non-selective superagonist superagonist iperoxo, iperoxo, rationally rationally developed developed as M2 subtype- as M2 subtype-selectiveselective ligands (( ligands5a,b-C7,8,9,10 ((5a,b-),C7,8,9,10 Figure 4),), Figure were tested4), were for tested the inhibitory for the inhibitory activity of activitythe AChE of and the AChEfound andto have found modest to have activity. modest The activity. potency The is influe potencynced is by inﬂuenced the length by of the th lengthe linker of chain the linker connecting chain connectingthe orthosteric the and orthosteric allosteric and sites. allosteric The longest sites. chains The longest result in chains a higher result activity: in ahigher the decamethylene activity: the decamethylenechain seems to span chain the seems AChE to binding span the gorge, AChE allo bindingwing gorge,the quaternary allowing ammonium the quaternary head ammonium of iperoxo headto be localized of iperoxo in the to bePAS localized region. Compounds in the PAS region.containing Compounds the naphthalimide containing portion the (( naphthalimide5b-C7,8,9,10), portionFigure 4) (( show5b-C7,8,9,10 stronger), Figureinhibition4) show than strongerthose containing inhibition the than phthalimide those containing part ((5a-C7,8,9,10 the phthalimide), Figure part4). When ((5a-C7,8,9,10 replacing), Figurethe iperoxo4). When moiety replacing with an the is iperoxooxazole moietyring, almost with anno isoxazole AChE/BuChE ring, almostinhibition no AChE/BuChEwas observed (( inhibition44-C4,6,8), was Figure observed 22; Table ((44-C4,6,8 1) [80].), Figure 22; Table1)[80].

FigureFigure 22.22. AChE inhibitor/ mAch mAchRR agonist agonist compounds. compounds.

Table 1. Cholinesterase activity and cytotoxicity of the M2 bitopic compounds and tacrine hybrids. BChE from equine serum, AChE from electric eel. [110].

BChE AChE Cytotoxicity Compound pIC50 [M] pIC50 [M] HEP G2 IC50 [μM] Tacrine (42) 8.57 7.60 111 (±2.28) Phth-7-iper (5a-C7) 5.19 4.83 n.d. 11 Phth-8-iper (5a-C8) 5.13 4.89 n.d. 11 Phth-9-iper (5a-C9) 4.88 4.80 n.d. 11 Phth-10-iper (5a-C10) 5.37 5.26 n.d. 11 Naph-7-iper (5b-C7) 6.69 6.04 n.d. 11 Naph-8-iper (5b-C8) 6.46 6.10 n.d. 11 Naph-9-iper (5b-C9) 6.49 6.44 n.d. 11 Naph-10-iper (5b-C10) 6.99 6.50 n.d. 11 Tac-7-Tac (43-C7) 9.14 10.48 <1.38 (±0.08) Tac-10-Tac (43-C10) 9.34 9.00 <1.25 (±0.00)

Molecules 2018, 23, 3230 18 of 28

Table 1. Cholinesterase activity and cytotoxicity of the M2 bitopic compounds and tacrine hybrids. BChE from equine serum, AChE from electric eel. [110].

BChE AChE Cytotoxicity Compound pIC50 [M] pIC50 [M] HEP G2 IC50 [µM] Tacrine (42) 8.57 7.60 111 (±2.28) Phth-7-iper (5a-C7) 5.19 4.83 n.d. 1 Phth-8-iper (5a-C8) 5.13 4.89 n.d. 1 Phth-9-iper (5a-C9) 4.88 4.80 n.d. 1 Phth-10-iper (5a-C10) 5.37 5.26 n.d. 1 Naph-7-iper (5b-C7) 6.69 6.04 n.d. 1 Naph-8-iper (5b-C8) 6.46 6.10 n.d. 1 Naph-9-iper (5b-C9) 6.49 6.44 n.d. 1 Naph-10-iper (5b-C10) 6.99 6.50 n.d. 1 Molecules 2018Tac-7-Tac, 23, x FOR (43-C7 PEER) REVIEW 9.14 10.48 <1.38 (±0.08) 19 of 29 Tac-10-Tac (43-C10) 9.34 9.00 <1.25 (±0.00) Phth-4-isoxoPhth-4-isoxo (44-C4 ()44-C4) 4.074.07 19.12% 19.12% inhib. inhib. at at 100 100 µMµM n.d. n.d.1 1 1 Phth-6-isoxoPhth-6-isoxo (44-C6 ()44-C6) 4.414.41 32.91% 32.91% inhib. inhib. at at 100 100 µMµM n.d. n.d.1 1 Phtal-8-isoxoPhtal-8-isoxo (44-C8 ()44-C8) 5.105.10 4.17 4.17 n.d. n.d.1 ± Tac-7-iperTac-7-iper (45-C7 ()45-C7) 8.298.29 8.76 8.76 >160 >160 (±0.00) ( 0.00) Tac-10-iper (45-C10) 8.75 9.81 32.2 (±0.41) Tac-10-iper (45-C10) 8.75 9.81 32.2 (±0.41) 1 1 n.d.:n.d.: not not determined.

Starting from this finding,finding, new hybrids with multitarget potential (( 45), Figure Figure 22)22) were synthesized consisting consisting of of a a tacrine tacrine and and an an iperoxo iperoxo mo moiety,iety, aiming aiming at higher at higher inhibitory inhibitory activity activity for the for theAChE AChE and andBChE BChE [111]. [111 The]. Theidea ideawas wasto combine to combine the iperoxo the iperoxo affinity affinity for the for M1 the and M1 M2 and receptor M2 receptor with withthe AChE the AChE inhibitory inhibitory activity. activity. Tacrine Tacrine can serve can as serve both as an both allosteric an allosteric modulator modulator for M receptor for M receptor and as andinhibitor as inhibitor of AChE of [112–114]. AChE [112 In–114 particular,]. In particular, Tac-10-iper Tac-10-iper ((45-C10 ((),45-C10 Figure), 22) Figure was 22 an) wasextremely an extremely potent potentinhibitor inhibitor with an with IC50 an value IC50 ofvalue 9.81 of(AChE 9.81 (AChE from electric from electric eel; tacrine eel; tacrine IC50 7.60) IC50 and7.60) 8.75 and (BChE 8.75 (BChE from fromequine equine serum; serum; tacrine tacrine IC50 8.57) IC50 which8.57) which is even is evenbetter better than thantacrine tacrine itself. itself. Moreover, Moreover, it shows it shows less lesscytotoxicity. cytotoxicity. Docking studies show a similar binding mode of compounds 5a-C105a-C10 andand 5b-C105b-C10 toto Tac-10-iper Tac-10-iper ((((45-C10)) inin thethe CASCAS andand PAS regionsregions ofof AChEAChE (Figure(Figure 2323).). The remarkably lower affinity affinity of 45-C10 isis probablyprobably duedue toto thethe additionaladditional quaternaryquaternary ammoniumammonium group of the Phtal- and Naph-compounds whichwhich causescauses aa muchmuch higherhigher desolvationdesolvation penaltypenalty inin comparisoncomparison toto thethe unchargeduncharged alkylenealkylene chain.chain.

Figure 23.23. (a(a)) Docking Docking pose pose of 45-C10of 45-C10(pink), (pink), with with the interaction the interaction distances distances shown shown in theCAS in the (orange) CAS and(orange) in the and PAS in (green). the PAS (b (green).) Docking (b) representation Docking representation of 5b-C10 (blue)of 5b-C10 and (blue)5a-C10 andwith 5a-C10 their interactionwith their distancesinteraction shown distances in yellow. shown Reproduced in yellow. Re withproduced permission with frompermission [110]. from [110].

A dual mechanism of action which appears to be the right innovation to fight ﬁght the AD AD pathology pathology may alsoalso bebe possible possible due due to to the the high high afﬁnity affinity for M1for andM1 M2and receptors. M2 receptors. Furthermore, Furthermore, functional functional studies (investigationstudies (investigation of Ga and of PLC-b3 Ga and proteins PLC-b3 interaction proteins through interaction Split-Luciferase through assays)Split-Luciferase demonstrated assays) the demonstrated the ability of Tac-7-Iper and Tac-10-Iper ((45-C7, 45-C10, Figure 22) to activate the M1 muscarinic receptor in a nanomolar range (Volpato et al., unpublished results). Further studies are necessary for developing uncharged molecules that could cross the BBB; so far, it is promising to see that compounds with a single positive charge are associated with better activity than compounds with two charges [110,111].

Molecules 2018, 23, 3230 19 of 28 ability of Tac-7-Iper and Tac-10-Iper ((45-C7, 45-C10, Figure 22) to activate the M1 muscarinic receptor in a nanomolar range (Volpato et al., unpublished results). Further studies are necessary for developing uncharged molecules that could cross the BBB; so far, it is promising to see that compounds with a single positive charge are associated with better activity than compounds with two charges [110,111].

5.1.3. Tacrine/Xanomeline Hybrids A similar dualsteric strategy was previously employed by the Decker group [114]. They designed and synthesized tacrine and xanomeline hybrid compounds ((46a), Figure 24). The rational design of these hybrids was aimed at a triple mechanism of cooperative action: activation of the M1 receptor through the xanomelinic molecular portion ((18), Figure9), positive allosteric modulation regarding orthosteric agonists at the M1 receptor subtype thanks to the allosteric properties of tacrine, and increasingMolecules 2018 the, 23, x amount FOR PEER of REVIEW ACh in cholinergic synapses due to the AChE inhibiting activity20 [of114 29]. Xanomeline is an M1 agonist but unfortunately also shows some affinity to the M4 receptor. Therefore, parasympatheticsalivation occur. Binding side effects studies including on cloned bradycardia, human muscarinic increased receptors gut motility, did not and detect salivation the selective occur. Bindingbinding capacity studies on between cloned M1 human and muscarinicM4. Xanomeline receptors is well-known did not detect for its the anti-dementia selective binding properties capacity in betweenvivo, and M1the andattenuation M4. Xanomeline of cognitive is well-knowndecline in 6 formonths its anti-dementia clinical trial human properties patientsin vivo proved, and theits attenuationactivity against of cognitive AD [115]. decline The metabolic in 6 months instabilit clinicaly trialdue humanto an extensive patients first-pass proved its metabolism, activity against the ADwell-known [115]. The cholinomimetic metabolic instability side effects, due and to its an un extensivedesirable first-passactivity for metabolism, the dopaminergic the well-known and 5-TH cholinomimeticreceptors (serotonin side receptors) effects, and rendered its undesirable the compound activity an for insufficient the dopaminergic clinical candidate and 5-TH [116,117]. receptors (serotoninXanomeline receptors) induces a rendered specific mode the compound of M1 receptor an insufficient activation clinical by interacting candidate with [116 a, 117site]. that Xanomeline does not inducesfully overlap a specific with the mode endogenous, of M1 receptor orthosteric activation bindin byg site. interacting Differences with in binding a site that kinetics does [118] not fullyand overlapa wash-persistent with the endogenous,binding, probably orthosteric arising binding from a hydrophobic site. Differences interaction in binding of xanomeline’s kinetics [118 O-hexyl] and a wash-persistentchain with the membrane binding, probably lipids surrounding arising from athe hydrophobic receptor [119,120], interaction may of xanomeline’sexplain its functional O-hexyl chainselectivity with [121]. the membrane lipids surrounding the receptor [119,120], may explain its functional selectivityThe xanomeline [121]. portion was connected to the tacrine with amide bridges of different length (10- 17 atomsThe xanomelinespacer). A clear portion increase was of connected AChE inhibiti to theon tacrine was observed with amide by increasing bridges of the different spacer length chain (10-17from pIC atoms50 of spacer).7.20 to 8.21. A clear The increase hybrids of inhibited AChE inhibition the AChE was with observed similar by or increasinghigher potency the spacer compared chain fromto tacrine pIC50 butof 7.20 did to not 8.21. activate The hybrids the M1 inhibited receptor. the Instead AChE withof a similarsimultaneous or higher allosteric/orthosteric potency compared tobinding, tacrine the but ligands did not seem activate to prefer the M1 a receptor.purely allosteric Instead binding of a simultaneous even in the allosteric/orthosteric orthosterically free binding,receptor. theThese ligands findings seem were to prefer supported a purely by allosteric in vivo studies binding in even rats inwith the a orthosterically representative free compound receptor. These((46a), findingsFigure 24). were The supported cognitive bydeficitsin vivo inducedstudies by in administration rats with a representative of scopolamine compound were enhanced ((46a), Figureby the 24purely). The M1 cognitive allosteric deficits interaction induced of bythe administration hybrid 46a counterbalancing of scopolamine its were high enhanced ChE-inhibitor by the purelyproperties. M1 allostericThe desired interaction triple mechanism of the hybrid of46a acticounterbalancingon unfortunately its did high not ChE-inhibitor take place with properties. these Thecompounds. desired triple This mechanismdemonstrates of actionthe difficulty unfortunately of predicting did not takethe placeintrinsic with activities these compounds. of the chosen This demonstratescomponent molecules the difficulty and ofthe predicting importance the of intrinsic their cooperativity, activities of the which chosen must component lead to amolecules balanced andcompromise. the importance of their cooperativity, which must lead to a balanced compromise.

FigureFigure 24.24. Dual-active AChE inhibitor/mAChRinhibitor/mAChR agonist agonist compounds. compounds.

5.1.4. 77-LH-28-1/Xanomeline Hybrids Hybrids As already mentioned above, compound 77-LH-28-1 (( 47), Figure 2525)) displaying mixed modes of orthosteric or or allosteric allosteric pharmacology pharmacology depending depending on onthe theexperimental experimental assay assay conditions conditions [122], [ 122was], wasincorporated incorporated into intoa series a series of ofallo/ortho allo/ortho hybrid hybrid ligands, ligands, hypothesizing hypothesizing a ashared shared function function of of the common chain ligand structures of xanomeline and 77-LH-28-1 (( 48a,b), Figure 25).25). With With a merging approach, the two scaffolds were fused into the new hybrids, aiming for a better understanding of the allosteric pocket and of the bitopic binding pose of 77-LH-28-1 itself.

Molecules 2018, 23, 3230 20 of 28 approach, the two scaffolds were fused into the new hybrids, aiming for a better understanding of the allostericMolecules 2018 pocket, 23, x FOR and PEER of the REVIEW bitopic binding pose of 77-LH-28-1 itself. 21 of 29

FigureFigure 25.25. Dual-active AChE inhibitor/ mAChR mAChR agonist agonist compounds. compounds.

The pharmacological investigation of these hybrid derivatives evidenced evidenced an an affinity affinity at at the the M1 receptor higherhigher thanthan 77-LH-28-1 and lower than xanomeline. In general, hybrid compounds showed either aa weakweak agonisticagonistic or or an an antagonistic antagonistic activity activity profile profile (M1 (M1 and and M4) M4) in allin theall the functional functional assays assays and theand activity/selectivity the activity/selectivity was relatedwas related to the lengthto the ofleng theth spacer. of the Thespacer. highest The affinity highest values affinity are values associated are withassociated a seven- with or a nine-atom seven- or nine-atom length carbon length chain, carb indicatingon chain, indicating that the two that pharmacophores the two pharmacophores probably interactprobably with interact two distinctwith two sites distinct located sites at alocated distance at a of distance 7 to 9 methylene of 7 to 9 methylene moieties. Xano-9-77-LH-28-1 moieties. Xano-9- exhibited77-LH-28-1 higher exhibited efficacy higher for theefficacyβ-arrestin2 for the β engagement-arrestin2 engagement in M1. In line in M1. with In the line reported with the functional reported propertiesfunctional ofproperties the aliphatic of the chain alipha moietytic chain in 77-LH-28-1,moiety in 77-LH-28-1, these results these indicate results that indicate the linker that the extension linker affectsextension the affects orthosteric the orthosteric binding site binding [89]. site [89].

6. Nicotinic Receptor Nicotinic receptors are transmembrane pentameric proteins that belong to the superfamily of ligand-gated ion channels, composed of α and β subunits assembled around a central hydrophilic pore ligand-gated ion channels, composed of α and β subunits assembled around a central hydrophilic + + 2+ thatpore mediates that mediates the ﬂow the of flow the cations of the Kcations, Na ,K and+, Na Ca+, and. Different Ca2+. Different combinations combinations of subunits of generate subunits a varietygenerate of nAChRa variety subtypes of nAChR with distinctsubtypes electrophysiological with distinct electrophysiological properties and brain properties localization and [123 brain,124]. nAChRslocalization are [123,124]. considered nAChRs to play anare important considered role toin play neuroprotection an important duerole to in a neuroprotection concentration dependent due to a β β interactionconcentration with dependent the -amyloid interaction peptide. with In the particular, β-amyloid the peptide. physiological In particular, concentration the physiological of A may directly stimulate the α nicotinic receptors while an increasing pathological concentration of Aβ concentration of Aβ may7 directly stimulate the α7 nicotinic receptors while an increasing pathological damages the cholinergic responses mediated by α and α β receptors. concentration of Aβ damages the cholinergic responses7 mediated4 2 by α7 and α4β2 receptors. 6.1. Design of Hybrid Compounds to Investigate the Nicotinic Receptor Design of Hybrid Compounds to Investigate the Nicotinic Receptor 1-(2-Methoxybenzyl)-Piperazine-Carbazole Hybrids 1-(2-Methoxybenzyl)-Piperazine-Carbazole Hybrids The mutual interaction between Aβ and cholinergic transmission was investigated. Following a ligand-basedThe mutual approach,interaction hybridbetween compounds Aβ and cholinergic addressing transmission both systems was investigated. were synthetized Following and a ligand-based approach, hybrid compounds addressing both systems were synthetized and subsequently pharmacologically studied. The molecules combine an α7 agonist and a carbazole moiety havingsubsequently AChE inhibitionpharmacologically properties studied. ((49a,b), FigureThe molecules 26)[125]. combine Substituted an carbazolesα7 agonist are and able a tocarbazole interact withmoiety the having narrow AChE inner inhibition part of the properties AChE enzyme ((49a,b gorge), Fig and 26) also[125]. present Substituted anti-amyloidogenic carbazoles are activity.able to interact with the narrow inner part of the AChE enzyme gorge and also present anti-amyloidogenic This moiety was connected with the α7 agonistic activity of 1-(2-methoxybenzyl)-piperazine [126]. Itactivity. was selected This moiety because was of connected the structural with similaritythe α7 agonistic with activity the ethyl-(2-methoxybenzyl)-amine of 1-(2-methoxybenzyl)-piperazine group recognizing[126]. It was the selected AChE because catalytic of site the [127 structural,128]. similarity with the ethyl-(2-methoxybenzyl)-amine groupThe recognizing resulting multitarget the AChE catalytic compounds, site in[127,128]. particular compound ((49b), Figure 26), presented AChE inhibition activity in a micromolar concentration range (compound with six methylene unit spacer IC50 = 0.773 µM). AChE inhibition was strongly dependent on the linker elongation. In particular,

Molecules 2018, 23, x FOR PEER REVIEW 22 of 29

MoleculesThe2018 resulting, 23, 3230 multitarget compounds, in particular compound ((49b), Figure 26), presented21 of 28 AChE inhibition activity in a micromolar concentration range (compound with six methylene unit spacer IC50 = 0.773 µM). AChE inhibition was strongly dependent on the linker elongation. In compoundsparticular, compounds with a shorter with chain a shorter in the chain series in the had series lower had IC 50lowervalues. IC50 Invalues. radioligand In radioligand binding binding assays, µ theassays, compounds the compounds exhibited exhibited a micromolar a micromolar affinity affinity profile withprofile K iwithvalues Ki values ranging ranging from 15 from to 120 15 toM 120 in 3 125 α α β α theµM displacement in the displacement of [ H]-epibatidine of [3H]-epibatidine and [ I]- and-bungarotoxin [125I]-α-bungarotoxin from 4 2 fromand 7αreceptor4β2 and α subtypes7 receptor in ratsubtypes cortex, in without rat cortex, discrimination without discrimination in the receptor in the subtype. receptor The subtype. action towardsThe action nAChRs towards was nAChRs tested bywas using tested two-electro by using two-electro voltage clamps. voltage Unfortunately,clamps. Unfortunately, the compounds the compounds only reached only reached 15–22% 15–22% of the µ α β α responseof the response (reference (reference 100 M100 Ach) µM Ach) at 4 at2 αreceptors.4β2 receptors. No No response response was was observed observed at at the the α77 receptor,receptor, possiblypossibly duedue toto itsits lower sensibility to ACh or its faster desensitization. Interestingly, their inhibition abilityability atat 1010 µµMM ofof AAββ fibrilfibril formation, proved with a ThT-based fluorimetric fluorimetric assay, is similar to the anti-aggregatinganti-aggregating andand blockingblocking fibrilfibril formation activity of curcumin [[129]129] (28-36% compared to 34.4% ofof curcumin).curcumin). Synergistically, aa weakweak perturbationperturbation of one of thethe systemssystems involved in the cholinergic neurotransmissionneurotransmission (AChE,(AChE, nAChRs) nAChRs) and and amyloid amyloid aggregation aggregation could could be sufficient be sufficient to modify to modify the entire the scenario.entire scenario. Therefore, Therefore, the compounds the compounds are a suitable are a su startingitable starting point for point further for further optimization. optimization.

FigureFigure 26.26. Dual-activeDual-active AChEAChE inhibitor/inhibitor/ nAChR agonist compounds.

7. Looking to the Future for AD Hybrids Tools 7. Looking to the Future for AD Hybrids Tools TheThe hybridhybrid compounds targeting different biologicalbiological proteins and dualstericdualsteric ligands addressing distinctdistinct partsparts ofof thethe samesame targettarget oror aa combinationcombination ofof bothboth strategiesstrategies provideprovide pharmacologicalpharmacological tools thatthat couldcould expandexpand horizonshorizons inin medicinalmedicinal chemistry.chemistry. All the hybrid examples reported provide additionaladditional usefuluseful andand importantimportant information regarding their target, showing that the development ofof bivalentbivalent compoundscompounds is notnot onlyonly possiblepossible butbut alsoalso promisingpromising inin offeringoffering newnew pharmacologicalpharmacological instrumentation.instrumentation. Poor drugability is the direct consequenceconsequence of the relatively highhigh molecularmolecular weight ofof bitopicbitopic ligandsligands whichwhich preventsprevents theirtheir progressionprogression inin drugdrug development.development. The bivalent compounds cancan provideprovide newnew usefuluseful informationinformation regardingregarding protein-ligandprotein-ligand interactionsinteractions anyway, understandingunderstanding thethe intricateintricate relationshiprelationship betweenbetween receptorreceptor activation,activation, conformational dynamics, signaling profile proﬁle in thethe cholinergiccholinergic system,system, andand thethe connectionconnection betweenbetween different hallmarks of AD. Advantages such as betterbetter selectivity,selectivity, signaling signaling bias, bias, or or reduced reduced toxicity toxicity of of some some hybrids hybrids can can be be further further exploited exploited through through a subsequenta subsequent fragment fragment based based process process trying trying to reduce to reduce physicochemical physicochemical issues butissues keeping but keeping the precious the improvementprecious improvement obtained. obtained. The simultaneous The simultaneous attack on atta severalck on fronts several seems fronts to provideseems to insights provide that insights may bethat the may key be to the opening key to new opening glimpses new inglimpses the cholinergic in the cholinergic ﬁeld of the field AD. of the AD.

Funding:Funding: D.V.D.V. receivedreceived financialfinancial supportsupport byby thethe internationalinternational doctoraldoctoral collegecollege “Receptor“Receptor Dynamics:Dynamics: Emerging ParadigmsParadigms forfor Novel Novel Drugs” Drugs” funded funded within within the the framework framework of the of Elite the NetworkElite Network of Bavaria of Bavaria (ENB). (Grant(ENB). number(Grant K-BM-2013-247). number K-BM-2013-247). Conflicts of Interest: The authors declare no conflict of interest. Conflict of Interest: The authors declare no conflict of interest. References

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