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Dave L Dixon,1 Leo F Buckley,1 Cory R Trankle,2 Dinesh Kadariya,3 Antonio Abbate2 1Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, 2Pauley Heart Center, 3Department of Internal Medicine, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA Abstract: Inhibition of PCSK9 is a novel therapeutic strategy aimed at reducing low-density-lipoprotein cholesterol (LDL-C) and cardiovascular risk. Evolocumab is a fully humanized monoclonal antibody that inhibits PCSK9, an enzyme that binds to LDL receptors and prevents them from recycling to the hepatocyte surface. Clinical trials have demonstrated 50%–70% reductions in LDL-C with evolocumab when used in combination with statin therapy. The recent FOURIER trial demonstrated that evolocumab further reduces cardiovascular events, but not mortality, in high-risk patients already receiving statin therapy. Furthermore, evolocumab did not affect neurocognitive function and was not associated with antidrug-antibody production in over 60,000 patient-years of drug exposure. Appropriate candidates for evolocumab primarily are individuals at high cardiovascular risk, including those with familial hypercholesterolemia and/or established cardiovascular disease, who are already on statin therapy. At this time, the use of evolocumab monotherapy seems appropriate only for individuals deemed statin-intolerant despite attempting several statins. Consideration must be given toward patient willingness to self-inject evolocumab and issues concerning third-party coverage, given the current costs of evolocumab. Keywords: evolocumab, PCSK9, familial hypercholesterolemia, hyperlipidemia

1 The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3′-phthalimidopropyl-ammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular α1-adrenoceptors (Gq), and β-adrenoceptors (Gs). 2 The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 μmol l-1 W84, 30 μmol l-1 alcuronium, 1000 μmol l-1 gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the α1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the β-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3 CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88±0.15 and 12.27±0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36±2% of the control value with alcuronium and to 42±2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and... [ABSTRACT FROM AUTHOR]

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2,control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,diss=27 µM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants KA for the binding of the allosteric modulators to free M2-receptors: KA,gallamine=100 nM, KA,alcuronium=450 nM, KA,W84=69 nM. In H1-receptors, more than 1000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 µM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors. [ABSTRACT FROM AUTHOR]

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM 1-5 ) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM 1 , hM 2 and hM 3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM 2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.e., biased) engagement of downstream effectors. However, the structural basis underlying ligand-dependent control of this essential allosteric mechanism is poorly understood. Here, we show that two sets of extended muscarinic acetylcholine receptor M 1 agonists, which only differ in linker length, progressively constrain receptor signaling. We demonstrate that stepwise shortening of their chemical linker gradually hampers binding pocket closure, resulting in divergent coupling to distinct G-protein families. Our data provide an experimental strategy for the design of ligands with selective G-protein recognition and reveal a potentially general mechanism of ligand-specific allosteric coupling., Competing Interests: The authors declare no competing financial interest.

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1'-(2-tolyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M 1 -selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M 1 positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M 1 -M 5 (hM 1 -hM 5 ) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [ 3 H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 μM of MB ranging from none in hM 3 to 4.6-fold in hM 2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [ 3 H]NMS (0.2 and 2.0 nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logK I (± S.E.) hM 3  = 8.71 ± 0.15, hM 1  = 8.68 ± 0.14, hM 5  = 8.58 ± 0.07, hM 2  = 8.27 ± 0.07 to hM 4  = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logK B : hM 1  = 9.53 ± 0.05, hM 4  = 9.33 ± 0.05, hM 5  = 8.80 ± 0.05, hM 2  = 8,79 ± 0.06, to hM 3  = 8.43 ± 0.04. In conclusion, MB, below 1 μM, binds competitively and non-selectively (except for the difference between hM 3 vs. hM 4 ) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM 1 and hM 4 .

A set of hybrid compounds composed of the fragment of allosteric modulators of the muscarinic receptor, i.e. W84 and naphmethonium, and the well-known AChE inhibitor tacrine on the one hand, and the skeletons of the orthosteric muscarinic agonists, iperoxo and isox, on the other hand, were synthesized. The two molecular moieties were connected via a polymethylene linker of varying length. These bipharmacophoric compounds were investigated for inhibition of AChE (from electric eel) and BChE (from equine serum) as well as human ChEs in vitro and compared to previously synthesized dimeric inhibitors. Among the studied hybrids, compound 10-C10 , characterized by a 10 carbon alkylene linker connecting tacrine and iperoxo, proved to be the most potent inhibitor with the highest pIC 50 values of 9.81 (AChE from electric eel) and 8.75 (BChE from equine serum). Docking experiments with compounds 10-C10 , 7b-C10 , and 7a-C10 helped to interpret the experimental inhibitory power against AChE, which is affected by the nature of the allosteric molecular moiety, with the tacrine-containing hybrid being much more active than the naphthalimido- and phthalimido-containing analogs. Furthermore, the most active AChE inhibitors were found to have affinity to M 1 and M 2 muscarinic receptors. Since 10-C10 showed almost no cytotoxicity, it emerged as a promising lead structure for the development of an anti-Alzheimer drug.

A series of new dibenzodiazepinone-type muscarinic receptor ligands, including two homo-dimeric compounds, was prepared. Sixteen representative compounds were characterized in equilibrium binding studies with [(3)H]N-methylscopolamine ([(3)H]NMS) at the muscarinic receptor subtype M2, and seven selected compounds were additionally investigated at M1, M3, M4 and M5 with respect to receptor subtype selectivity. The side chain of the known M2 preferring muscarinic receptor antagonist DIBA was widely varied with respect to chain length and type of the basic group (amine, imidazole, guanidine and piperazine). Most of the structural changes were well tolerated with respect to muscarinic receptor binding, determined by displacement of [(3)H]NMS. Compounds investigated at all subtypes shared a similar selectivity profile, which can be summarized as M2>M1≈M4>M3≈M5 (46, 50, 57, 62-64) and M2>M1≈M4>M3>M5 (1, 58). The homo-dimeric dibenzodiazepinone derivatives UNSW-MK250 (63) and UNSW-MK262 (64) exhibited the highest M2 receptor affinities (pIC50=9.0 and 9.2, respectively). At the M2 receptor a steep curve slope of -2 was found for the dimeric ligand 63, which cannot be described according to the law of mass action, suggesting a more complex mechanism of binding. In addition to equilibrium binding studies, for selected ligands, we determined pEC50,diss, an estimate of affinity to the allosteric site of M2 receptors occupied with [(3)H]NMS. Compounds 58 and 62-64 were capable of retarding [(3)H]NMS dissociation by a factor >10 (Emax,diss >92%), with highest potency (pEC50,diss=5.56) residing in the dimeric compound 64. As the monomeric counterpart of 64 was 100 times less potent (62: pEC50,diss=3.59), these data suggest that chemical dimerization of dibenzodiazepinone-type M receptor ligands can enhance allosteric binding., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aiming to design partial agonists for a G-protein-coupled receptor based on dynamic ligand binding, we synthesized three different series of bipharmacophoric ligands composed of the orthosteric building blocks iperoxo and 1 linked to allosteric modulators (BQCA-derived compounds, BQCAd; TBPB-derived compound, TBPBd). Their interactions were studied with the human muscarinic acetylcholine M1-receptor (hM1) with respect to receptor binding and Gq-protein signaling. Results demonstrate that iperoxo/BQCAd (2, 3) and 1/BQCAd hybrids (4) act as M1 partial agonists, whereas 1/TBPBd hybrids (5) did not activate M1-receptors. Among the iperoxo/BQCAd-hybrids, spacer length in conjunction with the pattern of substitution tuned efficacy. Most interestingly, a model of dynamic ligand binding revealed that the spacer length of 2a and 3a controlled the probability of switch between the inactive purely allosteric and the active bitopic orthosteric/allosteric binding pose. In summary, dynamic ligand binding can be exploited in M1 receptors to design partial agonists with graded efficacy.

G protein-coupled receptors (GPCRs) are involved in almost every (patho)physiological process, which explains their importance as drug targets. GPCRs have long been regarded as on/off-switches, which is reflected by direct activation or blockade of these receptors through the majority of marketed GPCR drugs. In recent years, however, our view of GPCRs has changed dramatically. GPCRs are now appreciated as integrative and highly dynamic signaling machines which can adopt numerous distinct conformations enabling them to initiate a highly ramified signaling network. We argue here that it may be possible to chemically encode distinct signaling profiles into ligands by rational ligand design. We exemplify our hypothesis by fine-tuning partial and biased agonism, thereby exploiting two new principles of GPCR modulation - dynamic and dualsteric ligand binding. We propose that the emerging understanding of the multiplicity of receptor dynamics will eventually lead to rationally designed new drugs which pilot the pulse; in other words, that stabilize distinct receptor states to fine-tune GPCR signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nine novel β- and γ-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M1 receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-γ-carboline (6) was the most potent (0.25 μmol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

A new series of analogues of the calabash curare alkaloid toxiferine I was prepared and pharmacologically evaluated at α7 and muscle-type nAChRs and the allosteric site of muscarinic M2 receptors. The new ligands differ from toxiferine I by the absence of one (2a-c) or two (3a-c) hydroxy groups, saturation of the exocyclic double bonds, and various N-substituents (methyl, allyl, 4-nitrobenzyl). At the muscle-type nAChRs, most ligands showed similar binding to the muscle relaxant alcuronium, indicating neuromuscular blocking activity, with the nonhydroxylated analogues 3b (Ki = 75 nM) and 3c (Ki = 82 nM) displaying the highest affinity. At α7 nAChRs, all ligands showed a moderate to low antagonistic effect, suggesting that the alcoholic functions are not necessary for antagonistic action. Compound 3c exerted the highest preference for the muscle-type nAChRs (Ki = 82 nM) over α7 (IC50 = 21 μM). As for the allosteric site of M2 receptors, binding was found to be dependent on N-substitution rather than on the nature of the side chains. The most potent ligands were the N-allyl analogues 2b and 3b (EC0.5,diss = 12 and 36 nM) and the N-nitrobenzyl derivatives 2c and 3c (EC0.5,diss = 32 and 49 nM). The present findings should help delineate the structural requirements for activity at different types of AChRs and for the design of novel selective ligands.

Bivalent ligands of G protein-coupled receptors have been shown to simultaneously either bind to two adjacent receptors or to bridge different parts of one receptor protein. Recently, we found that bivalent agonists of muscarinic receptors can simultaneously occupy both the orthosteric transmitter binding site and the allosteric vestibule of the receptor protein. Such dualsteric agonists display a certain extent of subtype selectivity, generate pathway-specific signaling, and in addition may allow for designed partial agonism. Here, we want to extend the concept to bivalent antagonism. Using the phthal- and naphthalimide moieties, which bind to the allosteric, extracellular site, and atropine or scopolamine as orthosteric building blocks, both connected by a hexamethonium linker, we were able to prove a bitopic binding mode of antagonist hybrids for the first time. This is demonstrated by structure-activity relationships, site-directed mutagenesis, molecular docking studies, and molecular dynamics simulations. Findings revealed that a difference in spatial orientation of the orthosteric tropane moiety translates into a divergent M2/M5 subtype selectivity of the corresponding bitopic hybrids.

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M₂ receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo - unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M₂ and M₄ receptor in the picomolar range. Probing ternary complex formation on the M₂ receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M₂, the preferred Gi protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM₂ cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We present a new concept of partial agonism at G protein-coupled receptors. We demonstrate the coexistence of two functionally distinct populations of the muscarinic M2 receptor stabilized by one dynamic ligand, which binds in two opposite orientations. The ratio of orientations determines the cellular response. Our concept allows predicting and virtually titrating ligand efficacy, which opens unprecedented opportunities for the design of drugs with graded activation of the biological system.

Cell-membrane-spanning G protein coupled receptors (GPCRs) belong to the most important therapeutic target structures. Endogenous transmitters bind from the outer side of the membrane to the "orthosteric" binding site either deep in the binding pocket or at the extracellular N-terminal end of the receptor protein. Exogenous modulators that utilize a different, "allosteric", binding site unveil a pathway to receptor subtype-selectivity. However, receptor activation through the orthosteric area is often more powerful. Recently there has been evidence that orthosteric/allosteric, in other words "dualsteric", hybrid compounds unite subtype selectivity and receptor activation. These "bitopic" modulators channelreceptor activation and subsequent intracellular signaling into a subset of possible routes. This concept offers access to GPCR modulators with an unprecedented receptor-subtype and signaling selectivity profile and, as a consequence, to drugs with fewer side effects., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M(2) acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling.

The occurrence of orthosteric and allosteric binding sites is a characteristic common feature of several acetylcholine- binding proteins, like acetylcholinesterase or the nicotinic and muscarinic acetylcholine receptors. These proteins are involved in a number of neurological disorders, such as Alzheimer's disease, and represent important therapeutic targets for the development of heterodimeric ligands addressing both of their binding sites. Among the pharmacophores, which have been combined in such heterodimers, the tetrahydroacridine derivative tacrine has attracted particular interest. This review discusses the chemistry behind the linker connection of tacrine to other pharmacophores and summarizes the types of linkers established to date. Especially, the development of a hydrazide linker for tacrine-derived heterodimers is highlighted by applications in the inhibition of cholinesterases, the bivalent binding to nicotinic and muscarinic acetylcholine receptors, as well as the histochemical imaging of acetylcholinesterase and amyloid-β., (© 2011 Bentham Science Publishers)

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu(173) or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter-bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein-coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated.