Bitopic Ligands and Metastable Binding Sites: Opportunities for G

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Bitopic Ligands and Metastable Binding Sites: Opportunities for G Protein-Coupled Receptor (GPCR) Medicinal Chemistry Philipp Fronik,† Birgit I. Gaiser,† and Daniel Sejer Pedersen* Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark ABSTRACT: G protein-coupled receptors (GPCRs) belong to a large superfamily of membrane receptors mediating a variety of physiological functions. As such they are attractive targets for drug therapy. However, it remains a challenge to develop subtype selective GPCR ligands due to the high conservation of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding sites. Computational studies on ligand binding to GPCRs have revealed transient, low-affinity binding sites, termed metastable binding sites. Metastable binding sites may provide a new source of allosteric binding sites that could be exploited in the design of bitopic ligands. Unlike the bitopic ligands that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites.

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INTRODUCTION G protein-coupled receptors (GPCRs), also referred to as seven-transmembrane (7TM) receptors, belong to a large superfamily of receptors that are divided into five families, termed: rhodopsin, secretin, glutamate, adhesion, and frizzled/ TAS2.1 GPCRs respond to a wide variety of ligands ranging from photons, ions, and small molecules to peptides.2 The physiological functions of GPCRs are wide-ranging and include smell, taste, vision, and the reaction to neurotransmitters and hormones that affect cardiovascular, neurological, and reproductive functions among others.3,4 The abundance of physiological functions of GPCRs has made them an attractive target for drug discovery, and approximately 30% of all FDAapproved drugs act on GPCRs.1,5 However, upon closer inspection of marketed GPCR drugs, it becomes apparent that the vast majority of these drugs target the aminergic receptors belonging to the rhodopsin family. Thus, to date medicinal chemists have only scratched the surface with respect to exploiting the full therapeutic potential of GPCRs. The biggest challenge facing medicinal chemists is to develop subtype selective GPCR ligands. Orthosteric binding sites are highly conserved and hence do not offer many options in terms of selectivity. Allosteric binding sites in contrast are generally distant from the orthosteric site and have been altered significantly throughout evolutionary processes. Targeting allosteric sites to achieve subtype selectivity has been a successful strategy, but it is hampered by the lack of knowledge regarding their location and structure.6,7 Thus, any advances in the prediction and identification of new allosteric binding sites are highly desirable. Bitopic ligands are yet another class of © 2017 American Chemical Society

ligands introduced to address the subtype selectivity problem. They contain two distinct pharmacophores which are connected by a linker, thus allowing concomitant binding to the orthosteric and an allosteric binding site of the same receptor monomer. In addition to the benefit of inducing selectivity into the orthosteric ligand, bitopic ligands can provide advantages such as higher affinity, improved off-rates, etc.,8−13 and can induce signaling bias (i.e., functional selectivity).14,15 With increasing computational power, computational chemists have undertaken extensive molecular dynamics simulations to probe the mechanism of action for an orthosteric ligand’s entry and exit from the binding pocket. Several independent reports have shown that various orthosteric ligands often engage in stable interactions at the entrance of the receptor and adopt energetically stable conformations before either continuing toward the binding pocket or dissociating. These transient binding sites have been termed metastable binding sites (or meta-binding sites).16−21 Metastable binding sites may provide a new source of allosteric binding sites that could be exploited in the design of allosteric modulators and bitopic ligands. Herein, we provide an overview of bitopic ligands targeting different GPCR subfamilies to showcase the opportunities the approach provides within medicinal chemistry. Moreover, the current knowledge on metastable binding sites and how these potential allosteric binding sites could be used in the design of bitopic ligands is discussed. Received: October 28, 2016 Published: January 31, 2017 4126

DOI: 10.1021/acs.jmedchem.6b01601 J. Med. Chem. 2017, 60, 4126−4134

Journal of Medicinal Chemistry

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Figure 1. Bitopic/dualsteric ligands target the orthosteric and an allosteric binding site in a single receptor and commonly are heterobivalent. Dimeric ligands target two separate receptors/protomers simultaneously, here illustrated for a GPCR homodimer. Metastable binding sites may provide a new source of allosteric binding sites that could be targeted by homobivalent bitopic ligands.

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BITOPIC LIGANDS Bivalent ligands are defined as consisting of two pharmacophores that simultaneously engage a combination of two allosteric and/or orthosteric binding sites.22−24 Dimeric ligands (ligands that bind to two separate receptors) and bitopic ligands (also termed dualsteric) are subclasses of bivalent ligands (Figure 1). The term bitopic ligand describes a molecule able to simultaneously bind to both the orthosteric and an allosteric binding site of a single receptor monomer. The two pharmacophores are connected by a linker adapted to fit the target. This idea dates back to Porthogese’s messageaddress concept that was applied to the development of opioid receptor dimer antagonists.25 All bivalent ligands can be further categorized as homo- or heterobivalent, depending on whether the same pharmacophore is engaging both binding sites or not.26−29 Bitopic ligands in theory combine the advantages of orthosteric and allosteric ligands by combining high affinity and subtype selective pharmacophores, respectively (Figure 2).

can be designed as orthosteric (inverse) agonists or antagonists connected to either positive or negative allosteric modulators (PAMs or NAMs), in theory providing access to many regulatory options. However, it is difficult to predict the outcome for such ligands. Chen et al. combined a PAM of the M1-muscarinic acetylcholine receptor (mAChR) with a full agonist, but rather than increasing potency a partial agonist was created.31 Mismatches such as a NAM-agonist hybrid can result in partial loss of affinity, presumably because each pharmacophore stabilizes a different receptor conformation.32 The M1-Muscarinic Acetylcholine Receptor (mAChR). The history of bitopic ligands dates back to 1961, when 1 (McN-A-343, Figure 3) was first described.34 At the time, the

Figure 3. Examples of bitopic mAChR ligands 1,34 2,37 3,38 4,39 5,40 6,41 and 7.42

Figure 2. Generally observed trends of orthosteric, allosteric, and bitopic ligands. Inspired by Kruse et al.33

ligand was believed to be a selective agonist of the mAChR M1 subtype. In the 1980s, evidence suggested that the compound was an allosteric ligand,35 however, recently it was shown that 1 in fact is bitopic. Progressively truncating 1 led to the identification of both an orthosteric and an allosteric fragment, and the pharmacological results were supported by molecular modeling studies. The allosteric fragment was found to positively modulate orthosteric antagonists, while negatively modulating orthosteric agonists, and was considered respon-

Moreover, bitopic ligands bear the potential to induce biased signaling, which for example has been shown for an adenosine A1 receptor ligand, an orthosteric-allosteric hybrid derived from the unbiased orthosteric ligand adenosine (vide infra).30 When designing bitopic ligands, both the orthosteric and the allosteric fragments need to be selected carefully and the linker must be optimized for the target with respect to its attachment point, length, composition, and flexibility.12,13 Bitopic ligands 4127



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A1AR ligands are rarely used as drugs because the same receptor mediates bradycardia as a significant side effect.43 Thus, it is desirable to develop biased ligands that will induce the beneficial effect of cytoprotection while eliminating bradycardia side effects. The first bitopic ligand for A1AR was reported in 2010 in an effort to locate the allosteric binding site of the receptor.57 The agonist adenosine was linked to a PAM via the exocyclic amino group of the nucleobase, and the linker length was increased systematically. The bitopic ligands were evaluated pharmacologically in orthosteric radioligand displacement studies and in functional assays in the presence and absence of the PAM. The ligand 8 (LUF6258) with a linker length of nine atoms displayed the best overall properties (Figure 4).57 Later, Lane et al. reanalyzed the original data on 8

sible for both the partial agonism and the functional selectivity of 1. It was further hypothesized that 1 in addition to its bitopic mode of action acts as an allosteric modulator by only binding to the allosteric binding site when the orthosteric pocket is already occupied by another ligand. On the basis of these findings, 1 was classified as a bitopic, functionally selective mAChR partial agonist.36 In recent years, several new and selective mAChR agonists have been discovered, particularly for the M1 subtype. Just as in the case of 1, the selectivity for these ligands were ascribed to targeting allosteric binding sites, but later a bitopic mode of action was revealed. When 2 (AC-42)37 and 3 (77-LH-28-1)38 were discovered, they were believed to have a bitopic mode of action because their pharmacological profiles were similar to that of 1.36,43 However, this was only confirmed several years later based on further pharmacological evidence and molecular modeling.44 As in the case of 1, these ligands were found to adopt different orientations depending on whether an orthosteric ligand was present or not. The bitopic mode of action of 2 was probed and substantiated further by FRET experiments with fluorescent 2 derivatives and a fluorescent protein-fused mAChR.45,46 Further examples of such ligands are the selective M1 mAChR agonists 4 (VU0364572),39 5 (VU0357017),40 and several analogues, which were found to be bitopic during an optimization study.47,48 Likewise, 6 (TBPB) was first described as a novel, highly selective allosteric M1 mAChR agonist41 but later shown to be a bitopic ligand by a truncating study similar to that reported for 1.15,49 Since the discovery of their bitopic mode of action, the above-mentioned ligands have been used as tool compounds to investigate mAChR pharmacology. Site-directed mutagenesis, analytical pharmacology, and molecular modeling studies with 1−3 and 6 led to the identification of key receptor areas responsible for selective binding, signaling efficacy, and pathway-specific receptor conformations, i.e., functional selectivity.49−51 Taken together, these studies highlight the potential of targeting allosteric pockets, which can promote unique receptor states and provide biased ligands.51 The knowledge gained from the discovery of bitopic ligands and their pharmacological characterization has later been applied in the rational design of bitopic ligands. Disingrini et al. reported the first de novo synthesis of ligands that were designed to target the orthosteric and an allosteric pocket simultaneously.52 They combined nonselective, high affinity agonists with subtype selective allosteric ligands and obtained a new class of agonists with a pharmacological profile similar to that of the allosteric parent compound and described them as hybrid compounds. Several examples of rationally designed bitopic ligands have followed since then.31,42,53 One of these, 7 (THRX-160209), nicely illustrates that careful design, i.e., the appropriate combination of orthosteric and allosteric fragments, and choice of linker, can give rise to ligands with notable improvements in affinity and selectivity.42 To study receptor conformational transformations Bock et al. designed a series of bitopic agonists as chemical probes and found that the allosteric vestibule of GPCRs controls their functional selectivity.54 In 2014, the principle was extended to include inactive receptors when the first antagonist hybrid derived from an orthosteric antagonist and an allosteric antagonist was reported.55 The Adenosine A1 Receptor (A1AR). The adenosine A1 receptor (A1AR) is an important target within tissue protection, in particular in the context of cardiac diseases.56 However,

Figure 4. Bitopic A1AR ligands 857 and 9,30 both composed by the orthosteric agonist adenosine (blue) linked to a PAM (green).

and its analogues with shorter linkers and found a clear signaling bias toward cytoprotection for 8 in comparison to its shorter counterparts.29 Hence this study demonstrates that by careful design of bitopic ligands it is possible to access unique receptor conformations that can result in functionally selective compounds. This study was followed up by Valant et al., who developed 9,43 which fused adenosine with the PAM (2-amino4-(2-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone (VCP171, structure not disclosed).30 The latter was a known ligand that had been shown to bias cytoprotection at A1AR, and it was hoped that the bradycardia signaling pathway could be reduced further in this manner. As anticipated, the bitopic ligand 9 was found to induce significant biased cellular signaling in favor of cytoprotection. The finding was confirmed in a more recent study in which 9 was profiled pharmacologically against a series of known agonists.14 The Dopamine D2-Like Receptor Family. The dopamine D2-like family is associated with many neuropsychiatric disorders as well as drug addiction, and in particular the D3 receptor (D3R) has been investigated extensively as a drug target.58−61 The first selective bitopic ligand 10 (SB269652, Figure 5) for D3R was published by Stemp et al. in 2000, but its bitopic mode of action was only determined recently.62,63 In the same study, 10 was shown to exert allostery at D2R as well as across a D2R dimer. This led to the hypothesis that bitopic 4128



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Figure 6. 1376 and 1474 are bitopic ligands for the S1P3 receptor.

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METASTABLE BINDING SITES During the past decade, it has become possible to perform wide-spanning molecular dynamics (MD) simulations that include entire receptors, cell membranes, and the surrounding solvent. Consequently, many computational studies that characterize ligand recognition as well as entry and exit pathways from a protein’s binding site have been reported. On the basis of MD simulations, it has on multiple occasions been reported that ligands and proteins form loose, transient complexes before the ligand enters the binding site.16,18−21,77−80 These transient binding sites have been referred to as “metastable binding sites” or “meta-binding sites”. Moreover, the resulting complexes have been termed “metastable state”, “loose binding”, “persistent intermediate conformations”, etc.19−21,77,80,81 The current hypothesis regarding metastable binding sites is that they function as selectivity filters. Thus, upon binding of a suitable ligand to the metastable binding site, the receptor will undergo a conformational change that allows the ligand to move further into the receptor to engage the orthosteric binding pocket. Other molecules that do not have the required features are denied access to the receptor and diffuse into the surroundings.82,83 Metastable Binding Sites in GPCRs. The existence of metastable binding sites was first suggested in 1999.84 However, it took more than a decade before experimental evidence of metastable binding sites was provided by Dror et al. based on MD simulations on the β1- and β2-adrenergic receptors (β1AR and β2AR).16 These two receptors represent some of the best characterized GPCRs with >15 crystal structures of the receptors alone or in complex with an agonist or antagonist reported.85,86 Dror et al. found that the structurally related βblockers alprenolol, dihydroalprenolol (15), and propranolol, and the β-agonist isoproterenol upon entering the β2AR occupy a similar metastable binding site in the extracellular vestibule of the receptor (Figure 7).16 The ammonium groups of the ligands were found to make a key interaction with Asp300ECL3 and resided in the vestibule for several hundred nanoseconds (pose 2) before moving on to the orthosteric binding pocket (pose 5). When the same MD simulation was performed with 15 on the β1AR, a similar result to that observed for β2AR was obtained. By a similar method, González et al. studied ligand exit from the β2AR by the antagonist/partial inverse agonist carazolol.17 The existence of a metastable binding site in the extracellular vestibule and the involvement of Asp300ECL3 among other key residues was recapitulated. Two channels for ligand entry for the endogenous agonist histamine at the histamine H4 receptor

Figure 5. Structures of bitopic dopamine receptor ligands 10,63 11,66 and 12.67 In the dopamine area, some research groups have defined PAMs and NAMs as allosteric ligands, whereas other binding sites (not including the orthosteric binding site) have been classified as “secondary binding pockets (SBP)”.68−70 Herein, we describe all other binding sites than the orthosteric as allosteric binding sites. Moreover, a bitopic ligand is defined as a ligand that concomitantly binds to the orthosteric and an allosteric binding site in a single receptor monomer.

binding of 10 at one protomer of a D2R dimer allosterically modulates the binding of dopamine at the other protomer. Through fragmentation of 10 closely related and more potent bitopic antagonists were discovered, but no data on selectivity was reported.64,65 In 2009, Newman et al. reported 11 (R-22) to be a 400-fold selective bitopic ligand for D3R (over D2R).66 In a molecular modeling study, 11 was docked into the D3R crystal structure and was found to bind in a bitopic mode.68 Several studies were performed to determine what induced the marked selectivity for D3R, ranging from computational methods to mutagenesis studies and functional assays with 11 fragments.71−73 The size and shape of extracellular loop (ECL) 1 was found to be an important determinant for subtype selectivity. Unlike in D3R, the ECL1 in D2R is relatively short and thus cannot accommodate the allosteric pharmacophore of 11.71 Moreover, the short ECL1 for D2R influences the orientation of transmembrane helix 2 unfavorably.72 Vass et al. reported a fragment based docking approach on D3R, which resulted in the synthesis of a new type of bitopic ligands (e.g., 12).67 Subnanomolar affinities were achieved and some of the ligands showed moderate selectivity for D2R over D3R. Other GPCR Targets. The majority of bitopic GPCR ligands have been reported for muscarinic acetylcholine, adenosine, and dopamine receptors. However, one successful example of a bitopic ligand developed for another target, the sphingosine-1-phosphate receptor (S1PR), illustrates that the principle is applicable across the GPCR landscape.74 The S1PRs are implicated in vasculogenesis and immune cell regulation and development, and the nonselective S1PR modulator fingolimod is used in the treatment of multiple sclerosis.75 Two closely related bitopic ligands, 13 (SPM-242) and 14 (SPM-354) (Figure 6), have been reported for the S1PR subtype 3 (S1P3), for which the latter has improved potency and in vivo efficacy.74,76 Both ligands occupy the allosteric pocket while competing with sphingosine-1-phosphate for the orthosteric binding site. 4129



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studies have also been reported for nonrhodopsin-like GPCRs, with comparable results. A metastable binding site was discovered for the agonist strychnine at the TAS2R46 bitter taste receptor in the vicinity of ECL1.89 The data is not directly comparable to that outlined above for the rhodopsinlike GPCRs, however, it does fortify the hypothesis that metastable binding sites act as selectivity filters for ligand recognition and binding across GPCR families. Metastable Binding Sites in Other Systems. Recognition pathways similar to those described for GPCRs have also been discovered in other protein ligand systems, thus suggesting that metastable binding sites are prevalent across large parts of the human proteome. Other receptors, transporters, and enzymes alike display a variety of low affinity metastable complexes with their respective ligands. The entry pathways of the inhibitor DADMe-immucillin-H at the enzyme purine nucleoside phosphorylase were simulated by MD.77 It was discovered that the ligand can adopt two separate metastable poses before reaching its final binding site. In a similar manner, trypsin binds its ligand benzamidine via two metastable states.18 At the enzyme Src kinase, the situation appears to be similar, as the ligands PP1 and dasatinib enter the active center via multiple “persistent intermediate conformations”.80 However, in this study, the binding positions do not seem to be well-defined. Other reports do not explicitly mention transient low affinity binding sites but do acknowledge the presence of certain points of interaction and recognition on or near the surface of proteins. Hasenhuetl et al. proposed a “selectivity filter” in the entry pathways of the dopamine and serotonin transporters based on binding kinetic experiments.82 Another computational study dealt with two separate enzyme ligand systems: the haloalkane dehalogenase LinB and its ligand 1,2-dichloroethane, as well as levansucrase and its ligands glucose and sucrose.90 These ligands appear to have defined entry pathways along their respective enzyme’s surfaces, thus suggesting that key interactions are made along the ligands’ trajectory.

Figure 7. Process of 15 approaching the orthosteric binding pocket of β2AR via several metastable binding sites.16 The timeline for the simulation is indicated in μs for each binding pose. (1 (red)) The ligand moves from bulk solvent and attaches to the receptor. (2 and 3 (green)) The ligand pauses at two metastable binding sites in the extracellular vestibule. (4 and 5 (blue)) The ligand at the orthosteric binding site. Pose 4 is a long-lived metastable binding site overlapping with the final orthosteric binding pose 5. Pose 5 matches that seen in the X-ray crystal structure of β2AR in complex with alprenolol (PDB: 3NYA). The figure was adapted from Dror et al.16

were recently described by Wittmann and Strasser.87 Similarly to that described above for the β2AR they reported that Glu160ECL2 served as a point of first ligand attachment for both possible pathways. Metastable binding sites for the adenosine A2A receptor have also been proposed by Sabbadin et al. for several antagonists as well as the orthosteric agonist adenosine.20,21 In every case, Glu169ECL2 was seen to form a persistent interaction with the ligand and Asn2536.55 was also suggested as a ligand anchoring point upon receptor entry. Computational studies on the mAChR family showed comparable results to those for the A2A receptor. Ligands paused near known allosteric binding sites in the extracellular vestibule when entering and exiting the orthosteric binding pocket.54,78,79,88 For the mAChR M3 subtype, the large inverse agonist tiotropium bound at a metastable site interacting with Phe221ECL2 and Leu225ECL2, which coincidentally correlates with a known allosteric site in the extracellular vestibule reported by Dror et al.88 The small M3 (partial) agonists acetylcholine and arecoline paused in two separate “clusters”. One cluster was located just above the orthosteric binding site maintaining most of the key orthosteric interactions. The second cluster was found to be in the extracellular vestibule with important interactions to Phe1242.60 and Tyr1272.63. Tiotropium binds to the M2 subtype of mAChR in a similar manner as seen for M3 with key interactions to Tyr177ECL2 and Phe181ECL2 (corresponding to Phe221ECL2 and Leu225ECL2 in M3). Supervised MD simulations on the P2Y12 receptor with antagonist ticagrelor also indicate the presence of metastable binding sites.19 For the ligand to enter the orthosteric binding pocket of the P2Y12 receptor, a stable salt bridge must dissociate. The ligand interaction with the metastable binding site may be the driving force necessary for this energetically unfavorable rearrangement to occur. Some computational

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CONCLUSION AND FUTURE DIRECTIONS In recent years, the development of bitopic ligands has transcended the subfamily of mAChRs and moved on to new targets such as the adenosine and dopamine receptors. The ability of such ligands to induce signaling bias, as illustrated with the A1AR ligand 9, showcases one of many unique properties that ligands of this class can possess. Unprecedented subtype selectivity can also be achieved with bitopic ligands and recent advances with dopamine receptors show promising results for the future. With the discovery of new allosteric binding sites in spatial proximity to the orthosteric binding site, the potential for bitopic ligands will increase and the first therapeutically promising bivalent ligands may see the light of day. When the presence of metastable binding sites in the extracellular vestibule was first computationally determined by Dror et al., they suggested that these transient binding sites could serve as targets for allosteric modulators.16 The presence of allosteric binding sites in the extracellular vestibule is now well established, and the PAM 3-amino-5-chloro-N-cyclopropyl-4-methyl-6-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)thieno[2,3-b]pyridine-2-carboxamide (LY2119620, structure not disclosed) serves as a prominent example of such a ligand targeting the mAChR M2 subtype.91 Besides in silico studies, no experimental evidence for the existence of metastable binding 4130



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human β2AR in complex with the neutral antagonist alprenolol and docked 15 into both the orthosteric binding pocket and the metastable binding site in the extracellular vestibule (Figure 8) to obtain poses for both ligands in agreement with that published by others. The two docked ligands (15) are positioned in such a way that they may in principle be connected by a linker either via the channel that the ligand normally traverses in MD simulations (left)16 or alternatively through the wider channel (right). The idea of developing homobivalent bitopic ligands targeting the orthosteric and a metastable (i.e., allosteric) binding site simultaneously is in principle easily probed by the design of ligands based on known orthosteric ligands (e.g., 15, Figure 8). Work in our laboratories and those of others are underway to explore this strategy on the adrenergic and adenosine receptors and will be reported in due course.

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AUTHOR INFORMATION

Corresponding Author

Figure 8. 15 (dark blue) docked into: (i) the orthosteric binding site of β2AR (PDB: 3NYA) overlaid with the crystallized ligand alprenolol (cyan), and (ii) the metastable binding site (dark blue).16 Dotted yellow lines highlight the two channels via which one could imagine connecting the ligands to create a potential homobivalent bitopic ligand. In MD simulations, ligands normally traverse the narrow channel to the left.16 The ligands were docked using the Schrödinger software suite with the OPLS3 force field.

*Phone: +45 35 33 60 00. Fax: +45 35 33 60 01. E-mail: daniel. [email protected]. ORCID

Daniel Sejer Pedersen: 0000-0003-3926-7047 Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

sites has been provided to date. However, the presence of metastable binding sites appears likely based on the independent observation in silico by many different research groups on a diverse range of receptors. Interestingly, if the orthosteric ligand for a receptor is known it follows that the same ligand should bind to the metastable binding sites, thus providing a template for medicinal chemists. One could imagine elaborating on an orthosteric ligand to obtain an allosteric ligand binding in the extracellular vestibule. Alternatively, metastable binding sites may provide an entry to homobivalent bitopic ligands that simultaneously target the orthosteric and a metastable (i.e., allosteric) binding site (Figure 1). In theory, homobivalent bitopic ligands combine the benefits of high affinity and selectivity by targeting both the highly conserved orthosteric binding site and an evolutionarily less conserved metastable binding site. To the best of our knowledge, no one has reported this ligand design strategy. It should be mentioned that Gmeiner and co-workers published the synthesis and pharmacological evaluation of homodimeric ligands targeting D2-like receptor dimers.92 They discovered three ligands with increased affinity and enhanced selectivity on the D3 subtype, displacing only 1 equiv of radioligand. Thus, they reasoned that they were targeting the orthosteric binding pocket and an undefined allosteric binding site within one receptor rather than bridging the two orthosteric pockets on the GPCR homodimer. However, when considering the very long linker employed in this study, it is unlikely a metastable binding site is engaged. More likely the second pharmacophore is interacting with the extracellular surface of either one of the receptors. Jörg et al. reached a similar conclusion when targeting the same receptor with another class of bivalent ligands.93 Thus, the bivalent D2R ligands reported in these two instances are probably not binding to a metastable binding site in the extracellular vestibule. On the basis of the original reports by Dror et al. and González et al., we utilized the X-ray crystal structure of the

Biographies Philipp Fronik received his B.Sc. degree in Molecular Biotechnology from the University of Applied Sciences “FH Campus Wien” in Vienna, Austria (2013). In 2016, he completed a double M.Sc. degree in Medicinal Chemistry at the University of Copenhagen, Denmark/ VU University Amsterdam, The Netherlands. Birgit I. Gaiser obtained her B.Sc. in Chemistry (2011) from the University of Stuttgart, Germany, and her M.Sc. in Medicinal Chemistry (2014) from the University of Copenhagen, Denmark, where she currently is undertaking her graduate studies under the supervision of Associate Professor Daniel Sejer Pedersen. Daniel Sejer Pedersen received his M.Sc. degree under the supervision of Associate Professor Otto Dahl in 1999 (University of Copenhagen), and his Ph.D. degree under the supervision of Professor Stuart Warren in 2009 (University of Cambridge). He worked for several years in the biotech industry and as a Postdoctoral research fellow both in Australia and Denmark. In 2010, he was appointed Associate Professor in medicinal chemistry at the Department of Drug Design and Pharmacology, University of Copenhagen.

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ACKNOWLEDGMENTS The Lundbeck Foundation, The Carlsberg Foundation, and the Faculty of Health and Medical Sciences, University of Copenhagen, are thanked for financial support.

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ABBREVIATIONS USED A1AR, adenosine A1 receptor; ABS, allosteric binding site; AR, adrenergic receptor; D2R, dopamine D2 receptor; D3R, dopamine D3 receptor; ECL, extracellular loop; GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; MBS, metastable binding site; MD, molecular dynamics; NAM, negative allosteric modulator; OBS, orthosteric binding site; OPLS, Optimized Potentials for Liquid Simulations; PAM, positive allosteric modulator; S1PR, 4131



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sphingosine-1-phosphate receptor; S1P3, sphingosine-1-phosphate receptor 3; SAR, structure−activity relationship; TM, transmembrane

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REFERENCES

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