Tactical Approaches to Interconverting GPCR Agonists and

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Tactical Approaches to Interconverting GPCR Agonists and Antagonists Peter I. Dosa*,† and Elizabeth Ambrose Amin‡ †

Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota 55414, United States ‡ Department of Medicinal Chemistry and Minnesota Supercomputing Institute for Advanced Computational Research, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota 55414, United States ABSTRACT: There are many reported examples of small structural modifications to GPCR-targeted ligands leading to major changes in their functional activity, converting agonists into antagonists or vice versa. These shifts in functional activity are often accompanied by negligible changes in binding affinity. The current perspective focuses on outlining and analyzing various approaches that have been used to interconvert GPCR agonists, partial agonists, and antagonists in order to achieve the intended functional activity at a GPCR of therapeutic interest. An improved understanding of specific structural modifications that are likely to alter the functional activity of a GPCR ligand may be of use to researchers designing GPCR-targeted drugs and/or probe compounds, specifically in cases where a particular ligand exhibits good potency but not the preferred functional activity at the GPCR of choice.

1. INTRODUCTION G protein-coupled receptors (GPCRs) have garnered a great deal of attention as popular and important therapeutic targets. A 2006 study found that over 25% of all U.S. FDA-approved drugs targeted GPCRs, while in 2008 seven of the top 15 prescription drugs acted through mechanisms involving these receptors.1−3 GPCRs continue to be an active area of research focus for many pharmaceutical companies, with 63 GPCRtargeted drugs launched between 2000 and 2009, representing about 25% of total new drugs reaching the market during that time period.4,5 One theme frequently encountered in drug discovery research focusing on GPCRs is that small modifications to the structure of GPCR ligands can lead to major changes in functional activity, switching agonists to antagonists or vice versa. In many cases, these dramatic shifts in functional activity are accompanied by only minor variations in binding affinity. Perhaps the best known historical example of this phenomenon occurs with morphine and its derivatives. Morphine is an analgesic obtained from the opium poppy (Papaver somniferum) that is an agonist of the μ-opioid receptor, with good selectivity for the μ-receptor over the related δ- and κ-opioid receptors.6,7 A small structural change, replacing the N-methyl group of morphine with an N-allyl, results in the potent μ-opioid receptor antagonist nalorphine (Figure 1). This modification has a relatively small effect on the binding affinity of the compounds at the μ-receptor, with a Ki of 0.53 nM reported for morphine and 0.36 nM for nalorphine.6 By contrast, this structural modification has the effect of significantly increasing binding affinity for the k-opioid receptor from 120 to 2 nM.6 Functional assays demonstrate that © XXXX American Chemical Society

Figure 1. Change in N-substitution converts the selective μ-opioid agonist morphine into the μ-antagonist/κ-agonist nalorphine. Further modifications (shown in blue) furnish naloxone, which is an antagonist at both receptors.

nalorphine has significant partial agonist character at this receptor.6 Nalorphine can, in turn, be converted to naloxone, which is an antagonist at both receptors, with additional structural modifications. The functional activity of morphine derivatives does not vary linearly with the size of the substituent on the basic nitrogen. The N-demethylated derivative normorphine has analgesic properties in animal models and was reported to be approximately one-fourth as effective as morphine in patients with postoperative pain.8,9 While the allyl derivative nalorphine is an antagonist at the μ-opioid receptor, the larger analogue N-benzylnormorphine is a weak analgesic and N-phenethylnormorphine has several times the analgesic potency of morphine.8 This phenomenon is not uncommon, and several Received: June 24, 2015

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DOI: 10.1021/acs.jmedchem.5b00982 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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other examples of similar nonlinear relationships between functional activity and the size of substituents will be presented later in this perspective. While methods that researchers have used to interconvert agonists and antagonists have been discussed in previous publications,10,11 there have been few if any extensive reviews on this topic. The purpose of our perspective is to provide a practical guide for medicinal chemists who need to perform such transformations by presenting an overview of structural modifications that have proven to exhibit the desired effects. We have focused primarily on cases published in 2000 or later where a single structural modification changed the functional activity of a ligand at a GPCR without significantly affecting its binding affinity at the receptor. However, we also discuss several examples where multiple structural alterations were necessary to fully switch between agonists and antagonists; our objective here is to underscore the utility of building into a ligand increasing (or decreasing) amounts of partial agonism in a stepwise manner. Case studies described in Section 2 are grouped by the type of structural modification used to alter functional activity, although it should be noted that several of them could have been placed in multiple categories. In Section 3 of this perspective, we will present our conclusions and discuss how structural modifications that have been used to transform functional activities of ligands at GPCRs differ from those effecting this change at other types of receptors.

Figure 2. N-Ethylation converts ghrelin receptor agonist 1 into antagonist 2.

potent in the radioligand binding assay, with a Ki of 20 nM. While the compound itself displayed poor bioavailability, the investigators were able to synthesize a series of structurally related N-alkylated ghrelin receptor antagonists with improved DMPK properties which demonstrated the desired in vivo effects of suppressed food intake and improved glucosedependent insulin secretion. 2.1.2. Conversion of a CCR3 Antagonist into an Agonist. The CC chemokine-3 (CCR3) receptor is naturally activated by several different endogenous chemokines and is responsible for helping to control eosinophil chemotaxis. As part of a program to develop CCR3 antagonists as potential treatments for asthma, researchers at Schering Plough identified compound 3 (Figure 3) as a high affinity ligand for the receptor (Ki = 23 nM), with the desired antagonist activity in a GTPγS functional assay.13 However, while exploring the effects of structural modifications to the central bipiperidine core of the scaffold, Ting and co-workers found that the addition of a methyl group at the 3′-position of the 1,4′-biperidine (midway between the two basic nitrogens) increased binding potency at the receptor but also unexpectedly led to a ligand that was an agonist (compound 4, Ki = 7.3 nM). This effect was specific to that point of attachment, as compound 5, where the added methyl group is shifted to the 4′-position, showed very little agonism in a GTPγS functional assay, with an Emax of only 8%. 2.1.3. Changing Functional Activity at mGlu2 and mGlu3 Receptors by Addition of a Methyl Group. Eglumegad (Ly354740) is an agonist of the metabotropic glutamate receptors mGluR2 and mGluR3 that has been investigated clinically for its anxiolytic properties (Figure 4).14 Dominguez and co-workers at Eli Lilly synthesized methyl-substituted analogues of eglumegad as part of ongoing SAR studies around their clinical candidate.15 These researchers found that incorporating a methyl group at the C3-position of eglumegad unexpectedly led to a compound (6) that was a functional antagonist at both mGluR2 and mGluR3. Interestingly, the effect of incorporating a methyl group on functional activity appeared to diminish with distance from the basic amino group attached at C2. Addition of a methyl group at the C4-position syn relative to the amino led to compound 7, which was a mGluR2 agonist/mGluR3 antagonist, while addition of a methyl group on the same carbon but anti to the amino group yielded compound 8, which was an agonist at both mGluR2 and mGluR3, similar to eglumegad. Using the X-ray structure of glutamate bound to rat mGluR1 as a structural guide, these workers manually generated binding modes for compounds 6−8 in the active sites of both mGluR2 and mGluR3. They found that predicted binding modes revealed a hydrogen bonding network that closely resembled that which

2. RECENT EXAMPLES OF MINOR STRUCTURAL ALTERATIONS THAT INTERCONVERT GPCR AGONISTS AND ANTAGONISTS 2.1. Steric Modifications near a Basic Nitrogen. One of the most commonly used approaches to interconverting GPCR agonists and antagonists is altering the steric environment in the vicinity of a basic amine. This is the general method used to convert morphine analogues from μ-opioid agonists into antagonists (Figure 1). It is interesting to note that while each of the cases discussed in this section feature the incorporation or modification of an alkyl group in close proximity to a basic amine, the examples presented here are roughly equally divided between alterations that increase or decrease agonism. 2.1.1. Ghrelin Receptor Modulators. One of the most straightforward recent examples of a minor structural modification converting an agonist into an antagonist was discovered by scientists at Bayer, who were working with ligands targeting the ghrelin receptor. Ghrelin is an endogenous peptide hormone that stimulates appetite through its action on the growth hormone secretagogue type 1a receptor (GHS-R1A). Ghrelin has also been shown to suppress insulin secretion from islet cells. It has been suggested that antagonists of GHS-R1a could help reduce obesity through appetite suppression while also treating diabetes by increasing insulin secretion and glucose tolerance. As a result, many pharmaceutical companies have shown interest in developing GHS-R1a antagonists.12 A high-throughput screen conducted at Bayer targeting GHS-R1A identified quinazoline derivative 1 as a potential hit (Figure 2).12 Follow-up assays confirmed that while 1 exhibited high affinity for GHS-R1A (Ki = 36 nM) in a radioligand binding assay, in a GTPγS functional assay it acted as an agonist instead of the desired antagonist. However, the Bayer researchers determined it was possible to obtain an antagonist simply by synthesizing the N-ethylated analogue of 1, compound 2. This analogue also proved to be more B



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Figure 3. Methylation midway between the two basic nitrogens in the bipiperidine core changes an antagonist into an agonist, but methylation at an adjacent carbon does not.

Figure 4. Incorporation of a methyl group at different positions on the eglumegad scaffold leads to ligands with a variety of functional activities at mGluR2 and mGluR3.

determining the functional activity of a ligand at a GPCR can be found in the work of Zaveri and colleagues at SRI and the Naval Research Laboratory. These researchers were attempting to identify agonists and antagonists targeting the nociceptin (NOP) receptor. NOP agonists are of interest as anxiolytics and drug abuse treatments, while NOP antagonists may potentiate analgesia and modulate morphine tolerance development.17 Zaveri’s investigations began with the identification of piperidine 9 from a screening campaign. This compound is a weak partial agonist at the NOP receptor, with Ki = 201 nM in a receptor binding assay and 18% maximal response in a GTPγS functional assay (Figure 5). An initial SAR study determined that replacing the N-benzyl group of 9 with a cyclooctylmethyl group yielded a compound (10) with greatly improved potency (Ki = 6.0 nM) and selectivity over opioid receptors. This analogue was a full antagonist in a functional assay. Zaveri also discovered that by deleting the methylene between the basic nitrogen of the piperidine and the lipophilic cyclooctyl group, the resulting compound 11 had improved binding potency (Ki = 1.4 nM) but acted as an agonist at the NOP receptor. Further SAR work varying the substituents on the piperidine determined that attaching a cyclic, saturated lipophilic substituent directly to the piperidine resulted in an agonist, whereas an antagonist was obtained when an extra methylene was added between the two pharmacophores. The phenomenon of an added methylene altering functional activity

was observed in the mGluR1 crystal structure, with the methyl group of 6 targeting a hydrophobic region and thereby impeding closure of the two sections of the glutamate binding site, a conformational change which is necessary to induce an agonist response. In the proposed binding mode for 8, the methyl group was predicted to interact with residues located on the same lobe of the glutamate binding site, which did not preclude closure of either the mGluR2 or mGluR3 active sites and therefore did not interfere with adoption of a fully closed, agonist configuration. In the case of compound 7, which demonstrated agonist activity at mGluR2 and acted as an antagonist at mGluR3, the researchers concluded from structural information that slight movement of a mGluR2 side chain due to binding of 7 did not adversely affect the ability of the glutamate binding site in that receptor to adopt an agonist conformation, while displacement of an amide functionality in the mGluR3 active site would not allow this conformational change, resulting in the consequent antagonist activity. Note that these results highlight the difficulties inherent in predicting how structural modifications may affect functional activity, as two receptors with high homology (∼70%)16 exhibit different functional responses to 7 but behave similarly with other ligands (6, 8, and eglumegad). 2.1.4. Converting a Nociceptin Agonist into an Antagonist by Incorporating an Extra Methylene. Another example of the steric environment near a basic nitrogen playing a key role in C



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Figure 5. Addition of a methylene between the piperidine nitrogen and a cyclooctyl group switches functional activity at the NOP receptor.

dopamine agonist that is also a 5-HT2B agonist. However, unlike pergolide, cabergoline is still used clinically, although its use has been significantly restricted to indications that require low doses, such as the treatment of prolactinomas. Continuing their investigation into the SAR of 5-HT2B agonism in ergot alkaloids, Pertz and co-workers synthesized a series of cabergoline analogues, focusing on varying the substitution on the basic nitrogen at the 6-position.22 In a similar result to what was found with pergolide, replacing the 6-allyl of cabergoline with a less bulky methyl group (6-methylcabergoline in Figure 7) led to a compound that was an antagonist at

occurs fairly frequently, and several related examples are discussed in Section 2.4. 2.1.5. Eliminating 5-HT2B Agonism in Ergot Alkaloids: Pergolide. The dopamine agonist pergolide was withdrawn from the U.S. market in 2007 due to its tendency to cause heart valve damage. This potentially deadly side effect has been observed with many, but not all, ergot alkaloids used in the clinic and has been linked to activation of the 5-HT2B receptor.18 As part of a research program exploring the triggers of 5-HT2B agonism in ergot alkaloids, Pertz and co-workers synthesized a series of pergolide analogues and determined their pharmacology at the 5-HT2B and D2 receptors.19 For functional assays, the researchers used porcine pulmonary arteries to investigate the 5-HT2B receptor-mediated effects of their compounds, while they used recombinant human receptors expressed in Chinese hamster ovary (CHO) cells to study dopamine D2 receptor activity. It was found that replacing the N-propyl group of pergolide with an N-methyl group switched functional activity at the 5-HT2B receptor but not at the D2 receptor (Figure 6). The same pattern held when the −SMe

Figure 7. Replacing the 6-allyl group of cabergoline with a methyl reduces functional agonism at porcine and human 5-HT2B receptors but to a differing extent. Methylation of the indole of 6-methylcabergoline leads to an antagonist at the human receptor (see Section 2.2.1).

5-HT2B receptors in porcine pulmonary arteries. However, by the time of Pertz’s publication in 2011, we (the Dosa group) had become interested in developing a safer alternative to cabergoline as a possible treatment for depression and sexual dysfunction and had independently synthesized 6-methylcabergoline.23 We found that in two different types of functional assays using human 5-HT2B receptors, 6-methylcabergoline was a partial agonist instead of an antagonist. We believe that this discrepancy is most likely due to interspecies differences in the 5-HT2B receptor, but there have also been cases of functional activity varying with the type of assay or tissue used to assess functional activity.19 In particular, different levels of receptor expression have been found to lead to variations in the measured intrinsic activity of a ligand.24,25 These results highlight the critical importance of evaluating the functional activity of a ligand at both the human receptor and the receptor of the animal species used for pharmacological studies, as the respective results may vary significantly. Remarkably, almost none of the reports addressed in this review present functional data in the animal species used for in vivo studies, although presumably (and hopefully) this data was available to the researchers in many cases. 2.2. Change in Functional Activity by Methylation of an Indole. 2.2.1. A Further Modification to the Cabergoline

Figure 6. Small change in N-substitution in pergolide or O-pergolide switches functional activity at the 5-HT2B receptor.

moiety of pergolide was replaced with an −OMe group: the N-propyl analogue was a 5-HT2B agonist and the N-methyl analogue was an antagonist (D2 activity was not reported for these compounds). The researchers suggested that 6-methylpergolide could potentially be used as a replacement for pergolide clinically, as only dopamine agonists that are also 5-HT2B receptor agonists are believed to cause heart valve damage in patients.20,21 2.1.6. Reducing 5-HT2B Agonism in Ergot Alkaloids: Cabergoline. Like pergolide, cabergoline is an ergot alkaloid D



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Scaffold Completely Eliminates 5-HT2B Agonism. In the example discussed in the previous section, replacing the 6-allyl group of cabergoline with a 6-methyl group reduced, but did not completely eliminate, agonism at the human 5-HT2B receptor (Figure 7). However, we were able to obtain an antagonist via a second modification, N-methylation of the indolic N−H in cabergoline. Two other compounds with the same substitution patterns at the 1- and 6-positions, amide 12 and acetal 13, also showed minimal agonism in two different functional assays at the human 5-HT2B receptor (Figure 8).23

showed similar potency to 16 in binding assays but was a partial agonist in a functional assay. On the basis of molecular modeling, and the different orientations exhibited by the methyl groups of isomers 15 and 17, the authors concluded that these functional changes were most likely due to steric interactions rather than specific electrostatic interactions between the added methyl groups (or the removed indolic hydrogen) and key residues on the receptor. 2.3. Changes in Aniline Nitrogen Substitution. 2.3.1. V2 Receptor Modulators. After developing the vasopressin V2 receptor antagonist tolvaptan, which received U.S. FDA approval in 2009 for the treatment of hyponatremia in patients with heart failure, scientists at Otsuka Pharmaceutical Company became interested in developing V2 receptor agonists for the management of urinary incontinence and central diabetes insipidus. They began the search for agonists with the tolvaptan analogue 18 (Figure 10), which they had identified in earlier research as a partial agonist but that elicited only 9% of maximal cAMP accumulation (PMA) in a functional assay.27 As part of their SAR studies, these researchers synthesized a series of monoalkyl substituted anilines related to 18 (Figure 11). While the binding affinity of these analogues at the V2 receptor varied remarkably little (with IC50s ranging from 18 to 35 nM), the functional activity of these compounds showed a dramatic dependence on the length of the alkyl chain, with the propyl analogue demonstrating the most agonism in a functional assay (79% PMA). Similarly, incorporating the aniline nitrogen into a pyrrolidine ring (compound 19 in Figure 10) furnished a strong partial agonist, while enlarging the ring by a single methylene gave piperidine analogue 20, which showed minimal functional agonism. These results suggest that there is an optimal size for alkyl substituents on the aniline for maximal agonist activity at the V2 receptor and that agonism drops off sharply if the substituents are too large or small, even if binding affinity remains relatively unchanged.

Figure 8. Amide 12 and acetal 13 are also 5-HT2B antagonists.

2.2.2. CCK2 Receptor Agonist and Antagonists. Examples in other systems also exist where methylation of an indole leads directly to a change in functional activity. While exploring the SAR of a series of CCK2 receptor antagonists, Kalindjian and co-workers synthesized the N-methylated analogue of the potent antagonist 14 in order to determine if the hydrogen connected to the indolic nitrogen of 14 formed a key hydrogen bond with the CCK2 receptor (Figure 9).26 The resulting compound 15 was roughly equipotent with 14 in binding assays but was a partial agonist in a functional assay. Similarly compound 17, the methylated analogue of antagonist 16,

Figure 9. Methylation of CCK2 antagonists 14 and 16 leads to partial agonists 15 and 17.

Figure 10. V2 antagonist tolvaptan and partial agonists 18−20. E



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at the α2-adrenergic receptor, even if effects on binding affinity are negligible. 2.3.3. Functional Activity at the MT1 and MT2 Receptors Varies with the Size of Substituents on an Aniline. As part of a research program focused on identifying agonists and antagonists selective for the two melatonin receptor subtypes MT1 and MT2, Gilberto Spadoni and colleagues in Italy and Canada prepared a series of N-(substituted-anilinoethyl)amido derivatives (Figure 13).29 The methyl derivative in this series, 29, was roughly equipotent at MT1 and MT2 and a full agonist at both receptors. The researchers found that increasing the steric bulk on the aniline nitrogen increased selectivity for MT2 over MT1 and lowered intrinsic activity at both receptors. Thus, the phenyl derivative 30 was a partial agonist at both receptors with good selectivity for MT2, while the even bulkier β-naphthyl analogue 31 was an antagonist at MT2 and showed weak partial agonism with poor binding affinity at MT1. It is likely that the reduction in intrinsic activity seen with 30 and 31 compared to 29 results from a steric effect rather than an electronic effect due to the incorporation of the nitrogen into a second anilinic system, as benzyl derivative 32 also displayed partial agonism at both MT1 and MT2. 2.4. Changing the Distance or Angle between Pharmacophores. 2.4.1. Shifting a Guanidinium Group Changes a κ-Opioid Receptor Antagonist into an Agonist. A common technique utilized to modify functional activity is to alter the distance or angle between key pharmacophores in a ligand. One elegant example of this approach was published in 2001 by the Portoghese group at the University of Minnesota, who were investigating analogues of the potent and selective κ-opioid receptor antagonist 5′-guanidinonaltrindole (GNTI, Figure 14). To explore the interaction of the 5′-guanidinium group of GNTI with a key residue in the κ-receptor (Glu297), the researchers synthesized a set of derivatives where the guanidinium group was moved to the 4′-, 6′-, or 7′- positions.30 While the 4′-derivative exhibited poor opioid receptor activity and the 7′-derivative behaved primarily as a δ-opioid receptor antagonist, the 6′-derivative 33 was a potent κ-selective agonist. On the basis of the results of the κ-opioid receptor functional assay and a binding study with a mutant (Glu297Ala) κ-receptor, the authors hypothesized that the carboxylate group of Glu297 may form a salt bridge with the guanidinium moiety of the ligand, thereby stabilizing the receptor in either the inactive (with the 5′-guanidinium) or the active (with the 6′-guanidinium) state. The researchers also found that

Figure 11. Functional agonism at the V2 receptor in a series of monoalkylated aniline derivatives varies greatly with the length of the alkyl substituent.

The development of V 2 receptor agonists from V 2 antagonists by Kondo and associates at Otsuka illustrates a commonly used research strategy for that purpose: evaluate a library of antagonists in functional assays for any evidence of partial agonism (even if weak) and then enhance agonist activity in the partial agonists identified from the compound series. Another example of this stepwise approach featuring bradykinin B2 receptor modulators will be discussed in Section 2.8.3. 2.3.2. α2-Adrenoceptor Ligands. Further evidence of alterations in functional activity resulting from small differences in aniline alkyl substitution was discovered by Rodriguez and co-workers using ligands for the α2-adrenergic receptor.28 The researchers synthesized two related series of ligands, one based on 2-aminoimidazoline (Figure 12, top row) and the other based on guanidine (bottom row). Almost all compounds synthesized were full agonists; however, one analogue in each series unexpectedly displayed antagonist activity. In the 2-aminoimidazoline-based series, the N,N-dimethylaniline derivative 21 was an antagonist, while the very closely related derivatives 22−24 were all full agonists. In the guanidine-based series, the N-ethylaniline derivative 25 was an antagonist, while 26−28 were full agonists. The functional activity SAR of these compound series is difficult to elucidate, as the two antagonists appear to more closely structurally resemble the agonists in the respective series than they do each other.28 It is apparent, however, that even very subtle structural changes in ligands can result in large changes in functional activity

Figure 12. Minor differences in alkyl substitution can alter the functional activity of ligands at the α2-adrenergic receptor. F



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Figure 13. Intrinsic activity at the MT1 and MT2 receptors diminishes with the size of substituents on aniline moieties.

Figure 15. Increasing the distance between the imidazole and piperidine moieties of immepip decreases functional agonism at the H3 receptor. Intrinsic activity (IA) is reported relative to the full agonist histamine.

Figure 14. Shifting the guanidinium group from the 5′- to 6′-position of naltrindole switches functional activity at the κ-opioid receptor.

interactions with Glu297 may play a unique role in receptor function, as the helix it is located on is adjacent to the inner loop 3 (IL3) that is involved in G-protein activation. One hypothesis is that conformational variations in TM6 would contribute to agonist vs antagonist effects, in which movement of TM6 would in turn affect the configuration of IL3; interactions between different ligands are therefore likely to result in different conformations of TM6 due to the positioning of the 5′- or 6′-guanidinium groups. 2.4.2. H3 Agonism Decreases as the Distance between Two Pharmacophores Increases. In 2003, the Leurs group at Vrije University in The Netherlands, working with collaborators in Belgium and Thailand, identified a set of ligands whose functional activity changed significantly depending on the distance between two key pharmacophoric features.31 These researchers were exploring derivatives of immepip as potential modulators of the histamine H3 receptor. Immepip is a histamine analogue comprised of a piperidine moiety linked to an imidazole by a one-carbon tether (Figure 15) and is a potent full agonist at the H3 receptor with pKi = 9.32 in a radioligand binding assay. Adding an extra methylene to the tether of immepip yields a partial agonist (34) that is significantly less potent than immepip in the binding assay (pKi = 7.70). However, increasing the distance between the imidazole and piperidine pharmacophores by an additional methylene unit furnishes 35, which is more potent than 34 in a binding assay (pKi = 8.35) as well as an antagonist. Leurs and co-workers also synthesized a series of immepip analogues where a 3-piperidinyl group was separated from an imidazole by a carbon chain of varying length (Figure 16), and these workers evaluated compound activity at the H3 receptor in binding and functional assays. They found the same trend of

Figure 16. H3 receptor agonism decreases with chain length in a series of 3-piperidinyl analogues of immepip.

decreasing agonism with increasing chain length that had previously been observed with the 4-piperidinyl analogues. In addition, the relative rank order of potency in a binding assay was the same in both cases (1 carbon chain >3 > 2), although the 3-piperidinyl analogues were consistently less potent than the 4-piperidinyl compounds. 2.4.3. Functional Activity at the AT 2 Receptor is Determined by the Distance between Two Pharmacophores. Working with collaborators from the University of Sherbrooke in Quebec, Canada, the Alterman group at Uppsala University in Sweden explored the dependence of functional activity on pharmacophoric feature distances for a group of AT2-targeted ligands. These researchers were interested in AT2 receptor agonists as potential anti-inflammatory agents and as therapeutics for improving heart function after myocardial infarction.32 In addition, an AT2 receptor antagonist has shown efficacy in a phase II clinical study against neuropathic pain.33 In a study published in 2012, the Alterman group found that G



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most straightforward hypothesis, the altered position of the methylene imidazole substituent in 40 leaves this group in a poor position to stabilize the active conformation of the receptor. Alternatively, and perhaps less obviously, the methylene imidazole substituents of 39 and 40 could bind to the same location on the receptor, forcing the rest of the scaffold, especially the lipophilic isobutyl groups, to shift position. Initially, the primary evidence supporting the second hypothesis was a 1997 study showing that in a set of ligands structurally related to 39, a small change in alkyl substitution (isobutyl to ethyl) was sufficient to transform functional activity at the AT1 receptor (Figure 18).35 Alterman and co-workers suggested that the binding interaction between the lipophilic side chain and the AT2 receptor may be an important determinant of functional activity, as it is with the AT1 receptor. To test these two hypotheses, the researchers synthesized a set of ligands for the AT2 receptor, including the regioisomers 41−43 (Figure 19). In biphenyl 41, the methylene imidazole moiety and the lipophilic isobutyl side chain are found in similar locations and exhibit similar orientations as they do in para-substituted compound 39. Unsurprisingly, a functional assay determined that biphenyl 41 is an agonist. Similarly, in biphenyl 42, the relative positions of the methylene imidazole and isobutyl groups are roughly the same as in meta-substituted analogue 40, yielding an antagonist. Up to this point, both hypotheses hold up fairly equally concerning which group has a greater effect on functional activity, the methylene imidazole moiety or the isobutyl group. However, when the methylene imidazole remains in the meta-position on the top ring of the biphenyl but the isobutyl is shifted to the para position in the bottom ring, the resulting compound 43 is an agonist at the AT2 receptor. This result clearly demonstrates that moving the methylene imidazole moiety to the meta-position of the top phenyl ring is not in itself enough to change functional activity from agonist to an antagonist. The authors therefore concluded that the key structural feature that is responsible for determining functionality is not the location of the isobutyl and the imidazole pharmacophoric elements themselves but rather the relative spatial relationship between them. 2.4.4. Swapping a Benzyl for a Phenyl Transforms the Functional Activity of a Ligand. The next two examples discussed in this section feature a common theme: the addition of a methylene between two aromatic rings results in a more flexible ligand while at the same time converting an agonist into an antagonist. This situation is analogous to that discussed in Section 2.1.4, where a methylene added between a basic amine

the AT2 receptor agonist 39 could be converted into AT2 receptor antagonist 40 by migrating the methylene imidazole substituent from the para to the meta position on the central benzene ring (Figure 17).34 This result is analogous to that

Figure 17. In a pair of AT2 receptor ligands, para-substitution leads to an agonist while meta-substitution yields an antagonist.

reported by the Portoghese group with GNTI derivatives (Section 2.4.1).30 Subsequently, these researchers chose to revisit this class of ligands to better elucidate the determining factors of agonist/ antagonist functional activity. In a recently published study, the authors presented two distinct hypotheses that could potentially explain the data from their 2012 paper.32 In their

Figure 18. Lipophilic side chain plays a key role in determining functional activity at the AT1 receptor.35

Figure 19. Spatial relationship between the imidazole and the isobutyl groups contributes to determining AT2 functional activity.

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flipped from antagonism to agonism. However, these researchers found that 48, the benzyl analogue of phenyl derivative 47, was an α2 receptor antagonist. A similar difference in functional activity between phenyl and benzyl analogues was also found in compounds incorporating a methyl on the oxymethylene bridge of 46, as phenyl derivative 49 was an agonist, while benzyl analogue 50 was an antagonist (Figure 22). Pigini and co-workers also synthesized the metaand para-substituted regioisomers of 49 and determined that the effect of adding an extra phenyl on functional activity depended on its point of attachment, as the meta-substituted analogue 51 was a partial agonist while para-substituted analogue 52 was an antagonist. Related examples of how addition of a phenyl group alters functional activity when introduced at a strictly specific location are discussed in Section 2.8. 2.5. Adding or Removing a Hydrogen Bond. One of the most effective ways to change the functional activity of a ligand is to add a hydrogen bonding moiety; however, determining the optimal location for substitution continues to pose a challenge. To date, the most common (though not the most heralded) strategy for finding good attachment points has been trial and error, although as additional X-ray crystal structures of GPCRs interacting with ligands become available,2,4,38 targeted modification of hydrogen bonding interactions will undoubtedly become more feasible. Examples of using structural biology to guide the interconversion of agonists and antagonists with receptors other than GPCRs are already present in the literature. For instance, scientists at GlaxoSmithKline used structural information to identify a key hydrogen bond between the acid group of agonist GW409544 (Figure 23) and the Y464 residue of the PPARα nuclear receptor, an interaction that proved essential for stabilizing the receptor in its active conformation.39 These results were then applied to the design of compound GW6471 (Figure 23), which is incapable of forming this hydrogen bond and is therefore an antagonist at PPARα. 2.5.1. Converting a GPR119 Antagonist into an Agonist by Adding a Hydrogen Bond Acceptor. An example of adding a hydrogen bond acceptor to change the functional activity of a ligand was published by Semple and co-workers at Arena Pharmaceuticals, who used this approach to identify smallmolecule agonists of the GPR119 receptor as potential therapeutics to treat diabetes.40 A high-throughput screen of Arena’s compound library identified compound 53 as a modulator of the GPR119 receptor, but unfortunately it was an inverse agonist rather than the desired agonist (Figure 24). In initial optimization studies, the trifluoromethyl pyrazole of 53 was replaced by a series of aryl ethers, leading to a family of reasonably potent inverse agonists such as 54. However, when a hydrogen-bond accepting group was incorporated at the 4-position of the aryl ring, a series of agonists including 55 was

and a cyclooctyl ring converts the nociceptin receptor agonist 11 into antagonist 10. In the first example, Wallez and co-workers investigated a series of benzofuran analogues designed to function as bioisosteres of melatonin.36 This work included synthesizing a variety of 2-substituted benzofurans such as phenyl derivative 44, which is a nonselective, highly potent agonist of both the MT1 and MT2 melatonin receptors (Figure 20). As the overall

Figure 20. Phenyl for benzyl transformation altered functional activity at both the MT1 and MT2 receptors and improved selectivity for MT2.

goal of this research program was to develop MT2-selective ligands, additional compounds were prepared including benzyl derivative 45. This compound showed much better MT2 selectivity in binding assays but displayed antagonist activity at the MT2 receptor. The phenyl to benzyl substitution also affected functional activity at MT1. At this receptor, 45 acted as a partial agonist with an Emax of 48% (compared to the full agonist melatonin). The second example involved ligands for the α2-adrenoreceptor. In this case, Maria Pigini and a group of collaborators in Italy and France synthesized a set of analogues of imidazoline 46 with the purpose of identifying derivatives with improved selectivity for α2 over the α1 and I2 imidazoline receptors (Figure 21).37 They discovered that by introducing a phenyl ring at the ortho position of 46, α2 functional activity was

Figure 21. Attaching a phenyl group to the ortho position of 46 furnished an agonist, while the benzyl analogue was an antagonist.

Figure 22. Same phenyl/agonist benzyl/antagonist pattern was seen with derivatives 49 and 50. Functional activity also varied depending on the regiochemistry of the biphenyl analogues. I



Perspective

Figure 23. Structural information was used to identify which hydrogen bond donor to remove to convert a PPARα agonist into an antagonist.

Figure 24. Optimization of initial screening hit 53 led to compounds such as inverse agonist 54. Incorporation of a hydrogen-bond acceptor at the 4position of the phenyl ring yielded agonist 55.

Figure 25. Removal of the hydrogen bonding ethanone moiety of GPER agonist 56 leads to antagonist 57. Further optimization led to selective GPER antagonist 58.

(Figure 25).42 With a selective agonist now in hand, the investigators also sought to obtain a GPER antagonist as a probe to use in their studies. Hypothesizing that the ketone moiety of 56 might be engaging in key hydrogen bonds facilitating activation of the GPER receptor, the researchers subsequently synthesized 57, an analogue lacking this functional group.41 A radioligand binding assay demonstrated that 57 exhibited similar affinity (20 nM) for GPER as compound 56 (7 nM), while functional assays showed that 57 was an antagonist. In a later study, the same researchers synthesized 58, which was also a GPER antagonist but exhibited improved selectivity over other estrogen receptors. As 56 and 58 are similar in overall size, this result provides evidence that the switch in functional activity from agonist to antagonist occurred due to removal of a hydrogen bonding interaction rather than because of steric effects.43 2.5.3. Adding a Hydrogen Bond Donor Switches Functional Activity in a Family of 5-HT7 Ligands. Leopoldo and colleagues at the University of Bari in Italy discovered another instance of how altering hydrogen bonding properties can change the functional activity of ligands at a GPCR. These researchers were investigating a series of arylpiperazine derivatives (Figure 26) as potential modulators of the 5-HT7 receptor,44 which is a therapeutic target for a range of diseases

obtained. Further SAR work confirmed that the most favorable agonist activity was obtained when the hydrogen-bond accepting group was attached to the 4-position of the phenyl group instead of at the 2- or 3-positions. Notably, agonist activity was unpredictable, as not all compounds synthesized with a hydrogen-bond acceptor attached at the 4-position were agonists. 2.5.2. Removing a Hydrogen Bond Acceptor Converts a GPER Agonist into an Antagonist. While finding the right location to add a hydrogen-bond acceptor or donor to alter functional activity at a receptor can be challenging, it is also possible to effect this change by removing an existing hydrogenbonding functionality. A major advantage of this technique compared to adding hydrogen-bonding functionality is that potential locations for such alterations are more limited. Prossnitz and co-workers at the University of New Mexico used this method to identify an antagonist of G protein-coupled estrogen receptor 1 (GPER), also referred to as GPR30. The elucidation of the multiple physiological roles that this receptor plays has been complicated by the frequent interaction of GPER ligands with other estrogen receptors.41 As a tool to aid in better understanding the in vivo actions of GPER, these researchers developed 56 as an agonist of GPER, which displayed good selectivity over classical estrogen receptors J



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Figure 26. 2-hydroxy derivative 59d was an antagonist at the 5-HT7 receptor, while related analogues were all agonists.

from depression45 to inflammatory diseases.46 The authors found that the most potent compounds were all substituted at the 2-position of the aryl ring connected to the piperazine. Surprisingly, while most of the 2-substituted analogues were agonists, the 2-hydroxy derivative proved to be an antagonist. The researchers hypothesized that this difference was due to the hydrogen bond donating capacity of this substituent. 2.5.4. Adding a Hydrogen Bond Acceptor Changes Functional Activity in a Set of 5-HT1A Ligands. Interestingly, the above example is not the only instance where altering the hydrogen-bonding capability of the substituent at the 2-position of the aryl group results in modifying the functional activity of a family of arylpiperazine-based ligands at a serotonin receptor. Monge and co-workers in Spain and Austria encountered a similar occurrence while studying modulators of the 5-HT1A receptor. These workers were interested in this receptor because of its connection to the delayed onset of activity of SSRIbased antidepressants.47 After synthesizing a set of arylpiperazine derivatives with different aryl substituents, they evaluated the activity of the resulting compounds in radioligand binding and GTPγS functional assays. While many of the resulting compounds had favorable binding affinity in the nanomolar range, the functional activity varied from antagonists like 60 to agonists such as 61 (Figure 27). The authors suspected that differences in the hydrogen bonding capabilities of the ligands accounted for their different functional activities. Pharmacophoric analyses supported this theory; the researchers used the CATALYST pharmacophore hypothesis package to generate a series of models based on (a) the common feature pharmacophore of all 19 5-HT transport inhibitors reported in the manuscript, and (b) common feature models starting from four potent agonists and the five partial agonists reported. The first model was initially able to return 18 out of 19 active compounds reported; after refinement and removal of one feature, it retrieved all 19 actives. From among the models obtained in (b), the best antagonist pharmacophore hypothesis consisted of six key features: two hydrophobic functionalities, one positively charged feature, one H-bond donor, one H-bond acceptor, and one aromatic ring. In a computational database filtering study,

Figure 27. Variations in hydrogen bonding lead to different functional activities in compounds with potent biological activities.

the researchers found that this model accurately returned only receptor antagonists. The five-featured partial agonist model was also capable of returning partial agonists but with somewhat lower fit values, indicating that it was less selective overall than the antagonist model. In all models, hydrogen bonding was found to play a central role. 2.5.5. Fine-Tuning Hydrogen Bonding Capabilities of a Heterocyclic Substituent Alters Functional Activity in a Set of C3a Receptor Modulators. Reid and co-workers at the University of Queensland in Australia identified a series of modulators of the C3a receptor whose functional activity varied with the hydrogen bonding capacity of particular substituents. The C3a receptor plays an important role in regulating an inflammatory response in humans.48 The researchers synthesized a set of ligands that contained a variable five-membered ring heterocycle connected to a diphenylmethane on one side and an arginine on the other (generic structure 62 in Figure 28). They found that the binding affinities of the resulting compounds correlated well with the calculated H-bond interaction energy between water and the heteroatom of the incorporated heterocycle, with the most potent compounds possessing the most favorable hydrogen-bonding moieties.48 Functional assays revealed both agonist and

Figure 28. A series of compounds with the generic structure 62 were synthesized. Several, like imidazole derivative 63, are agonists, while others such as furan 64 are antagonists. K



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Figure 29. Diverse functional activity at the 5-HT4 receptor of a family of pyrrolizidine compounds may reflect differences in the ability to form strong intramolecular H-bonds.

antagonists among the active compounds. After further SAR studies involving methylated heterocyclic derivatives, the authors determined that the functional activity correlated well with the H-bond acceptor ability of the substituent at position X in the generic structure 62. For instance, imidazole analogue 63, which can form a strong H-bond at the nitrogen in the X position, is an agonist, while furan 64, whose oxygen at position X is a weaker H-bond acceptor, is an antagonist. 2.5.6. A Potential Case of Switching Receptor Functionality by Altering Intramolecular Hydrogen Bonding in a Ligand. The examples discussed above illustrate how functional activity can change due to altered ligand−receptor hydrogen bonding interactions. However, hydrogen bonding within a ligand can change its preferred conformation, which in turn may lead to different functional activity at a receptor. One potential example of this phenomenon was published in 2006 by Becker and colleagues at Pfizer, who explored ligands targeting the 5-HT4 receptor as potential therapeutics for gastrointestinal motility disorders.49 In their initial research, these workers identified pyrrolizidine amide 65 (Figure 29) as a potent partial agonist with Ki = 5.2 nM in a binding assay and 80% efficacy in a 5-HT4 functional assay (as compared to the full agonist serotonin). Becker concluded that the methoxy group of 65 may form a key intramolecular hydrogen bond with the amide NH in that compound, helping to maintain a planar relationship between the amide and the aromatic ring of the ligand. In turn, this planar relationship was thought to be crucial for maintaining agonist efficacy. As evidence supporting this hypothesis, he noted that the ester analogue 66, which is not capable of forming such an intramolecular hydrogen bond, showed reduced efficacy (57%) compared to 65 in a functional assay despite improved activity in a binding assay. The researchers then synthesized a set of heterocyclic analogues of 65. One of the most potent of the resulting compounds was imidazopyridine 67, which was designed to maintain an intramolecular hydrogen bond between the amide NH and the imidazopyridine. However, this molecule acted as an antagonist in functional assays. It is unclear if this result is due to the decreased ability of the imidazopyridine moiety to form a strong intramolecular hydrogen bond or if that moiety instead engages in another type of interaction with the receptor, thereby stabilizing the receptor in an inactive state. 2.6. Changing a Stereocenter. 2.6.1. One Enantiomer Yields Agonists and the Other Results in Antagonists in a Family of 5-HT6 Receptor Modulators. One common and straightforward method for switching the functional activity of a ligand is to change the stereochemistry around one or more chiral centers. A dramatic instance of stereocenters affecting functional activity was published by Derek Cole and co-workers at Wyeth in 2005.50 These researchers synthesized a family of 5-arylsulfonamido-3-(pyrrolidin-2-ylmethyl)-1H-indoles

Figure 30. All pyrrolidine analogues containing an (R)-stereocenter acted as 5-HT6 agonists, while those with an (S)-stereocenter were mostly antagonists.

(Figure 30) and found that many of the compounds displayed good activity at the 5-HT6 receptor. As the pyrrolidine moiety in these compounds contains a chiral center, both enantiomers were synthesized. After testing the resulting compounds in 5-HT 6 binding and functional assays, one interesting correlation emerged: all compounds containing an (R)stereocenter tested in functional assays were agonists, while all but one of the analogues with a (S)-stereocenter were antagonists (the one exception was a weak partial agonist). 2.6.2. Flipping a Chiral Center Switches Functional Activity at the 5-HT1A Receptor. Ao Zhang and co-workers were investigating modulators of another serotonin receptor: 5-HT1A. Modulators of this receptor have been extensively studied for their potential to treat a range of psychiatric conditions including anxiety and depression.51 Zhang and coworkers synthesized a set of aporphine derivatives designed to be potent at the 5-HT1A receptor while minimizing dopamine receptor activity. Their most potent compound 69 was originally synthesized as a mix of cis and trans diastereomers and showed antagonistic activity when evaluated in a GTPγS functional assay (Figure 31). However, after separating the two

Figure 31. Changing the configuration at one chiral center alters the functional activity of aporphine derivative 69 at the 5-HT1A receptor.

diastereomers by chiral column and using X-ray crystallography to determine the absolute configuration of the compounds, Zhang discovered that the cis-isomer of 69 was a 5-HT1A L



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Figure 32. CCK agonist (S)-70 could be converted into an antagonist either by inverting its chiral center (red) or by replacing the isopropyl group on its amide with a methyl (blue).

Figure 33. (3S,4R)-Diastereomers of a pair of trans-4-phenylpyrrolidine-3-carboxamides (72a and 73a) were agonists, while the (3R,4S)diastereomers 72b and 73b were antagonists.

agonist while the trans-isomer was an antagonist. Zhang’s research also clearly demonstrates the pitfalls of evaluating mixtures of compounds in functional assays, as the cis/trans mixture of 69 did not have the same functional activity as its cis component. 2.6.3. A Pair of Enantiomeric Photoaffinity Probes Exhibit Different Functional Activities at the CCK Receptor. Laurence Miller and colleagues at the Mayo Clinic, Vanderbilt University, Glaxo-SmithKline, and CGI Pharmaceuticals prepared a set of agonist and antagonist photoaffinity probes in order to investigate the binding locations of these probes at the type A cholecystokinin receptor.52 One of these probes, compound 70, was originally synthesized as a racemic mixture that had overall agonist activity at the CCK receptor. However, when the enantiomers of 70 were separated, the (S)-enantiomer was an agonist while the (R)-enantiomer was an antagonist (Figure 32), underscoring the risk of assessing the functional activity of racemic compound mixtures. The researchers also identified a second small modification that could change functional activity: replacing the isopropyl group attached to the amide of (S)-70 with a methyl resulted in an antagonist. Computational models were also constructed for ligand−CCK receptor complexes using the agonist and antagonist photoaffinity probes and were generally in good agreement with experimental photoaffinity labeling data. As a starting point for modeling, the researchers used previously obtained models of receptor−peptide complexes. Docking calculations supported the importance of the key leucine residue (Leu88) that had been covalently labeled by the antagonist probe compound, and

these simulations also pinpointed two key intermolecular hydrogen bonds with Asn334 and Ser364. 2.6.4. Diastereomers Exhibit Different Functional Activities at the MC4 Receptor. In the cases discussed above, altering the configuration around one chiral center was sufficient to transform functional activity. However, in other systems, changing multiple stereocenters may be necessary to achieve the same effect. For example, Chen and co-workers at Neurocrine, who were looking for selective melanocortin-4 (MC4) receptor agonists as potential treatments for obesity,53,54 synthesized a set of trans-4-phenylpyrrolidine-3-carboxamides and evaluated the resulting compounds in binding and functional assays (Figure 33). They found that the (3S,4R)diastereomers of compounds 72 and 73 were agonists, while the (3R,4S)-diastereomers were antagonists. Interestingly, a mixture of the two trans-73 compounds acted as a potent full agonist (IA = 121%) while the combined trans-72 compounds displayed only partial agonist activity (IA = 35%). The investigators at Neurocrine also synthesized a compound where only one chiral center was modified, a cis4-phenylpyrrolidine-3-carboxamide analogue of 73 where the N-tetrahydropyran moiety was replaced with an i-Pr. However, this compound proved to be 20-fold less potent in a binding assay than its trans analogue (90 nM compared to 4.7 nM) and its functional activity was therefore not determined. 2.7. Conformational Restriction. 2.7.1. Identifying the Bioactive Conformation of an Agonist and Antagonist at an Opioid Receptor. Another technique that can be used to interconvert agonists and antagonists is altering the extent of M



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Figure 34. Constrained analogues of 74 were prepared. Dotted lines represent bonds that were synthesized with different stereochemistries.

conformational restriction in a molecule. This can involve a number of approaches, including rigidifying a flexible molecule or locking an already constrained molecule into a different position. This technique is widely used in SAR studies because a significant increase in potency may be elicited if a molecule is locked in its bioactive conformation. In addition, an increase in selectivity can also occur if the resulting molecule is locked out of a conformation necessary for activity at an off-target receptor.55 A major advantage of altering conformational constraints to change functional activity is that this technique can help to determine differences in the bioactive conformations of agonist and antagonist ligands. In 2006, Bertrand Le Bourdonnec and colleagues at Adolor Corporation and the University of Delaware56 sought to determine the bioactive conformation of the previously identified μ-opioid antagonist 74 (Figure 34). The phenethyl side chain of 74 is highly flexible; therefore, it was not possible to readily identify how this moiety binds the μ-opioid receptor relative to the piperidine core of the molecule in the absence of structural biology data. To better elucidate the active conformation of 74 as an antagonist of the μ-opioid receptor, these scientists synthesized four analogues of 74 (differing in their stereochemistry) where the benzylic position of the phenethyl moiety was linked by two carbons to the 2-position of the piperidine (structure 76 in Figure 34). They then synthesized four additional analogues where the benzylic position of the phenethyl moiety was linked to the 6-position of the piperidine instead (structure 75). While most of the resulting compounds showed significantly reduced potency compared to 74 in a binding assay at the μ-opioid receptor, one analogue from each set showed improved potency: compounds 77 (Ki = 0.62 nM) and 78 (Ki = 0.90 nM). However, in functional assays, 77 was an antagonist while 78 was an agonist (Figure 35). The researchers hypothesized that opioid receptor binding affinity as well as antagonist activity depended on trans positioning of the 3- and 4-methyl moieties on the central piperidine ring and that pure antagonist activity was dependent upon equatorial conformation of the hydroxyphenyl group at opioid receptors. Conformational analyses were subsequently performed to identify global energy minima for compounds 74, 77, and 78. These simulations supported the above hypotheses and also indicated that very small changes in ligand structure and conformation led to fundamental changes in binding affinity as well as efficacy and agonist versus antagonist activity. 2.7.2. Reducing Flexibility Converts an A3 Adenosine Receptor Agonist into an Antagonist. In the example

Figure 35. One of the constrained analogues of 74 was a potent antagonist, while another was a potent agonist.

discussed above, conformational restriction was used to lock a ligand into active agonist and antagonist conformations. This approach can also be used to lock out a ligand from certain orientations, with the result that key interactions responsible for causing either agonism or antagonism are lost. Kenneth Jacobson and co-workers published an example of this phenomenon in 2002.57 These researchers examined methods to convert agonists of the A3 adenosine receptor into antagonists. They were able to effect this transformation by incorporating the freely rotating amide group of agonist 79 into a five-membered ring, obtaining antagonist 80 (Figure 36).

Figure 36. Restricting the free rotation of an amide group transformed an A3 adenosine receptor agonist into an antagonist.

Computer modeling was subsequently used to support the hypothesis that this difference in functional activity was due to loss of a H-bonding interaction with receptor residue T94: the flexible alkylamide of 79 could engage in this interaction, but N



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Figure 37. Chlorination at the 2-position of adenine reduces functional agonism, except in the case of compound 85.

Figure 38. Varying functional activities at H4 due to structural modifications and removal of constraints. Notably, removing the constraining bonds in red from antagonist 87 results in agonist 88.

the rigid pyrrolidine-based moiety of 80 could not. Loss of this hydrogen bond may also explain why compound 80 (Ki = 29.3 nM) binds to the A3 adenosine receptor with lesser affinity than 79 (Ki = 1.8 nM). Jacobson et al. also discovered a different method to convert A3 adenosine receptor agonists into antagonists: by adding a lipophilic chlorine group at the adenine 2-position (Figure 37). This addition converted agonist 81 into antagonist 82, as well as partial agonist 83 into antagonist 84. However, the ability of the added halogen to reduce agonism was influenced by other substituents on the ligand, as compound 85, the 2-Cl derivative of 79, proved to be a full agonist. 2.7.3. Removal of a Constraint Turns an H4 Antagonist into an Agonist. Brad Savall and co-workers at Johnson & Johnson uncovered an interesting example of how conformational constraints can alter the functional activity of ligands while working on identifying histamine H4 receptor modulators as possible therapeutics for inflammatory diseases.58 The starting point for their study was compound 86, which was a partial agonist at the H4 receptor (Figure 38). Replacing the N-methylpiperazine of 86 with a series of constrained diamines led to ligands with a wide range of activity at the H4 receptor, with small variations in constraints leading to large differences in binding and functional activity. For example, the 5,6-pyrrolopiperidine derivative 87 was an H4 antagonist (Ki = 14 nM), while its enantiomer showed negligible activity (Ki = 691 nM). Most interestingly, removing part of the pyrrolidine moiety from 87, allowing for more rotational freedom, resulted in compound 88, a potent full agonist. 2.8. Adding an Aromatic Group. 2.8.1. Using a Trigger Identified while Studying Peptides. Many of the structural alterations leading to functional changes featured in this review were discovered unexpectedly or via trial and error approaches. In contrast, the Miura group took advantage of an

agonist/antagonist trigger they had identified in a study of peptide/receptor interactions to convert a small-molecule antagonist into an agonist. In earlier studies, these researchers determined that an interaction between Tyr4 of the octapeptide angiotensin II and Asn111 of the AT1 receptor was critically important in order to activate the receptor.59 The Miura team used mutations of both the AT1 receptor and the angiotensin II octapeptide to determine that this effect was likely due to an amino-aromatic bonding interaction60 between the two residues. While the initial investigations conducted by the Miura group into the factors that determine AT1 receptor functional activity were conducted using peptide ligands, more recently these researchers shifted their focus to small-molecule ligands. As a starting point for their study, Miura et al. used the known AT1 inverse agonist olmesartan (Figure 39).61 Replacing the carboxyl group of olmesartan with a carbamoyl yielded the neutral agonist 89. Subsequently, the researchers derivatized this compound, incorporating the 4-hydroxybenzyl group of the tyrosine agonist trigger into olmesartan and 89. Interestingly, while the addition of this moiety converted the carbamoylcontaining neutral antagonist 89 into an agonist (90), it was not sufficient enough to effect a functional change when added to the carboxyl-containing parent inverse agonist olmesartan as the resulting compound 91 was also an inverse agonist. Molecular modeling, guided by site-directed mutagenesis experiments, confirmed that key conformational changes take place in transmembrane helix 3 (TM3) upon olmesartan binding, and that ligand-induced changes in TM3 conformation affect GPCR activation and therefore agonist versus antagonist activity. Putative binding modes of various ligands in the AT1 receptor generated in silico indicated that binding of 90 not only disrupts ligand−receptor interactions that stabilize the inactive state of the enzyme but also stabilizes key receptor O



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Figure 39. Changes to the olmesartan scaffold can yield agonists, neutral antagonists, or inverse agonists of the AT1 receptor.

identification of FR165649 as a bradykinin B2 antagonist (Figure 41).64−66 The researchers were also interested in obtaining nonpeptide B2 agonists and were able to convert their starting antagonist into a full agonist by making several staged modifications. They began by replacing the hydrogen at the quinolone 4-position with an ethoxy group, resulting in a compound (95) that was a partial agonist but which only elicited an increase in inositol phosphates formation of about one-tenth the maximum effect produced by bradykinin. From there, they substituted the 4-position of the quinoline with the larger 2-pyridylmethoxy group in place of the ethyoxy. The resulting compound, 96, was more potent in a binding assay with increased, though still partial, agonistic activity (59.5% of maximum effect). After further optimization of the rest of the scaffold, they were able to identify the potent, full agonist 97. 2.8.4. Replacing an Isopropyl with a Phenyl Switches Functional Activity at the CB2 Receptor. Interestingly, both agonists and antagonists of the CB2 receptor have been investigated as potential anti-inflammatory agents. As part of a research program studying modulators of this receptor, Corelli and co-workers synthesized a series of 6-substituted 4quinolone-3-carboxamides (Figure 42).67,68 Many of the resulting compounds were potent in binding assays, with good selectivity for CB2 over the related CB1 receptor. However, small differences in substitution at the 6-position led to major differences in functional activity. For instance, in an in vivo study, isopropyl derivative 98 demonstrated analgesic behavior consistent with a CB2 agonist,67 while phenyl analogue 99 was an inverse agonist in a GTPγS functional assay.69 One noteworthy aspect about the series of papers from Corelli and co-workers is that they highlight how hazardous it can be for readers of an article to infer functional activity SAR when only binding data are presented. Compound 99 was originally disclosed in a 2008 paper entitled “Synthesis and Structure−Activity Relationship of Potent and Selective Cannabinoid-2 Receptor Agonists Endowed with Analgesic Activity in Vivo”.67 On the basis of the article title, and the fact that the closely structurally related compound 98 was disclosed to be an agonist, a researcher interested in CB2 modulators in 2008 could be excused for assuming that 99 was also an agonist. It was not until two years later, in a paper focused on describing the pharmacology of 99, that the inverse agonist behavior of this compound was disclosed.69 2.9. Structural Changes near an Amide Bond. 2.9.1. MC4 Receptor Revisited. Another technique that can lead to alterations in functional activity is changing amide bond participants. This approach has the major advantage that large

conformations in the active (agonist) state. In this case, the subtle yet specific variations in stabilization of TM3 by olmesartan vs 90 were sufficient to result in differences in agonist activity. 2.8.2. Adding Phenyl Groups Switches Functional Activity at the M2 and M3 Muscarinic Receptors. Piergentili and colleagues in Italy reported a similar example of using a known trigger to alter ligand functional activity, working with muscarinic receptor modulators. These researchers had previously synthesized compound 92, which is an agonist at the M2 and M3 receptors and bears a methyl group at the 6-position of a central 1,4-dioxane ring (Figure 40).62

Figure 40. Added steric bulk at the 6-position converts agonist 92 into antagonist 94.

Piergentili then set out to identify analogues of 92 that were antagonists at those receptors as possible therapeutics for chronic obstructive pulmonary disease, overactive bladder, and irritable bowel syndrome. Previously published studies on other muscarinic agonists such as ACh or muscarine had shown that replacing a methyl substituent with a bulkier group could lead to antagonists; therefore, Piergentili and co-workers synthesized analogues of 92 in which the methyl group was replaced with larger substituents.63 The 6-monophenyl analogue of 92, compound 93, was indeed an antagonist at both the M2 and M3 receptors but exhibited only weak activity in functional assays. These researchers, however, were able to significantly increase antagonist activity by adding additional steric bulk at the 6-position, as diphenyl derivative 94 was a more potent antagonist at both receptors. Interestingly, the same trend of functional activity also held true for the enantiomers of the compounds 92 and 94, as (2S,6R)-92 was an agonist at both receptors while (S)-94 was an antagonist. 2.8.3. From Antagonists to Partial Agonists to Full Agonists at the Bradykinin B2 Receptor. The human kinin bradykinin is an endogenous nonapeptide that is a potent agonist of the bradykinin B2 receptor. As bradykinin has strong proinflammatory properties, modulators of the B2 receptor have been investigated for their potential use as therapeutics for conditions such as asthma and hyperalgesia.64 Initial SAR studies by scientists at Fujisawa Pharmaceutical Co. led to the P



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Figure 41. B2 functional activity varied with substituents on the quinoline ring 4-position (in red) and was further optimized by modifying the right side of the molecule (in blue).

An elegant illustration of this technique was reported by Chen and co-workers at Neurocrine, the same group of researchers who modified chiral centers to alter functional activity at the MC4 receptor (Section 2.6.4). Returning to MC4 receptor modulators, the Chen group synthesized a library of amides including those in Figure 43.70 These amides ranged in functional activity from the full agonist 100a to partial agonist 100c (IA = 17%, with antagonist activity against α-MSH). Notably, Chen et al. found that while modifying the substituents on the amide portion of the molecule (highlighted in red in Figure 43) significantly affected functional activity, changing the substituents at the 2-position of the aniline (highlighted in blue) did not: a library of 16 analogues bearing alkylamines on the blue-colored region in Figure 43 and a Ticgroup on the red-colored area were all agonists with high intrinsic activities (>80%).

Figure 42. Changing an isopropyl to a phenyl moiety alters CB2 functional activity.

libraries of amides are often relatively synthetically accessible. Examples of this method have been discussed previously in Sections 2.5.5 and 2.5.6.

Figure 43. Changes in the red portion of the scaffold strongly affected functional activity at the MC4 receptor, while changes in the blue portion showed little effect. Q



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Contemporaneously, researchers at Amgen investigated similar molecules, with one of the amides of the scaffold replaced by a reverse amide and incorporating an additional methylene (Figure 44).71 These ligands also showed significant

changes in MC4 functional activity, from full agonism to antagonism depending on the amide substituent (highlighted in red), again underscoring the importance of interactions between the right (red) side of the scaffold and the MC4 receptor in determining functional activity. The researchers at Amgen also saw no sign of variation in functional activity with changes in substitution at the 2-position of the aniline (highlighted in blue), although it should be kept in mind that only a single pair of direct analogues was prepared. 2.10. Changes in Aromatic Substitution. 2.10.1. Cannabinoid Receptor Modulators. While modifications to ligands causing changes in functional activity at GPCRs are frequently found in close proximity to well-defined structural features such as basic amines or chiral centers, this is not always the case, and it remains difficult to predict which location(s) in a particular system will be most sensitive to modifications affecting agonism and/or antagonism. In 2012, Rempel and coworkers synthesized a series of 7-alkyl-3-benzylcoumarins, with a variety of substitutions on the benzyl ring, in an attempt to find ligands that were potent and selective for the CB1 and CB2 receptors (Figure 45).72 The o-methyl derivative 103 was found to be potent antagonist at the CB1 receptor, with a Ki of 22 nM in a binding assay. It also acted as a partial agonist at CB2 but with weaker binding affinity (Ki = 405 nM). An o-chloro analogue also displayed a similar activity profile. In contrast, however, the o-methoxy derivative 104 was a full and potent agonist at both the CB1 (Ki = 32 nM) and the CB2 (Ki = 22 nM) receptors. It would be interesting to examine the functional activity of an o-ethyl analogue of 103 and 104 to help determine whether the observed changes in activity are due to the increased size of the methoxy derivative and/or due to another factor such as either the strong electron donating ability of the methoxy group or hydrogen bonding between the receptor and the methoxy.73 However, no such derivative has been reported to date. 2.10.2. How an Added Methoxy Group Changes the Functional Activity of a Ligand at the Ghrelin Receptor Depends on the Substitutions Elsewhere on the Molecule. Working from a peptide starting point, Fehrentz and coworkers developed a ligand for the ghrelin receptor containing a central 1,2,4-triazole scaffold linked to three major pharmacophores: two indole rings and a basic amino group coupled to the rest of the scaffold via an amide bond (Figure 46). In an initial manuscript featuring these molecules, an α-aminoisobutyryl moiety (shown in blue in Figure 46) was utilized as their basic amino group, yielding compounds including 105 and 106.74 This amino group is a common structural component of many known small molecule ghrelin receptor modulators.75 Most of the analogues featured in the manuscript

Figure 44. Modifications to the red portion of the scaffold again determined functional activity at the MC4 receptor.

Figure 45. Substituting a methoxy in place of a methyl moiety significantly increases agonism at both cannabinoid receptors.

Figure 46. Substitution pattern on the benzyl ring in red determined functional activity at the ghrelin receptor in a family of α-aminoisobutyryl containing 1,2,4-triazoles.

Figure 47. Identity of the amine in blue also was important in determining functional activity. R



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Table 1. Examples of Small Structural Modifications that Convert GPCR Agonists into Antagonists

S



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Table 1. continued

T



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Table 1. continued

U



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Table 1. continued

V



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Table 1. continued

W



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Table 1. continued

a

Structural differences between agonist and antagonist occur near a nitrogen (γ-position or closer) (N); a basic nitrogen (γ-position or closer) (B); an oxygen (γ-position or closer) (O); a sulfonamide moiety (S), and/or an alkene or alkyne (A).

3. DISCUSSION The overall goal of this study has been to provide a practical guide for researchers interested in converting GPCR agonists to antagonists (or vice versa). As can be seen from cases presented in Section 2, a wide variety of structural modifications exist that have been successfully implemented to perform this transformation, with changes in certain moieties affecting functional activity more than others. To help prioritize structural changes in attempts to convert the functional activity of a ligand, we have compiled 42 examples from Section 2 into Table 1, generally limiting the entries in this Table to one example per scaffold unless multiple distinct changes could be made to the same scaffold to alter functional activity. We were also interested in determining how applicable our findings would be to non-GPCR receptors. We therefore identified 20 examples of small structural changes that interconvert agonists and antagonists at these (mostly nuclear) receptors; these are shown in Table 2. The data shown in Tables 1 and 2 reveal that the size of the ligand appears to play a much larger role in determining functional activity at non-GPCRs than at GPCRs. For our selected GPCR ligand agonist/antagonist pairs in Table 1, there was no clear correlation between functional activity and size: in 18 cases the antagonists were larger (as measured by molecular weight), in 16 cases the agonists were larger, while in 8 cases they were the same size. However, with respect to the nonGPCR ligand pairs in Table 2, antagonists tended to be larger than agonists, as 75% of the antagonists (15 of 20) had a higher molecular weight while only 25% of the agonists (5 of 20) were larger. One key conclusion to be drawn is that most structural changes that interconvert agonists and antagonists at GPCRs take place near a nitrogen. In 71% of the examples in Table 1, there is a structural difference between agonist and antagonist that occurs at the γ- position to a nitrogen or closer (changes in ortho substituents of anilines are also included in this total). We have listed these in Table 1 as belonging to category N. By

also contained a benzyl group connected to the 4-position of the triazole (in red in Figure 46). The substitution pattern at the 4-position of this benzyl ring played a key role in determining functional activity at the ghrelin receptor. Specifically, the unsubstituted benzyl derivative 105 was a potent partial agonist (Ki = 15 nM, IA = 73%), while the 4-methoxybenzyl analogue 106 was an antagonist (Ki = 6 nM). When the indole group on the right side of the molecule (shown in green in Figure 46) was replaced with other groups such as a phenyl, similar reductions in agonism were seen when a 4-methoxy group was added to the benzyl ring at the 4-position of the triazole. However, a subsequent report by these investigators showed that this correlation between substitution on the benzyl ring and functional activity disappeared when the α-aminoisobutyryl moiety was replaced with other types of amines.75 When 4-piperidine was chosen as the basic amino moiety on the left side of the molecule (blue in Figure 47), the 4-methoxy derivative 108 actually demonstrated higher agonist efficacy than benzyl analogue 107, a partial agonist. Nevertheless, in several cases including the proline derivative 109, 4-methoxybenzyl-containing compounds remained antagonists (no unsubstituted benzyl analogue of 109 was reported). As in the earlier study,74 the substitution pattern on the right side of the ligand (shown in green in Figure 47) played a negligible role in determining functional activity: phenyl analogues of 108 and 109 were an agonist and an antagonist, respectively. The results published by Fehrentz and co-workers demonstrate that functional activity trigger points, such the 4-position on the benzyl ring of the α-aminoisobutyryl containing analogues in Figure 46, can vary or even disappear depending on the nature of substitutions elsewhere on the scaffold. The results also indicate that certain ligand moieties, in this case the basic amino group in blue in Figure 46 and Figure 47, affect functional activity more than others, such as the part of the ligands colored in green in these figures. X



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Table 2. Examples of Small Structural Modifications that Convert Non-GPCR Agonists into Antagonists

Y



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Table 2. continued

Z



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Table 2. continued

a Structural differences between agonist and antagonist occur near a nitrogen (γ-position or closer) (N); a basic nitrogen (γ-position or closer) (B); an oxygen (γ-position or closer) (O); a sulfonamide moiety (S), and/or an alkene or alkyne (A).

In particular, it is clear that previously identified ligands with undesired functional activity at a GPCR of interest can serve as feasible starting points in the search for ligands with preferred modes of activity. Similarly, when designing HTS screening campaigns to identify new hits, we recommend that researchers consider incorporating dual-mode HTS assays capable of detecting both agonists and antagonists, rather than limiting searches exclusively to compounds exhibiting the desired functional activity. The cases presented in this perspective also illustrate the potential hazard of relying solely on binding data to drive lead optimization because functional activity can and often does shift without any significant changes in binding affinity. It is also problematic to assume a given functional activity for a published compound if only binding data are reported, even if functional data are available for related ligands. Finally, while this review has focused on modifications to molecules that are presumed to bind to orthosteric sites of GPCRs, small structural changes have also been reported to alter the functional activity of allosteric76−78 and biased GPCR ligands.79 As increasing numbers of these types of transformations are identified, it should prove interesting to compare the structure−functional activity relationships for those ligands to the cases reported in this perspective.

contrast, in only 50% of the non-GPCR cases in Table 2 are structural modifications leading to agonist/antagonist interchange found in proximity to a nitrogen. This distinction becomes even more evident when modifications near basic nitrogens only are considered. As illustrated in Tables 1 and 2, over a third (36%) of the GPCR agonist/antagonist ligand pairs fall into category “B”, where structural modifications take place near a basic amine, guanidine, or imidazole (not conjugated to an electron withdrawing group), while for non-GPCR agonist/ antagonist ligand pairs, this number is zero. While nitrogens, especially basic nitrogens, play a more prominent role with GPCRs than with non-GPCRs as sites for modifications that alter functional activity, the opposite holds true for certain other structural features. For example, 20% of the cases in Table 2 feature modifications near sulfonamides (listed as belonging to category S in the Table), while no such cases exist in Table 1. Similarly, 20% of the examples in Table 2 involve changes to or near alkenes or alkynes (listed as category A) while only one such case (2%) occurs in Table 1. Structural changes altering functional activity that occur in proximity to an oxygen were also quite common with respect to ligands for both GPCRs and non-GPCRs. In 57% of the GPCR ligands in Table 1, a structural difference between the agonist and the antagonist occurs at the γ- position to an oxygen or closer (listed as category O in Tables 1 and 2). Similarly, 65% of the non-GPCR examples in Table 2 fall into the same category. It is noteworthy that only one case in Table 1, the CB2 modulators developed by Corelli and co-workers, involves a structural modification that does not occur in close proximity to either an oxygen or a nitrogen. This is likely because these heteroatoms are typically involved in key interactions that determine the functional activity of a ligand at a GPCR, and therefore modifying a ligand near these heteroatoms is more likely to affect functional activity. Thus, it is our recommendation that investigators looking to interconvert GPCR agonists and antagonists begin by focusing on modifying areas of the ligand near either a nitrogen or an oxygen. In conclusion, the scientific literature contains many examples of employing small structural changes to interconvert agonists and antagonists, underscoring the importance of this technique as part of a viable GPCR drug discovery strategy.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1-612-625-7948. Fax: 1-612-626-6318. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Peter I. Dosa received his A.B. from Princeton University in 1995 and an M.S. in Chemistry from MIT in 1998. He earned his Ph.D. in Chemistry in 2002 from the University of California, Berkeley, under the direction of Prof. Peter Vollhardt. From 2002 to 2009, he worked as a medicinal chemist at Arena Pharmaceuticals, where his research focused on small-molecule therapeutics targeting GPCRs including the 5-HT2A and 5-HT2C receptors. Since 2009, he has worked at the University of Minnesota, where he is currently Associate Program Director of the Institute for Therapeutics Discovery & Development AA



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James, A. B., Ed.; Academic Press: New York, 1997; Vol. 32, Chapter 28, pp 277−283. (11) Fujioka, M.; Omori, N. Subtleties in GPCR Drug Discovery: a Medicinal Chemistry Perspective. Drug Discovery Today 2012, 17, 1133−1138. (12) Rudolph, J.; Esler, W. P.; O'Connor, S.; Coish, P. D. G.; Wickens, P. L.; Brands, M.; Bierer, D. E.; Bloomquist, B. T.; Bondar, G.; Chen, L.; Chuang, C.-Y.; Claus, T. H.; Fathi, Z.; Fu, W.; Khire, U. R.; Kristie, J. A.; Liu, X.-G.; Lowe, D. B.; McClure, A. C.; Michels, M.; Ortiz, A. A.; Ramsden, P. D.; Schoenleber, R. W.; Shelekhin, T. E.; Vakalopoulos, A.; Tang, W.; Wang, L.; Yi, L.; Gardell, S. J.; Livingston, J. N.; Sweet, L. J.; Bullock, W. H. Quinazolinone Derivatives as Orally Available Ghrelin Receptor Antagonists for the Treatment of Diabetes and Obesity. J. Med. Chem. 2007, 50, 5202−5216. (13) Ting, P. C.; Umland, S. P.; Aslanian, R.; Cao, J.; Garlisi, C. G.; Huang, Y.; Jakway, J.; Liu, Z.; Shah, H.; Tian, F.; Wan, Y.; Shih, N.-Y. The Synthesis of Substituted Bipiperidine Amide Compounds as CCR3 Ligands: Antagonists versus Agonists. Bioorg. Med. Chem. Lett. 2005, 15, 3020−3023. (14) Bergink, V.; Westenberg, H. G. M. Metabotropic Glutamate II Receptor Agonists in Panic Disorder: a Double Blind Clinical Trial with LY354740. Int. Clin. Psychopharmal. 2005, 20, 291−293. (15) Dominguez, C.; Prieto, L.; Valli, M. J.; Massey, S. M.; Bures, M.; Wright, R. A.; Johnson, B. G.; Andis, S. L.; Kingston, A.; Schoepp, D. D.; Monn, J. A. Methyl Substitution of 2-Aminobicyclo[3.1.0]hexane 2,6-Dicarboxylate (LY354740) Determines Functional Activity at Metabotropic Glutamate Receptors: Identification of a Subtype Selective mGlu2 Receptor Agonist. J. Med. Chem. 2005, 48, 3605− 3612. (16) Woolley, M. L.; Pemberton, D. J.; Bate, S.; Corti, C.; Jones, D. N. C. The mGlu2 but not the mGlu3 Receptor Mediates the Actions of the mGluR2/3 Agonist, LY379268, in Mouse Models Predictive of Antipsychotic Activity. Psychopharmacology 2008, 196, 431−440. (17) Zaveri, N. T.; Jiang, F.; Olsen, C. M.; Deschamps, J. R.; Parrish, D.; Polgar, W.; Toll, L. A Novel Series of Piperidin-4-yl-1,3Dihydroindol-2-ones as Agonist and Antagonist Ligands at the Nociceptin Receptor. J. Med. Chem. 2004, 47, 2973−2976. (18) Rothman, R. B.; Baumann, M. H.; Savage, J. E.; Rauser, L.; McBride, A.; Hufeisen, S. J.; Roth, B. L. Evidence for Possible Involvement of 5-HT2B Receptors in the Cardiac Valvulopathy Associated with Fenfluramine and Other Serotonergic Medications. Circulation 2000, 102, 2836−2841. (19) Goernemann, T.; Huebner, H.; Gmeiner, P.; Horowski, R.; Latte, K. P.; Flieger, M.; Pertz, H. H. Characterization of the Molecular Fragment that is Responsible for Agonism of Pergolide at Serotonin 5Hydroxytryptamine2B and 5-Hydroxytryptamine2A Receptors. J. Pharmacol. Exp. Ther. 2007, 324, 1136−1145. (20) Hofmann, C.; Penner, U.; Dorow, R.; Pertz, H. H.; Jähnichen, S.; Horowski, R.; Latté, K. P.; Palla, D.; Schurad, B. Lisuride, a Dopamine Receptor Agonist With 5-HT2B Receptor Antagonist Properties: Absence of Cardiac Valvulopathy Adverse Drug Reaction Reports Supports the Concept of a Crucial Role for 5-HT2B Receptor Agonism in Cardiac Valvular Fibrosis. Clin. Neuropharmacol. 2006, 29, 80−86. (21) Zanettini, R.; Antonini, A.; Gatto, G.; Gentile, R.; Tesei, S.; Pezzoli, G. Valvular Heart Disease and the Use of Dopamine Agonists for Parkinson’s Disease. N. Engl. J. Med. 2007, 356, 39−46. (22) Kekewska, A.; Hübner, H.; Gmeiner, P.; Pertz, H. H. The Bulky N(6) Substituent of Cabergoline Is Responsible for Agonism of This Drug at 5-Hydroxytryptamine (5-HT)2A and 5-HT2B Receptors and Thus Is a Determinant of Valvular Heart Disease. J. Pharmacol. Exp. Ther. 2011, 338, 381−391. (23) Dosa, P. I.; Ward, T.; Walters, M. A.; Kim, S. W. Synthesis of Novel Analogs of Cabergoline: Improving Cardiovascular Safety by Removing 5-HT2B Receptor Agonism. ACS Med. Chem. Lett. 2013, 4, 254−258. (24) Kenakin, T. Differences Between Natural and Recombinant G Protein-coupled Receptor Systems with Varying Receptor/G Protein Stoichiometry. Trends Pharmacol. Sci. 1997, 18, 456−464.

and a Research Assistant Professor in the Department of Medicinal Chemistry. Elizabeth Ambrose Amin is Associate Professor of Medicinal Chemistry, Scientific Computation, and Biomedical Informatics and Computational Biology in the College of Pharmacy at the University of Minnesota and is a Fellow of the Minnesota Supercomputing Institute for Advanced Computational Research (MSI). She earned her Ph.D. in Chemistry from the University of Missouri in 2002 under the direction of Prof. William J. Welsh and has both academic and industrial experience in drug design and discovery at Biosafety Level 3 (BSL-3) and with Select Agents. Her laboratory’s research program focuses on homeland security and designing countermeasures to biological and chemical warfare agents. The Amin laboratory also focuses on central nervous system, GPCR-targeted, and psychiatric drug development, specifically therapeutics for neurological and mood disorders.

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ACKNOWLEDGMENTS We gratefully acknowledge Dr. Gunda Georg, Dr. Michael Walters, and Sara Coulup for helpful discussions.

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ABBREVIATIONS USED 5-HT, serotonin; AT2, angiotensin II receptor type 2; C3a, complement component 3a receptor; CB2, cannabinoid receptor type 2; CCK2, cholecystokinin-2 receptor; CCR3, CC chemokine-3 receptor; CHO, Chinese hamster ovary; GHS-R1A, growth hormone secretagogue type 1a receptor; GNTI, 5′-guanidinonaltrindole; GPCR, G protein-coupled receptor; GPER, G protein-coupled estrogen receptor; IA, intrinsic activity; MC4, melanocortin receptor 4; mGlu, metabotropic glutamate receptor; NOP, nociceptin receptor; PMA, percentage of maximal cAMP accumulation; PPARα, peroxisome proliferator-activated receptor alpha

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