Mutagenesis Analysis Reveals Distinct Amino Acids of the Human

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Mutagenesis analysis reveals distinct amino acids of the human serotonin 5-HT2C receptor underlie the pharmacology of distinct ligands Yue Liu, Clinton E. Canal, Tania C. Cordova-Sintjago, Wanying Zhu, and Raymond G. Booth ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00124 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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ACS Chemical Neuroscience

Mutagenesis analysis reveals distinct amino acids of the human serotonin 5-HT2C receptor underlie the pharmacology of distinct ligands Yue Liua, Clinton E. Canala, Tania C. Cordova-Sintjagoa,b, Wanying Zhua, and Raymond G. Bootha,b a

Center for Drug Discovery, Department of Pharmaceutical Sciences and Department of

Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA b

Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA

Running title (60 characters): Drug design targeting human 5-HT2C receptors.

Key words: serotonin, 5-HT2C receptor, mutagenesis, pharmacology, drug discovery

Corresponding author: Yue Liu, E-mail address: [email protected] Work address: Center for Drug Discovery, Department of Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA, USA

A list of nonstandard abbreviations: 4-phenyl-2-dimethylaminotetralin: PAT

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Abstract: While exploring the structure activity relationship of 4-phenyl-2dimethylaminotetralins (PATs) at serotonin 5-HT2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT2C receptors. In HEK293 cells expressing human 5-HT2C-INI receptors, for example, (–)-trans-3’-Br-PAT and (–)-trans-3’-ClPAT are agonists regarding Gαq-inositol phosphate signaling, whereas (–)-trans-3’-CF3-PAT is an inverse agonist. To investigate the ligand receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (Ki) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC50 functional potencies of PATs. We discovered that when residue(s) S5.43 and/or N6.55 are mutated to alanine, (–)-trans-3’-CF3PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (– )-trans-3’-Br-PAT switches from agonist to inverse agonist function. Notably, most pointmutations that affected PAT pharmacology did not significantly alter affinity (KD) of the antagonist radioligand [3H]mesulergine, but, every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially-available 5-HT2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids, and that some 5HT2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.


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Introduction: Serotonin 5-HT2C receptors belong to the large family of G protein-coupled receptors (GPCRs), and are one of three 5-HT2 receptor subtypes, which also include 5-HT2A and 5-HT2B receptors. 5-HT2C agonists have attracted attention in the pharmaceutical industry as potential medications to treat obesity, addiction, schizophrenia, depression, and anxiety 1-4. Notable, the selective 5-HT2C agonist, lorcaserin (Belviq®), was recently approved for obesity, and clinical trials with lorcaserin as a treatment for cocaine addiction are underway5. Human 5-HT2C receptors share 76% sequence homology with 5-HT2A, and 68% sequence homology with 5HT2B in the transmembrane (TM) domain, and the 5-HT2 family receptors primarily couple to Gαq proteins, which when active, stimulate phospholipase C (PLC) and PKC signaling pathways. The high sequence homology shared by 5-HT2A, 5-HT2B, and 5-HT2C creates a challenge for designing 5-HT2C agonists that do not also activate 5-HT2A and/or 5-HT2B, which if activated, can lead to hallucinations and cardiotoxicity, respectively6-7. Previously, we reported the discovery of (2S, 4R)-(–)-trans-4-phenyl-N,N-dimethy1,2,3,4 tetrahydronaphthalene-2-amine ((–)-trans-PAT), a unique compound possessing 5-HT2C receptor agonist activity combined with 5-HT2A and 5-HT2B inverse agonist activity 8. In the process of lead optimization, we designed a series of PAT analogs with various substituents at the 3’ position on the 4-phenyl ring in order to improve the potency of PATs at 5-HT2C receptors (Figure 1). Among the 3’-substituted PATs, (–)-trans-3’-Br-PAT showed both enhanced binding affinity and potency compared to (–)-trans-PAT as a 5-HT2C agonist. Meanwhile, (–)-trans-3’Br-PAT behaved as a 5-HT2A competitive antagonist, and 5-HT2B inverse agonist in in vitro function assays 9. In vivo, (–)-trans-3’-Br-PAT (also coined “(–)-MBP”) displayed preclinical efficacy for psychosis, without liability for weight gain, motoric side effects, or obesity 9. Further



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investigation of 4-phenyl 3’ analogs of PAT led to the discovery of our first 5-HT2C inverse agonist in this series, the 3'-CF3 analog of PAT, (–)-trans-3’-CF3-PAT. There are numerous examples in the literature that show how small structural modifications can convert agonists to antagonists (or visa versa) at GPCRs 10, such as what we observed with a 3'-Br- to a 3'-CF3modification of PAT. The opposite pharmacology of (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3PAT steered us to investigate the specific molecular interactions between the two 3’-substituted PAT ligands and the 5-HT2C receptor that contribute to stabilization of agonist versus inverse agonist receptor conformations. We previously reported that the protonated amine moiety of PATs forms a salt bridge to the side chain carbonyl of the 5-HT2C receptor aspartate residue, D3.32, and this interaction is crucial for PAT binding, similar to 5-HT and other monoamine neurotransmitters at aminergic GPCRs 8, 11-20. The salt bridge is likely reinforced by a hydrogen bond network formed by D3.32 with residues S3.36 and Y7.4315, 21. Based on molecular modeling results, we subsequently investigated the impact of three TM domain aromatic residues, W6.48, F6.51 and F6.52, on the binding of PATs at 5-HT2C. The W6.48A mutation significantly reduced the affinity of PATs, and all three mutations significantly decreased the potency of PATs to activate the receptor 22. Using the representative 5-HT2C partial agonist (–)-trans-3’-Br-PAT and the inverse agonist (–)-trans-3’-CF3-PAT as tools, the major goal of the current study was to delineate the molecular determinants governing receptor activation and inactivation of 5-HT2C receptors by the PAT class of ligands. Site-directed mutagenesis, radioligand binding, and cell-based Gαq signaling assays were performed to identify amino acids in the 5-HT2C receptor orthosteric binding pocket important for binding and function of 3’-substituted PAT ligands. We pinpointed several amino acids that play key roles in the binding and/or function of (–)-trans-3’-Br-PAT, (–


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)-trans-3’-CF3-PAT, and other PAT analogs. In addition, we screened the effects of these mutations on commercially-available, synthetic 5-HT2C ligands, including agonists and antagonists, to determine whether effects extended to different chemotypes. Finally, we tested the effects of these mutations on 5-HT pharmacology, and with 5-HT, observed a general phenomenon. All amino acids mutated significantly and negatively impacted 5-HT binding affinity, suggesting a likely example of receptor evolution that produces highly efficient interactions with its cognate ligand.

Results and Discussion 1. (–)-trans-3’-CF3-PAT is a 5-HT2C receptor inverse agonist at the WT receptor. Although we recently reported an affinity value of (–)-trans-3’-CF3-PAT at WT 5-HT2C receptors 21, 23, herein we report for the first time its activity as a 5-HT2C receptor inverse agonist. (–)-trans-3’-CF3-PAT decreased basal activity in a dose-dependent manner. Its pIC50 was 7.7 ± 0.1 M (mean ±SEM), and it decreased basal activity by 24.5±3.3% (Figure 2 and Table 2). In Figure 2, IP1 accumulation data were normalized based on basal activity (100%) for each assay (i.e., not based on 5-HT efficacy). Potency and efficacy values were determined by IP1 accumulation and percent basal activity (100%) of each mutant receptor. In the present studies, the binding affinity (pKi±SEM) of (–)-trans-3’-CF3-PAT at wild type (WT) 5-HT2C receptor was determined to be 7.3 ± 0.1, an affinity consistent with our initial report 21. 2. [3H]mesulergine saturation binding at WT and mutant 5-HT2C receptors The selection of amino acids for site-directed mutagenesis was based on available GPCR crystal structures, and from molecular mechanism of action studies of multiple biogenic amine GPCRs



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12, 14, 16-20, 24-27

. The point mutant 5-HT2C receptors were expressed in HEK293 cells, and the

effect of mutations on receptor binding site densities (Bmax) and KD values was determined by [3H]mesulergine saturation binding (Table 1). Bmax values of WT and mutant receptors ranged from 1.0 fmol/mg to 3.9 pmol/mg of protein. The mean ± SEM KD value of [3H]mesulergine at the WT 5-HT2C receptor was 2.0 ± 0.27 nM, in line with established values reported in the literature. None of the mutants with the exception of S5.43A and F5.47A significantly affected [3H]mesulergine binding, suggesting the mutations did not greatly affect receptor structure; S5.43A and F5.47A decreased affinity of mesulergine modestly by ~2.6 and 2.3-fold, respectively (Table 1). The binding affinities (Ki) of ligands at the mutant receptors were calculated using the KD value of [3H]mesulergine determined experimentally at each receptor, based on the Cheng-Prusoff equation: Ki = IC50 / ( 1+ L/ KD), where L is the concentration of the radioligand; IC50 is the concentration of ligand which causes 50% inhibition of radioligand binding 28. 3. Effects of amino acid residue mutations of 5-HT2C on the pharmacology of PATs and 5-HT Based on the crystal structures of aminergic receptors, with a focus on the recently reported 5HT1B and 5-HT2B receptors (PDB codes 4IAQ and 4IB4), binding affinities, functional potencies, and efficacies of PATs and 5-HT were assessed at 14 point-mutated 5-HT2C receptors to provide new insights into 5-HT2C ligand-receptor interactions. The effects of 5-HT2C receptor mutations on the pharmacology of PATs can be categorized in three groups: 1) mutations that affected affinity and functional potencies; 2) mutations that affected functional, but not affinity potencies; 3) mutations that reversed the function of PATs from either inverse agonist to agonist, or the converse.


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3a. Mutations of 5-HT2C residues S3.36, T3.37, and F5.47 decrease both binding affinity and functional potency of PATs and 5-HT The binding affinity and functional potency of (–)-trans-3’-Br-PAT, (–)-trans-3’-CF3-PAT, and the endogenous agonist 5-HT at WT, S3.36A, T3.37A and F5.47A point-mutated receptors are shown in Table 2, Figure 3, and Figure 4. Relative to WT, S3.36A, T3.37A, and F5.47A mutations decreased the affinity of both (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT by 4-10 fold (Figure 3 and Table 2). These mutations also negatively impacted the function of both PAT ligands. For (–)-trans-3’-Br-PAT, S3.36A, T3.37A, and F5.47A decreased the functional potency (EC50) by 14, 4, and 54-fold, respectively, as assessed by 5-HT2C-Gαq-mediated IP1 accumulation (Figure 4A). For (–)-trans-3’-CF3-PAT, these mutations completely abolished inverse agonist activity (Figure 4B). We also observed, in extended studies, that these three mutations decreased the binding affinity and potency of other PAT partial agonists, including (– )-trans-PAT, (–)-trans-3’-Cl-PAT, and (–)-trans-3’-NO2-PAT (Table S2). In addition to PATs, the S3.36A, T3.37A, and F5.47A mutations had very robust, negative effects on the affinity of 5-HT (Figure 3C, 4C and Table 2). The rank order of mutations that affected 5-HT affinity was T3.37A (82-fold increase in Ki) > S3.36A (41-fold) > F5.47A (33fold) relative to WT 5-HT2C. A similarly robust effect was observed on the potency of 5-HT, where all mutations markedly decreased 5-HT potency: T3.37A (144-fold increase in EC50) > F5.47A (126-fold) > S3.36A (19-fold), relative to WT 5-HT2C (Table 2). To construct a docking pose of PATs bound at the 5-HT2C receptor, a homology model of the 5HT2C receptor was built from the crystal structure of human 5-HT2B 20-21. In our previous work, we showed in modeling and observed in mutagenesis experiments that D3.32 is critical for PATs



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binding, forming the ionic contact between the positively charged nitrogen of PATs and the negatively charged carboxylate side chain of D3.32. The hydroxyl group of residue S3.36 also interacts with the protonated amine. In addition, the p-hydroxy moiety at Y7.43 forms a hydrogen bond with the carboxylate of D3.32. Thus, the pharmacology of (–)-trans-PAT and 5HT are negatively impacted when residues D3.32, S3.36 and Y7.43 are mutated to alanine15 29. In the crystal structures of human 5-HT1B and 5-HT2B in complex with ergotamine, residue C/S3.36 forms a hydrophobic cleft for ergotamine binding20. Here, we show that residue T3.37 is critically important for the binding and function of 5-HT (Figure 3 and Table 2). This result is supported by the crystal structure of 5-HT1B and 5-HT2B, where Wang and colleagues showed that T3.37 forms a hydrogen bond with the indole nitrogen in 5-HT, ergotamine, and LSD20. Mutating T3.37 to alanine decreased the affinity of 5-HT at 5HT1B and 5-HT2B receptors by one to two orders of magnitude 20, a result in line with our finding at 5-HT2C receptors (Figure 3 and Table 2). In addition, the crystal structure of human β2AR shows that T3.37 forms a hydrogen bond with the inverse agonist timolol 30, and in the crystal structure of the histamine H1 receptor, T3.37 forms a hydrogen bond with the antagonist dexepin 12

.

The T3.37A mutation also negatively impacted PATs binding and function (Figure 3 and Table 2). In our 5-HT2C model with PATs docked, T3.37 does not appear to interact with PATs directly. Rather, the side chain of T3.37 can interplay via a hydrogen bond with the hydroxyl group of S4.53, in the interface of TM3-TM4, possibly contributing to the receptor configuration and modification of the binding pocket 21. The discrepancy in the role of S3.36 and T3.37 between the 5-HT2B crystal structure and our 5-HT2C homology model is likely due to the molecular structures of PAT ligands that lack nearby hydrogen bond donors or acceptors to


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effectively interact with residues S3.36 and T3.37 in the 5-HT2C binding pocket. Thus, PATs can stabilize different binding pocket structures than those of ergotamine. Similarly, residue F5.47 is not close enough to directly interact with either (–)-trans-3’-Br-PAT or (–)-trans-3’-CF3-PAT in 5-HT2C, notwithstanding, it is noted that the phenyl ring of F5.47 in TM5 is in the TM5-TM6 interface, close to the phenyl ring of F6.52 in TM6. Thus, π-π and hydrophobic interactions can form in the binding pocket, contributing to stabilization of the TM bundle structure 21. The π-π stacking interaction is also observed in the crystal structure of 5HT2B and β2AR receptors (4IB4 and 2RH1). This interaction is abolished in the F5.47A 5-HT2C point-mutated receptor, and concomitantly impacts receptor conformation to affect ligand binding. In support of this, we observed a negative impact of the F5.47A mutation on PATs, 5HT, and mesulergine (the KD of [3H]mesulergine was significantly decreased at F5.47A mutation) (Table 1 and 2), indicating the impact of F5.47A mutation binding and signaling was not ligand specific. π-π stacking between F5.47 and F6.52 was also reported in a 5-HT2A model based on rhodopsin 31. In addition, the S3.36A, T3.37A, and F5.47A mutations caused a significant decrease in basal (constitutive) activity compared to the WT receptor (Figure 4D). The basal activities of 5-HT2C S3.36A, T3.37A and F5.47A mutant receptors were 48 ± 16 %, 55 ± 7%, and 47 ± 5% of WT 5HT2C receptors. S3.36A, T3.37A and F5.47A also lead to super-agonist effects (efficacies above 5-HT Emax at WT) of 5-HT and (–)-trans-3’-Br-PAT (Figure 3C). This effect is mainly due to the decreased basal activities at the mutant receptors, because Emax was calculated as fold increase from the basal activity of each receptor. The negative impact of mutations on basal activity suggests that the mutations likely altered the overall receptor structure and caused less efficient Gαq protein coupling in the absence of ligand binding. G protein coupling which occurs in the



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intracellular part of the receptor can modulate ligand binding pocket structure, possibly changing ligand binding and function26, 32-33. Thus we are cautious about interpreting data for specific ligand-receptor interactions at mutated receptors with compromised constitutive activity. The basal activities of point-mutated receptors were not normalized to their expression level, because there was not a direct correlation between the two. For example, the S3.36A, T3.37A and F5.47A point-mutated receptors all demonstrated much lower basal activity compared to the WT receptor, but, only the S3.36A receptor showed lower expression than the WT receptor. The decreased basal activity of the T3.37A and F5.47A receptors may reflect decreased Gαq coupling efficiency. Meanwhile for the N6.55Q, M6.47A, M6.47AC7.45A receptors that showed higher expression compared to WT, basal IP1 production was not significantly different than the WT receptor. The compromised basal activity may also account for the (–)-trans-3’-CF3-PAT functional change, because some ligands can behave as agonists, neutral antagonists, or inverse agonists at the same receptor depending on the basal receptor conformations (a protean ligand) 34

. This discovery that certain ligands can behave like protean ligands is of particular relevance

not only for designing new compounds targeting 5-HT2C receptors, but also may apply to other GPCRs with high constitutive activity similar to the 5-HT2C receptors. 3b. Mutations of 5-HT2C residues F6.44, M6.47, C7.45, and S7.46 minimally impact binding affinity, but decrease functional potency of PATs. We identified four amino acids located in TM6 and TM7 (F6.44, M6.47, C7.45, and S7.46) that when mutated to alanine did not negatively impact affinity, but dramatically decreased potency or abolished function of (–)-trans-3’-Br-PAT and/or (–)-trans-3’-CF3-PAT (Table 3). Mutation of C7.45S was also tested in addition to C7.45A. Serine is the native residue at position 7.45 in 5-HT2A and 5-HT2B receptors, and could provide information to guide design of selective or


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specific 5-HT2 subtype ligands. Each of these mutations substantially decreased the potency of (– )-trans-3’-Br-PAT (Table 3). For (–)-trans-3’-CF3-PAT, M6.47A and C7.45A decreased the potency while F6.44A, C7.45S, and S7.46A mutations abolished its inverse agonist properties, turning it to a neutral antagonist (defined here as a ligand that binds to the receptor without affecting Gαq-IP1 functional activity). Analysis of data from crystal structures of class A GPCRs reveals that amino acids in positions 6.47 and 7.45 form interhelical interactions regulating the physical interface of TM6 and TM7 35. Therefore, we made a M6.47A C7.45A double mutant receptor to study the role of this putative 5-HT2C intramolecular interaction. The double mutation M6.47A C7.45A did not affect the affinity of (–)-trans-3’-Br-PAT or (–)-trans-3’-CF3-PAT, but it abolished the activity of (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT (Table 3). For the endogenous ligand 5-HT, F6.44A, M6.47A, C7.45A, C7.45S, S7.46A mutants and the M6.47A C7.45A double mutant caused consistent decreases in affinity and functional potency, but with variability in the degrees of change. F6.44A, M6.47A, C7.45A, and C7.45S showed 2-4 fold decreases in affinity, and 2-8 fold decreases in potency. The M6.47A C7.45A double mutation decreased 5-HT affinity by 5-fold, and it decreased the potency of 5-HT by 34-fold. The S7.46A mutation decreased 5-HT binding by 18-fold, and it decreased the potency of 5-HT by 82-fold (Table 3). Because the S7.46 residue has been reported to play an important role in receptor signaling by stabilizing the conserved NPxxY motif in TM7 and the binding pocket residues of TM6 36, we tested three other 5-HT2C agonists lorcaserin, Ro 60-0175, and WAY161503 at the S7.46A mutation. As would be predicted based on results from other class A GPCRs, this mutation dramatically decreased the potency of each agonist (Figure S1), further supporting a critical role of S7.46 residue on signal transduction and integrity of the 5-HT2C receptor structure. We also discovered that when residue Y7.53 of the NPxxY motif is mutated



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to alanine at 5-HT2C receptor, neither 5-HT nor any of the PATs activate the receptor (data not shown), suggesting residue Y7.53 is important for 5-HT2C receptors to adopt the activated conformational states. Residue F6.44, together with residues P5.50 and I3.40 form the conserved PIF motif, and conformational changes in this motif are associated with receptor activation 19, 24, 37. The mutation of F6.44 to alanine presumably impedes the receptor to adopt an agonist conformation, thus decreases the potency or abolishes the activity of ligands. These residues likely regulate signal transduction by modulating intramolecular interactions. Losing these interactions could lead to conformational changes in the TM interfaces, decreasing the receptor’s ability to adopt activated or inactivated conformations. The basal activities of F6.44A, M6.47A, C7.45A, C7.45S and M6.47A C7.45A double mutations were not changed significantly compared to the WT [81 ± 4 %, 87 ± 11%, 96 ± 10 %, 119 ± 10%, and 89 ± 23 %] (Figure 5). However, the S7.46A point-mutated 5-HT2C receptor showed significantly reduced basal activity by nearly 70% (34 ±12%) compared to WT (Figure 5), consistent with the literature report on the function of S7.46 to stabilize GPCR interactions with G-proteins 36. The role of residues F6.44, M6.47, C7.45, and S7.46 on PATs binding and function was investigated using molecular docking and MD simulations at WT 5-HT2C homology model embedded in a lipid POPC membrane to elucidate their involvement in binding 21. Residues F6.44, M6.47, C7.45, and S7.46 do not interact directly with the ligands, explaining they had little or no impact on PATs affinity (Table 4). Replacement by alanine, however, caused a


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conformational change in the orientation of TM6 and TM7, which can have an effect on function. 3c. N6.55A and S5.43A mutations of 5-HT2C switch the pharmacology of PAT 5-HT2C inverse agonists to partial agonists, and the N6.55L mutation switches PAT 5-HT2C partial agonists to inverse agonists. The 5-HT2C inverse agonist (–)-trans-3’-CF3-PAT, switched to a partial agonist at both S5.43A and N6.55A point-mutated receptors, while the function of (–)-trans-3’-Br-PAT as a partial agonist was unchanged at these mutant receptors (Figure 6 and Table 4). To obtain more insight into the molecular interactions between residues S5.43 and N6.55 and the PAT ligands, PAT pharmacology was assessed at N6.55L, N6.55Q, and N6.55A S5.43A double mutations. Unexpectedly, the partial agonist (–)-trans-3’-Br-PAT switched to an inverse agonist at the N6.55L mutant, while the inverse agonist activity of (–)-trans-3’-CF3-PAT was unchanged at this mutant (Figure 6 and Table 4). The mutation of N6.55 to render glutamine (N6.55Q), which has similar physicochemical properties as asparagine (N), did not significantly change the binding affinity or flip the function of (–)-trans-3’-Br-PAT or (–)-trans-3’-CF3-PAT, but it had a negative impact on the potency for both PATs analogs, suggesting residue Q is less efficient in signaling transduction compared to residue N. Similar to the single alanine mutations, the S5.43A N6.55A double mutation turned the inverse agonist (–)-trans-3’-CF3-PAT to a partial agonist (Figure 6 and Table 4). Affinity of (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT increased (decreased Ki) at the N6.55A receptor by 7.0- and 7.2-fold, at the N6.55L receptor by 2.5- and 3.2-fold, and at the S5.43A N6.55A receptor by 3.9- and 1.5- fold, respectively. The N6.55A single and S5.43A



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N6.55A double mutations increased the potency of (–)-trans-3’-Br-PAT by 5.0- and 1.9- fold, consistent with the increased affinity. Affinities of both ligands were unaffected by the S5.43A point mutant (Table 4 and Figure 7). The increased or unchanged affinity of PATs at N6.55A point-mutated receptor implies that N6.55 residue is not crucial for PATs binding. In fact, for all the mutations tested in this study, we found very similar effects on the binding affinities of both (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT, suggesting the two PAT compounds are likely binding to the receptor in a very similar fashion, consistent with (–)-trans-3’-Br-PAT and (–)trans-3’-CF3-PAT having very similar overall molecular structure. However, minor binding differences between (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT can cause distinct conformational changes in TM domains, leading to opposite functional outcomes.

We then tested the effects of S5.43 and N6.55 mutations on the binding and function of other PATs that are partial agonists or inverse agonists at WT 5-HT2C receptors in order to obtain more information regarding the impact of these residues on PAT pharmacology to potentially elucidate whether various PATs share common binding poses. These compounds included the partial agonists (–)-trans-PAT, (–)-trans-3’-Cl-PAT, and (–)-trans-3’-NO2-PAT, and the inverse agonist (–)-trans-3’-C6H5-PAT. Results were generally consistent with those we obtained from (–)trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT. Like (–)-trans-3’-Br-PAT, the N6.55L mutation switched (–)-trans-PAT, (–)-trans-3’-Cl-PAT, and (–)-trans-3’-NO2-PAT from agonists to inverse agonists (Figure 8B and Table S2). N6.55A had a dramatic positive effect on the binding affinity and potency for (–)-trans-PAT (Figure 8A and Table S2). No change in either Ki or EC50 values was observed at the S5.43A and N6.55Q mutations for (–)-trans-PAT (Table S2). Similar to (–)-trans-3’-CF3-PAT, the inverse agonist (–)-trans-3’-C6H5-PAT converted to an agonist at


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the N6.55A receptor and also at the S5.43A N6.55A double mutant receptor. However, S5.43A failed to switch the function of (–)-trans-3’-C6H5-PAT, unlike (–)-trans-3’-CF3-PAT (data not shown). Intriguingly, the function-switching effects of N6.55 mutations predictively affected distinct classes of PATs. For example, all PAT partial agonist analogs tested were inverse agonists at the N6.55L receptor, but this mutant receptor did not affect the inverse agonist function of any of the PAT inverse agonists. These data provide strong support that multiple PAT partial agonists stabilize a similar 5-HT2C active conformation, and multiple PAT inverse agonists stabilize a similar inactive conformation. That such closely related chemical structures can bind to the 5HT2C receptor in a very similar orientation, but result in different functional outcomes argues that only subtle changes in receptor structure can switch the balance from inactive to active or active to inactive. Our previous work supports this claim. A single residue difference between human (S5.46) and mouse (A5.46) 5-HT2A receptors resulted in different functions of the PAT analog, (+)-trans-6-OH-7-Cl-PAT. At 5-HT2A S5.46, (+)-trans-6-OH-7-Cl-PAT was a partial agonist, but at 5-HT2A A5.46 (and at 5-HT2C A5.46), it was a neutral antagonist; these observations were confirmed by point-mutagenesis38. To test whether N6.55 is important for the function of other classes of ligands, we tested the function of the marketed antipsychotic drugs clozapine and olanzapine (both are inverse agonists at WT 5-HT2C) at the N6.55A and N6.55L mutations, and found that neither mutation affected their inverse agonist properties (Figure S2). The new 5-HT2C agonist, anti-obesity drug, lorcaserin, remained an agonist at the N6.55L mutation (Figure S2). Collectively, these results suggest that the functional importance of N6.55 on function is specific for the PAT class of ligands, suggesting other classes of ligands such as the dibenzodiazepines and benzazepine



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derivatives (clozapine, olanzapine, and lorcaserin) employ distinct molecular signaling mechanisms leading to the activation or inactivation of the receptor. For the endogenous ligand 5-HT, the N6.55 and S5.43 mutations decreased the binding affinity and functional potency, consistent with the general negative impact of mutations on 5-HT pharmacology. The greatest negative impact was observed at the N6.55A mutation (Table 4). Notably, 5-HT remained an agonist at all the mutations, and the IP1 accumulation induced by 5HT, and basal activity at the mutant receptors were comparable to the WT (Figure 6C and Figure 6D). The unchanged basal activity of N6.55 and S5.43 point-mutant receptors implies that these mutant receptors were able to maintain the overall structure and G protein coupling similar to the WT receptor.

To explain our pharmacological results that the S5.43 and N6.55 series of mutations switch the pharmacology of PATs at 5-HT2C receptors, we conducted a ligand docking and molecular dynamics study 21. However, the modeling results do not support direct interactions between either PATs ligand and N6.55 or S5.43. The human 5-HT2B-receptor crystal structure in complex with the biased agonist ergotamine provided some insights on the possible involvement of S5.43 and N6.55 in receptor activation. In this biased ligand bound structure, a hydrogen bond between S5.43 and N6.55 was observed, and this hydrogen bond is postulated to prohibit the rearrangement of TM 6 and impede the rotation of F6.44, thus disfavoring G protein coupling 1920

. This observation implies that (–)-trans-3’-Br-PAT and (–)-trans-3’-CF3-PAT can stabilize

different receptor conformations, and that the presence or absence of the hydrogen bond between S5.43 and N6.55, is critical for PAT derivatives function. N6.55 and S5.43 hydrogen bonding has also been reported in the human β2AR and the turkey the β1AR structures in complex with


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agonist, antagonist, or inverse agonist, suggesting the existence of this hydrogen bond may also be ligand specific 14, 17-18, 24-25, 39-40. When residue N6.55 is mutated to alanine, or leucine, this interaction is lost and the distance between these two residues increases, impacting the conformation of TM6, and ligand-mediated rearrangement of TM5 and TM6. The result that N6.55L switched the pharmacology of (–)-trans-3’-Br-PAT implies that, although the N6.55L mutation no longer forms a hydrogen bond between N6.55 and S5.43, it allows the receptor to adopt an alternative inverse agonist conformations. In summary, the most parsimonious explanation to account for the 5-HT2C agonist activity of (–)-trans-3’-Br-PAT and inverse agonist activity (–)-trans-3’-CF3-PAT appears to involve differential modulation of the S5.43 and N6.55 microdomain, which likely impacts TM helical conformational changes associated with 5-HT2C receptor activated vs. inactivated conformational states. It is worth noting that many GPCRs have been shown to signal via multiple pathways. A current challenge in drug discovery is understanding how to design a ligand to elicit a specific signaling profile relevant to desired in vivo efficacy and safety. In part, this requires characterizing compound behavior in a wide variety of different assay systems measuring multiple outcomes to identify which system(s) has the appropriate translational power. While it has been shown that 5HT2C receptors primarily couple to Gαq and mediating IP signaling, other putative 5-HT2C GPCR signaling pathways almost certainly contribute to therapeutic and untoward effects associated with receptor activation. Herein, our scope was limited to measuring effects of PATs on 5-HT2Cmediated Gαq /IP signaling to rationalize ligand chemical structural interactions with receptors residues that lead to agonist vs. inverse agonist functional activity. To our knowledge, this study represents the first extensive mutagenesis investigation of the 5HT2C receptor. Results on specific ligand-receptor interactions can guide drug design for more 5-



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HT2C receptor selective agonists. We observed that residue N6.55 is a key molecular determinant for the agonist and inverse agonist activity of PAT analogs at 5-HT2C receptors, and this effect is possibly regulated by a hydrogen bond between S5.43 and N6.55. Thus when we are designing new PAT-type 5-HT2C agonists, these data support that we should avoid disturbing the hydrogen bond between S5.43 and N6.55, which will disfavor receptor activation. Based on the discovery that residues F6.44, M6.47, C7.45 and S7.46 are important for the potency of PATs through intramolecular interactions and receptor signaling, when designing PATs antagonists, direct ligand receptor interactions with F6.44, M6.47, C7.45 and S7.46 can be considered to avoid receptor activation. Data obtained with other chemically diverse 5-HT2C ligands herein (lorcaserin, clozapine, olanzapine, Ro 60-0175, WAY161503) indicate that the functional outcome(s) of GPCR ligands often involve receptor amino acid interactions that are unique, i.e., ligand-specific (see Supplementary Figure 1 and 2). For example, an amino acid necessary for agonist activity of one ligand may be important for inverse agonist activity of another ligand—there likely is not a single agonist pharmacophore and a single antagonist (or inverse agonist) pharmacophore. Thus, the data reported in this paper confirm and add to the now large body of literature that supports the tenet that GPCR ligand molecular determinants for function are ligand specific, for example, impacted by organic and physical chemical properties of the ligand. Interpretation of this phenomenon has been extended to suggest that each GPCR ligand stabilizes a unique receptor conformation 41that can lead to a common signaling outcome (e.g. Gαq-PLC activation), and/or, a unique signaling outcome to effect a physiological response42. Thus, reliably successful design of GPCR agonists or antagonists (or inverse agonists), a priori, seems to require further


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understanding of how ligand chemistry impacts GPCR conformation and how receptor conformation impacts G protein coupling type and activity.

Materials and methods: Compounds The synthesis of the 4-phenyl-2-dimethylaminotetralin (PAT) compounds used in this study has been reported 21, 23. Lorcaserin hydrochloride was purchased from Chem Scene (Monmouth Junction, NJ). Clozapine and olanzapine were obtained from Sigma-Aldrich (St. Louis, MO). Ro 60-0175 and WAY161503 were purchased from Tocris (Bristol, United Kingdom). Site-directed mutagenesis Mutations of the human 5-HT2C receptor were generated by polymerase chain reactions (PCR) using QuikChange II Site-Directed mutagenesis kit (Agilent, Santa Clara, CA) according to the manufacturer’s protocol. The wild type (WT) human 5-HT2C-INI receptor cDNA, cloned in pcDNA3.1+ vector, was purchased from Missouri S&T cDNA Resource Center (http://www.cdna.org/). Mutagenesis primers (Table S1) were obtained from Life Technologies (Carlsbad, CA). The PCR reaction was performed as detailed previously38. Subsequently, each amplification reaction was digested by 10 U of Dpn I restriction enzyme at 37 °C for 1 hour. The digested product was then transformed into XL1-Blue supercompetent cells and plated on LB agar plates containing 100 µg/ml ampicillin, and incubated at 37 °C overnight. Several colonies were picked for screening. The DNA sequences containing the desired mutation were verified using the Sanger sequencing method (Genewiz, South Plainfield, NJ).



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Cell culture, transfection and radioligand binding assays HEK293 cells (CRL-1573, ATCC, Manassas, VA) under pass 20. [Pass one was defined as the first plate of cells derived from our liquid nitrogen stock.] were grown in DMEM (10-013CV, Mediatech, Manassas, VA) supplemented with 5% dialyzed fetal bovine serum (FBS) (35071-CV, Mediatech, Manassas, VA) and 1% of antibiotics (SV30079.01, Thermo Scientific, Waltham, MA) in 10 cm dishes. When cells reached ~70% confluency, they were transfected with 10 µg of WT or mutant DNA using 20 µl of Turbofect (Thermo Scientific, Waltham, MA) in DMEM (10-013-CV). 24-48 h after transfection, cells were collected by centrifugation at 3000 × g at 4 °C for 15 min, homogenized in ice cold assay buffer containing 50 mM Tris, 10 mM MgCl2 and 0.1 mM EDTA, and the membrane was collected by centrifugation twice at 3000 × g at 4 °C for 15 min, and stored at −80 °C until use. Radioligand saturation isotherm and competitive displacement binding assays were performed in 96-well plates as previously described8. For saturation binding assays, 0.02 to 20 nM [3H]mesulergine (PerkinElmer, Waltham, MA) was used to obtain KD and Bmax values at the WT and mutant receptors. For competition binding assays, 1–2 nM [3H]mesulergine (~KD concentration) was used to obtain the Ki values for unlabeled ligands. Non-specific binding was determined in the presence of 10 µM mianserin hydrochloride (Sigma-Aldrich, St. Louis, MO). The reaction mixtures were incubated for 90 min at room temperature to reach equilibrium, and then were rapidly passed through GF/B filters using a Brandel cell harvester (Gaithersburg, MD) and subsequently washed with 5 volumes of 50 mM Tris–HCl at room temperature. Filter discs containing bound [3H]mesulergine were placed in vials containing 2 ml ScintiVerse scintillation cocktail (Thermo Scientific, Waltham, MA), incubated overnight, and then counted for


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scintillation using a PerkinElmer Tri-Carb 2910TR Liquid Scintillation Analyzer (Waltham, MA). Each assay was performed a minimum of three times. Inositol phosphate accumulation assay 5-HT2C-Gαq-mediated inositol-1-phosphate (IP1) production was measured by Cisbio IPOne HTRF assay kit (Bedford, MA) as previously described38. HEK293 cells transiently transfected with 5-HT2C WT or mutant receptors were incubated in serum-free DMEM (SH30604.01, Thermo Scientific, Waltham, MA) for 1 hour before harvest. Cells were then pelleted by centrifugation at 200 × g at 37 °C for 5 min and resuspended in serum-free DMEM. Cells number was counted using a hemacytometer (Hausser Scientific, Horsham, PA). Cells were diluted to 1000 cells/µl. Five µl of cell suspension and 5 µl of test compounds diluted in stimulation buffer (containing 50 mM LiCl) or stimulation buffer alone (for the detection of basal activity) was added to wells in a 384-well plate (Greiner Bio-one, Monroe, NC). The plate was incubated at 37 °C, 5% CO2, 95% humidity, for 2 hours. After incubation, the reaction was terminated by adding 5 µl each of the donor and acceptor fluorescent conjugates in lysis buffer. The plate was incubated at room temperature for 1 hour to reach equilibrium. The amount of IP1 accumulation induced by ligands or produced by the unliganded receptors (basal activity or constitutive activity) were assessed by the ratio of fluorescence emission at 665 nM and 620 nM using Synergy H1 reader (BioTek, Winooski, VT). IP1 levels were calculated from a standard curve with known IP1 concentrations. Each assay was performed a minimum of three times. Statistics



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All experiments were performed at least 3 times. Data derived from WT and mutant receptors were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test using Prism software version 6.0. A P value < 0.05 was considered statistically significant.

Supporting Information Supporting Information Available: 1.

Primers sequences used for site-directed mutagenesis

2.

Binding and function of PATs analogs at 5-HT2C WT and mutant receptors

3.

Sequence alignment of the mutated amino acids across human 5-HT receptors

4.

Lorcaserin, clozapine and olanzapine activity at 5-HT2C N6.55A and N6.55L mutations

5.

Lorcaserin, Ro 60-0175, WAY161503 activity at 5-HT2C S7.46A mutant

This material is available free of charge via the Internet at http://pubs.acs.org.

Abbreviations: GPCR(G-protein coupled receptor)

Author Information *Northeastern University, Center for Drug Discovery, 360 Huntington Ave., 211A Mugar Life Sci. Bldg., Boston, MA 02115. Corresponding Author: Yue Liu Tel: 3528713132; Email: [email protected]

Author Contribution


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YL, CEC, TSC, and RGB designed experiments. YL initiated, conducted, and managed experiments with assistance from WZ. YL collected and analyzed data. YL, CEC, and RGB interpreted data. YL and CEC wrote the manuscript.

Funding Sources This work was funded by NIH R01MH081193 and R01DA030989 awarded to RGB.

Conflict of Interest The authors declare no conflicts of interest.



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32. Manglik, A.; Kim, T. H.; Masureel, M.; Altenbach, C.; Yang, Z.; Hilger, D.; Lerch, M. T.; Kobilka, T. S.; Thian, F. S.; Hubbell, W. L.; Prosser, R. S.; Kobilka, B. K., Structural insights into the dynamic process of beta2-adrenergic receptor signaling. Cell 2015, 161 (5), 1101-11. 33. DeVree, B. T.; Mahoney, J. P.; Velez-Ruiz, G. A.; Rasmussen, S. G.; Kuszak, A. J.; Edwald, E.; Fung, J. J.; Manglik, A.; Masureel, M.; Du, Y.; Matt, R. A.; Pardon, E.; Steyaert, J.; Kobilka, B. K.; Sunahara, R. K., Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature 2016, 535 (7610), 182-6. 34. Kenakin, T., Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2001, 15 (3), 598-611. 35. Olivella, M.; Caltabiano, G.; Cordomi, A., The role of Cysteine 6.47 in class A GPCRs. BMC structural biology 2013, 13, 3. 36. Hulme, E. C., GPCR activation: a mutagenic spotlight on crystal structures. Trends Pharmacol Sci 2013, 34 (1), 67-84. 37. Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G.; Choi, H. J.; Devree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K., Structure and function of an irreversible agonistbeta(2) adrenoceptor complex. Nature 2011, 469 (7329), 236-40. 38. Canal, C. E.; Cordova-Sintjago, T.; Liu, Y.; Kim, M. S.; Morgan, D.; Booth, R. G., Molecular pharmacology and ligand docking studies reveal a single amino acid difference between mouse and human serotonin 5-HT2A receptors that impacts behavioral translation of novel 4-phenyl-2dimethylaminotetralin ligands. Journal of Pharmacology and Experimental Therapeutics 2013, 347 (3), 705-716. 39. Verlinde, C. L.; Hol, W. G., Structure-based drug design: progress, results and challenges. Structure 1994, 2 (7), 577-87. 40. Lebon, G.; Warne, T.; Edwards, P. C.; Bennett, K.; Langmead, C. J.; Leslie, A. G.; Tate, C. G., Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 2011, 474 (7352), 521-5. 41. Kenakin, T., Functional selectivity and biased receptor signaling. The Journal of pharmacology and experimental therapeutics 2011, 336 (2), 296-302. 42. Canal, C. E.; Booth, R. G.; Morgan, D., Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4iodoamphetamine elicited head-twitch response model. Neuropharmacology 2013, 70, 112-121.


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N 2S 4R

R R=H, Br, NO2, Cl, CF3, C6H5

Fig 1. Structures of PAT ligands



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Fig 2. Representative agonist and inverse agonist functional activity (mean ± SEM) of (–)-trans3’-Br-PAT and (–)-trans-3’-CF3-PAT at WT 5-HT2C receptor.


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Table 1. Effect of mutations on the KD and Bmax of 5-HT2C receptors

KD

BMAX

5-HT2C WT

2.0±0.27

1.7±0.12

S3.36A

2.1±0.50

1.0±0.28

T3.37A

3.8±0.27

2.0±0.50

S5.43A

5.2±0.61*** 1.9±0.38

F5.47A

4.6±0.76*

2.3±0.15

F6.44A

2.9±0.38

2.2±0.50

N6.55A

1.1±0.30

2.3±0.43

N6.55L

0.6±0.14

3.0±0.88

N6.55Q

3.5±0.57

3.8±0.93

S5.43AN6.55A

1.4±0.06

2.1±0.13

M6.47A

2.0±0.33

3.4±0.47

M6.47AC7.45A

1.3±0.25

3.9±0.86

C7.45A

2.6±0.37

2.2±0.33

C7.45S

1.9±0.44

2.7±0.46

S7.46A

3.1±0.27

1.5±0.20

Table 1. KD (nM) and Bmax values (pmol/mg protein) (mean ± SEM) determined using [3H]mesulergine binding in HEK293 cell membranes expressing 5-HT2C WT and mutant receptors. * P < 0.05 and *** P < 0.001



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Table 2. Amino acids important for binding and function of PATs

Table 2. Binding affinity (pKi), potency (pEC50 or pIC50), and IP1 production (% basal activity) of (–)-trans-3’-Br-PAT, (–)-trans-3’-CF3-PAT, and 5-HT at WT and mutant receptors (mean ± SEM).


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A

B

C

Fig 3. Representative binding curves of (–)-trans-3’-Br-PAT (A); (–)-trans-3’-CF3-PAT (B); and 5-HT (C) at WT and S3.36A, T3.37A and F5.47A mutations (mean ± SEM).



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A

C

B

D

Fig 4. Representative functional curves for IP1 accumulation induced by (–)-trans-3’-Br-PAT (A); (–)-trans-3’-CF3-PAT (B); and 5-HT (C) in HEK293 cells transiently expressing human 5HT2C WT and S3.36A, T3.37A, and F5.47A mutant receptors (mean ± SEM). Basal activity is reduced, relative to 5-HT2C WT, at S3.36A, T3.37A and F5.47A mutated receptors (D) , which likely accounts for the super-agonist-like effects in (A) and (C). ** P < 0.01, and *** P < 0.001


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Table 3. Amino acids important for functional potency of PAT

Table 3. Binding affinity (pKi), potency (pEC50 or pIC50), and IP1 production (% basal activity) of by (–)-trans-3’-Br-PAT, (–)-trans-3’-CF3-PAT, and 5-HT at WT and mutant 5-HT2C receptors (mean ± SEM).



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Fig 5. Basal activity of the TM6 and TM7 mutant receptors expressed as % of basal activity of 5HT2C WT receptors. ** P < 0.01.


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Table 4. Amino acids important for the function of PATs

Table 4. Binding affinity (pKi), potency (pEC50 or pIC50), and IP1 production (% basal activity) of (–)-trans-3’-Br-PAT, (–)-trans-3’-CF3-PAT, and 5-HT at WT and mutant receptors (mean ± SEM).



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A

B

C

Fig 6. Representative binding curves of (–)-trans-3’-Br-PAT (A); (–)-trans-3’-CF3-PAT (B); and 5-HT (C) at 5-HT2c WT and S5.43A, N6.55A, N6.55L, N6.55Q single, and S5.43A N6.55A double mutations (mean ± SEM).


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A

B

C

D

Fig 7. Representative functional curves for IP1 accumulation induced by (–)-trans-3’-Br-PAT (A), (–)-trans-3’-CF3-PAT (B), and 5-HT (C) in HEK293 cells transiently expressing human 5HT2C WT and S5.43A, N6.55A, N6.55L, N6.55Q single, and S5.43A N6.55A double mutations (mean ± SEM). Basal activity of 5-HT2C WT and S5.43A, N6.55A, N6.55L, N6.55Q single, and S5.43A N6.55A double mutations (D).



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A

B

Fig 8. Representative binding curves of (–)-trans-PAT at 5-HT2C WT and S5.43A, N6.55A, N6.55L, N6.55Q single, and S5.43A N6.55A double mutations (mean ± SEM) (A); Representative functional curves for IP1 accumulation induced by (–)-trans-PAT at 5-HT2C WT and S5.43A, N6.55A, N6.55L, N6.55Q single, and S5.43A N6.55A double mutations (mean ± SEM) (B).


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200

human 5-HT2C WT N6.55A N6.55L

200


300


100

50

150 100

50

IP1 formation (% basal)

150

IP1 formation (%basal)

IP1 formation (%basal)

Page 39ACS of 39Chemical Neuroscience ACS Paragon Plus Environment 250 200 150 100

1

0 -11 -10 -9 -8 -7 -6 -5 -4 Log [(-)-trans-3'-Br-PAT] / M

0 -12 -11 -10 -9 -8 -7 -6 -5 Log [(-)-trans-3'-CF3-PAT] / M

-13 -12 -11 -10 -9 -8 -7 Log[5-HT] / M

-6

-5

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human

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