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Article Cite This: J. Med. Chem. 2017, 60, 8716-8730

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Click-Chemistry-Mediated Synthesis of Selective Melanocortin Receptor 4 Agonists Daniel Palmer,*,† Juliana P. L. Gonçalves,† Louise V. Hansen,† Boqian Wu,‡ Helle Hald,† Sanne Schoffelen,† Frederik Diness,† Sebastian T. Le Quement,§ Thomas E. Nielsen,∥,⊥,# and Morten Meldal*,† †

CECB, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark Aquaporin A/S, Ole Maaløes Vej 3, 2200 Copenhagen, Denmark § CMC API Development, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark ∥ Protein & Peptide Chemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark ⊥ Department of Immunology and Microbiology, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark # Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, SG 637551, Singapore ‡

S Supporting Information *

ABSTRACT: The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC50 values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.

E

MCRs have distinct, but overlapping, tissue distributions and have demonstrated receptor-specific roles in an array of physiologic processes.7,8 While MC1R has long been known to play an important role in the process of skin pigmentation,9 MC2R has an important role in steroidogenesis in the adrenal cortex,10 and MC5R, although expressed in several tissues, is best characterized for its governance of skin secretions, including that of sebum.11 By way of comparison, MC3R and MC4R are expressed in the central nervous system (CNS), among other tissues, and have attracted significant interest as therapeutic targets owing to their involvement in obesity, metabolism, feeding behavior, and sexual dysfunction.4,5,12,13 The proposed metabolic role for MC4R is, in part, based on observations that the receptor displays robust expression in a discrete region of the brain, the paraventricular nucleus of the hypothalamus, which governs appetite and energy expenditure, and is further

ven by conservative estimates, G-protein-coupled receptors (GPCRs) are targets for approximately 30% of marketed pharmaceuticals and represent an attractive class of targets for the development of novel therapeutics.1 Within the rhodopsin subfamily of GPCRs resides the melanocortin receptor (MCR) family of five receptors (MC1R−MC5R),2,3 of which all are activated by endogenous peptide agonists derived from the proopiomelanocortin (POMC) gene.4 Through post-translational modifications, several peptide agonists for the MCRs are generated, including adrenocorticotropic hormone (ACTH) as well as α-, β-, and γ-melanocyte-stimulating hormones (α-, β-, and γ-MSH, respectively). MCRs have evolved such that varied agonist specificity exists across the family: MC2R is activated solely by ACTH, whereas MC1R and MC3−5R have varying degrees of selectivity for the different MSH variants, with α-MSH displaying activity toward all MCRs except MC2R.3,5,6 This agonist selectivity agrees with the homology comparisons across the MCR family, with MC2R differing substantially from the other MCRs and MC3−5R displaying higher degrees of amino acid sequence identity (see Figure S1 in Supporting Information). © 2017 American Chemical Society

Received: March 20, 2017 Published: October 3, 2017 8716

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Figure 1. (a) Structure of the parent “macrocyclic” ligand, compound 1, which was used as a basis for analogs 2−17 as compared with other reference cyclic peptides. (b) Melanocortin 4 receptor (the extracellular face at the top of the model), with the relaxed NOE-constrained triazolecontaining macrocyclic compound 1 in the receptor binding pocket. The model was adapted from Pogozheva et al.32 by replacement of α-MSH with

culminated in the development of several candidate therapeutics targeted toward MC4R,16 no molecule has been granted approval for any indication at present owing to inconsistencies in therapeutic efficacy1 and undesirable side effects (including elevations in blood pressure).17−20 This, coupled with the

reinforced by the obese, hyperphagic phenotype displayed by MC4R knockout mice.13,14 Additionally, certain polymorphisms in the human MC4R gene correlate with an obese phenotype and have generated sustained interest in this receptor as a target for ameliorating metabolic disorders.15 While this interest has 8717

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Table 1. Primary Sequences of Reference Molecules and Linear Precursors to Cyclic Peptide Compounds 1−17 compda

precursor/peptide structureb

MW, calcd (Da)

MW, obsd (Da)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 NDP-MSH α-MSH melanotan-II (MT-II) setmelanotide

K-DapN3‑H-DPhe-R-W-Pra-M K-DapN3-H-DPhe-R-W-Pra-A K-DapN3-H-DPhe-R-Bta-Pra-M K-DapN3-H-F-R-W-Pra-M K-DapN3-H-DPhe-K-W-Pra-M R-DapN3-H-DPhe-R-W-Pra-M K-DapN3-H-DPhe-Orn-W-Pra-M K-DapN3-H-DPhe-R-A-Pra-M K-DapN3-H-DPhe-A-W-Pra-M K-DapN3-H-DAla-R-W-Pra-M K-DapN3-A-DPhe-R-W-Pra-M A-DapN3-H-DPhe-R-W-Pra-M K-DapN3-H-DIle-R-W-Pra-M K-DapN3-W-DPhe-R-H-Pra-M K-DapN3-H-DPhe-R-Nal-Pra-M Orn(N3)-H-DPhe-R-W-Pra-M N3R-H-DPhe-R-W-Pra-M Ac-S-Y-S-Nle-E-H- DPhe-R-W-G-K-P-V-NH2 Ac-S-Y-S-M-E-H-F-R-W-G-K-P-V-NH2 Ac-Nle-cyclo[D-H-DPhe-R-W-K]-NH2 Ac-R-cyclo[C-DAla-H-DPhe-R-W-C]-NH2

1111.5372 1051.5338 1128.4983 1111.5372 1083.5310 1139.5433 1069.5154 996.4950 1026.4732 1035.5059 1045.5154 1054.4788 1077.5523 1111.5372 1122.5414 1011.4730 1053.4950

1111.5416 1051.5306 1128.4945 1111.5432 1083.5370 1139.5392 1069.5137 996.4922 1026.4541 1035.4985 1045.5339 1054.4683 1077.5386 1111.5339 1122.5255 1011.4606 1053.4955

a Purified by HPLC in yields of 40−80% (see Figures S5−S21). bThese ligands were cyclized through the CuAAC reaction between azide (Dap(N3), N3R or Orn(N3)) and alkyne (Pra) functional groups. All peptides utilized were purified to >95% purity. Where appropriate, D-amino acids are indicated within the sequence. Compounds 2−17 were modifications of compound 1 as indicated in bold. DapN3, S-2-amino-3-azidopropanoic acid; Pra, L-propargylglycine; Bta, benzothien-3-ylalanine; Orn, L-ornithine; Nal, naphth-1-ylalanine; Orn(N3), L-5-azido-2-aminopentanoic acid; Nle, norleucine.

increasing prevalence of obesity on a global scale,21 speaks to the need for further refinement of MC4R pharmacophores for both research and therapeutic molecules Upon activation by MSH variants, MC4R, like all members of the MCR subfamily, couples to the stimulatory heterotrimeric G-protein complex, Gs, resulting in an increase in intracellular cAMP as the dominant signaling system.22 Assays screening for MCR function have, therefore, typically utilized end points related to cAMP signaling. All endogenous ligands for MC4R, including α-MSH (Ac-SYSMEHFRWGKPV-NH2), contain a conserved core motif HFRW, which is also the minimum fragment believed to be required for activation with synthetic peptide ligands and was initially characterized by several early structure−function relationships.6,23−26 Predicated on this motif, a variety of ligands has been prepared for studies of MC4R.26−50 Upon binding of endogenous ligands, this motif has been determined to present a reverse β-turn, placing the pharmacophore HFRW motif optimally for interaction with residues mainly in the transmembrane helices TM2−TM6 of MC4R (modeled in Figure 1, Figure S2, and Tables S1 and S2).32,51,52 Interestingly, the core HFRW motif has lesser activity on its own as compared to being placed within the context of longer peptide variants.24 Replacement of L-phenylalanine (F) with D-phenylalanine (DPhe) and M with norleucine (Nle) in the α-MSH analog NDP-MSH (Ac-SYS-Nle-EH-DPhe-RWGKPV-NH2), as well as cyclization of truncated peptide analogs (e.g., melanotan II (MT-II); Ac-Nle-c[DH-DPhe-RWK]-NH2),17 has been demonstrated to induce this reverse β-turn and increase activity at MC4R in a ring size-dependent manner.33,52−55 Peptide cyclization also facilitates pharmacophore scans by creating a rigid, structural framework to determine the relative importance of the different residues as it relates to binding affinity and efficacy.

Furthermore, cyclized peptides exhibit improved metabolic stability compared to the linear counterpart, potentially aiding drug development.56 Additionally, several studies have evaluated the influence of the length and rigidity of a cyclization linker in stabilization of the active conformations of HfRW on the MC1R, MC3R, MC4R, and MC5R with efficacious macrocycle chain lengths possessing 20−26 atoms, depending on the chemical properties of the macrocycle.6,29,43,44,53,55,57 For this study, we devised a solid-phase method to obtain i − i + 5 cyclic peptide ligands for the MCR family by the Cu(I)mediated azide−alkyne cycloaddition (CuAAC) reaction. We evaluated the ability of the cyclic ligands to stimulate the receptor using cAMP measurements and transcriptional reportergene assays in cells overexpressing human MC3R, MC4R, or MC5R, the MCRs with the highest mutual identity (Figure S1). With an aim toward identifying cyclic peptide ligands matching or exceeding the potency and efficacy of α-MSH and in order to use analogs of this ligand in structure−activity studies, dose− response experiments for ligands 1−17 (Figure 1a, Table 1) were conducted using the two assays. Over the course of these studies, we identified several peptides that display marked selectivity for MC4R, with amino acids flanking the cyclized structure providing an extension to the established pharmacophore. Additionally, ligands displayed reduced potency in the cAMP assay as compared with those observed with the CRE (cAMP-response element) reporter assay such that robust reporter protein expression was often observed at concentrations significantly lower than those observed for the detection of appreciable cAMP levels, arguing for particular care to be taken in assay selection for agonist screening.

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RESULTS Synthesis of Cyclized Melanocortin-Derived Peptides by CuAAC. A synthetically tractable approach to generate 8718

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Table 2. Potency and Efficacy Data for MCR Peptide Agonists Determined by CRE Assaya MC3R

α-MSH NDP-MSH melanotan II (MT-II) setmelanotide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

MC4R

EC50 ± SEM (nM)

max response (% of max α-MSH)

EC50 ± SEM (nM)

16.7 ± 2.9 (n = 12) 6.8 ± 1.6 (n = 3) 33.9 ± 25.2 (n = 3) 30.9 ± 26.7 (n = 3) 122.6 ± 38.6 (n = 5) 161.7 ± 64.2 (n = 3) 821.3 ± 112.9 (n = 3) >3000 (n = 3)g >3000 (n = 3)g 290.6 ± 49.1 (n = 6) >3000 (n = 3)g >3000 (n = 5)g,h >3000 (n = 6)g >3000 (n = 3)g 633.1 ± 112 (n = 8)b 19.4 ± 5.9 (n = 3) >3000 (n = 3)g >3000 (n = 3)g 721.6 ± 93.6 (n = 5) >3000 (n = 6)g 160 ± 77.9 (n = 4)

99% 124% 83% 119% 105% 107% 96% 32% at 3000 nM 40% at 3000 nM 102 12% at 3000 nM 17% at 3000 nM 10% at 3000 nM 33% at 3000 nM 104% 111% 0% at 3000 nM 0% at 3000 nM 84% 16% at 3000 nM 112%

9.2 ± 2.0 (n = 14) 0.4 ± 0.1 (n = 4) 8.6 ± 5.5 (n = 3) 5.7 ± 3.7 (n = 3) 1.8 ± 0.3 (n = 5) 2.3 ± 0.5 (n = 5) 7.6 ± 2.5 (n = 3) 677.5 ± 563.2 (n = 4) 12.7 ± 1.6 (n = 3) 11.4 ± 3.6 (n = 6)e,i 317.9 ± 18.2 (n = 4) >3000 (n = 6)g 257.7 ± 102.3 (n = 7) 112.3 ± 56.7 (n = 4) 2.8 ± 0.4 (n = 6)e 1.4 ± 0.4 (n = 3) >3000 (n = 3)g >3000 (n = 5)g 6.4 ± 1.6 (n = 4) 184.6 ± 44.4 (n = 7) 3.2 ± 0.9 (n = 4)

MC5R

max response (% of max α-MSH)

EC50 ± SEM (nM)

104% 35.3 ± 8.6 (n = 10) 101% 1.1 ± 0.3 (n = 3) 120% 33.9 ± 22.3 (n = 3) 97% 109.7 ± 93.6 (n = 3) 114% 298.7 ± 104.7 (n = 3) 103% 635 ± 113.6 (n = 3)e,f 110% >3000 (n = 3)g 100% >3000 (n = 3)g 103% >3000 (n = 3)g 103% 752.3 ± 97.6 (n = 3)d,e,f 112% >3000 (n = 3)g 19% at 3000 nM >3000 (n = 5)g,j 131% >3000 (n = 6)g 127% >3000 (n = 3)g 108% 375.2 ± 65.7 (n = 5)e,f,k 120% 137 ± 30 (n = 3) 42% at 3000 nM >3000 (n = 3)g 48% at 3000 nM >3000 (n = 3)g 124% >3000 (n = 3)g 119% >3000 (n = 6)g 125% 916 ± 105.9 (n = 3)d,e,f

max response (as % of max α-MSH) 98% 117% 140% 148% 120% 126% 59% at 3000 nM 1% at 3000 nM 13% at 3000 nM 85% 9% at 3000 nM 18% at 3000 nM 5% at 3000 nM 8% at 3000 nM 130% 123% 0% at 3000 nM 0% at 300 nM 30% at 3000 nM 3% at 3000 nM 106%

a EC50 values are depicted as an average of n independent determinations from concentration−response series ± standard deviation. bP < 0.05 vs MC3R-compound 1. cP < 0.05 vs MC4R-compound 1. dP < 0.05 vs MC5R-compound 1. eP < 0.05 vs corresponding compound on MC3R fP < 0.05 vs corresponding compound on MC4R. gValues represent an estimate as the concentration−response curves were incomplete and lacking a maximal response plateau. Data from these estimates were not included in statistical comparisons hIn one additional independent experiment, an EC50 of 1910 nM was obtained. All others were beyond the concentration range tested. iIn three additional independent experiments, EC50 values were unable to be determined due to a lack of an observable maximum value. Data from these estimates were not included in statistical comparisons. jIn one additional independent experiment, an EC50 of 809 nM was obtained. All others were beyond the concentration range tested. k In one additional independent experiment, an EC50 value was unable to be determined due to a lack of an observable maximum value. Data from these estimates were not included in statistical comparisons.

triply charged ions were observed for most of the compounds in their respective, validatory MS spectra. To probe the importance of constituent amino acid residues in defining agonist potency and selectivity, a panel of 17 peptides was synthesized using the scheme in Figure S3. The reference compound, predicted by molecular modeling (using a novel modeled structure predicated on an activated, Gs-coupled β2-adrenergic receptor)63 to have similar potency as endogenous agonists and denoted as compound 1, harbors the key pharmacophore residues H-DPhe-RW within a macrocycle derived from a triazole product of Pra-DapN3 “clicking” and flanked by K and M residues at the amino and carboxyl termini, respectively (Figure 1a and Figure S4). To improve the predictive power of the model, a solution structure was determined using NOESY and ROESY NMR (Figure 1b, Tables S3 and S4, Figures S22−S27). The structure of 1 shares a general similarity toward known cyclic peptide MC4R agonists such as melanotan-II and setmelanotide20,64,65 (Ac-R-c[C-DAla-H-DPhe-RWC]-NH2, a cyclic peptide currently in clinical trials for the treatment of POMC-deficiency and Prader−Willi syndrome) but differs in the mechanism of ring closure (a triazole bridge as compared to a disulfide bridge or lactam bridge) and ring size (21 atoms in 1 vs 23 atoms in the other structures) (Figure 1a). Sixteen analogs with substitutions of the different residues in 1 were prepared to explore the structure−activity relationship between the pharmacophore elements, as well as azide-containing residues and extracyclic residues, and receptor activation. Of note,

constrained cyclic peptides is a valuable starting point for screening peptide ligands for their conformational preference in ligand−receptor interactions. The CuAAC reaction has found numerous applications, notably in peptide cyclization reactions.58−62 The reaction is operationally facile and provides full chemical compatibility with both unprotected peptides and aqueous reaction conditions. Furthermore, it can be effectively performed on solid support, thereby providing a convenient path to synthesize cyclic peptide ligands. Linear peptides were synthesized using solid-phase synthesis, namely, Fmoc-based synthesis on a hydroxymethylbenzamidefunctionalized PEGA resin, deprotected, and subjected to a copper-mediated intramolecular cyclization reaction, facilitating nearly quantitative formation of the cyclic peptides. To facilitate the CuAAC reaction, nonproteinogenic amino acids were incorporated within the linear peptide sequence to provide the necessary alkyne and azide moieties for the copper(I)-catalyzed macrocyclization. Specifically, L-propargylglycine (Pra) was introduced to provide the alkyne functional group, whereas azide moieties were supplied by (S)-2-amino-3-azido-propanoic acid (DapN3), L-5-azido-2-aminopentanoic acid (Orn(N3)), or α-azidoarginine (N3R). The peptides were deprotected and cyclized on the support resin. The resulting 1,4-disubstituted 1,2,3-triazole-containing macrocycles were cleaved off the resin in high yields, purified, and characterized by LC−MS (Table 1, Figures S3 and S4 with validatory MS data found in Figures S5−S21). In addition to the molecular ion, doubly and 8719

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Figure 2. Comparison of agonist potency for MC3R, MC4R, and MC5R determined by measurements of EYFP expression in the CRE reporter assay. EC50 (nM) determinations were made for 11-point concentration−response curves (performed in independent triplicates for each peptide−receptor combination). Data are displayed as the mean ± SEM from at least three independent experiments. The lower Y-axis segment encompasses EC50 values between 0 and 1500 nM, whereas the higher segment encompasses 2000−3000 nM. Data points corresponding to the columns in this figure can be found in Table 2.

remarkable degree of selectivity for MC4R (70-fold vs MC3R and 170-fold vs MC5R). It has been established previously that the core pharmacophore residues of H, DPhe, R, and W are important, if not essential, for ligand activity toward MC4R.17 The H-residue, buried in a deep receptor pocket, influences the agonist selectivity exhibited by the different MCRs.27,28,31,49 Of note, our model of 1 complexed with the activated receptor predicts that the H residue is coordinated by ionic interactions with E1002x60, using the generic GPCR helix nomenclature as devised by Ballesteros and Weinstein.69 The predicted interaction is further stabilized by hydrophobic interactions with T1012x61 and I1042x64 (Figure S2). Remarkably, replacing H with A (11) exerted negligible effects on the potency for MC4R activation (EC50 of 2.8 nM), whereas 11 exhibited reduced activation of MC3R (EC50 = 633.1 nM) and MC5R (EC50 = 375.2 nM). Switching W and H (14) essentially ablated all potency for the ligand toward all three receptor subtypes. Substituting DPhe with the natural enantiomer F markedly decreases the potency for MC4R (385-fold; 4 vs 1). Of further interest, 4 possessed negligible agonist activity when tested on MC3R and MC5R (with EC50 values beyond the highest concentration tested). Exchanging DPhe with D-alanine (DAla) (10) decreases the peptide potency to approximately 112.3 nM against MC4R and, as with 4, results in minimal activity toward the other MCRs. When substituted for DPhe, the bulky D-isoleucine (DIle) residue, which also lacks a bulky aromatic ring, results in a similarly poor agonist (13) for all receptors tested. The R residue of the agonist pharmacophore has strong interactions with carboxylates from aspartate residues in MC4R helix TM3 as well as a proximal asparagine residue (Figure 1, Figure S2, Table S2).16 Specifically, D1223x25, D1263x29, and N1233x26 are predicted to form important interactions with the R residue of the agonist (Figure S2b), in agreement with point

alterations were introduced at the amino-terminal K residue (6 and 12), azide source (16 and 17), pharmacophore residues (3−5,7−11,13, and 15), and carboxy-terminal M residue (2) (Table 1, Figure S4). Evaluation of Cyclic Peptide Ligands Using CRE Reporter Transcription Assay. Synthesized, purified ligands were screened for activity using a reporter gene assay as described previously and summarized in Figure S28.66 Briefly, when Gs-coupled GPCRs are activated, cAMP levels are increased, resulting in the cAMP-dependent protein kinase (PKA) mediated phosphorylation of the transcription factor CREB (cAMP-response element binding protein), and binding of phospho-CREB to consensus sites, comprised of nine nucleotides (CREs), upstream of target genes (Figure S28a).67 This canonical pathway was exploited, using engineered plasmids that permit activation of reporter genes, and used as a surrogate of receptor activation. The system utilized in this study and validated in our laboratory (see Figures S29 and S30 for representative results)66 uses stably transfected cells harboring a single plasmid containing the GPCR of interest for agonist screening and a EYFP reporter gene preceded by nine CREs and a minimal promoter unit (Figure S28a,b). Using this reporter system, we cross-referenced agonist activity toward the human MC4R against the activity of the same molecules toward MC3R and MC5R. In the CRE-driven reporter assay, an endogenous agonist for MC3−5R, α-MSH, exhibited respective EC50 values of 16.7, 9.2, and 35.3 nM for MC3R, MC4R, and MC5R (Table 2, Figure 2) that are comparable to previously reported values.68 Similarly, the synthetic linear peptide agonist, NDP-MSH, was very potent on all receptors tested. Reference cyclic peptide agonists MT-II and setmelanotide showed the expected potency as well as selectivity toward MC4R (setmelanotide)64 or lack thereof (MT-II).44 The CuAAC-cyclized reference molecule, 1, displayed strong potency (EC50 of 1.8 nM) and a 8720

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mutant-based, structure−function studies and previous efforts at modeling agonist−receptor interactions.32,70 Exchange of the basic R residue with A (9) markedly reduced MC4R activity (EC50 = 257.7 nM) and abolished activity toward the other MCRs. To evaluate the nature and location of the charge in the side chain, other basic residues, K (5) and Orn (7), were introduced in place of R. While K reduced MC4R activity modestly (7-fold reduction as compared with 1), Orn substitution resulted in a 181-fold reduction. Replacement of W with A (8) generated an inactive ligand, (EC50 > 3000 nM) on all cells tested, confirming the critical nature of this residue. In contrast, more subtle alterations, such as exchanging nitrogen with sulfur in the W indole ring (benzothien-3-ylalanine, 3) or utilizing another bulky planar ring structure (1-naphthylalanine, 15), yielded peptides that were roughly equipotent as compared to 1 when tested on MC4R. Additionally, a point of divergence between MC3R and MC5R emerged, as 3 and 15, while still possessing reduced activity against MC3R (EC50 = 821.3 nM and 721.6 nM, respectively), were essentially inert against MC5R (EC50 > 3000 nM). The structure−activity relationship between macrocycle ring tension and constraints was evaluated by utilizing different azido-containing residues. Specifically, DapN3 in compound 1 was substituted with Orn(N3) (16) and N3R (17) to evaluate the ring size and triazole position relative to the peptide backbone and thus the flexibility of the macrocyclic ring. The larger ring scaffold synthesized via the linkage from Pra and Orn(N3) in 16 resulted in a diminished MC4R agonist (nearly 105-fold less potent vs 1) with no activity toward MC3R or MC5R. Alternatively, the more rigid ring structure, 17, with the triazole formed between Pra and N3R was nearly equipotent with 1 (EC50 = 3.2 nM) with reduced potencies toward MC3R (EC50 = 160 nM) and MC5R (EC50 = 916 nM). The extracyclic residues flanking the macrocycle were also evaluated in order to determine the key structural features of this novel analog series. Compound 1 contains flanking residues of K and M at the amino and carboxyl termini, respectively. To explore the significance of the amino-terminal K, we exchanged K with R (6) and A (12). Compound 6 resulted in a MC4R agonist almost equipotent with 1. The potency of 6 was reduced for both MC3R (EC50 = 290.6 nM) and MC5R (EC50 = 792.3 nM). The replacement of the charged amino-terminal residue proximal to the macrocycle, e.g., exchange of the K residue for A (12), resulted in a peptide that had moderateto-strong potency toward all three receptors tested, with the potency being best for MC4R and weakest for MC5R. By way of comparison, the substitution of the carboxyl M for A diminished the agonist (2) potency against MC3R and MC5R while leaving the potency for MC4R relatively unaltered. Measurement of cAMP Levels and Differences between cAMP and CRE-Based Assays. As a means of validating the results obtained with the CRE reporter assay, cAMP levels were measured using an immunologically based enzymatic complementation assay (see Experimental Section). Given that activity toward MC4R was of primary interest for this study, measurements were obtained for all compounds using MC4R-expressing cells (Table 3 and Figure 3). While the potency data from the two assays correlated well (R2 = 0.5934; P < 0.002, Figure 3a), there was an upward shift in the EC50 for the cAMP measurements relative to the CRE reporter assay, save for the reference compounds MT-II and setmelanotide. Consistent with such correlation, the agonist activities were

Table 3. Potency and Efficacy of MC4R Agonists As Determined by cAMP Accumulation Assaysa compd α-MSH NDP-MSH melanotan-II (MT-II) setmelanotide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

EC50 ± SEM (nM)

max response (% of max α-MSH)

24.4 ± 8.2 (n = 12) 6.4 ± 2.7 (n = 3) 3.1 ± 0.1 (n = 3)

102% 109% 92%

1.4 ± 0.2 (n = 3) 9.2 ± 2.5 (n = 3) 17.9 ± 7.3 (n = 3) 63.7 ± 11.6 (n = 5) 792 ± 243 (n = 2)e 124.2 ± 34 (n = 4) 43.3 ± 18.2 (n = 6) 730 ± 148.3 (n = 4) >3000 (n = 6)d 684.5 ± 60 (n = 5)e 943 ± 584.1 (n = 4)b,c 28.8 ± 12 (n = 6)f 2.0 ± 1.0 (n = 3) >3000 (n = 3)d >3000 (n = 3)d 89.3 ± 9.9 (n = 3) 720.4 ± 131.6 (n = 5)b 9.6 ± 2.6 (n = 3)

93% 101% 96% 98% 78% 92% 101% 80% 16% at 3000 nM 73% 87% 87% 87% 13% at 3000 nM 28% at 3000 nM 87% 98% 102%

a EC50 values are depicted as an average of n independent determinations from concentration−response series ± standard deviation. bP < 0.05 vs compound 1 (cAMP measurements). cP < 0.05 vs corresponding compound (CRE reporter assay). dSome values represent an estimate as the concentration−response curves were incomplete and lacking a maximal response plateau. Data from these estimates were not included in statistical comparisons. eIn three additional independent experiments, EC50 values were unable to be determined due to a lack of an observable maximum value. Data from these estimates were not included in statistical comparisons. fIn one additional independent experiment, EC50 values were unable to be determined due to a lack of an observable maximum value. Data from these estimates were not included in statistical comparisons.

highly comparable in terms of examining the effect of amino acid substitution on the two assays. There were, however, points with a reduced concordance such that several compounds displayed an approximate 5- to 15-fold reduction in potency in the cAMP measurements vs the CRE reporter assay.

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DISCUSSION Collectively, the data presented here describe novel MC4Rselective peptide agonists derived from H-DPhe-RW-containing macrocycles. Using a simple and efficacious synthetic method based on the CuAAC click reaction, this study has identified analogs that largely preserve the intrinsic chemical properties of the endogenous ligand while conferring upon these peptides strong selectivity toward MC4R over MC3R and MC5R. Further, in a comparison of the agonist activity of this panel of compounds, important insight has been gained on how to refine a pharmacophore based on a macrocycle containing the H-DPhe-RW motif for improved potency and specificity. Numerous studies have utilized various facets of peptide chemistry to synthesize modified MSH analogs with the aim of offering a potent and receptor subtype selective molecule. Urea-based peptidomimetic scaffolds,71 β-amino acid containing linear peptides,72 alternative heterocyclic peptides,73 peptides derived from features of both agouti peptide and the 8721

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Figure 3. Relationship between independent measures of MC4R activation using second messenger accumulation and transcriptional reporter assays. (a) Positive correlation between average potency determinations for increases in cAMP vs CRE-driven EYFP expression. As an index of correlation between the two parameters, the R2 value was calculated and determined to be highly statistically significant (P < 0.0002). (b) Direct comparison of EC50 values for peptides using cAMP and CRE reporter assay. EC50 (nM) determinations were made for 11-point concentration−response curves (performed in independent triplicates for each peptide−receptor combination). Data are displayed as the mean ± SEM from at least three independent experiments. The lower Y-axis segment encompasses EC50 values between 0 and 1500 nM, whereas the higher segment encompasses 2000−3000 nM.

agonist pharmacophore,74 N-methylation of the peptide backbone,29,75 and even MT-II analogs derived by CuAAC-catalysis have been characterized previously.57,76 While high potency agonists were sometimes obtained, strong selectivity was more elusive for several of these classes of compounds. Here, the combination of the CuAAC chemistry with a simple peptide gave rise to a rigid macrocycle with a structure similar to that of the β-turn in native α-MSH, thereby preserving the agonist potential.

The H-DPhe-RW motif, based on the conserved native HFRW sequence, and particularly the DPhe-RW residues, has been shown to have important roles in governing agonist activity. Consistent with this, our data confirmed that the H residue is dispensable for MC4R activity within our CuAAC-catalyzed macrocycle scaffold. This broadly agrees with previous studies that have demonstrated the limited effects on macrocycle peptide potency toward MC4R upon exchanging the H residue for the neutral A or other polar residues.42,45,77 Interestingly, 8722

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derived macrocycle in 16, reduced activity toward MC4R and abolished activity toward the other tested MCRs. It is tempting to suggest that the size of the macrocycle chain correlates with specificity. Expanding the range of azide and alkyne side chain length, as described in Testa et al.,57 can be used to explore this variable. Extracyclic residues can also modulate the activity of candidate agonists.44 Within the context of the panel of candidate ligands, substituting the flanking K and M residues yielded only modest effects. In replacing the K residue with A (12), agonist activity was improved for all receptors suggesting that some interaction with MC4R is mediated by the basic functional group proximal to the macrocycle. This is supported by the observation that changing K for R (6) results in a molecule that was a marginally worse MC3R and MC5R agonist while remaining roughly equipotent to 1 against MC4R. As with 6, exchange of the carboxyl-terminal M residue with A (2) resulted in an equipotent MC4R agonist with modest reductions in the potency toward MC3R and MC5R. In order to understand and visualize the receptor binding, molecular dynamics (MD) were performed in Molecular Operating Environment (MOE) by applying the Mosberg homology model of the α-MSH-bound state of MC4R to the structure of the activated β2-adrenergic receptor coupled to Gs (see Experimental Section).32,63 A solution structure model was generated using NOESY and ROESY spectra-based assignments for compound 1 to be used to model a ligand-bound receptor complex. Like the MT-II solution structure and another CuAAC generated macrocycle,57 a β-turn is adopted by 1 in its NMR structure. Differences in agonist selectivity and potency between 1 and the reference cyclic peptides are therefore likely explained by the ring size and rigidity. Transmembrane and intracellular domains were maintained in a fixed state while residues in close contact with the ligand and extracellular domains were allowed to relax during MD simulations. Critical residues in 1 were used to replace α-MSH/agouti protein in the model. The critical amino-terminal domain was engineered onto the receptor, optimized through a series of MD calculations, and allowed to converge toward a single conformation.79 The extracellular surface was soaked with water, and the model with fixed transmembrane domains was subjected to 96 h of MD. Finally, the entire structure was energy minimized to release strain imposed by the original homology modeling. It is expected that this novel model, predicated on a characterized structure of a receptor in an active conformation, will provide the means for structural refinement when paired with the functional cell-based assays. While mutational analysis of residues in the transmembrane helices of MC4R broadly validates the model developed for this study,32,70 these residues predicted to mediate ligand− receptor interaction are notably conserved across all three MCRs evaluated in this study, arguing these residues are key for binding of the H-DPhe-RW motif and that residues beyond these regions are ostensibly important in conferring the selectivity these receptors display with respect to agonist interaction. The cell-based assay utilized in this study is a sensitive assay that allows for transformation of cells, using a single construct housing the relevant receptor and reporter gene with regulatory elements and sensitive fluorescence measurements normalized to cell density as the final readout.66 As GPCRs that share the cAMP signaling mechanism can still evoke differential CREmediated transcriptional responses owing to intrinsic receptor localization and membrane dynamics, as well as any additional signaling pathways resulting from the activation of a given

this alteration within the context of the characterized macrocycle markedly (130- to 220-fold) reduced agonist activity toward MC3R and MC5R, in contrast to a larger MT-II macrocycle,45 which may suggest that the turn imposed by the ring is more dynamically altered in the triazole-based ring. The interchanging of the H and W residues (14) indicates that the hydrophobicity and bulkiness of the W residue are required for activity. This is consistent with our model that argues for significant hydrophobic interactions occurring between the W residue and F2616x51, Y2686x58, F2847x34, and L2907x40. However, substitution of the core DPhe, R, and W residues with the corresponding DAla and A (8, 9, and 10) had varying negative effects on agonist potency toward MC4R, with W exerting the most dominant contribution within the present macrocycle. These modifications also diminished MC3R and MC5R activity, further emphasizing their importance. These data are consistent with previous studies probing the effect of A substitutions at key pharmacophore residues in cyclic peptide structures.45 Interestingly, subtle modifications of these residues had wide ranging effects on both potency and receptor selectivity. Exchanging DPhe for F diminished the potency of 4 by approximately 385-fold for the MC4R and ablated all activity at the other MCRs tested, in agreement with a previous characterization of ligand binding following F to DPhe substitution.38 This accentuates the importance of chirality on the secondary structure of the peptide and, ultimately, the potency of the pharmacophore. Similarly, changing the basic residue R to K (5) or to Orn (7) decreased MC4R potency 7- and 181-fold, respectively. Remarkably, these changes completely abolished the agonist activity at MC3R and MC5R at all concentrations investigated. Within the context of the MT-II macrocycle structure, R to K substitution also resulted in a similar decrease in agonist potency with greater effects on MC3R and MC5R as compared with MC4R.42 These data could be explained by the relative location of the charge within these functional groups relative to the D residues in MC4R helix TM3. Specifically, shorter side chains within the residues subsituted for R in the ligand likley have reduced electrostatic interactions with TM3 due to their further distance from the D carboxylates and, thus, reduced avidity. Comparatively, conservative replacement of W with analogs (3 and 15) yielded equipotent MC4R agonists that displayed reduced MC3R activity and a marked loss of activity toward MC5R. A linear peptide variant that incorporated these conservative analogs in place of W displayed greatly diminished binding, suggesting that the conformations adopted by the macrocycle are more potent than those of a similar linear structure.78 Collectively, while these data further support the argument that the core motif residues are key mediators of agonist activity, they offer opportunities to generate highly selective agonists without incurring serious penalties against potency. Notably, conservative alterations at R and W within our scaffold provide some opportunities to achieve MC4R-selective, potent agonists. Beyond the core motif, our data demonstrate that the triazole linkage of the macrocycle and the flanking amino acids also impacts agonist potency and selectivity. Specifically, the choice of azide gave rise to peptides with different agonist acitivties. While the macrocycle derived by triazole formation between Pra and DapN3 (1) had MC4R potency roughly equivalent to NDP-MSH, it was less potent toward MC3R and MC5R. Changing the size of the macrocycle to larger (16) or smaller (17) both changed the activity. While N3R substitution in 17 only seemed to reduce MC5R agonism, the extended Orn(N3) 8723

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GPCR (see Figure S31), this assay is robust, sensitive, flexible and reflects actual gene transcription. To validate the data generated by the reporter assay, the data from this assay were cross-referenced with cAMP measurements in the same cells in parallel experiments. A caveat to comparing reporter gene expression with cAMP levels, or other second messengers, relates to the intrinsic design of the gene reporter assay. Reporter systems possess robust sensitivity because they measure a distal response proceeding over several steps in a pathway from an initial signal, so measurement of the initial signal is not certain to yield high levels of correlation with related end points. Indeed, weak agonists by cAMP concentration determinations can induce significant CRE-reporter responses,80,81 consistent with signal amplification dictating the quality of the response. Our results showed a general lowered potency/sensitivity of the cAMP assay compared to the CRE-based assay. Functional selectivity, characterized as receptors adopting conformations with distinct signaling properties, which are stabilized by ligand binding, could play a role here.82 Given that the CRE reporter has been demonstrated to be activated by alternative pathways in addition to cAMP signaling,83 agonists that favor, for example, both Gs and Gq/11 pathways might display a higher potency in a CRE reporter assay as opposed to a cAMP accumulation assay. As some MC4R ligands display functional selectivity,84 this phenomenon is potentially relevant to in vivo effects and may provide a better understanding of α-MSH mimetic activity. Interestingly, a recent study showed that even within the cAMP cascade, signaling specifics for CREB activation may well be context dependent.85 Thus, while the results from both assays show strong correlation, further characterization of agonist signaling is warranted to select the best assay that reflects physiologically relevant behavior. In conclusion, these data provide novel pharmacological insight into the activation and signaling of MC3R, MC4R, and MC5R. Specifically, the ligands that performed best presented activities similar to α-MSH and the synthetic analog NDPMSH. Further, agonists that balance the preferred outcomes of potency and selectivity were achieved by (1) making subtle changes to the R and W residues of the core H-DPhe-RW motif, (2) altering the size of the macrocycle formed by the CuAAC reaction, and (3) selecting the flanking residues at the amino and carboxyl termini. Changing several of these features in concert may be an effective approach to obtain highly selective MC4R agonist molecules. Observed discrepancies in measured potencies from the cAMP and reporter assays argue for caution in selecting screening approaches to evaluate novel ligands for Gs-coupled GPCRs. The eventual suitability of these molecules as therapeutics is subject to future investigation in animal models and/or clinical evaluations.

■

reactions was performed after cleaving the products as their free acids from the resin. Analytical and preparative reverse-phase HPLC separations were performed on a Waters HPLC system using analytical Chromolith SpeedROD RP-18E (50 mm × 4.6 mm) and delta PAK (25 mm × 200 mm) columns with a flow rate of 5 and 10 cm3/min, respectively. Detection was done at 215 nm on a multiwavelength detector (Waters 490E) for analytical purposes, and a photodiode array detector (Waters M991) was used for preparative separations. A solvent gradient system consisting of (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile−water (9:1) was used. All peptides utilized were purified to >95% purity NMR Acquisition. NOESY and ROESY NMR spectra were acquired using standard pulse sequences on a Bruker Avance 800 spectrometer at 298 K. The sample was dissolved in 0.5 mL of H2O/D2O/ CD3CO2D (88/10/2). Both presaturation and WATERGATE pulses were used for water suppression, and the resulting spectra were compared to ensure that the suppression did not affect the spectral density of, for example, NOESY spectra. Acquisition conditions were as follows: 1D 1H: spectrometer frequency (sf) = 799.80 MHz, spectral width (sw) = 9615.38 Hz, Fourier transform size (si) = 32768, acquisition time (aq) = 1.70 s, relaxation delay (rd) = 1.0 s, number of scans (ns) = 32, number of dummy scans (ds) = 8. NOESY: sf = 799.80 MHz, sw = 7978.7 Hz, si = 1024, number of increments (ni) = 512, aq = 0.213 s, rd = 2.5 s, ns = 16, ds = 32 COSY, HSQC, and HMBC acquired pulse sequences using a Bruker 500 spectrometer at 298 K. The sample was dissolved in D2O. Acquisition conditions were as follows. HSQC: sf = 500.13 MHz; sw = 9.61 (1H); sw = 165.65 (13C); aq = 0.106 s; ns = 32; ds = 16. HSQC: sf = 500.13 MHz; sw = 9.61 (1H); sw = 222.09 (13C); aq = 0.43 s; ns = 64; ds = 16 The NOESY and ROESY spectra were processed by phase and baseline correction by a fifth degree polynomial function. 1D: number of points (np) = 65K, windows function (wdw) = exponential, line broadening (lb) = 0.3. 2D: np(F1) = 1K, np(F2) = 8K, wdw (F1, F2) = sine squared, sine bell shift (sbs) (F1, F2) = 2. Buildup of NOEs was obtained by using ROESY delays of 50, 100, 200, 300, 400, and 600 ms. All the mixing times were found to be within the linear range when extended as suggested by Macur et al.86 Considering these conditions, we find that all relative nuclear Overhauser effect (NOE) intensities are constant with the mixing time. The proportionality of the build-up and the lack of transferred NOEs suggest that the initial rate approximation was held true in the experiments. Distances were then obtained by the isolated spin pair approximation (ISPA). Using correlation time theory from previous work,86,87 it is given that for short mixing times and homonuclear NOEs eq 1 is valid.

k=

⎞ ⎛ μ0 ⎞2 ℏ2γ 4 ⎛ 6τc ⎜ ⎟ ⎜ − τc⎟ 2 2 ⎝ 4π ⎠ 10 ⎝ 1 + 4ω τc ⎠

(1)

For a given experiment, the values of μ0, γ, and ω are fixed. For different spin pair, if the value of τc is assumed equal, for each pair InS k is also constant. Then the ratio of the intensities of a NOE is directly proportional to the ratio of the internuclear distances, simplified by eq 2. ηI1S

EXPERIMENTAL SECTION

ηI 2S

General Remarks. ESI-MS were recorded on a Micromass Global Ultima instrument or a Bruker Solarix ICR instrument, including HRMS. Novel compounds prepared on PEGA800 resins were cleaved off and characterized by HRMS (ESI) in positive mode using a mixture of A buffer and B buffer; see below. MS (ESI) in negative mode was performed in a solvent mixture of water and acetonitrile (1:1) on a Bruker Esquire 3000 Plus mass spectrometer. For all reactions on solid support, PEGA800 resin (0.30 mmol/g, VersaMatrix A/S) in a solid phase vial array was used. Prior to use, the resin was washed with methanol (×6), acetonitrile (×6), DMF (×6), and CH2Cl2 (×6). Each portion of solvent corresponds to the volume of the resin bed (1 vol). All commercially available reagents were used as received without further purification. Analysis of solid-phase

=

rI1S −6 rI 2S −6

(2)

From eq 2, provided correlation time is constant, if one distance is known (e.g., for a methylene or aromatic proton pair), the distance between other proton pairs can be obtained by integration of NOE correlation volumes. For our calculations, the NH and H2 of the Trp-indole was used as reference with a fixed distance of 2.55 Å. Table S3 shows the bond distance for the NOEs found. Solid Phase Synthesis. The following is a general procedure to MC4R triazole cyclized ligand analogs 1−17 (see Figures S3−S21, Table 1). Attachment of the 4-hydroxymethylbenzoic acid (HMBA linker) to the amino-functionalized resin (PEGA800) was carried out by following the standard amino acid coupling procedure (Fmoc-AA-OH, TBTU, NEM, DMF). The HMBA-linker (3 equiv), N-ethylmorpholine 8724

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β2-Adr were first manually mutated to those of the MC4R; all residues in the structure were corrected for any structure violations (cis peptide bonds, ring intersections, etc.), the positions of α-carbons were fixed, and the rest of the receptor energy was minimized. Recursive MD (molecular dynamics) calculations with alternate fixation of the backbone and side chain positions with intermediate minimizations were performed. Helixes 5 and 6 were extended with helical fragments, and these were fused in the turn region to form the third intracellular loop. A central loop bond was broken in each of the remaining loops in need of insertions or deletions. The β-2-Adr loop was extended or shortened at breakpoints (helix or extended as appropriate), and residues were mutated to fit the sequence of MC4R residues. A long MD calculation with annealing from 1000 to 400 K was conducted with fixed transmembrane domains and constraints to facilitate residue proximity at breakpoints of the loop regions. The missing amide bonds at breakpoints were reintroduced and the ligand 1 inserted into the binding site to align f, R, and W with that previously obtained by docking of agouti to a MC4R model. A minimization of energy and a MD calculation with fixed positions for Cα carbons were performed to accommodate the ligand. MD annealing at 1000−400 K with fixed positions of transmembrane Cα‘s gave an initial MC4R lacking the N- and C-terminal loop peptides. These were both subject to several rounds of MD calculations with annealing to derive probable and similar structures from diverse starting conformations. The N- and C-terminal domains were attached, and while having fixed transmembrane Cα′s, the loop regions were subjected to MD-annealing for 5 days. The structure was then soaked in water at termini and an MD calculation at 298 K was performed. The energy was minimized and the structure saved. Minimization was continued at 298 K, without constraints or fixed atoms, allowing for some minor adjustments of the transmembrane helices and to obtain optimal placement of ligand 1 at the binding site. This initial model overlaid well with the inactive β-2-adrenergic receptor. All subsequent calculations were performed on the receptor soaked in water initially maintaining the lipids in contact with the transmembrane domain of the crystal structure. The active state of the receptor structure was used to establish the G-protein bound complex by fitting to the recent crystal structure by Rasmussen et al.63 First, the initial structure was overlaid with 3SN6 to provide an optimal fit of TM-1−4 and -7. By application of restrained dynamics, TM-5 and -6 were gently pulled away from the rest of the receptor (fixed in space) to the position of helixes 5 and 6 in the 3SN6 crystal structure. This activated state model was fitted to the 3SN6 crystal structure bound to the G-protein complex. It followed a series of molecular dynamics calculations with annealing from 800 K, with all atoms fixed in space except for those of the solvent, ligand, lipids, the extracellular domains of the receptor, and residues within 7 Å of the interface between receptor and the G-protein complex. The protein complex was resolvated and subjected to several rounds of molecular dynamics at (400 and 300 K, Amber12-ETH force field) in MOE, maintaining the major part of the G-protein complex fixed in space. The intracellular loop between helixes 5 and 6 hinged onto the helix of the Gα-subunit in contact with the receptor. The C-terminal part of the receptor folded into the interface, exposing the farnesylated cysteine, and interacted C-terminally with Arg-38 of the Gs-protein. The final structure was stable and did not change over 1 week of restraints free molecular dynamics at 298 K. The final structure was again overlaid with the β-2-adrenergic receptor crystal structure 3SN6, and deviations were marginal in the transmembrane region of the two receptors. The topological arrangement and position of the G-protein complex matched perfectly with the crystal structure (see Figure 1, Figure S2, Tables S1 and S2; a PDB file of the resulting theoretical structure is included with the manuscript and is also available from the authors upon request ([email protected])). Phylogenetic Comparison Using GPCRdb.org. All GPCRs with verified peptide agonists, including the entire melanocortin receptor family, were analyzed using the phylogenetic tool provided by www.gpcrdb.org/phylogenetic_trees/targetselection (see Figure S1).89 Full sequences were used with no bootstrapping, and neighbor joining was used as a distance calculation method. Data are presented in a

(NEM, 2.9 equiv), and N-[1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide (TBTU, 2.88 equiv) were mixed in DMF (1 vol) and allowed to react for 5 min. The reaction mixture was added to a preswollen PEGA800 resin (0.1 g/mL) and allowed to react for 2 h, followed by washing with DMF (6 vol) and CH2Cl2 (6 vol). The linker was esterified with Fmoc-methionine (4 equiv) by preactivation (3 min) with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT, 4 equiv) and 1-methylimidazole (3.5 equiv) in CH2Cl2 (1 vol).62 The resin was washed with CH2Cl2 (6 vol) and DMF (6 vol). The Fmoc-protecting group was subsequently removed by two times addition of 20% piperidine in DMF: 2 and 18 min, 1 vol each, with gentle agitation. Subsequently, the resin was washed with a standard wash with DMF (6 vol). The resin was aliquoted into a multicolumn peptide synthesizer (20 column MCPS).61 Each column contained 1 mL of swollen resin (0.03 mmol). Fmoc-propargylglycine (Fmoc-Pra-OH, 3 equiv (30 mg, 0.09 mmol)/column) was subsequently coupled to the resin in each column using the standard TBTU procedure above. After the standard DMF washing procedure, the Fmoc group was removed with piperidine and washed with DMF as described above. The free amine was immediately coupled with the subsequent Fmocamino acid according to sequences in Table 1 using the standard TBTU coupling procedure in order to minimize diketopiperazine formation. The coupling/deprotection cycle was repeated using the same strategy, according to the designed sequence, and the success of each step was verified by Kaiser test.88 After removal of the last Fmoc group, the resin was washed with CH2Cl2 (6 vol) and dried under nitrogen flow. Side chain protecting groups were removed in a 4 h reaction by directly swelling the resin in TFA (trifluoracetic acid) containing ethane dithiol (2%), water (2%), and triisopropylsilane (2%). The TFA was removed and the resin washed with CH2Cl2 (6 vol), DMF (6 vol), and a mixture of t-BuOH and water (7:2). The resins were subjected to peptide cyclization as described below. CuAAC Cyclization (Compound 1). The CuAAC reaction was carried out in a sealed vial by swelling the PEGA800 resin (0.3 mmol/g) displaying the “click” ligand precursor (1.5 mL, 0.045 mmol, 1 equiv) in degassed t-BuOH/H2O (7:2, 2 mL). The mixture was then bubbled through with nitrogen (20 min), and to it was added a solution of CuSO4 (5 mg, 0.031 mmol, 0.7 equiv) and sodium ascorbate (71 mg, 0.36 mmol, 7 equiv) in water (0.5 mL). The reaction mixture was then left under N2 for 12 h with occasional stirring. The resin was filtered off and washed with water (×6), aqueous EDTA solution (1 mM, ×2), and water (×6). Release of the product was achieved by treatment with 0.1 M NaOH (aq, 2 mL) for 2 h. This was followed by neutralization with 0.1 M HCl (aq, 2 mL). The solution was filtered off, and the resin was washed with MeCN (×4) and CH2Cl2 (×2). The combined filtrates were evaporated in vacuo to provide 49 mg (0.044 mmol, 98%) of the crude cyclic product 1. The crude product was purified by preparative HPLC to give 40 mg (80% yield) of pure product, which was characterized by LC−MS and LC−MS/MS, (Table 1, Table S1, Figures S5−S21. Cyclopeptides 2−17 were prepared similarly with comparable yields. All peptides utilized were purified to >95% purity. Molecular Homology Modeling of the Activated State of MC4R. Homology modeling was performed in Molecular Operating Environment (MOE), using version 2014.0901, and the Amber12-ETH force field. The rhodopsin based theoretical MC4R model (2IQP PDB code)32 was superimposed on the crystal structure of A2a and β2-adrenergic receptors (β2-Adr), and both gave an acceptable fit with the β2-Adr being best. The crystal structure of β2-Adr with ligand (3D4S PDB code)63 was therefore used for the homology modeling of MC4R after removal of the auxiliary protein added as an appendage to helixes 5 and 6 for crystallization. First, the sequences were aligned starting with the transmembrane helices. These were optimally aligned, and the alignment was extended, as feasibly as possible, from either end of the helical regions taking into account identical residues, conservative substitutions, structural amino acids, small amino acids, and hydrophobicity (Table S2). Maintaining the crystal structure of the β2-Adr transmembrane domain throughout the procedure, a MC4R homology model was derived as follows: the residues of the transmembrane domains of 8725

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circular format with equal branch lengths. Phylogenetic dendrograms were generated using the programs PHYLIP (http://evolution. genetics.washington.edu/phylip.html) and jsPhyloSVG (http://www. jsphylosvg.com/). Identity refers to shared identical amino acids at a given loci in the primary sequence, whereas similarity refers to the presence of amino acids with similar chemical properties (e.g., acidic, basic, nonpolar aromatic, polar uncharged, etc.) at the same loci. Single Vector−Melanocortin Receptor−EYFP Reporter Constructs. Reporter constructs containing both the receptor of interest and EYFP reporter regulated by nine CRE sequences were synthesized using a core vector, pX-9CRE-d2EYFP such that pMC4R-9CRE-d2EYFP containing the full length human MC4R (accession number AY236539.1) was generated as described previously.66 Plasmids containing full length human cDNAs of MC3R (MC3R-pcDNA3.1+; accession number AY227893) and MC5R (MC5R-pcDNA3.1+; accession number NM_005913), for use as templates for PCR amplification and subcloning, were obtained from the UMR cDNA Resource Center (www.cDNA.org). PCR primers, containing engineered restriction sites for FseI and AscI for facile, directional subcloning of the products into pX-9CRE-d2EYFP, were designed to amplify both receptor cDNAs (MC3R forward: 5′-TTTAAGGCCGGCCACCATGAGCATCCAAAAGACG-3′. MC3R reverse: 5′-AATTTGGCGCGCCCTATCCCAAGTTCATGCCGT-3′. MC5R forward: 5′-TTTAAGGCCGGCCGGATCCACCATGAA-3′. MC5R reverse: 5′-AATTTGGCGCGCCTCTAGACTCGAGTT-3’). PCR amplifications were carried out using Phusion high fidelity DNA polymerase (New England Biolabs). Both vector and purified PCR products were digested with FseI and AscI and ligated with T4 DNA ligase (enzymes from New England Biolabs). After transformation of ligation products into DH10B Escherichia coli (Thermo Fisher), positive clones were identified. Plasmid DNA was purified and sequenced to validate the successful generation of pMC3R-9CRE-d2EYFP and pMC5R-9CRE-d2EYFP, respectively. Stably Expressing EYFP Reporter Gene Cell Lines. As host to all reporter constructs, HEK293 (human embryonic kidney 293) cells (American Type Culture Collection, ATCC) were used. Cells were grown at 37 °C in a 5% CO2, humidified environment with Dulbecco’s modified Eagle’s medium (DMEM), supplemented with heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich; 10% v/v) as well as the antibiotics penicillin (Sigma-Aldrich, 100 U/mL) and streptomycin (Sigma-Aldrich, 100 μg/mL), used as a growth substrate. HEKs expressing the MC4R receptor had been generated previously.66 Transfection of HEKs with pMCXR-9CRE-d2EYFP vectors was achieved using the Nucleofect II electroporation system (Lonza Group). Posttransfection, cells were grown under puromycin-driven selective pressure (Sigma-Aldrich, 0.6 μg/mL). Following expansion of the resistant population, sorting of cells using a FACSAria (BD Biosciences) was conducted at the FACS facility at Biotech Research and Innovation Center (BRIC), at the University of Copenhagen. Cells were first sorted to remove cells with high basal expression of the reporter and then for cells that responded robustly to α-MSH (Sigma-Aldrich, 10 nM) in order to obtain monoclonal cell lines with optimal signal-tonoise ratios, as described previously in Hald et al.66 Cells were later grown in a low-fluorescence medium (Fluorobrite DMEM, Thermo Fisher) with the above supplements to further improve the detection of cellular fluorescence while assaying reporter gene expression. Concentration−Response Relationships for CRE Reporter Assays. To evaluate the potential of the studied compound to act as agonists for the melanocortin receptors of interest, destabilized EYFP fluorescence was quantified as an index of receptor activation.90 Cells expressing MC3R, MC4R, and MC5R, in low-fluoresence medium, were seeded, 50 000 cells per well to provide 30−50% confluence, in a 96 well plate with an opaque-walled well, tissue-culture treated and optically clear bottom (PerkinElmer). After 24 h, the cells were treated with reference compounds (α-MSH, MT-II, NDP-MSH; SigmaAldrich and setmelanotide; MedChemExpress), test compounds, or vehicle (DMSO, Sigma-Aldrich, 0.5% v/v) for 16−20 h. For each independent experiment, 11-point concentration−response series, using 3-fold serial ligand dilutions from 3.3 μM to approximately 0.5 nM, were added (1 μL of concentrated agonist solution into 100 μL) to the wells for each agonist in triplicate. After removal of the incubation medium,

one wash of PBS was done. Fluorobrite supplemented with HEPES buffer (Thermo Fisher; 10 mM) was added to each well. To permit subsequent image analysis, a bright-field and a fluorescence image (Olympus IX73 microscope with EYFP filter cube: excitation, 500/20 nm; emission, 530/30 nm) of the cells in each well was recorded. The images were subjected to image analysis using custom software (Cell Image Analyzer, available from http://cecb.ki.ku.dk) that determines the fluorescence intensity of cells, normalized to the cellular coverage in the bright-field component of the image set with an additional background correction for the basal fluorescence within the images (see Figure S29). With cellular confluency at approximately 70−80%, this analysis provided an accurate measure of the ligand induced expression of EYFP. The normalized fluorescence was corrected for fluorescence in unstimulated cells and then normalized to the percent maximal response observed for α-MSH in that experiment to limit variability between experiments. Concentration−response relationships, prepared in GraphPad Prism (La Jolla, CA), for a given compound were evaluated for at least three independent experiments. Agonist activity curves were fitted using a four-parameter variable slope model and least-squares fitting methodology. The curves were constrained to a minimal value of 0, with 10 000 iterations and the strictest possible convergence criteria for the iterations. Where possible an EC50 value corresponding to the fitted curve was obtained and the maximal value of the curve for α-MSH-treated cells was selected. Concentration−Response Relationships for cAMP Measurements. To validate the CRE reporter data with an independent assay system, cAMP levels were also measured following test compound stimulation of HEKs expressing MC4R. Levels of cAMP were determined using a luminescent, enzymatic complementation-based assay, according to the manufacturer’s protocol (HitHunter assay platform, DiscoveRx). Briefly, 50 000 cells were added to each well of a completely opaque plate. After 24 h, the medium was replaced (30 μL/well) with HBSS (Hanks balanced salt solution), supplemented with 3-isobutyl1-methylxanthine (IBMX, to inhibit cyclic nucleotide degradation, Sigma-Aldrich, 500 μM). Cells were equilibrated in the HBSS medium while agonist dilutions and cAMP standard curve solutions were prepared. Compounds 1−17 were added in 11-point concentration− response series, as described for the CRE reporter assay in triplicate. After 30 min, cells were lysed and lysates were incubated at room temperature in the dark with enzyme fragments and substrate over at least 4 h. In the presence of cAMP, restoration of a functional luciferase enzyme was achieved through complementation such that a linear relationship existed between the cAMP levels and the luciferase activity. Enzymatic activity was measured on Synergy 4 (BioTek) and Spectramax i3x (Molecular Devices) plate readers. As with the CRE reporter assay, activity determinations were corrected for background and normalized to the maximal response for α-MSH in that experiment. Concentration−response relationships, prepared in GraphPad Prism (La Jolla, CA), for a given compound were evaluated for at least three independent experiments. EC50 determinations were obtained as described above. Statistical Analysis. All statistical analyses were conducted using Prism 7.0 (GraphPad, La Jolla, CA, USA). For comparison of agonists across receptor subtypes or between assay platforms (whose concentration−response curves yielded valid EC50 values), two-way ANOVA with Tukey post hoc analysis was employed to compare the statistical significance of EC50 differences. A p-value of 0.05 or less was considered significant. For the evaluation of correlation between variables, R2 values were calculated with a p-value of