Discovery of selective hexapeptide agonists to human neuromedin U receptors types 1 and 2.

Article pubs.acs.org/jmc

Discovery of Selective Hexapeptide Agonists to Human Neuromedin U Receptors Types 1 and 2 Kentaro Takayama,† Kenji Mori,‡ Koji Taketa,† Akihiro Taguchi,† Fumika Yakushiji,† Naoto Minamino,§ Mikiya Miyazato,‡ Kenji Kangawa,‡ and Yoshio Hayashi*,† †

Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan ‡ Department of Biochemistry and §Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan S Supporting Information *

ABSTRACT: Neuromedin U (NMU) are bioactive peptides with a common C-terminal heptapeptide sequence (FLFRPRN-amide, 1a) among mammals, which is responsible for receptor activation, namely NMU receptor types 1 (NMUR1) and 2 (NMUR2). Among the various physiological actions of NMU, the anorexigenic effect has recently attracted attention in drug discovery efforts for treating obesity. Although several structure−activity relationship (SAR) studies have been reported, receptor-selective small peptide agonists have yet to be disclosed. Herein a SAR study of 1a-derived peptide derivatives is described. We initially screened both human NMUR1- and NMUR2-selective peptides in calcium-mobilization assays with cells transiently expressing receptors. Then we performed a precise assay with a stable expression system of receptors and consequently discovered hexapeptides 8d and 6b possessing selective agonist activity toward each respective receptor. Hexapeptide 6b, which selectively activates NMUR2 without significant NMUR1 activation, should aid in the development of anorexigenic drugs as well as advance NMU-related endocrinological research.

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increases in the intracellular calcium concentration ([Ca2+]i) in cells expressing either NMUR1 or NMUR2 with comparable potency and efficacy.7 Additionally both exhibit a variety of biological behaviors (e.g., appetite suppression,2,8 reduction in body weight,9 increase of body temperature,9 regulation of cardiovascular function through the sympathetic nervous system,10,11 suppression of gastric acid secretion via CRH,12 regulation of circadian rhythms,7,13 promotion of mast cellmediated inflammation,14 regulation of puberty onset,15 antidiuresis via vasopressin release,16 increase in milk secretion via oxytocin release,17 and regulation of glucose homeostasis18). These pharmacological actions of NMU and NMS have been confirmed by administration of nonselective agonists (NMU and NMS) or by use of mice with genetic defects. Among the various physiological actions of NMU, the anorexigenic effect has received attention as a potential drug to treat obesity. For instance, poly(ethylene glycol) (PEG) or human serum albumin (HSA) conjugated hNMU has been reported to display long-lasting and potent anorexigenic activity in mice when administered peripherally.19,20 In addition, these studies indicated that both NMUR1 and NMUR2 engage in the anorexigenic activity of peripherally administrated hNMU conjugates.

INTRODUCTION Human neuromedin U (hNMU, Figure 1A) is a linear bioactive peptide, which consists of 25 amino acid residues with a Cterminal amide structure. Its receptors belong to the class A family of G-protein coupled receptors (GPCRs), and are designated as NMU receptors types 1 (NMUR1) and 2 (NMUR2), which were previously identified as the orphan GPCRs FM-3/GPR66 and FM-4/TGR-1, respectively.1 The NMUR1 mRNA is widely expressed in peripheral tissues, including the intestinal tract and lungs, while the NMUR2 mRNA is expressed in the central nervous system (CNS) at high levels, particularly in the hypothalamic paraventricular nucleus (PVN).2−4 In 2003, Hanada et al.5 demonstrated that intracerebroventricularly administrated rat NMU stimulated the secretion of corticotropin-releasing hormone (CRH), which is produced in PVN, and results in the CRH-dependent regulation of feeding behavior and energy homeostasis. Via selective knockdown of NMUR2 in rat PVN, Benzon et al.6 recently found that the NMUR2 signaling in PVN plays an important role in the anorexigenic response to high-fat food intake. Therefore, the NMU in CNS is believed to mediate NMUR2. On the other hand, neuromedin S (NMS), which is a Cterminal-amidated linear bioactive peptide consisting of 33 amino acid residues (in the case of human NMS), has been identified as another novel ligand for the NMU receptors (Figure 1A).7 NMU and NMS induce dose-dependent, robust © 2014 American Chemical Society

Received: April 17, 2014 Published: July 7, 2014 6583

dx.doi.org/10.1021/jm500599s | J. Med. Chem. 2014, 57, 6583−6593

Journal of Medicinal Chemistry

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revealed three features: (1) Aromatic Phe0 is important for the contractile activity. (2) Substitution of Phe2 by Tyr increases activity. (3) Arg5 and the C-terminal Asn6-amide are indispensable for receptor binding and activation (Figure 1B).24−26 An alanine scan study of tyrosyl-1a demonstrated that Arg3 and Arg5 are necessary for the agonistic activity in murine NMU receptors.27 Substituting Arg3 with Ala (EC50 = 2411 and 156 nM for NMUR1 and NMUR2, respectively) decreases the agonistic activity of the peptide toward mouse NMUR1 (EC50 = 9.5 nM) and NMUR2 (EC50 = 3 nM) receptors, suggesting that Arg3 is a critical residue for activity, whereas the drastic reduction of agonistic activity observed only in NMUR1 receptor would be a useful clue to distinguish between two receptors of NMUR1 and NMUR2 (Figure 1B). In consideration of these previous findings, we thought that there still remains room for discovering a small peptidic agonist that selectively activates each hNMU receptor by a precise follow-up SAR study. Because with nonselective agonists it is generally difficult to distinguish the receptor types responsible for biological actions, the development of a receptor-selective agonist may elucidate the molecular mechanism and biological phenomena of hNMU. In this study, we developed a screening system to identify selective agonistic activity at human NMUR1 or NMUR2 by use of human embryonic kidney 293 (HEK293) cells with a transient expression of these receptors. A series of synthetic peptides designed from lead peptide 1a was initially screened. Then the agonistic activities of selected peptides were further evaluated by use of receptors stably expressed in Chinese hamster ovary (CHO) cells to obtain PC50 values and identify NMUR1- and NMUR2-selective hexapeptidic agonists.

Figure 1. (A) Structures of human neuromedin U (hNMU) and human neuromedin S (hNMS). Underlines indicate the common amidated heptapeptide sequence in C-termini of NMUs. (B) Structure of hNMU-derived heptapeptide 1a and the SAR from previous studies. Arrows indicate the structural entities for potent biological activity of heptapeptide 1a.22−26 Asterisk under Arg3 indicates which residue should be useful to distinguish the selectivity toward NMUR1 and NMUR2.27

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RESULTS AND DISCUSSION Peptide Synthesis. The peptide derivatives were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (see Experimental Section).28,29 Pure peptides were prepared by treating protected peptide resins with trifluoroacetic acid (TFA)−m-cresol−thioanisole−1,2ethanedithiol (EDT) (4.0 mL, 40:1:1:1) for 150 min at room temperature and subsequent preparative reversed-phase (RP) HPLC purification in a 0.1% aqueous TFA−CH3CN gradient system. To prepare guanydinate peptides 3a−c, synthesized peptide derivatives 2a−c with aminoalkyl side chains at residue 3 were treated with 1H-pyrazole carboxamidine hydrochloride in the presence of DIEA (N,N-diisopropylethylamine) at room temperature for 24 h in N,N-dimethylformamide (DMF) followed by HPLC purification (Scheme 1).30,31 All peptide derivatives were characterized by electrospray ionization timeof-flight mass spectrometry (TOF MS ES+), and key peptides

These two bioactive peptides (NMU and NMS) share a common heptapeptide sequence (H-Phe0-Leu1-Phe2-Arg3-Pro4Arg5-Asn6-NH2, 1a) at their C-termini (Figure 1B). Hence, the C-terminus is presumed to play a key role in the activation of both NMUR1 and NMUR2,7,21 which is consistent with the fact that an endogenous C-terminal peptide, tyrosyl-1a (porcine NMU-8, pNMU-8), shows excellent agonistic activity.21 Sakura et al.22 conducted a structure−activity relationship (SAR) study to develop potent peptidic agonists; an N-terminal modified peptide, pyroglutamyl (Pyr)-1a (dog NMU-8), shows improved aminopeptidase resistance and increased agonistic activity compared to tyrosyl-1a (pNMU-8) in a contractility assay using isolated smooth muscle from a chicken crop. Their later study, which was based on the peripheral avian NMU receptor, found more potent derivatives, succinyl- and glutaryl1a.23 Additional SAR studies on the peripheral avian receptor Scheme 1. Guanidinylation of Hexapeptide Derivatives 2a−ca

a

Reagents and conditions: (a) 1H-pyrazole carboxamidine hydrochloride, DIEA, DMF; (b) preparative RP-HPLC. 6584



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Figure 2. (A) Structures of 1a−l. (B) Effect of substitution at the N-terminal moiety (residue 0) of derivative 1a on the agonistic activity to the human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentrations, 10 and 100 nM; positive controls, hNMU (activity at 1000 nM = 100%), pNMU-8, and succinyl-1a.23

hexapeptide 1b (3-phenylpropionyl-Leu1-Phe2-Arg3-Pro4-Arg5Asn6-NH2), in which the α-amino group of the N-terminus Phe0 in 1a was eliminated, and heptapeptide 1c, in which Phe0 was cyclized to (S)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl group through a methylene linker (Figure 2A). The agonistic activities of 1b toward human NMUR1 and NMUR2 were nonselective and less than those of hNMU and pNMU-8 (tyrosyl-1a) (Figure 2B). However, the activity of 1a was only slightly lower, suggesting that the N-terminal α-amino group is not important for agonistic activity. On the other hand, heptapeptide derivative 1c had a much lower activity than 1a, implying that the conformational position of the phenyl group in Phe0 is unfavorable for the activity. Next, the 3-phenylpropionyl group in hexapeptide 1b was substituted with various acyl groups (1d−l, Figure 2A). Although many derivatives decreased the agonistic activity,

were analyzed by NMR (see Supporting Information). Each peptide had a purity >95% (RP-HPLC analysis at 230 nm, see Supporting Information) and was solubilized in dimethyl sulfoxide (DMSO) as a 20 mM stock solution for biological assays. Substitution Effect at the N-terminal Phe Residue of Acyl Structures in Peptide 1a. The agonistic activities of peptide derivatives for both human NMUR1 and NMUR2 transiently expressed in HEK293 cells were evaluated by calcium-mobilization assays. Figures 2−5 and S1 and S2 (Supporting Information) show the results for each tested peptide with concentrations between 1 and 1000 nM as a relative value (%) compared to the activity of hNMU at a concentration of 1000 nM (=100%). Because aminopeptidases generally degrade bioactive peptides from the N-terminus, we initially synthesized desamino 6585



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phenylacetyl derivative 1e and nitrogen heterocycle derivative 1g (3-pyridinepropionyl) displayed moderate NMUR1 selectivity. In addition, derivatives 1f (4-phenylbutanoyl), 1i (3cyclohexanepropionyl), and 1j (cyclohexanebutanoyl) displayed NMUR2 selectivity (Figure 2B). Although the structure−activity correlation among these derivatives was unremarkable, the existence of aryl groups in the α- or βposition of the acyl group of hexapeptide 1 tended to increase the NMUR1 selectivity, while the presence of aliphatic acyl groups tended to increase the NMUR2 agonistic activity. In particular, the NMUR2 selectivities of 3-cyclohexyl derivatives 1i, which is a reduced form of the 3-phenylpropionyl group, and 1j were remarkably enhanced (Figure 2B). Effect of Amino Acid Substitutions at Residues 3−6 of Hexapeptide 1b. To understand the effect of amino acid substitution on agonistic activity, we conducted a SAR study based on nonselective agonist 1b. A previous study26 using isolated chicken crop smooth muscles reported that the Cterminus is important for activity (Figure 1B). Thus, we initially evaluated the importance of C-terminal Asn6-amide. All synthesized Asn6-amide derivatives possessing Ala (S1a), Gln (S1b), Asp (S1c), Dab (S 1d; Dab = α,γ-diaminobutyric acid), and Leu (S1e) at residue 6 drastically decreased the activity (Figure S1A,C, Supporting Information), which is consistent with the hypothesis that Asn6 is an important residue at this position to exert agonistic activities. Although this residue has been also reported to be indispensable for peripheral NMU receptor activation in an avian receptor study,25 the importance of Arg5 was examined by substituting a series of other basic amino acid residues with amino or guanidino side chains of different lengths (S2a−c and S3a−c; Scheme S1, Supporting Information). Most derivatives drastically decreased the activity (Figure S1C, Supporting Information), confirming the importance of the original Arg5 in our hNMU receptor assay. Next to elucidate the importance of Pro4, we evaluated three 1b derivatives containing Hyp (S4a; Hyp = 4-hydroxyproline), homoPro (S4b), and 1-NH-cPropn (S4c; 1-NH-cPropn = 1aminocyclopropanecarboxylic acid) at residue 4 (Figure S1B, Supporting Information). Only the structurally similar homoPro-derivative S4b exhibited a slightly lower activity compared to 1b, but it did not have a sufficiently selective agonistic activity (Figure S1C, Supporting Information), suggesting that Pro4 was another critical residue for the activity. These results suggested that the C-terminal three amino acid residues, Pro4, Arg5, and Asn6-NH2, strictly form the core structure to mutually activate both NMUR1 and NMUR2. Because receptor selectivity via modification of the Cterminal three amino acid residues was difficult, we then focused on Arg3 by preparing a series of derivatives in a manner similar to that for Arg5. These derivatives were aminoalkyl sidechain derivatives 2a−c, which contained Dap (α,β-diaminopropanoic acid), Dab, and Lys at residue 3, and their corresponding guanidinate derivatives 3a−c (Scheme 1, Figure 3A). Interestingly, the NMUR2 selectivity increased upon substitution of Arg3 with Dap (2a) or Dab (2b), which had shortened aminoalkyl side chains. Moreover, these peptides exhibited similar NMUR2 agonistic activity as derivative 1b (Figure 3B). However, corresponding guanidinate derivatives 3a and 3b exhibited moderate NMUR2 selectivities. On the contrary, the Lys-substituted derivative 2c with an extended side chain showed nonselectivity and a marked decrease in activity. Additionally, corresponding guanidinate derivative 3c (homoArg) showed nonselective activity. These findings agree

Figure 3. (A) Structures of 2a−c and 3a−c. (B) Effect of chemical structure at residue 3 (Arg) of derivative 1b on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentrations, 1−100 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 1b.

well with the data from the aforementioned experiment using Ala scanning (Figure 1B)27 and indicate that side-chain substitution from guanidino to amino group in residue 3 may improve the NMUR2 selectivity. Thus, modifying residue 3 with an aminoalkyl group consisting of a proper side-chain length should help realize high NMUR2 selectivity. Discovery of NMUR2-Selective Hexapeptide Agonist. In terms of residue 2, a previous report on the peripheral receptor using avian smooth muscle mentioned that substituting Phe with Tyr at residue 2 typically increased the contractile activity.24 However, our evaluation with a series of aromatic amino acids [i.e., Tyr (4a), Phg (4b; phenylglycine), and homoPhe (4c) at residue 2] decreased the activity (Figure 4), indicating that human receptors and the observed avian receptor completely differ. On the other hand, when Phe2 6586



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cyclohexyl ring in the side chain of Phe2 increases the selectivity for NMUR2. Next, residue 3 (Arg) of 5a was substituted by Dap (5b, Figure 5A). As expected, derivative 5b (3-cyclohexylpropionylLeu1-Cha2-Dap3-Pro4-Arg5-Asn6-NH2) exhibited higher selectivity and agonistic activity for NMUR2 compared to 1i and 5a (Figure 5B). Derivative 5c, with Dab instead of Dap at residue 3, also showed almost the same activity toward hNMU receptors (Figure 5A,B). Finally, we optimized the structure of 5b with regard to residue 2 using a series of amino acid residues with alkyl side chains, such as Chg (6a; cyclohexylglycine), Leu (6b), Ile (6c), Nle (6d; norleucine) and Nva (6e; norvaline) instead of Cha2 (Figure 5C). All these peptide derivatives dominantly activated only NMUR2. However, derivatives 6a (Chg) and 6c (Ile) with β-branched side chains exhibited lower agonistic activity toward NMUR2 than 5b (Figure 5D). In contrast, derivative 6b (Leu), with an isobutyl side chain, showed the best NMUR2 selectivity among the synthesized peptide derivatives and higher agonistic activity, which was around 70% and 88% that of hNMU at concentrations of 1 and 10 nM, respectively (Figure 5D). Derivatives 6d (Nle) and 6e (Nva) showed similar activities to 5b. Thus, preliminary screening of agonists to hNMU receptors indicates that derivative 6b (3-cyclohexylpropionyl-Leu1-Leu2Dap3-Pro4-Arg5-Asn6-NH2) is the most NMUR2-selective and has potent hNMU agonistic activity. Discovery of NMUR1-Selective Hexapeptide Agonist. To develop a NMUR1-selective agonist, we focused on 3pyridinepropionyl derivative 1g, which showed moderate NMUR1 selectivity with similar potency as hexapeptide 1b (Figure 2). When residue 1 (Leu) of 1g was substituted with Phe (7a, Figure 6A), derivative 7a showed higher selectivity to NMUR1 with more potent agonistic activity (Figure 6B). Thus, we then optimized the N-terminal acyl structure of 7a. However, 6-quinolinecarboxyl derivative 7b and 2-quinolinecarboxyl derivative 7c showed a marked decrease in activity, suggesting that the conformational position of the aromatic ring at the N-terminal acyl group is unfavorable, as observed in derivative 1c. Because 1e (a phenylacetyl derivative of 1b) displayed comparable NMUR1 selectivity as 1g (Figure 2), we prepared one-carbon-shorter nitrogen heteroaromatic derivatives 7d−g (Figure 6A). These derivatives 7d−g exhibited similar NMUR1 selectivities to 7a (Figure 6B), suggesting that the heteroaromatic ring structure at the N-terminus is crucial to develop a NMUR1-selective agonist. On the basis of the structure of derivative 7a, we further investigated Phe1 substitution on the NMUR1 selectivity using a series of sterically bulky aromatic amino acid residues. Among the synthesized derivatives bearing His (8a), Trp (8b), 1-Nal (8c; 1-naphthylalanine) and 2-Nal (8d; 2-naphthylalanine) (Figure 6C), derivatives 8b−d displayed stronger agonistic activity toward NMUR1 than 7a (Figure 6D). These results suggest that the bulky aromatic structure at residue 1 in combination with the N-terminal nitrogen-containing aromatic acyl moiety is favorable for NMUR1 selectivity. Derivative 8d [3-pyridinepropionyl-(2-Nal)1-Phe2-Arg3-Pro4-Arg5-Asn6-NH2] was a potent agonist toward human NMUR1, exhibiting about 63% and 84% NMUR1 agonist activity compared to that of hNMU at concentrations of 1 and 10 nM, respectively, in the transient expression system (Figure 6D). Measurement of Agonistic Activities for NMUR1 and NMUR2. In the aforementioned preliminary SAR study, we screened the C-terminal hexapeptide derivatives derived from

Figure 4. (A) Structures of 4a−d. (B) Effect of chemical structure at residue 2 (Phe) of derivative 1b on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentrations, 1 and 10 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 1b.

was substituted with an aliphatic Cha (cyclohexylalanine) (4d), NMUR2-selective agonistic activity was observed in the calcium-mobilization assay (Figure 4). Therefore, to develop more selective NMUR2 agonists, we synthesized derivative 5a, which is a 1i derivative possessing Cha at residue 2 (Phe2) (Figure 5A). Derivative 5a exhibited at least 3-fold higher NMUR2 selectivity compared to 1i (Figure 5B). With regard to substitution at residue 1, the original amino acid residue Leu was better than ether aliphatic substituents S5a−e (Figure S2, Supporting Information). These SAR studies of amino acid substitutions in residues 1− 6 provided three findings. (1) The C-terminal three amino acid residues, Pro4, Arg5, and Asn6-NH2, should be maintained to appropriately recognize both NMUR1 and NMUR2. (2) Substitution of guanidino group by amino group (Dap or Dab) in the side chain of Arg3 increases selectivity for NMUR2. (3) Substitution of the aromatic phenyl ring by an aliphatic 6587



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Figure 5. (A) Structures of 5a−c. (B) Effect of chemical structure at residue 2 (Phe) and residue 3 (Arg) of derivative 1i on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentration, 10 and 100 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 1i. (C) Structures of 6a−e. (D) Effect of chemical structure at residue 2 (Cha) of derivative 5b on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentrations, 10 and 100 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 5b.

NMUR1−9 and CHO/NMUR2−8 cell lines were used because quantitative polymerase chain reaction (PCR) analysis revealed that these cell lines showed high-level expression of NMUR1 (1.39 ± 0.28 copies/pg total RNA, mean ± SD) and NMUR2 (5.26 ± 0.17 copies/pg of total RNA), respectively (Figure S3, Supporting Information). Stock solutions of the

hNMU to develop selective agonists that activate specific hNMU receptors, and we obtained NMUR1-selective agonist 8d and NMUR2-selective agonist 6b (Figure 7). To evaluate the PC50 values of these derivatives, we established new CHO cells that stably expressed human NMUR1 or NMUR2 using a previously reported method.7 In this study, two types of CHO/ 6588



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Figure 6. (A) Structures of 7a−g. (B) Effect of chemical structure at the N-terminal moiety (3-pyridinepropionyl group) and residue 1 (Leu) of derivative 1g on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calciummobilization assay). Peptide concentrations, 1−100 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 1g. (C) Structures of 8a−d. (D) Effect of chemical structure at residue 1 (Phe) of derivative 7a on agonistic activity toward human NMUR1 (black bars) and NMUR2 (gray bars) transiently expressed on HEK293 cells (calcium-mobilization assay). Peptide concentrations, 1−100 nM; positive controls, hNMU (activity at 1000 nM = 100%) and 7a. 6589



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Figure 7. Development of NMUR1-selective agonist 8d and NMUR2-selective agonist 6b by derivatization of lead hexapeptide 1b.

Figure 8. In vitro agonistic activities of hexapeptide derivatives 1b, 8d, and 6b toward human NMUR1 and NMUR2 stably expressed on CHO cells (calcium-mobilization assay). Peptide concentrations, 10−12−10−6 M; reference compound, hNMU. Data were determined in triplicate.

Table 1. Receptor Agonistic Activities of Hexapeptide Derivatives 1b, 8d, and 6b PC50a (nM) compd

NMUR1

hNMU 1b 8d 6b

0.43 ± 0.03 20 ± 5 5.1 ± 1 >1000

EC50b (nM)

selectivity c

NMUR2

NMUR1

± ± ± ±

5.8 0.90 49 152

NMUR1

NMUR2

0.43 ± 0.03 9.0 ± 3 (71%) 2.2 ± 1 (79%) NAe

2.5 ± 0.5 8.4 ± 1 (82%) NAe 6.4 ± 1 (98%)

PC50, concentration of a tested peptide derivative that has a 50% response induced by the positive control (hNMU at 1000 nM). Means ± SD were determined in triplicate. bIntrinsic activity (%) compared to the standard agonist activity of the endogenous ligand hNMU is shown in parentheses. c NMUR1 selectivity was calculated by dividing the PC50 value for NMUR2 by the PC50 value for NMUR1 for each peptide. dNMUR2 selectivity was calculated by dividing the PC50 value for NMUR1 by the PC50 value for NMUR2 for each peptide. eNA, not applicable. a

respectively). Additionally, derivative 1b with EC50 values of 9.0 and 8.4 nM (ia = 71% and 82%) for NMUR1 and NMUR2, respectively, was a partial agonist (Figure 8 and Table 1). On the other hand, derivative 8d showed 50-fold higher NMUR1 selectivity with a PC50 value of 5.1 nM, which was 4-fold increased activity compared to 1b. Because the activity of 8d was still about 10-fold less than that of hNMU, it should be improved in the future. Additionally, derivative 8d with an EC50 value of 2.2 nM (ia = 79%) for NMUR1 was also a partial agonist (Figure 8 and Table 1).

peptide derivatives (20 mM in DMSO) were diluted in an assay buffer and applied at 10−12−10−6 M (final concentration) to the established CHO cell lines stably expressing the receptors. Figure 8 shows the agonistic activities of peptide derivatives 1b, 8d, 6b, and hNMU (positive control), while Table 1 lists their PC50 values, receptor selectivities, and EC50 values including intrinsic activities (ia). The PC50 values of nonselective hexapeptide derivative 1b for NMUR1 and NMUR2 were 20 and 18 nM, respectively, which were about 50- and 7-fold decreased in potency compared to hNMU (0.43 and 2.5 nM, 6590



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cell lines were grown on 100 mm dishes and incubated at 37 °C under 5% CO2 to approximately 70% confluence. Screening of Synthetic Peptide Derivatives (CalciumMobilization Assays). HEK293 cells (5.0 × 105 cells) were seeded into 100 mm dishes in 10 mL of culture medium, and then transfected 18 h after seeding with expression plasmids7 by use of FuGENE6 as per the manufacturer’s instructions. Eighteen hours after transfection, the transfected cells were seeded (4.0 × 104 cells/well) into 96-well black-wall plates with clear bottoms coated by poly-D-lysine. Eighteen hours after incubation, the cells were loaded for 40 min with 4 μM Fluo-4-AM fluorescent indicator dye in an assay buffer [HBSS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 2.5 mM probenecid, pH 7.4] with 1% FCS, and washed four times with assay buffer without FCS. Then the intracellular calcium change was assayed on a fluorometric imaging plate reader (Molecular Device, Sunnyvale, CA). The tested peptide derivatives were dissolved in an assay buffer containing 0.05% bovine serum albumin (BSA) and 0.001% Triton X-100 and prepared at 1−1000 nM. The efficacy of the tested peptide derivative was determined from the maximal value. Establishment of Cell Lines Expressing NMUR1 and NMUR2. CHO cells stably expressing human NMUR1 or NMUR2 were newly established as previously reported.7 Briefly, CHO cells were transfected with the expression plasmid, and then the cells were maintained in a culture medium containing 1 mg/mL G418 to select for cells expressing the neomycin-resistance gene. After 2 weeks, 12 colonies/ plasmid were picked up, and the expression of receptor gene was validated by quantitative PCR as described previously.32 The primer set used for human NMUR1 was 5′-TGTGGAGCGTCGTGTCACAG-3′ and 5′-CTGGAAGGTCTCTCGGAAGC-3′, while that for human NMUR2 was 5′-GAGTATCTGGCCTTCCTCTG-3′ and 5′GTGGGCGTCTTCATAGCCTG-3′. The stably expressing cell lines (CHO/NMUR1−9 and CHO/NMUR2−8) were obtained, and the function of the recombinant receptors expressed in these cell lines was confirmed by a calcium-mobilization assay with human NMU. Determination of PC50 Values. The CHO cells stably expressing receptors were seeded (2.0 × 104 cells/well) into 96-well black-wall plates with clear bottoms. A calcium-mobilization assay was performed as described above. The tested peptide derivatives were dissolved at concentrations of 10−12−10−6 M. The receptor agonistic activities of the tested peptide derivatives were determined in triplicate at each concentration. Then the PC50 value, which is the concentration of a tested peptide derivative at which the response is 50% of the response induced by the positive control (hNMU at 1000 nM),33 was calculated.

On the other hand, peptide derivative 6b showed more than 150-fold increase in the NMUR2 selectivity with a PC50 value for NMUR2 of 6.6 nM, which was only a 3-fold decrease in potency compared to hNMU (Table 1). Intriguingly, derivative 6b with an EC50 value of 6.4 nM (ia = 98%) for NMUR2 was a full agonist (Figure 8 and Table 1). Moreover, 6b did not show agonistic activity for NMUR1, at least at concentrations below 10−6 M (Figure 8). Consequently, peptide derivative 6b is the first example of a human NMUR2-selective agonist without significant NMUR1 activation.

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CONCLUSIONS A series of C-terminal core peptide derivatives of human neuromedin U (CPN) were synthesized for a detailed SAR study in a calcium-mobilization assay system. Hexapeptide agonists 8d (designated as CPN-124) and 6b (designated as CPN-116) selectively activated human neuromedin U receptors types 1 and 2, respectively. These selective small peptide agonists, especially NMUR2-specific activator 6b (CPN-116), have potential in the discovery and development of anorexigenic drugs and may result in innovative NMU-related biological and pharmacological research. In the future, the receptor binding mechanism will be investigated in order to develop a more specific activator for NMUR1 and a small molecule superagonist.

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EXPERIMENTAL SECTION

Materials. Reagents and solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan), Sigma−Aldrich (St. Louis, MO), Nakalai Tesque (Kyoto, Japan), Watanabe Chemical Industries (Hiroshima, Japan), and Tokyo Chemical Industries (Tokyo, Japan). All were used as received. Sterile Dulbecco’s modified Eagle’s medium (DMEM)-high glucose, α-modified Eagle minimum essential medium (α-MEM)-nucleoside, Hank’s balanced salt solution (HBSS), and fetal calf serum (FCS) were purchased from Life Technologies (Carlsbad, CA). Sterile 100 mm dishes and 96-well black-wall plates with clear bottoms were purchased from Iwaki (Tokyo, Japan) and Corning (Cambridge, MA), respectively. FuGENE 6 was purchased from Promega (Madison, WI). Synthesis of Peptide Derivatives. As previously reported,28,29 the Fmoc-NH-SAL resin (80−100 mg, 0.038−0.047 mmol) and Fmoc-amino acids/R-COOH (0.114−0.141 mmol) were sequentially coupled via a diisopropylcarbodiimide (DIPCI, 0.114−0.141 mmol)− 1-hydroxybenzotriazole (HOBt, 0.114−0.141 mmol) method for 2 h in DMF (1.0 mL) after removal of each Fmoc group with 20% piperidine−DMF (1.5 mL, 20 min) to obtain protected peptide− resins. The resins were treated with TFA−m-cresol−thioanisole−EDT (4.0 mL, 40:1:1:1) for 150 min at rt, followed by preparative RPHPLC purification in a 0.1% aqueous TFA−CH3CN system to obtain peptide derivatives as TFA salts, except for 3a−c. For preparation of derivatives 3a−c, 1H-pyrazole carboxamidine hydrochloride (0.021 mmol) was added to a solution of derivatives 2a−c (0.014 mmol) and DIEA (0.055 mmol) in DMF (0.14 mL), respectively.30,31 The reaction mixture was stirred at rt for 24 h, and subsequent preparative RP-HPLC purification in a 0.1% aqueous TFA-CH3CN system provided peptide derivatives 3a−c as TFA salts. High-resolution mass spectra (TOF MS ES+) were recorded on a micromass LCT (see Supporting Information). 1H NMR spectra were obtained on a Bruker Avance III spectrometer (400 MHz) with tetramethyulsilane (TMS) as an internal standard. The purity of all products was >95% (RPHPLC analysis at 230 nm). Cell Cultures. CHO cells were maintained in α-MEM-nucleoside with 10% heat-inactivated FCS. A subculture was performed every 3−4 days. HEK293 cells were maintained in DMEM-high glucose with 10% heat-inactivated FCS. A subculture was performed every 3−4 days. All

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ASSOCIATED CONTENT

S Supporting Information *

Analytical data for all peptide derivatives, analytical HPLC chromatograms, NMR spectra, one scheme showing guanidinylation of hexapeptide derivatives S2a−c, and three figures showing structures of S1a−e and S4a−c and effect of chemical structure at residues 6 (Asn), 5 (Arg), and 4 (Pro) of derivative 1b on agonistic activity toward NMUR1 and NMUR2, structures of S5a−e and effect of chemical structure at residue 1 (Leu) of derivative 1i on agonistic activity toward NMUR1 and NMUR2, and exogenous expression of human neuromedin U receptors types 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone +81 42 676 3275; fax +81 42 676 3279; e-mail [email protected]. Notes

The authors declare no competing financial interest. 6591



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Article

(8) Ida, T.; Mori, K.; Miyazato, M.; Egi, Y.; Abe, S.; Nakahara, K.; Nishihara, M.; Kangawa, K.; Murakami, N. Neuromedin S is a novel anorexigenic hormone. Endocrinology 2005, 146, 4217−4223. (9) Nakazato, M.; Hanada, R.; Murakami, N.; Date, Y.; Mondal, M. S.; Kojima, M.; Yoshimatsu, H.; Kangawa, K.; Matsukura, S. Central effects of neuromedin U in the regulation of energy homeostasis. Biochem. Biophys. Res. Commun. 2000, 277, 191−194. (10) Chu, C.; Jin, Q.; Kunitake, T.; Kato, K.; Nabekura, T.; Nakazato, M.; Kangawa, K.; Kannan, H. Cardiovascular actions of central neuromedin U in conscious rats. Regul. Pept. 2002, 105, 29−34. (11) Sakamoto, T.; Nakahara, K.; Maruyama, K.; Katayama, T.; Mori, K.; Miyazato, M.; Kangawa, K.; Murakami, N. Neuromedin S regulates cardiovascular function through the sympathetic nervous system in mice. Peptides 2011, 32, 1020−1026. (12) Mondal, M. S.; Date, Y.; Murakami, N.; Toshinai, K.; Shimbara, T.; Kangawa, K.; Nakazato, M. Neuromedin U acts in the central nervous system to inhibit gastric acid secretion via CRH system. Am. J. Physiol. Gastrointest. Liver Physiol. 2003, 284, G963−G969. (13) Nakahara, K.; Hanada, R.; Murakami, N.; Teranishi, H.; Ohgusu, H.; Fukushima, N.; Moriyama, M.; Ida, T.; Kangawa, K.; Kojima, M. The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. Biochem. Biophys. Res. Commun. 2004, 318, 156−161. (14) Moriyama, M.; Sato, T.; Inoue, H.; Fukuyama, S.; Teranishi, H.; Kangawa, K.; Kano, T.; Yoshimura, A.; Kojima, M. The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells. J. Exp. Med. 2005, 202, 217−224. (15) Fukue, Y.; Sato, T.; Teranishi, H.; Hanada, R.; Takahashi, T.; Nakashima, Y.; Kojima, M. Regulation of gonadotropin secretion and puberty onset by neuromedin U. FEBS Lett. 2006, 580, 3485−3488. (16) Sakamoto, T.; Mori, K.; Nakahara, K.; Miyazato, M.; Kangawa, K.; Sameshima, H.; Murakami, N. Neuromedin S exerts an antidiuretic action in rats. Biochem. Biophys. Res. Commun. 2007, 361, 457−461. (17) Sakamoto, T.; Mori, K.; Miyazato, M.; Kangawa, K.; Sameshima, H.; Nakahara, K.; Murakami, N. Involvement of neuromedin S in the oxytocin release response to suckling stimulus. Biochem. Biophys. Res. Commun. 2008, 375, 49−53. (18) Peier, A. M.; Desai, K.; Hubert, J.; Du, X.; Yang, L.; Qian, Y.; Kosinski, J. R.; Metzger, J. M.; Pocai, A.; Nawrocki, A. R.; Langdon, R. B.; Marsh, D. J. Effect of peripherally administrated neuromedin U on energy and glucose homeostatis. Endocrinology 2011, 152, 2644−2654. (19) Ingallinella, P.; Peier, A. M.; Pocai, A.; Marco, A. D.; Desai, K.; Zytko, K.; Qian, Y.; Du, X.; Cellucci, A.; Monteagudo, E.; Laufer, R.; Bianchi, E.; Marsh, D. J.; Pessi, A. PEGylation of neuromedin U yields a promising candidate for the treatment of obesity and diabetes. Bioorg. Med. Chem. 2012, 20, 4751−4759. (20) Neuner, P.; Peier, A. M.; Talamo, F.; Ingallinella, P.; Lahm, A.; Barbato, G.; Marco, A. D.; Desai, K.; Zytko, K.; Qian, Y.; Du, X.; Ricci, D.; Monteagudo, E.; Laufer, R.; Pocai, A.; Bianchi, E.; Marsh, D. J.; Pessi, A. Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide. J. Pept. Sci. 2013, 20, 7−19. (21) Minamino, N.; Kangawa, K.; Matsuo, H. Neuromedin U-8 and U-25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 1985, 130, 1078− 1085. (22) Sakura, N.; Kurosawa, K.; Hashimoto, T. Structure-activity relationships of neuromedin U. I. Contractile activity of dog neuromedin U-related peptides on isolated chicken crop smooth muscle. Chem. Pharm. Bull. 1995, 43, 1148−1153. (23) Hashimoto, T.; Kurosawa, K.; Sakura, N. Structure-activity relationships of neuromedin U. II. High potent analogs substituted or modified at the N-terminus of neuromedin U-8. Chem. Pharm. Bull. 1995, 43, 1154−1157. (24) Kurosawa, K.; Sakura, N.; Hashimoto, T. Structure-activity relationships of neuromedin U. III. Contribution of two phenylalanine residues in dog neuromedin U-8 to the contractile activity. Chem. Pharm. Bull. 1996, 44, 1880−1884.

ACKNOWLEDGMENTS This research was supported in part by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI, including Grants-inAid for Scientific Research (B) 23390029 (Y.H.) and 25293188 (M.M.).

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ABBREVIATIONS USED α-MEM, modified Eagle minimum essential medium; BSA, bovine serum albumin; [Ca2+]i, intracellular calcium concentration; Cha, cyclohexylalanine; Chg, cyclohexylglycine; CHO, Chinese hamster ovary; CNS, central nervous system; CPN, Cterminal core peptide derivative of neuromedin U; CRH, corticotropin-releasing hormone; Dab, α,γ-diaminobutyric acid; DMSO, dimethyl sulfoxide; Dap, α,β-diaminopropanoic acid; DIEA, N,N-diisopropylethylamine; DIPCI, diisopropylcarbodiimide; DMEM, Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; EDT, 1,2-ethanedithiol; ES, electrospray; FCS, fetal calf serum; Fmoc, 9-fluorenylmethoxycarbonyl; GPCR, G-protein coupled receptor; HBSS, Hank’s balanced salt solution; HEK293, human embryonic kidney 293; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOBt, 1hydroxybenzotriazole; HSA, human serum albumin; Hyp, 4hydroxyproline; (1)NHcPropn, 1-aminocyclopropanecarboxylic acid; Nal, naphthylalanine; Nle, norleucine; NMS, neuromedin S; NMU, neuromedin U; NMUR1, neuromedin U receptor type 1; NMUR2, neuromedin U receptor type 2; Nva, norvaline; PEG, poly(ethylene glycol); Phg, phenylglycine; PVN, paraventricular nucleus; Pyr, pyroglutamic acid; RP-HPLC, reversed-phase high-performance liquid chromatography; SAR, structure−activity relationship; TFA, trifluoroacetic acid; TMS, tetramethylsilane; TOF MS, time-of-flight mass spectrometry

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REFERENCES

(1) Brighton, P. J.; Szekeres, P. G.; Willars, G. B. Neuromedin U and its receptors: Structure, function, and physiological roles. Pharmacol. Rev. 2004, 56, 231−248. (2) Kojima, M.; Haruno, R.; Nakazato, M.; Date, Y.; Murakami, N.; Hanada, R.; Matsuo, H.; Kangawa, K. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem. Biophys. Res. Commun. 2000, 276, 435−438. (3) Fujii, R.; Hosoya, M.; Fukusumi, S.; Kawamata, Y.; Habata, Y.; Hinuma, S.; Onda, H.; Nishimura, O.; Fujino, M. Identification of neuromedin U as the cognate ligand of the orphan G protein-coupled receptor FM-3. J. Biol. Chem. 2000, 275, 21068−21074. (4) Raddatz, R.; Wilson, A. E.; Artymyshyn, R.; Bonini, J. A.; Borowsky, B.; Boteju, L. W.; Zhou, S.; Kouranova, E. V.; Nagorny, R.; Guevarra, M. S.; Dai, M.; Lerman, G. S.; Vaysse, P. J.; Branchek, T. A.; Gerald, C.; Forray, C.; Adham, N. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J. Biol. Chem. 2000, 275, 32452−32459. (5) Hanada, T.; Date, Y.; Shimbara, T.; Sakihara, S.; Murakami, N.; Hayashi, Y.; Kanai, Y.; Suda, T.; Kangawa, K.; Nakazato, M. Central actions of neuromedin U via corticotropin-releasing hormone. Biochem. Biophys. Res. Commun. 2003, 311, 954−958. (6) Benzon, C. R.; Johnson, S. B.; Mccue, D. L.; Li, D.; Green, T. A.; Hommel, J. D. Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight. Neuroscience 2014, 258, 270−279. (7) Mori, K.; Miyazato, M.; Ida, T.; Murakami, N.; Serino, R.; Ueta, Y.; Kojima, M.; Kangawa, K. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 2005, 24, 325−335. 6592



Article

(25) Sakura, N.; Kurosawa, K.; Hashimoto, T. Structure-activity relationships of neuromedin U. IV. Absolute requirement of the arginine residue at position 7 of dog neuromedin U-8 for contractile activity. Chem. Pharm. Bull. 2000, 48, 1166−1170. (26) Kawai, T.; Shibata, A.; Kurosawa, K.; Sato, Y.; Kato, S.; Ohki, K.; Hashimoto, T.; Sakura, N. Structure-activity relationships of neuromedin U. V. Study on the stability of porcine neuromedin U-8 at the C-terminal asparagine amide under mild alkaline and acidic conditions. Chem. Pharm. Bull. 2006, 54, 659−664. (27) Funes, S.; Hedrick, J. A.; Yang, S.; Shan, L.; Bayne, M.; Monsma, F. J., Jr.; Gustafson, E. L. Cloning and characterization of murine neuromedin U receptors. Peptides 2002, 23, 1607−1615. (28) Takayama, K.; Suehisa, Y.; Fujita, T.; Nguyen, J.-T.; Futaki, S.; Yamamoto, A.; Kiso, Y.; Hayashi, Y. Oligoarginine-based prodrugs with self-cleavable spacers for caco-2 cell permeation. Chem. Pharm. Bull. 2008, 56, 1515−1520. (29) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides. Chem. Commun. 2004, 1, 124−125. (30) López-Tudancam, P. L.; Labeaga, L.; Innerárity, A.; AlonsoCires, L.; Tapia, I.; Mosquera, R.; Orjales, A. Synthesis and pharmacological characterization of a new benzoxazole derivative as a potent 5-HT3 receptor agonist. Bioorg. Med. Chem. 2003, 11, 2709− 2714. (31) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. 1H-Pyrazole-1carboxamidine hydrochloride: An attractive reagent for guanylation of amines and its application to peptide synthesis. J. Org. Chem. 1992, 57, 2497−2502. (32) Mori, M.; Mori, K.; Ida, T.; Sato, T.; Kojima, M.; Miyazato, M.; Kangawa, K. Different distribution of neuromedin S and its mRNA in the rat brain: NMS peptide is present not only in the hypothalamus as the mRNA, but also in the brainstem. Front. Endocrinol. 2012, 3, 152. (33) Kawamura, Y.; Mutsuga, M.; Kato, T.; Iida, M.; Tanamoto, K. Estrogenic and anti-androgenic activities of benzophenones in human estrogen and androgen receptor mediated mammalian reporter gene assays. J. Health Sci. 2005, 51, 48−54.

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Discovery of potent hexapeptide agonists to human neuromedin u receptor 1 and identification of their serum metabolites.

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Isolation, structural characterization and pharmacological activity of dog neuromedin U.

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Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.