Discovery of a Human Neuromedin U Receptor 1-Selective

May 26, 2017 - Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a ...
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Discovery of a Human Neuromedin U Receptor 1‑Selective Hexapeptide Agonist with Enhanced Serum Stability Kentaro Takayama,† Kenji Mori,‡ Akiko Tanaka,¶ Erina Nomura,† Yuko Sohma,† Miwa Mori,‡ Akihiro Taguchi,† Atsuhiko Taniguchi,† Toshiyasu Sakane,# Akira Yamamoto,¶ Naoto Minamino,§ Mikiya Miyazato,‡ Kenji Kangawa,‡ and Yoshio Hayashi*,† †

Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan ‡ Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan ¶ Department of Biopharmaceutics, Kyoto Pharmaceutical University, 5 Misasaginakauchi-cho, Yamashina, Kyoto 607-8414, Japan # Laboratory of Pharmaceutical Technology, Kobe Pharmaceutical University, 4-19-1 Motoyamakitamachi, Higashinada, Kobe, Hyogo 658-8558, Japan § Omics Research Center, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan S Supporting Information *

ABSTRACT: Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure−activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice.

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INTRODUCTION Neuromedin U (NMU)1 is well-known as an anorexigenic peptide, inducing a variety of biological effects, including feeding suppression,2,3 reduction in body weight,4 regulation of energy homeostasis,5 increase in body temperature,4 and regulation of glucose homeostasis.6 The C-terminal amidated heptapeptide structure H-Phe0-Leu1-Phe2-Arg3-Pro4-Arg5-Asn6NH2 (1, Figure 1) of NMU, common to mammals, is core for the activation of two neuromedin U receptors, NMUR1 and NMUR2. The core structure (1) is conserved in neuromedin S (NMS), which is an NMU-related bioactive peptide possessing an anorexigenic effect.3,7 The majority of NMUR1 mRNA expression is observed in peripheral tissues, such as the intestinal tract and lungs, whereas NMUR2 mRNA is highly expressed in the central nervous system (CNS), particularly in the hypothalamic paraventricular nucleus (PVN).2,8,9 Hanada et al. demonstrated that centrally administered rat NMU suppresses food intake and regulates energy homeostasis via the secretion of corticotropin-releasing hormone from the PVN.5 Recently, a significant anorexigenic effect of peripherally administered human NMU (hNMU, 25 amino acid residues) was reported, suggesting the possibility of developing a drug to treat obesity.6,10,11 In 2015, Micewicz et al. reported that lipidconjugated NMU analogues based on the core structure (1) © 2017 American Chemical Society

displayed chronic anorexigenic effects in diet-induced obese mice.12 Previously, several structure−activity relationship (SAR) studies based on 1 acting with avian NMU receptors were conducted using chicken crop smooth muscle.13−16 NMU and NMS were identified in 1985 and 2005, respectively, but the first detailed SAR study directed at human NMU receptors was not reported until 2014 by our group. Our previous SAR study based on 1 afforded an NMUR2 selective hexapeptide agonist, 3-(3-cyclohexyl)propionyl-Leu 1-Leu 2 -Dap3 -Pro 4 -Arg5 -Asn6 NH2 in which Dap represents α,β-diaminopropionic acid (2, Supporting Information (SI), Figure S1).17 At concentrations below 10−6 M, the agonist 2 displayed highly selective activity toward human NMUR2 without inducing human NMUR1 activation in cultured cells expressing human NMU receptors and was only 3-fold less active than the original hNMU (25aa). In the same report, an NMUR1 selective agonist, 3-(3pyridyl)propionyl-Ala(2-Naph)1-Phe 2 -Arg 3-Pro 4-Arg5 -Asn6 NH2 in which Ala(2-Naph) is 2-naphthylalanine, was also disclosed (3, SI, Figure S1).17 This agonist 3 is 50-fold more selective toward NMUR1 but 20-fold less active than hNMU. Received: May 10, 2017 Published: May 26, 2017 5228

DOI: 10.1021/acs.jmedchem.7b00694 J. Med. Chem. 2017, 60, 5228−5234

Journal of Medicinal Chemistry

Brief Article

collected by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and displayed a purity of >95% in analytical RP-HPLC. All peptides were characterized by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS), and the key peptide 7b was also analyzed by NMR. A 20 mM stock solution of each peptide in DMSO was prepared for bioassays. In our previous report, the introduction of a fluorine atom at the para-position of Phe2 (i.e., agonist 4, Figure 1) was found to be tolerated, with a slight increase in the NMUR1 agonistic activity, although the serum stability was slightly reduced.18,19 A full SAR study involving this position had not previously been conducted, and consequently, we focused on this position in the present work, synthesizing a series of peptide derivatives 5a−j, bearing a variety of substituents (5a, 3-F; 5b, 2-F; 5c, 4Cl; 5d, 4-Br; 5e, 4-CN; 5f, 4-OMe; 5g, 4-NO2; 5h, 4-CF3; 5i, 4Ph; 5j, 4-Bz) on the phenyl ring of Phe2 (Figure 2A). The agonistic activities of these derivatives toward human NMUR1 and NMUR2, expressed in CHO cells, were then evaluated by calcium mobilization assays (Figure 2B). The activities of respective peptides are presented as a relative value (%) compared to that of hNMU at 1000 nM. Derivatives 5a−b, with a fluorine atom at the ortho- or metaposition of the phenyl ring, displayed agonistic activities toward both receptors similar to the activity of 4, which itself possessed a fluorine atom at the para-position, suggesting that the effect of a fluorine atom was limited. Other derivatives 5c−j, with halogen atoms and substituent groups at the para-position, were also prepared. Among these derivatives, 5d−j exhibit a tendency to decrease NMUR2 agonistic activities at 10 nM, with the exception of 5c which contains a chlorine atom. In particular, both 5i and 5j, bearing 4-phenyl and 4-benzoyl substituents, fail to display any significant NMUR2 agonistic activities at 10 nM, suggesting that the bulky side chain in residue 2 improves the NMUR1 selectivity. However, 5h−j show weak agonistic activities toward NMUR1 at 0.1 nM. In addition, we synthesized S1a (SI, Figure S2A), bearing a bulky side chain 3,3-diphenylalanine (Dph) at residue 2, and evaluated the agonistic activity of this derivative (SI, Figure S2B). S1a does not display agonistic activities toward either receptor at 100 nM, indicating that the introduction of the phenyl group at the β-position of Phe2 does not enhance the activity. We next synthesized the derivatives 6a−c, possessing bicyclic aromatic amino acids [6a, Trp; 6b, Ala(1-Naph); 6c, Ala(2Naph)] at residue 2 and evaluated their agonistic activities (Figure 2). These derivatives exhibit almost no significant NMUR2 agonistic activity at 10 nM, and thus are similar to 5i and 5j. Among the derivatives 5i, 5j, and 6a−c, derivative 6a displays the most potent agonistic activity toward NMUR1 at 0.1 nM, suggesting that a Trp moiety at residue 2 is consistent with the development of a highly NMUR1 selective agonist. Finally, we derivatized the Trp2 residue of 6a to form Ninand α-methylated Trp derivatives (7a and 7b) and a 3benzothiophenylalanine (S1b) (Figure 3A and SI, Figure S2A). As shown in Figure 3B and SI, Figure S2B, 7a and S1b showed a lower NMUR1 agonistic activity than 6a, suggesting that the 1H-indole structure in the side chain of residue 2 is important for the NMUR1 selective agonistic activity. On the other hand, 7b shows a higher potency and selectivity against NMUR1 than 6a (Figure 3B), indicating that α-methylation of Trp2 improves the agonistic activity and selectivity toward NMUR1.

Figure 1. Structures of the human neuromedin U-derived peptides 1 and 4.18 Arrows indicate the cleavage sites in rat or human serum.19

Subsequently, we identified a more potent NMUR1 agonist, 2thienylacetyl-Trp 1 -Phe(4-F) 2 -Arg 3 -Pro 4 -Arg 5 -Asn 6 -NH 2 in which Phe(4-F): 4-fluorophenylalanine, (4, Figure 1) that displays activity comparable to that of hNMU but only 30-fold selectivity toward NMUR1.18 These results obtained from our SAR studies suggested that (i) the 2-thienylacetyl group at the N-terminal acyl moiety contributes to the potent agonistic activities, (ii) the introduction of bicyclic aromatic amino acids at residue 1 increases the NMUR1 selectivity, and (iii) the aliphatic structures at the N-terminus, residues 1 and 2, supports the NMUR2 selectivity. We also identified the amide bonds of Phe(4-F) 2-Arg3 and Arg5-Asn6 in 4 as two biodegradation sites in rat and human serum (Figure 1).18 We demonstrated that serum thrombin is responsible for the rapid degradation of 4 at the latter site;19 this is a structure common to NMU and all agonists developed to date. Therefore, the development of an NMU receptor agonist that is stable against thrombin degradation would be indispensable in peripheral administration. In general, CNS-targeted drug development is usually hampered by various difficulties such as the blood−brain barrier, especially in peptide or protein drugs. In the present study, which was geared toward identification of a novel selective agonist to peripherally expressed NMUR1 rather than centrally expressed NMUR2, we carried out a series of SAR studies focused on residue 2, Phe(4-F) in 4, an appropriate site for structural derivatization. The derivative 7b, bearing an αmethylated Trp at residue 2 [(α-Me)Trp2], was obtained as a highly selective agonist toward human NMUR1, with enhanced serum stability and good pharmacokinetics in rats.

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RESULTS AND DISCUSSION All peptides derived from a lead peptide 4 were synthesized based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, as reported previously.17−19 The crude peptides were obtained by treating the prepared resin with a mixture of trifluoroacetic acid (TFA), m-cresol, thioanisole, and 1,2-ethanedithiol (EDT) (4.0 mL, 92.5:2.5:2.5:2.5) for 150 min at room temperature and subsequent ether precipitation. The pure peptides were 5229

DOI: 10.1021/acs.jmedchem.7b00694 J. Med. Chem. 2017, 60, 5228−5234

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Figure 3. (A) Structures of 7a and 7b. (B) Effect of Trp2-methylation on the agonistic activity with stably expressed human NMUR1 (black bar) and NMUR2 (gray bar) in CHO cells. The activity was measured using the calcium mobilization assay. Peptide concentrations: 0.1 and 100 nM; positive control, hNMU (activity at 1000 nM = 100%).

Figure 2. (A) Structures of 5a−j and 6a−c. (B) Effect of different side chains at residue 2 on the agonistic activity toward the stably expressed human NMUR1 (black bar) and NMUR2 (gray bar) in CHO cells. The activities were measured using a calcium mobilization assay. Peptide concentrations: 0.1 and 10 nM; positive control, hNMU (activity at 1000 nM = 100%).

The dose-dependent agonistic activities of 7b and hNMU toward both NMU receptors were evaluated, and the results are shown in Figure 4A. The EC50 values of these ligands toward NMUR1 were calculated using KaleidaGraph 4.5. The agonist 7b exhibits a dose-dependent agonistic activity toward NMUR1 with an EC50 value of 0.25 ± 0.05 nM (means ± SEM), whereas the corresponding activity of hNMU is 0.17 ± 0.03 nM (means ± SEM). These results indicate that 7b has a slightly lower agonistic activity than hNMU, with a value that is 1.5-fold lower than that of hNMU, although this activity is sufficient to provide full agonistic activity. However, 7b fails to display significant agonistic activity toward NMUR2, at least at concentrations below 10−7 M (Figure 4B), indicating that 7b is at least 1000 times more selective toward human NMUR1. These results clearly demonstrate that 7b is the most potent

Figure 4. In vitro agonistic activity in CHO cells of the hexapeptide derivative (7b) toward stably expressed human NMUR1 (A) and NMUR2 (B) as determined by the calcium mobilization assay. Peptide concentrations, 10−12−10−6 M; reference compound, hNMU. Data (means ± SD) were determined in triplicate. Curve fitting was performed using KaleidaGraph 4.5.

and selective hexapeptide agonist identified toward human NMUR1 identified to date. Next, the in vitro stabilities of the hexapeptide derivatives 4 and 7b were evaluated by incubating these peptides in rat or 5230

DOI: 10.1021/acs.jmedchem.7b00694 J. Med. Chem. 2017, 60, 5228−5234

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human serum, as reported previously.18,19 After the incubation in 25% rat or human serum at 37 °C,20,21 the quantities of intact peptides extracted from the sera at different time points, using the Sep-Pak C18 Plus cartridge, were analyzed by RPHPLC. As shown in Figure 5, the half-lives of derivative 4 are

improving the potency and selectivity of NMUR1 agonistic activity. To investigate the effects of the enhanced serum stability, we analyzed the pharmacokinetics of 4 and 7b using Wistar rats. After intravenous injection of peptides (1 mg), blood samples were collected at 1, 3, 5, 10, 15, 30, 60, 90, 120, 180, and 240 min, and the respective peptide concentrations were quantitated by LC-MS (Figure 6). The time course plots

Figure 5. Time course of the recovery rates of intact peptides 4 and 7b after incubation in 25% rat (A) or human (B) serum. Data (means ± SD) were determined in triplicate.

predictably short (less than 10 min) in both sera, whereas 7b is surprisingly stable because the recovery rates of intact 7b at 180 min after incubation in rat and human sera are 52 ± 0.5% and 45 ± 0.5%, respectively. These results were corroborated by the observation that 6a, bearing a Trp at residue 2, is rapidly degraded in both sera (56 ± 1.2% recovery at 10 min in rat serum; 64 ± 1.4% recovery at 5 min in human serum). These results suggest that α-methylation of Trp2 plays a key role in enhancing the serum stability of 7b. Moreover, we identified the metabolites 7b-m1 [2thienylacetyl-Trp 1 -(α-Me)Trp 2 -Arg 3 -Pro 4 -Arg 5 -OH; m/z 938.65 (M + H)+ (calcd 938.45), 469.85 (M + 2H)2+ (calcd 469.73)] (SI, Figure S3A,B) produced by serum thrombindependent cleavage of 7b, using RP-HPLC and mass spectrometric analysis, as reported previously.18 A similar metabolic cleavage was observed for 6a, producing 6a-m1 [2thienylacetyl-Trp 1 -Trp 2 -Arg 3 -Pro 4 -Arg 5 -OH] (SI, Figure S3A,B). These metabolites were also identified by chemical synthesis. The effect of α-methylation on the serum stability was investigated by incubation in rat serum in the same manner. As shown in SI, Figure S3C, metabolite 6a-m1 is further degraded to 52 ± 0.9% of its initial value at 120 min. As the biodegradation of 4 is known to proceed at two sites, Phe(4-F)2-Arg3 and Arg5-Asn6,18 6a-m1 is also degraded at the corresponding Trp2-Arg3 site to 6a-m2 [2-thienylacetyl-Trp1Trp2-OH] (SI, Figure S3D,E); however, 7b-m1 is stable during the incubation (101 ± 0.8%, SI, Figure S3C). In the present study, further metabolites derived from 6a or 7b were not detected. These results suggested that the biodegradation of 7b in serum proceeds predominantly in a thrombin-dependent manner at the Arg5-Asn6 site only, with no cleavage at the corresponding (α-Me)Trp2-Arg3 site. In addition, the extended stability of 7b in serum, as indicated in Figure 5, compared with the stabilities of 4 and 6a, suggests that the introduction of (αMe)Trp2 at residue 2 in 7b indirectly influences the thrombin degradation reaction. Because residue 2 corresponds to the P4 site, which is recognized by thrombin as an aryl-binding site,22 α-methylation at the P4-Trp2 residue may restrict the orientations of the indole ring to an unfavorable position. This effect may enable the successful discovery of an approach to reduction of thrombin recognition and the second degradation reaction in 7b, along with an approach for

Figure 6. Time course of the plasma concentrations of 4 and 7b intravenously injected into Wistar rats. Data (mean ± SEM) were determined in quadruplicate (4) or triplicate (7b).

were used to analyze the pharmacokinetics of each peptide using a two-compartment model. This analysis was performed on a Phoenix WinNonlin (Table 1). The area under the curve Table 1. Pharmacokinetic Parameters Obtained from 4 and 7ba compd

AUC (min·μg/mL)

CL (mL/min)

MRT (min)

4 7b

318 ± 47 653 ± 79

3.19 ± 0.46 1.55 ± 0.18

11.5 ± 6.1 33.6 ± 2.6

a Data (mean ± SEM) were determined in quadruplicate (4) or triplicate (7b) using the Phoenix WinNonlin software.

(AUC), clearance (CL), and mean residence time (MRT) of 7b were 653 ± 79 min·μg/mL, 1.55 ± 0.18 mL/min, and 33.6 ± 2.6 min, respectively. These values represent a 2.1-fold increase (p < 0.01), a 2.1-fold decrease (p < 0.01), and a 2.9fold increase (p < 0.01) compared to the corresponding values obtained from 4, yielding values of 318 ± 47 min·μg/mL, 3.19 ± 0.46 mL/min, and 11.5 ± 6.1 min, respectively. These results demonstrate that the pharmacokinetics of 7b are significantly better than those of 4. The improved pharmacokinetics seems to be attributed to the enhanced serum stability of 7b because small peptidic molecules, including hNMU,6 are easily eliminated from blood flow by renal excretion. Although the agonist 7b is a smaller molecule than hNMU, its pharmacokinetic parameters are significantly improved by structural derivatization. As described above,6,10−12 several groups have examined the peripheral administration of hNMU and its analogues. In those studies, an effort was made to obtain effective anorexigenic activity by improving the pharmacokinetics by conjugating the compounds to other molecules, such as poly(ethylene) glycol (PEG), human serum albumin, and various kinds of lipid molecules.10−12,23 In combination with other strategies using macromolecular modifications, our α-methylation strategy for 5231

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neuromedin U, bearing (α-Me)Trp2 (7b). Interestingly, in comparison with 4, 7b shows improved NMUR1 selectivity, serum stability, and pharmacokinetics. These observations are attributed to the presence of a bulky side chain and αmethylation of Trp2. This is the first report that the NMUR1 selective agonists such as 7b act as suppressants of body weight gain in mice. The agonist 7b is a key lead compound for the development of a peripherally administered antiobesity drug, and it represents a useful tool for NMU-related endocrinological and pharmacological researches.

developing NMU receptor agonists should be applicable to the development of practical drug candidates. Before the pharmacological study with mice, we evaluated the in vitro agonistic activities to mouse NMU receptors (SI, Figure S4), which are transiently expressed on human embryonic kidney 293 (HEK293) cells as reported previously.17 The agonist 7b exhibits a selective agonistic activity toward mouse NMUR1 with an EC50 value of 8.9 ± 0.9 nM (means ± SEM), whereas the corresponding activity of mouse NMU (mNMU) is 0.42 ± 0.06 nM (means ± SEM). These results indicated that 7b has a 21-fold lower agonistic activity than mNMU and provides a partial agonistic activity with a maximum of 39%. Meanwhile, 7b does not display significant agonistic activity toward mouse NMUR2, at least at concentrations below 10−6 M. Finally, the effect of agonist 7b on body weight gain in mice was investigated. The agonist 7b (6.8 mg/kg) or saline was subcutaneously injected to ddY (Deutschland, Denken, and Yoken) mice at 0, 24, and 48 h (0 h = just prior to 12 h dark period). The time course of body weight change in each group (n = 4) is shown in Figure 7. The agonist 7b significantly

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EXPERIMENTAL SECTION

Reagents and solvents were purchased from Wako Pure Chemical Ind. (Osaka, Japan), Sigma-Aldrich (St. Louis, MO), Nacalai Tesque (Kyoto, Japan), Watanabe Chemical Ind. (Hiroshima, Japan), and Tokyo Chemical Ind. (Tokyo, Japan). (S)-N-Fmoc-N′-Boc-α-methyltryptophan was provided by Nagase and Co. Ltd. (Osaka, Japan). All reagents were used as received. Sterile Alfa 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-walled plates with clear bottoms were purchased from Iwaki (Tokyo, Japan) and Corning (Cambridge, MA), respectively. Rat and human sera were purchased from Sigma-Aldrich. Sep-Pak C18 Plus cartridges were purchased from Waters (Milford, MA). Wistar rats and ddY mice were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). 1. Peptide Synthesis. As reported previously,17−19 Fmoc-amino acids/2-thienylacetic acid (0.126 M, 3 equiv) were sequentially coupled to an Fmoc-NH-SAL resin (0.56 mmol/g, 75 mg, 0.042 mmol) using the DIPCI (0.126 M, 3 equiv)−HOBt (0.126 M, 3 equiv) method over 2 h in DMF (1 mL). Respective coupling steps were performed after removal of each Fmoc group in 20% piperidineDMF (1.5 mL, 20 min) to obtain the resin-bound protected peptide. Crude peptides were obtained by treating the resin with TFA−mcresol−thioanisole−EDT (4.0 mL, 92.5:2.5:2.5:2.5) for 150 min at room temperature, followed by ether precipitation. The pure peptides were collected as TFA salts by preparative RP-HPLC purification in a 0.1% aqueous TFA−CH3CN system and subsequent lyophilization. The purity of all peptides was >95%, which was analyzed by RP-HPLC using a C18 reverse-phase column (Waters SunFire C18 5 μm (column 1) or COSMOSIL 5C18-AR-II (column 2)) using a binary solvent system delivered in a linear gradient of CH3CN (5−65%, 40 min) in 0.1% aqueous TFA at a flow rate of 0.9 mL/min (column 1) or 1.0 mL/min (columns 2), detected at 230 nm (columns 1) or 220 nm (column 2), respectively. High-resolution mass spectra (TOF MS ES+) were recorded on a micromass LCT. 1H NMR spectra were obtained on a Bruker Avance III spectrometer (400 MHz) using TMSP as an internal standard. Chemical data obtained from the newly synthesized peptides are presented in the SI. For in vivo studies, peptides 4 and 7b were treated with AG1-X8 anion exchange resins (BIO-RAD, Hercules, CA) to obtain the acetate salts. 2. Calcium Mobilization Assay. As reported previously,17−19 CHO cells stably expressing human NMUR1 or human NMUR2 were maintained in α-MEM-nucleotide media in the presence of 10% heatinactivated FCS and 1 mg/mL G418. Subcultures were performed every 3−4 days. Cells were grown to approximately 70% confluence on 100 mm dishes and were maintained at 37 °C under 5% CO2. CHO cells stably expressing receptors were seeded (2.0 × 104 cells per well) into 96-well black-walled plates with clear bottoms. Then 18 h later, the cells were loaded over 40 min with 4 μM Fluo-4 AM fluorescent indicator dye in an assay buffer (HBSS, 10 mM HEPES, 2.5 mM probenecid, pH 7.4) with 1% FCS, and were washed four times with the assay buffer without FCS. The intracellular calcium flux was then measured on a fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA). The peptide derivatives were dissolved in an assay buffer containing 0.05% BSA and 0.001% Triton X-100 and diluted to concentrations in the range 0.1−1000 nM. The activities of the

Figure 7. Effect of 7b on body weights in ddY mice. Arrows indicate the subcutaneous injections at 0, 24, and 48 h. Data (means ± SEM) were determined in quadruplicate. *p < 0.05, **p < 0.01.

suppresses body weight gain until 36 h after the first administration. Then the effect of 7b is weakened and a similar body weight gain to that of the control group is observed, resulting, however, in an overall decrease of body weight until the 60 h time point. The reason for the reduced response is possibly caused by its partial agonistic activity or emergence of a rapid tachyphylaxis, which has previously been reported in continuous administration of a lipidated NMU analogue.24 Recently, Inooka et al. reported nonselective PEGylated octapeptide agonists toward NMUR1 and NMUR2 which, conjugated with 20 kDa PEG, show a potent antiobesity effect.25 Although our small hexapeptide agonist 7b displayed 7fold shorter MRT than this PEGylated agonist in the intravenous injection,25 the significant suppression of body weight gain was confirmed. This is the first report that the NMUR1 selective agonist is a suppressant of body weight. Further in vivo studies assessing the possibility of practical use are currently under investigation.

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CONCLUSION In this paper, we report a SAR study, focused on the residue 2 of the previously reported agonist (4), which led to a novel NMUR1 selective and potent hexapeptide agonist designated CPN-267, CPN: C-terminal core peptide derivative of 5232

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peptide derivatives were determined from the maximal value. To determine the EC50 values, the peptide derivatives were dissolved at concentrations of 10−12−10−6 M and the agonistic activities of the peptide derivatives were determined in triplicate at each concentration. Curve fitting was performed using KaleidaGraph 4.5. Full-length cDNAs of mouse NMUR1 and NMUR2 were obtained by reverse transcription polymerase chain reaction using total RNAs prepared from mouse testis and brain, respectively. The primer set used for mouse NMUR1 was 5′-ATGGTCTGCAATATCAGTGA-3′ and 5′TCAGGAGGGGTCTGTCTCTT-3′, and that for mouse NMUR2 was 5′-ATGGGAAAACTTGAAAATGCTTCCTGGATCCACGA-3′ and 5′-TCATGGTACCTCTTCAACACA-3′. Amplified cDNAs were cloned into pcDNA3.1 vectors (Invitrogen, Carlsbad, CA), and these sequences were confirmed. The calcium mobilization assay was performed using HEK293 cells transiently expressing mouse NMU receptors as reported previously.17 3. Analysis of Metabolic Stability in Rat or Human Serum. As reported previously,18,19 each peptide stock solution (20 mM in DMSO) was diluted to 1 mM in RPMI-1640. An aliquot of the 1 mM peptide solution (20 μL, 20 nmol) was added to rat or human serum (100 μL) diluted with RPMI-1640 (280 μL) that had been preincubated at 37 °C for 15 min. The resulting 25% rat or human serum solution was incubated at 37 °C for the appropriate period of time. The incubation was stopped by adding ice-cold saline (400 μL) containing HCl (final conc 0.04 N). Samples were centrifuged at 3000 rpm at 4 °C for 15 min. Then an aliquot of the supernatant (640 μL) was loaded onto a Sep-Pak C18 Plus cartridge, and the cartridge was washed with saline and 10% CH3CN in 0.1% aqueous TFA. The intact peptide and its metabolites were eluted with 60% CH3CN in 0.1% aqueous TFA and collected into tubes containing a 0.1% Triton-X 100 solution (40 μL). The lyophilized samples were dissolved in 800 μL of 10% CH3CN in 0.1% aqueous TFA, and 20 μL of this solution was used for RP-HPLC on a C18 reverse-phase column (Chromolith Performance RP-18e 100−4.6 mm) using a binary solvent system delivered in a linear gradient of CH3CN (15−55%, 10 min) in 0.1% aqueous TFA at a flow rate of 2.5 mL/min. To clearly identify the metabolite 6a-m2, RP-HPLC was performed on a C18 reverse-phase column (4.6 mm × 150 mm; COSMOSIL 5C18-AR-II) using a binary solvent system: a linear gradient of CH3CN (10−55%, 90 min) in 0.1% aqueous TFA at a flow rate of 1.0 mL/min.18 Peaks at 220 nm were detected and analyzed by mass spectrometry. 4. Pharmacokinetic Analysis. Animal studies were approved by the Animal Research Committee of Kyoto Pharmaceutical University. Peptides (1 mg) dissolved in saline (100 μL) were intravenously injected into a cervical vein of male Wistar rats (220−250 g). At 1, 3, 5, 10, 15, 30, 60, 90, 120, 180, and 240 min, blood samples were collected from catheters implanted in a femoral artery in advance. The plasma concentration of the peptides at each time point was quantitatively measured by LC-MS. Pharmacokinetic analyses were performed using the Phoenix WinNonlin software (Certara, Princeton, NJ). 5. Pharmacological Study. At 0, 24, and 48 h, saline solutions (5 μL) in the presence or absence of peptide 7d (6.8 mg/kg) were subcutaneously injected on the back just prior to light off into male ddY mice (28−31 g), which maintained under an environment regulated by 12 h light/12 h dark lighting beginning at 8:00. Body weights were measured at 0, 12, 36, 60, 84, and 108 h. Statistical analyses were performed using a Student’s t-test.

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degradation of 7b and 6a, in vitro agonistic activity in HEK293 cells of the agonist 7b toward transiently expressed mouse NMUR1 and NMUR2 as determined by the calcium mobilization assay (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: +81 42 676 3275. Fax: +81 42 676 3279. E-mail: [email protected]. ORCID

Yoshio Hayashi: 0000-0002-7010-6914 Notes

The authors declare no competing financial interest.

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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) 15H04658 (K.T. and Y.H.). We thank Koji Taketa for peptide synthesis and Nagase and Co. Ltd. for providing (S)-N-Fmoc-N′-Boc-α-methyltryptophan.

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ABBREVIATIONS USED Ala(1-Naph), 1-naphthylalanine; Ala(2-Naph), 2-naphthylalanine; α-MEM, Alfa Modified Eagle Minimum Essential Medium; AUC, area under the curve; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CL, clearance; CNS, central nervous system; CPN, C-terminal core peptide derivative of neuromedin U; ddY, Deutschland, Denken, and Yoken; DIPCI, diisopropylcarbodiimide; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; Dph, 3,3-diphenylalanine; EDT, 1,2-ethanedithiol; ES, electrospray; FCS, fetal calf serum; Fmoc, 9-fluorenylmethoxycarbonyl; HBSS, Hank’s Balanced Salt Solution; HEK293, human embryonic kidney 293; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOBt, 1hydroxybenzotriazole; HSA, human serum albumin; MRT, mean residence time; NMS, neuromedin S; NMR, nuclear magnetic resonance; NMU, neuromedin U; NMUR1, neuromedin U receptor 1; NMUR2, neuromedin U receptor 2; Phe(4-F), 4-fluorophenylalanine; PEG, poly(ethylene) glycol; PVN, paraventricular nucleus; RP-HPLC, reversed phase highperformance liquid chromatography; SAR, structure−activity relationship; TFA, trifluoroacetic acid; TMSP, sodium 3(trimethylsilyl)propionate 2,2,3,3-d4; TOF MS, time-of-flight mass spectrometry

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REFERENCES

(1) Minamino, N.; Kangawa, K.; Matsuo, H. Neuromedin U-8 and U25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 1985, 130, 1078− 1085. (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) 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. (4) 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.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00694. Analytical data for all peptide derivatives and analytical HPLC chromatograms and NMR spectra; and structures of agonists 2, 3, S1a, and S1b, analytical RP-HPLC chromatograms showing the time-dependent metabolic 5233

DOI: 10.1021/acs.jmedchem.7b00694 J. Med. Chem. 2017, 60, 5228−5234

Journal of Medicinal Chemistry

Brief Article

(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) 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. (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. (8) 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. (9) 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. (10) 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. (11) Neuner, P.; Peier, A. M.; Talamo, F.; Ingallinella, P.; Lahm, A.; Barbato, G.; Di Marco, A.; 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. 2014, 20, 7−19. (12) Micewicz, E. D.; Bahattab, O. S.; Willars, G. B.; Waring, A. J.; Navab, M.; Whitelegge, J. P.; McBride, W. H.; Ruchala, P. Small lipidated anti-obesity compounds derived from neuromedin U. Eur. J. Med. Chem. 2015, 101, 616−626. (13) 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. (14) 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. (15) 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. (16) Sakura, N.; Kurosawa, K.; Hashimoto, T. Structure-activity relationships of neuromedin U. IV. Absolute requirement of the arginine residue at residue 7 of dog neuromedin U-8 for contractile activity. Chem. Pharm. Bull. 2000, 48, 1166−1170. (17) Takayama, K.; Mori, K.; Taketa, K.; Taguchi, A.; Yakushiji, F.; Minamino, N.; Miyazato, M.; Kangawa, K.; Hayashi, Y. Discovery of selective hexapeptide agonists to human neuromedin U receptors types 1 and 2. J. Med. Chem. 2014, 57, 6583−6593. (18) Takayama, K.; Mori, K.; Sohma, Y.; Taketa, K.; Taguchi, A.; Yakushiji, F.; Minamino, N.; Miyazato, M.; Kangawa, K.; Hayashi, Y. Discovery of potent hexapeptide agonists to human neuromedin U receptor 1 and identification of their serum metabolites. ACS Med. Chem. Lett. 2015, 6, 302−307. (19) Takayama, K.; Taguchi, A.; Yakushiji, F.; Hayashi, Y. Identification of a degrading enzyme in human serum that hydrolyzes a C-terminal core sequence of neuromedin U. Biopolymers 2016, 106, 440−445.

(20) Nguyen, L. T.; Chau, J. K.; Perry, N. A.; de Boer, L.; Zaat, S. A. J.; Vogel, H. J. Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs. PLoS One 2010, 5, e12684. (21) Powell, M. F.; Stewart, T.; Otvos, L., Jr.; Urge, L.; Gaeta, F. C. A.; Sette, A.; Arrhenius, T.; Thomson, D.; Soda, K.; Colon, S. M. Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum. Pharm. Res. 1993, 10, 1268−1273. (22) Bode, W.; Turk, D.; Karshikov, A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1992, 1, 426−471. (23) Dalbøge, L. S.; Pedersen, S. L.; van Witteloostuijn, S. B.; Rasmussen, J. E.; Rigbolt, K. T. G.; Jensen, K. J.; Holst, B.; Vrang, N.; Jelsing, J. Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake. J. Pept. Sci. 2015, 21, 85−94. (24) Dalbøge, L. S.; Pedersen, S. L.; Secher, T.; Holst, B.; Vrang, N.; Jelsing, J. Neuromedin U inhibits food intake partly by inhibiting gastric emptying. Peptides 2015, 69, 56−65. (25) Inooka, H.; Sakamoto, K.; Shinohara, T.; Masuda, Y.; Terada, M.; Kumano, S.; Yokoyama, K.; Noguchi, J.; Nishizawa, N.; Kamiguchi, H.; Fujita, H.; Asami, T.; Takekawa, S.; Ohtaki, T. A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects. Bioorg. Med. Chem. 2017, 25, 2307−2312.

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