Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Peptides 24 (2003) 1899–1908

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity Christine G. Joseph, Andrzej Wilczynski, Jerry R. Holder, Zhimin Xiang, Rayna M. Bauzo, Joseph W. Scott, Carrie Haskell-Luevano∗ Department of Medicinal Chemistry, University of Florida, P.O. Box 100485, Gainesville, FL 32610-0485, USA Received 11 August 2003; received in revised form 16 October 2003; accepted 17 October 2003

Abstract Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. ␣-Melanocyte stimulating hormone (␣-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4 -Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 ) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 ). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4 -Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-ValNH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands. © 2003 Elsevier Inc. All rights reserved. Keywords: Melanocortin; Melanotropin; Obesity; Agouti; Agouti-related protein; AGRP

1. Introduction Agouti-related protein (AGRP) is a 132 (human) amino acid peptide containing five disulfide bridges, and antagonizes the central brain melanocortin receptors (MC3R and MC4R) [38]. Agouti, a homologue of AGRP, contains a cysteine rich C-terminal region homologous with AGRP [32]. These two proteins are the only known naturally occurring antagonists of GPCRs reported to date, making them a unique family of peptides. Agouti (ASP), the first endogenous GPCR antagonist identified [35] was characterized as antagonizing the skin MC1R and the brain MC4R [32]. Interestingly, AGRP only antagonizes the brain melanocortin receptors MC3R and MC4R [38,59] and when ectopically expressed in transgenic mice, does not result ∗

Corresponding author. Tel.: +1-352-846-2722; fax: +1-352-392-8182. E-mail address: [email protected] (C. Haskell-Luevano).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.10.005

in the yellow coat color observed for the agouti mouse [14,38]. The cysteine rich C-terminus of both agouti and AGRP has been identified as possessing nM antagonistic properties at the melanocortin receptors, equipotent to the full length peptide [30,40,41,43,55,59]. Thus, suggesting the key structural and molecular recognition features are located in this C-terminal domain of AGRP and agouti. Both agouti and AGRP are members of the melanocortin pathway. The melanocortin system components includes five G-protein coupled receptors (MC1–5R) [8,9,11–13,36,45], endogenous agonists derived from post-translational modification of the proopiomelanocortin (POMC) gene transcript [42], endogenous antagonists agouti [32] and agouti-related protein (AGRP) [38], and auxiliary protein families (mahogany/attractin and syndecans) [15,21,37,44] that appear to regulate the function of the endogenous antagonists. The POMC derived melanocortin agonists contain a conserved His-Phe-Arg-Trp sequence postulated to be important for

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melanocortin receptor molecular recognition and stimulation. Structure-activity studies of ␣-melanocyte stimulating hormone (␣-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-ArgTrp-Gly-Lys-Pro-Val-NH2 ) using the classical frog and lizard skin darkening assays, resulted in the identification of two superpotent and prolonged acting ligands, NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-ProVal-NH2 ) [49] and MTII (Ac-Nle-c[Asp-His-DPhe-ArgTrp-Lys]-NH2 ) [1,2] that have been used extensively to characterize the melanocortin receptors, both in vitro and in vivo. Previous structure-activity studies of the agouti peptide identified the importance of the three amino acid motif ArgPhe-Phe [3,28,30,31,33] which is conserved in both agouti and AGRP peptides. A homology molecular model of agouti, based upon the NMR structures of ␻-conotoxin GVIA and ␻-agatoxin IVB, was generated which allowed for the visualization of how these antagonist Arg-Phe-Phe residues may be mimicking the agonist Phe-Arg-Trp residue interactions with the melanocortin receptors [31]. More recently, the NMR structure for the C-terminal AGRP region has been reported [3,28,33] These latter studies are the first structural reports for either of the GPCR antagonists agouti, or AGRP. It has been proposed by several laboratories that the conserved Arg-Phe-Phe motif found in both agouti and AGRP may be important for the antagonistic and molecular recognition properties of these two molecules at the melanocortin receptors. Mutation of these Arg-Phe-Phe residues results in notably less efficacious compounds [31,53]. Further extrapolation of the homology between the antagonist Arg-Phe-Phe motif and the endogenous melanocortin ligand conserved residues Phe-Arg-Trp, implies that the antagonist residues may be mimicking the agonist PheArg-Trp interactions with the melanocortin receptors. This hypothesis is supported by the results of a previous study that demonstrated that the hAGRP (109–118) decapeptide, Yc[CRFFNAFC]Y, possesses ␮M agonist activity at the skin MC1R and possesses ␮M binding affinities at the mouse MC1, MC3, MC4R, and MC5 receptors [18]. It has been previously reported that the melanocortin agonist peptides Ac-His-DPhe-Arg-Trp-NH2 and Ac-HisPhe-Arg-Trp-NH2 possess nM and ␮M potencies at the mouse melanocortin receptors [22], the tripeptide Ac-PheArg-Trp-NH2 possesses ␮M agonist activity only at the mMC1R [17], and that Ac-His-Phe-Arg-Trp-NH2 is the minimal fragment of melanocortin agonists required to produce a physiological response (␮M) in the classic frog and lizard skin bioassays [5,27]. The study presented herein was performed to evaluate the pharmacological activity of the NDP-MSH, MTII and AGRP chimeric peptides at the melanocortin receptors and to test the hypothesis that the melanocortin agonist (7–9) DPhe-Arg-Trp amino acids of NDP-MSH and MTII could be replaced by the hAGRP (111–113) Arg-Phe-Phe residues and still possess the necessary structural features to result in agonist activity.

2. Methods 2.1. Linear NDP-MSH peptide synthesis Linear NDP-MSH peptide synthesis was performed using standard Fmoc methodology [4,6] manually, or on an automated or semi-automated synthesizer (Advanced ChemTech 440MOS or LabTech, Louisville, KY). The amino acids Fmoc-Ser(tBu), Fmoc-Tyr(tBu), Fmoc-Nle, Fmoc-Glu(OtBu), Fmoc-His(Trt), Fmoc-Arg(Pbf), FmocDPhe, Fmoc-Trp(Boc), Fmoc-Gly, Fmoc-Lys(Boc), FmocPro, Fmoc-Val, and Fmoc-Phe were purchased from Peptides International (Louisville, KY). The peptides were assembled on Rink-amide-MBHA resin (0.40 meq/g substitution), purchased from Peptides International. All reagents were ACS grade or better. The synthesis was performed using a 40 well Teflon reaction block with a course Teflon frit. Approximately 200 mg resin (0.08 mmol) was added to each reaction block well. The resin was allowed to swell for 2 h in dimethylformamide (DMF) and deprotected using 25% piperidine in DMF for 5 min followed by a 20 min 25% piperidine incubation at 500 rpm. A positive Kaiser [29] test resulted indicating free amine groups on the resin. The growing peptide chain was added to the amide-resin using the general amino acid cycle as follows: 500 ␮l DMF is added to each reaction well to “wet the frit,” three-fold excess amino acid starting from the C-terminus is added (500 ␮l of 0.5 M amino acid solution containing 0.5 M HOBt in DMF) followed by the addition of 500 ␮l 0.5 M DIC in DMF and the reaction well volume is brought up to 3 ml using DMF. The coupling reaction is mixed for 1 h at 500 rpm, followed by emptying of the reaction block by positive nitrogen gas pressure. A second coupling reaction is performed by the addition of 500 ␮l DMF to each reaction vessel, followed by the addition of 500 ␮l of the respective amino acid (three-fold excess), 500 ␮l 0.5 M HBTU, 400 ␮l 1 M DIEA, the reaction well volume is brought up to 3 ml with DMF, and mixed at 500 rpm for 1 h. After the second coupling cycle, the reaction block is emptied and the resin-N␣-protected peptide is washed with DMF (4.5 ml five times). N␣-Fmoc deprotection is performed by the addition of 4 ml 25% piperidine in DMF and mixed for 5 min at 500 rpm followed by a 20 min deprotection at 500 rpm. The reaction well is washed with 4.5 ml DMF and the next coupling cycle is performed as described above. Deprotection of the amino acid side chains and cleavage of the amide-peptide from the resin was performed by incubating the peptide-resin with 3 ml cleavage cocktail (95% TFA, 2.5% water, 2.5% triisopropylsilane) for 3 h at 500 rpm. The cleavage product was emptied from the reaction block into a cleavage block containing 7 ml collection vials under nitrogen gas pressure. The resin was washed with 1.5 ml cleavage cocktail for 5 min and 500 rpm and added to the previous cleavage solution. The peptides were transferred to pre-weighted 50 ml conical tubes and precipitated with cold (4 ◦ C) anhydrous ethyl ether (up to 50 ml). The flocculent

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Table 1 Analytical properties of the peptides reported herein Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13

Structure

HPLC k (system 1)

HPLC k (system 2)

Purity (%)

Mass spectral analysis (M+1)

Ac-Ser-Tyr-Ser-Nle-Glu-His-Ala-Ala-Ala-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-Phe-Phe-Arg-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Phe-Phe-Arg-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-Phe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-DArg-Phe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-Phe-DPhe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]Tyr-Lys-Pro-Val-NH2 Ac-Nle-c[Asp-His-Ala-Ala-Ala-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-Phe-Phe-Lys]-NH2 Ac-Nle-c[Asp-His-DArg-Phe-Phe-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-DPhe-Phe-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-Phe-DPhe-Lys]-NH2

4.1 4.2 4.6 3.7 4.7 5.1 5.2 6.9

7.7 10.6 10.9 10.4 8.7 8.8 9.5 11.4

>99 >99 >95 >96 >98 >98 >99 >99

1370.6 1471.4 1608.8 1609.0 1608.7 1608.4 1607.7 2126.5

0.8 5.3 5.2 5.7 5.5

1.9 9.3 9.6 11.2 10.5

>98 >98 >98 >99 >99

749.4 985.9 985.8 985.4 985.7

HPLC k = [(peptide retention time-solvent retention time)/solvent retention time] in solvent system 1 (10% acetonitrile in 0.1% trifluoroacetic acid/water and a gradient to 90% acetonitrile over 35 min) or solvent system 2 (10% methanol in 0.1% trifluoroacetic acid/water and a gradient to 90% methanol over 35 min). An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rate of 1.5 ml/min. The percentage peptide purity is determined by HPLC at a wavelength of 214λ.

peptide was pelleted by centrifugation (Sorval Super T21 high speed centrifuge using the swinging bucket rotor) at 2000 rpm for 3 min, the ether was decanted off, and the peptide was washed one time with cold anhydrous ethyl ether and pelleted. The crude peptide was dried in vacuo 48 h. The crude peptide yields ranged from 60 to 90% of the theoretical yields. A 7–15 mg sample of crude peptide was purified by RP-HPLC using a Shimadzu chromatography system with a photodiode array detector and a semi-preparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 cm × 25 cm) and lyophilized. The purified peptide was >95% pure as determined by analytical RP-HPLC and had the correct molecular mass Table 1 (University of Florida protein core facility). 2.2. Cyclic lactam bridge MTII containing peptides Cyclic lactam bridge MTII containing peptides were synthesized using standard Boc methodology [34,51] on an automated synthesizer (Advanced ChemTech 440MOS, Louisville, KY). The amino acids Boc-Tyr (2ClBzl), Boc-Lys(Fmoc), Boc-diaminopropionic acid [Dpr(Fmoc)], Boc-Asp(OFm), Boc-DArg(Tos), Boc-Arg(Tos), Boc-Phe, Boc-His(Bom), Boc-DPhe, Boc-Trp(CHO), Boc-Asn, and Boc-Ala were purchased from Bachem (CA). The peptides were assembled on pMBHA resin (0.28 meq/g substitution), purchased from Peptides International. All reagents were ACS grade or better. The synthesis was performed using a 40 well Teflon reaction block with a course Teflon frit. Approximately 200 mg resin (0.056 mmol) was added to each reaction block well. Each peptide was synthesized in two separate reaction wells due to reaction volume limitations. The resin was allowed to swell for 2 h in 5 ml dimethylformamide (DMF) and the N␣-Boc group was deprotected using 4 ml 50% trifluroacetic acid (TFA), 2% anisole in

dichloromethane (DCM) for 3 min followed by a 20 min incubation at 500 rpm and washed with DCM (4.5 ml, 2 min, 500 rpm three times). The peptide-resin salt was neutralized by the addition of 4 ml 10% diisopropylethylamine (DIEA) in DCM (3 min, 500 rpm two times) followed by a DCM wash (4.5 ml, 2 min, 500 rpm four times). A positive Kaiser [29] test resulted indicating free amine groups on the resin. The growing peptide chain was added to the amide-resin using the general amino acid cycle as follows: 500 ␮l DMF is added to each reaction well to “wet the frit,” three-fold excess amino acid starting from the C-terminus is added (400 ␮M of 0.5 M solution in 0.5 M N-hydroxybenzotriazole (HOBt) in DMF) followed by the addition of 400 ␮l 0.5 M N,N -diisopropylcarbodiimide (DIC) in DMF and the reaction well volume is brought up to 3 ml using DMF. The coupling reaction is mixed for 1 h at 500 rpm, followed by emptying of the reaction block by positive nitrogen gas pressure. A second coupling reaction is performed by the addition of 500 ␮l DMF to each reaction vessel, followed by the addition of 400 ␮l of the respective amino acid (threefold excess), 400 ␮l 0.5 M O-benzotriazolyl-N,N,N ,N tetramethyluronium hexafluorophosphate (HBTU), 300 ␮l 1 M DIEA, the reaction well volume is brought up to 3 ml with DMF, and mixed at 500 rpm for 1 h. After the second coupling cycle, the reaction block is emptied and the resinN␣-protected peptide is washed with DCM (4.5 ml four times). N␣-Boc deprotection is performed by the addition of 4 ml 50% TFA, 2% anisole in DCM and mixed for 5 min at 500 rpm followed by a 20 min deprotection. The reaction well is washed with 4.5 ml DCM (four times), neutralized with 10% DIEA (3 min, 500 rpm two times) followed by a DCM wash (4.5 ml, 2 min, 500 rpm four times), and the next coupling cycle is performed as described above. The Fmoc and OFm protecting groups are removed from Lys (or Dpr) and Asp, respectively, by treatment with 4.5 ml

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25% piperidine in DMF (20 min at 500 rpm) with a positive Kaiser test resulting. The lactam bridge between the Asp and Lys (or Dpr) amino acids is formed using five-fold excess benziotriazolyloxy-tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) and six-fold excess DIEA as coupling agents and mixing at 500 rpm. The lactam bridges were formed (negative Kaiser test) after approximately 3 days at room temperature. Deprotection of the remaining amino acid side chains and cleavage of the amide-peptide from the resin was performed by incubation the peptideresin with anhydrous hydrogen fluoride (HF, 5 ml, 0 ◦ C, 1 h) and 5% m-cresol, 5% thioanisole as scavengers. After the reaction is complete and the HF has been distilled off, the peptide is ether precipitated (50 ml × 1) and washed with 50 ml cold (4 ◦ C) anhydrous ethyl ether. The peptide is filtered off using a course frit glass filter and dissolved in glacial acetic acid, frozen and lyophilized. The crude peptide yields ranged from 60 to 90% of the theoretical yields. A sample of crude peptide was purified by RP-HPLC using a Shimadzu chromatography system with a photodiode array detector and a semi-preparative reversed phase high performance liquid chromatography (RP-HPLC) C18 bonded silica column (Vydac 218TP1010, 1.0 cm × 25 cm) and lyophilized. The purified peptide was >95% pure as determined by analytical RP-HPLC and had the correct molecular mass Table 1 (University of Florida protein core facility).

to each well. Subsequently, 150 ␮l substrate buffer (60 mM sodium phosphate, 1 mM MgCl2 , 10 mM KCl, 5 mM ␤mercaptoethanol, 200 mg/200 ml ONPG) was added to each well and the plates were incubated at 37 ◦ C. The sample absorbance, OD405 , was measured using a 96-well plate reader (Molecular Devices). The relative protein was determined by adding 200 ␮l 1:5 dilution Bio Rad G250 protein dye:water to the 10 ␮l cell lysate sample taken previously, and the OD595 was measured on a 96-well plate reader (Molecular Devices). Data points were normalized both to the relative protein content and non-receptor-dependent forskolin stimulation. The antagonistic properties of these compounds were evaluated by the ability of these ligands to competitively displace the MTII agonist (Bachem) in a dose-dependent manner, at up to 10 ␮M concentrations [16]. The pA2 values were generated using the Schild analysis method [50]. 2.5. Data analysis EC50 and pA2 values represent the mean of duplicate experiments performed in triplicate, quadruplet, or more independent experiments. The results are not corrected for peptide content. EC50 and pA2 estimates and their associated standard errors were determined by fitting the data to a non-linear least-squares analysis using the PRISM program (v3.0, GraphPad Inc.).

2.3. Cell culture and transfection Briefly, HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum and seeded 1 day prior to transfection at 1 × 106 cell/100-mm dish to 2×106 cell/100-mm dish. Melanocortin receptor DNA in the pCDNA3 expression vector (20 ␮g) were transfected using the calcium phosphate method. Stable receptor populations were generated using G418 selection (1 mg/ml) for subsequent bioassay analysis. 2.4. cAMP reporter gene functional bioassay HEK-293 cells stably expressing the melanocortin receptors were transfected with 4 ␮g CRE/␤-galactosidase reporter gene as previously described [7]. Briefly, 5000–15,000 post-transfection cells were plated into 96 well Primera plates (Falcon) and incubated overnight. Forty-eight hours post-transfection the cells were stimulated with 100 ␮l peptide (10−4 –10−12 M) or forskolin (10−4 M) control in assay medium (DMEM containing 0.1 mg/ml BSA and 0.1 mM isobutylmethylxanthine) for 6 h. The assay media was aspirated and 50 ␮l of lysis buffer (250 mM Tris–HCl, pH 8.0, and 0.1% Triton X-100) was added. The plates were stored at −80 ◦ C overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 ␮l were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates, 40 ␮l phosphate-buffered saline with 0.5% BSA was added

3. Results The peptides reported herein were synthesized using standard fluorenylmethyloxy-carbonyl (Fmoc) [4,6] or tert-butyloxycarbonyl (Boc) [34,51] chemistries. The peptides were purified to homogeneity using semi-preparative reversed-phase high pressure liquid chromatography (RPHPLC). The peptides possessed the correct molecular weights, determined by mass spectrometry. The purity of these peptides were assessed by analytical RP-HPLC in two diverse solvent systems. Table 2 summarizes the pharmacological results of the chimeric AGRP-melanocortin peptides prepared in this study at the mouse melanocortin receptor MC1R, MC3–5R isoforms. 3.1. Linear NDP-MSH-based template Table 2 summarizes the chimeric peptides generated using the NDP-MSH linear tridecapeptide and cyclic MTII peptide templates, were the hAGRP (111–113) Arg-Phe-Phe amino acids have been substituted for the NDP-MSH and MTII agonist DPhe-Arg-Trp (7–9, ␣-MSH numbering) residues. The control peptide 1 had the agonist DPhe-Arg-Trp residues replaced with Ala’s to verify the hypothesis that these residues are critical for melanocortin receptor molecular recognition and agonist activity. Peptide 1 resulted in a complete loss of agonist activity at up to 100 ␮M concentrations and was

Table 2 Pharmacology of the chimeric melanocortin-hAGRP peptides using the NDP-MSH and MTII templates with the melanocortin agonist DPhe-Arg-Trp amino acids replaced by the hAGRP (111–113) Arg-Phe-Phe residues at the various indicated positions. Structure

mMC1R, EC50 (nM)

mMC3R, EC50 (nM)

mMC4R, EC50 (nM)

mMC5R, EC50 (nM)

␣-MSH NDP-MSH MTII hAGRP (109–118) 1 2

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Ala-Ala-Ala-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-Phe-Phe-Arg-Gly-Lys-Pro-Val-NH2

0.55 ± 0.09 0.038 ± 0.012 0.020 ± 0.003 5,120 ± 3,040 >100,000 Partial agonist (19,900 ± 5,100) 7,220 ± 2,200

0.79 ± 0.14 0.098 ± 0.013 0.16 ± 0.03 >100,000 >100,000 >100,000

5.37 ± 0.62 0.21 ± 0.03 0.087 ± 0.008 pA2 = 6.8 ± 0.24 >100,000 >100,000

0.44 ± 0.09 0.071 ± 0.012 0.16 ± 0.03 >100,000 >100,000 >100,000

Partial agonist (20,500 ± 6,800) 480 ± 49 >100,000 6,210 ± 990 >100,000 pA2 = 6.2 ± 0.3

>100,000

14,600 ± 4,000

930 ± 120 >100,000 17,000 ± 12,000 >100,000 pA2 = 6.2 ± 0.1

327 ± 118 Slight agonist 450 ± 110 Slight agonist >100,000

>100,000 >100,000

>100,000 >100,000

>100,000 >100,000

>100,000 14,400 ± 3,700 Slight agonist

>100,000 5,000 ± 900 >100,000

>100,000 3,600 ± 200 Slight agonist

3

Ac-Ser-Tyr-Ser-Nle-Glu-His-Phe-Phe-Arg-Gly-Lys-Pro-Val-NH2

4 5 6 7 8 9 10

Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-Phe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-DArg-Phe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-Phe-DPhe-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]Tyr-Lys-Pro-Val-NH2 Ac-Nle-c[Asp-His-Ala-Ala-Ala-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-Phe-Phe-Lys]-NH2

11 12 13

Ac-Nle-c[Asp-His-DArg-Phe-Phe-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-DPhe-Phe-Lys]-NH2 Ac-Nle-c[Asp-His-Arg-Phe-DPhe-Lys]-NH2

59.9 ± 8.1 Slight agonist 7.28 ± 0.76 3,630 ± 320 1,960 ± 500 >100,000 Partial agonist 38,300 ± 8,700 >100,000 440 ± 49 2,630 ± 830

C.G. Joseph et al. / Peptides 24 (2003) 1899–1908

Peptide

The indicated errors represent the standard error of the mean determined from at least three independent experiments. The antagonistic pA2 values were determined using the Schild analysis and the agonist MTII. >100,000 indicates that the compound was examined but lacked agonist or antagonist properties at up to 100 ␮M concentrations. Slight agonist denotes that some stimulatory response was observed at 100 ␮M concentrations, but not enough to determine an EC50 value.

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unable to bind to the MC3R or MC4R more than 25% total specific binding (data not shown), verifying the importance of the DPhe-Arg-Trp residues for melanocortin receptor activity. The precise orientation of substituting the AGRP ArgPhe-Phe residues into the agonist template for agonist activity was explored by presenting these residues in both the Arg-Phe-Phe and Phe-Phe-Arg directions. Peptide 2, containing the AGRP residues in the Phe-Phe-Arg orientation and the removal of the His6 residue (␣-MSH numbering) resulted in a lack of agonist or antagonist activity at up to 100 ␮M at the MC3–5 receptors, but was a partial agonist at the MC1R at up to 100 ␮M concentrations. Peptide 3, containing the His6 amino acid and the AGRP residues in the Phe-Phe-Arg orientation resulted in full ␮M agonist potency at the MC1R and MC5R, partial agonist activity at the MC3R (at up to 100 ␮M concentrations), no activity (agonist or antagonist) at the MC4R (Fig. 1). Upon incorporation of the AGRP Arg-Phe-Phe residues in the same linear orientation as in hAGRP into the NDP-MSH template, peptide 4, nM agonist activity resulted at all the melanocortin receptors examined in this study (Fig. 1). Systematic stereochemical inversion of the hAGRP (111–113) Arg-Phe-Phe residues were performed in attempts to correlate a DPhe position that may correspond to the melanocortin agonist DPhe residue and to determine if the melanocortin receptors prefer

-Galactosidase Normalized to Protein & Forskolin

1.25

0.75 0.50 0.25

the Arg residue in the D-configuration for enhanced potency. Generally, as observed for peptides 5–7, stereochemical inversion of these Arg-Phe-Phe residues in the NDP-MSH template resulted in dramatic decreased melanocortin receptor potency, with the exception of 6 (Fig. 2) that possessed 1.25

1.00 0.75 0.50 0.25 -5 -4

1.00 0.75 0.50 0.25

0.00 -13 -12 -11 -10 -9 -8 -7

-3

Log Peptide Concentration (M)

1.00

1.25

NDP-MSH Peptide 3 Peptide 4

0.75 0.50 0.25

1.00

NDP-MSH Peptide 3 Peptide 4

0.75 0.50 0.25 mMC5R

mMC4R 0.00 -13 -12 -11 -10 -9 -8 -7

-6 -5 -4 -3

Log Peptide Concentration (M)

-6 -5 -4 -3

Log Peptide Concentration (M) -Galactosidase Normalized to Protein & Forskolin

-Galactosidase Normalized to Protein & Forskolin

1.25

NDP-MSH Peptide 3 Peptide 4

mMC3R

mMC1R -7 -6

-3

Fig. 2. Illustrates the full agonist pharmacology of peptide 6 at the mouse melanocortin receptor isoforms indicated. Interestingly, peptide 6 is a potent mMC1R agonist that is 850-fold MC1R versus MC3R selective, 2300-fold MC1R versus MC4R, and 62-fold MC1R versus MC5R selective.

NDP-MSH Peptide 3 Peptide 4

0.00 -13 -12 -11 -10 -9 -8

1.00

mMC1R mMC3R mMC4R mMC5R

0.00 -11 -10 -9 -8 -7 -6 -5 -4 Log Peptide 6 Concentration (M)

-Galactosidase Normalized to Protein & Forskolin

-Galactosidase Normalized to Protein & Forskolin

1.25

Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 (6)

0.00 -13 -12 -11 -10 -9 -8 -7

-6 -5 -4 -3

Log Peptide Concentration (M)

Fig. 1. Summary of the agonist pharmacology of peptide 3 (Ac-Ser-Tyr-Ser-Nle-Glu-His-Phe-Phe-Arg-Gly-Lys-Pro-Val-NH2 ) and peptide 4 (Ac-SerTyr-Ser-Nle-Glu-His-Arg-Phe-Phe-Gly-Lys-Pro-Val-NH2 ), compared to NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 ) at the mouse melanocortin receptors. Peptide 3 has partial agonist activity, enough to determine an EC50 value, and it is speculated would reach maximal stimulation, relative to forskolin control and NDP-MSH, if tested at higher concentrations. At the mMC4R, peptide 3 did not stimulate the receptor and did not possess antagonist pharmacology (data not shown).

C.G. Joseph et al. / Peptides 24 (2003) 1899–1908

Ac-Nle-c[Asp-His-Arg-DPhe-Phe-Lys]-NH2 (12) -Galactosidase Normalized to Protein & Forskolin

1.25 1.00 0.75

mMC1R mMC3R mMC4R mMC5R

0.50 0.25 0.00 -11 -10 -9 -8 -7 -6 -5 -4 Log Peptide 12 Concentration (M)

-3

Fig. 3. Illustrates the full agonist pharmacology of peptide 12 at the indicated mouse melanocortin receptors.

eight-fold increased MC1R potency, as compared with peptide 4, respectively. Finally, peptide 8 was designed such that the N-terminal Ser-Tyr-Ser-Nle and C-terminal LysPro-Val amino acids of the melanocortin agonist NDP-MSH were added to the respective peptide termini of a hAGRP (109–118) decapeptide, and resulted in ca. equipotent ␮M MC1R agonist activity and MC4R antagonist activity as the hAGRP (109–118) peptide, but gained experimentally detectable MC3R antagonist pharmacology. 3.2. Cyclic MTII-based template Table 2 summarizes the melanocortin receptor pharmacology of the AGRP-melanocortin chimeric peptides using the cyclic MTII peptide template. Consistent with the above NDP-MSH peptide template, peptide 9 was generated with the MTII DPhe-Arg-Trp residues substituted with Ala’s to verify the importance of these ligand residues for melanocortin receptor binding and activity. Since it was previously determined using the linear NDP-MSH template that the Arg-Phe-Phe AGRP residues, when incorporated into the agonist template in this orientation, resulted in more potent ligands than the Phe-Phe-Arg orientation, the former was incorporated into the MTII template, peptides 10–13. Unexpectedly, peptide 10 resulted in a loss of full agonist activity at the MC3–5 receptors and only possessed ␮M partial agonist activity at the MC1R (at up to 100 ␮M concentrations). Similar to the NDP-MSH chimeric peptides described above, a D-amino acid scan was performed using the cyclic MTII template. Peptide 11 with the DArg-PhePhe motif lost the ability to generate a full agonist response at up to 100 ␮M concentrations at the melanocortin receptors examined herein. Interestingly, peptide 12 containing the Arg-DPhe-Phe motif resulted in full agonist activity at the mMC1 and mMC3–5 receptors (Fig. 3). Peptide 13, containing the Arg-Phe-DPhe motif resulted in slight agonist activity at 100 ␮M at the MC3R and MC5R, but was a ␮M MC1R agonist.

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4. Discussion It has been proposed by several laboratories that the conserved Arg-Phe-Phe motif in hAGRP (111–113) is critical for the antagonistic pharmacology and molecular recognition at the melanocortin receptors [18,53]. The hAGRPderived decapeptide Yc[CRFFNAFC]Y retained the antagonistic activity at the MC4R, and interestingly, possessed agonist activity at the MC1R [18]. Additional hAGRP (109–118)-based decapeptides have been synthesized and a novel MC1R antagonist has been reported [52]. Structureactivity relationships (SAR) indicated that the melanocortin agonist or antagonist activities can be switched only by small and local structural changes [16,25,52], which lead to different conformational families. To test the hypothesis that the antagonist hAGRP (111–113) Arg-Phe-Phe residues may be mimicking the melanocortin-based agonist Phe-ArgTrp amino acid interactions with the mouse melanocortin receptors, the design, synthesis, and pharmacological characterization of chimeric AGRP-melanocortin-based ligands using linear and cyclic peptide templates was undertaken. To determine if substitution of the hAGRP (111–113) Arg-Phe-Phe antagonist amino acids would confer agonist or antagonist activity to the melanocortin-based agonists, two peptide templates were selected. The potent linear tridecapeptide NDP-MSH [49] and the cyclic heptapeptide MTII [1,2] (Table 2) agonist templates were selected to examine the effect of replacing the agonist DPhe-Arg-Trp amino acids with the hAGRP (111–113) Arg-Phe-Phe residues. Since the precise orientation of AGRP (109–118) ligand-receptor interactions are unknown, chimeric NDP-MSH-AGRP peptides with both the Arg-Phe-Phe and Phe-Phe-Arg orientations were synthesized. Comparing peptides 3 and 4 (Fig. 1), the preferred orientation for the melanocortin receptors examined in this study is the Arg-Phe-Phe alignment, based upon the increased potencies of analogue 4 at these receptors. Interesting, at the mMC4R, peptide 3 possessed neither agonist or antagonist activity supporting the hypothesis that the Phe-Phe-Arg orientation in NDP-MSH does not mimic the melanocortin agonist Phe-Arg-Trp interactions with this receptor. At the mMC3R, peptide 3 possessed some agonist activity at 100 ␮M concentrations and may generate a maximal response at higher concentrations. Alanine scanning of ␣-MSH has previously been reported at the MC1R using B16 mouse melanoma cells [47]. These studied resulted in 82-, 509-, 2010-, and 2000-fold decreased binding affinities at the mMC1R when the His6 , Phe7 , Arg8 , and Trp9 amino acids, respectively, were substituted with the Ala residue [46]. Alanine scanning studies of the Ac-HisDPhe-Arg-Trp-NH2 tetrapeptide was performed and characterized at the mouse melanocortin receptors used herein [22–24]. Substitution at the DPhe7 position in the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 sequence with Ala resulted in essentially a lose of agonist activity at the mMC3–5Rs, with full agonist activity at the mMC1R (EC50 = 30 ␮M) [22]. Substitution at the Arg8 and Trp9 positions of the Ac-

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His-DPhe-Arg-Trp-NH2 tetrapeptide still retained full agonist ␮M potencies at the melanocortin receptors, with the exception of the Ac-His-DPhe-Arg-Ala-NH2 peptide that lost full agonist activity at the mMC4R [23,24]. Peptide 4, containing the Arg-Phe-Phe residues instead of the NDPMSH DPhe-Arg-Trp residues resulted in a 1600-fold decrease in potency at the mMC1R, similar in the order of magnitude to the substitution of either the Arg8 or Trp9 residues of ␣-MSH by Ala [46]. Incorporation of the hAGRP (111–113) residues into the NDP-MSH template in the Arg-Phe-Phe (4) versus the Phe-Phe-Arg (3) orientation resulted in ligands with nM melanocortin receptor agonist potency. Interestingly, peptide 6 (Ac-Ser-Tyr-Ser-Nle-GluHis-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 ), resulted in a potent nM mMC1R agonist possessing high nM agonist activity at the mMC5R, ␮M agonist activity at the mMC3R and mMC4R. Thus, peptide 6 is a 850-fold MC1R versus MC3R selective, 2300-fold MC1R versus MC4R, and 62fold MC1R versus MC5R selective peptide (Fig. 2). These results discussed herein, support the hypothesis that the hAGRP (111–113) Arg-Phe-Phe antagonist residues mimic the agonist Phe-Arg-Trp amino acids in a similar topographical orientation. Classical studies of the melanocortin peptides identified that inversion of chirality of Phe7 to DPhe7 of ␣-MSH resulted in 10- to 1000-fold increased potency [10,19,26,48,49]. Thus, in attempts to correlate which hAGRP (112–113) Phe residue might correspond to the melanocortin agonist Phe7 amino acid in regards to putative ligand-receptor interactions, stereochemical inversion was performed at the Phe residues in the melanocortin agonist templates. Interestingly, peptide 6 (NDP-MSH linear template) containing the DPhe that might putatively correspond to the hAGRP Phe112 residue, resulted in only an eightfold increase in mMC1R agonist potency, a 13- and 18-fold decreased mMC3R and MC4R agonist potency, respectively, and equipotent mMC5R agonist potency, compared with peptide 4. However, peptide 12 (MTII cyclic template) containing the DPhe that might putatively correspond to the hAGRP Phe112 residue, resulted in converting the peptide with the corresponding L-Phe isomer (peptide 10) from a mMC1R partial agonist into a nM full agonist, and a ligand possessing full ␮M agonist activity at the mMC3–5 receptors, Fig. 3. These results suggest that the antagonist hAGRP Phe112 may be mimicking the melanocortin agonist DPhe7 interactions with the receptor in the cyclic MTII peptide template, in terms of enhancing general melanocortin receptor agonist potency. Although this latter speculation remains to be experimentally verified, this is the first experimental evidence suggesting that the hAGRP Phe112 antagonist residue may topographically correlate with the melanocortin agonist Phe7 amino acid in terms of putative ligand–mMC4R interactions. Three-dimensional homology molecular modeling [20,54] and melanocortin receptor mutagenesis studies have been performed to putatively identify which recep-

tor residue(s) are interacting with the melanocortin ligand Phe-Arg-Trp amino acid side chains, and AGRP/agouti melanocortin receptor interactions [16,39,56–58]. The data presented herein describe experiments, from a ligand perspective, that test the hypothesis that the hAGRP (111–113) Arg-Phe-Phe amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp residue interactions with the melanocortin receptors. The findings in this study support this latter hypothesis and provide new insights into melanocortin structure–function relationships that may potentially be used to design selective therapeutic agents for diseases associated with the melanocortin system.

5. Conclusions The study presented herein provides experimental evidence supporting the hypothesis that the antagonist hAGRP (111–113) Arg-Phe-Phe residues may be mimicking the melanocortin agonist Phe-Arg-Trp amino acid interactions at the melanocortin receptors. Specifically, the hAGRP (111–113) antagonist amino acids Arg-Phe-Phe can be substituted in the melanocortin agonist NDP-MSH peptide template and result in agonist activities at the melanocortin receptors. These data demonstrate the hAGRP (111–113) Arg-Phe-Phe versus the Phe-Phe-Arg orientation substituted into NDP-MSH resulted in enhanced potencies and agonist activities at the mMC1 and mMC3–5 melanocortin receptors. Additionally, preliminary data is presented that might correlate the antagonist hAGRP Phe112 residue to the melanocortin agonist Phe7 (␣-MSH numbering) residue in topographical orientation of these Phe side chains with the mMC4R. These studies have also resulted in the identification of the peptide Ac-Ser-Tyr-Ser-Nle-Glu-His-Arg-DPhePhe-Gly-Lys-Pro-Val-NH2 that is a 850-fold MC1R versus MC3R selective, 2300-fold MC1R versus MC4R, and 62-fold MC1R versus MC5R selective, providing a novel melanocortin template to design receptor selective ligands.

Acknowledgments This work has been funded by NIH Grants DK57080, DK64250 and a University of Florida Opportunity Fund Grant. Carrie Haskell-Luevano is a recipient of a Burroughs Wellcome fund Career Award in the Biomedical Sciences and an American Diabetes Association Research Award. References [1] Al-Obeidi F, Castrucci AM, Hadley ME, Hruby VJ. Potent and prolonged acting cyclic lactam analogues of ␣-melanotropin: design based on molecular dynamics. J Med Chem 1989;32:2555–61. [2] Al-Obeidi F, Hadley ME, Pettitt BM, Hruby VJ. Design of a new class of superpotent cyclic ␣-melanotropins based on quenched dynamic stimulations. J Am Chem Soc 1989;111:3413–6.

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