Potent and selective agonists of α-melanotropin (αMSH) action at human melanocortin receptor 5; linear analogs of α-melanotropin

peptides 28 (2007) 1020–1028

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/peptides

Potent and selective agonists of a-melanotropin (aMSH) action at human melanocortin receptor 5; linear analogs of a-melanotropin Maria A. Bednarek a,*, Tanya MacNeil b, Rui Tang b, Tung M. Fong b, M. Angeles Cabello c, Marta Maroto c, Ana Teran c a

Department of Medicinal Chemistry, Merck Research Laboratories, R50G-140, P.O. Box 2000, Rahway, NJ 07065, USA Department of Obesity and Metabolic Disorders, Merck Research Laboratories, Rahway, NJ, USA c Centro de Investigacion Basica, Merck Sharp and Dohme de Espana, S.A., Madrid, Spain b

Dedicated to the memory of Prof. Dr. Miklos Bodanszky.

article info

abstract

Article history:

a-Melanotropin, Ac-Ser1-Tyr-Ser-Met-Glu-His6-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13-NH21, is a

Received 10 November 2006

non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in

Received in revised form

various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic

8 February 2007

analogs of aMSH, including a broadly used and more potent the NDP-aMSH peptide, Ac-Ser1-

Accepted 12 February 2007

Tyr-Ser-Nle4-Glu-His6-D-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13-NH2, are also not particularly

Published on line 17 February 2007

selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report

Keywords:

herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-aMSH

a-Melanotropin

which are highly potent and specific agonists for the human MC5R. The new linear peptides, of

aMSH

structures and solubility properties similar to those of the endogenous ligand aMSH, are

Melanocortin peptide

exemplified by compound 7, Ac-Ser1-Tyr-Ser-Met-Glu-Oic6-D-4,40 -Bip7-Pip8-Trp9-Gly-Lys-

NDP-aMSH

Pro-Val13-NH2 (Oic: octahydroindole-2-COOH, 4,40 -Bip: 4,40 -biphenylalanine, Pip: pipecolic

Melanocortin receptor 5

acid), shortly NODBP-aMSH, which has an IC50 = 0.74 nM (binding assay) and EC50 = 0.41 (cAMP

Agonist

production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the

Binding affinity

melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-aMSH: Ac-Nle-Glu-

cAMP accumulation assay

Oic6-D-4,40 -Bip7-Pip8-Trp9 -NH2 (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-GluOic6-D-4,40 -Bip7-Pip8 segment was found to be critical for high agonist potency, while the Cterminal Trp9 residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R. # 2007 Elsevier Inc. All rights reserved.

1.

Introduction

Melanocyte-stimulating hormones (melanotropins): aMSH, bMSH and gMSH, and adrenocorticotropic hormone, ACTH,

are produced in the pituitary and also in peripheral sites such as the skin, as the result of the post-translational cleavage of the pre-pro-opiomelanocortin gene product (POMC) [6,7,11,13,18,25,29]. These peptide hormones, melanocortin

* Corresponding author. Tel.: +1 732 594 4798; fax: +1 732 594 8080. E-mail address: [email protected] (M.A. Bednarek). Abbreviations: 4,40 -Bip, 4,40 -biphenylalanine; Nal(20 ), 20 -naphthylalanine; Oic, octahydroindole-2-COOH; Pip, pipecolic acid; Tic, tetrahydroquinoline-3-COOH 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.02.011

peptides 28 (2007) 1020–1028

peptides, are involved in numerous physiological functions in the central nervous system and in peripheral tissues [6,7,11,13,18,25,29]. For example, a-melanotropin, aMSH, AcSer1-Tyr-Ser-Met-Glu-His6-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13NH21, modulates behavior processes such as learning, feeding and sexual function, regulates various anti-immune, antiinfective and antipyretic responses, and affects pigmentation (skin-darkening in amphibians and other lower vertebrates), thermoregulation, pain perception and other processes [6,7,11,13,18,25,29]. Biological functions of melanocortin peptides are mediated by specific trans-membrane spanning proteins called melanocortin receptors (MCRs) [8,10,19,28]. The receptors are coupled to G-proteins and signal through several pathways in the cells (cAMP production, protein kinase A activation and [Ca2+]i elevation) [8,10,19,28]. aMSH is an endogenous ligand for melanocortin receptors 1, 3, 4 and 5 (MC1,3-5) but not for melanocortin receptor 2 (MC2R) for which ACTH is a selective compound. aMSH displays the highest affinity for the MC1R, found in melanocytes and several types of immune and inflammatory cells, and the lowest for the MC5R, found mainly in periphery, in exocrine glands, sebaceous glands, adipocytes, and others (also detected in the brain, cortex and cerebellum) [8,10,19,28]. This peptide hormone binds with an intermediate affinity to the melanocortin receptors 3 and 4 which are both widely distributed in the central nervous system (MC3R is also found in gastrointestinal tract, placenta and others) [8,10,19,28]. A potency order of aMSH at the melanocortin receptors is thus: MC1R > MC3R = MC4R > MC5R. aMSH and the other natural melanocortin peptides encompass in their structures a peptide segment His6-Phe7Arg8-Trp9 considered to be crucial to interactions with the melanocortin receptors [16]. This ‘‘essential core’’ consists of two bulky aromatic amino acids (Phe7 and Trp9) and two basic hydrophilic amino acids (His6 and Arg8.) The aromatic side chains of Phe7 and Trp9 appear to be indispensable to molecular recognition; they presumably interact with a putative hydrophobic binding pocket on the receptors, most likely through stacking of the aromatic side chains [2,4]. The hydrophilic basic residues His6 and Arg8 are regarded to be less critical to the formation and stabilization of complexes between the melanocortin peptides and the melanocortin receptors [2–4]. The ‘‘essential core’’ of melanocortin is also present in most synthetic peptidic ligands for the melanocortin receptors. One of those compounds is the NDP-aMSH peptide, AcSer1-Tyr-Ser-Nle4-Glu-His6-D-Phe7-Arg8-Trp9-Gly-Lys-ProVal13-NH2, an analog of aMSH which is more potent and enzyme-resistant than the parent compound [21]. Its structure differs from that of aMSH only in position 4 where Met has been replaced with Nle, and in position 7 where Phe has been substituted with D-Phe. NDP-aMSH, a non-specific, superpotent agonist at the melanocortin receptors, is approximately 10-fold more potent at hMC3R and hMC4R, and 50-fold more 1

Throughout this report, the numbering of the amino acid residues in aMSH, Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8Trp9-Gly10-Lys11-Pro12-Val13-amide, has been retained for all linear and cyclic peptides.

1021

potent at hMC5R than aMSH [3,4,21]. A similarly highly potent agonist at the melanocortin receptors is another aMSH analog, the MTII peptide, Ac-Nle4-cyclo(Asp5-His6-D-Phe7-Arg8-Trp9Lys10)-NH2 [1]. The lactam ring of this small cyclic peptide also encompasses the ‘‘essential core’’ (His6-Phe7-Arg8-Trp9) of the melanocortin peptides. Another peptide with the similar lactam scaffold but with D-Nal(20 ) in place of D-Phe7 is the SHU9119 peptide, Ac-Nle4-cyclo(Asp5-His6-D-Nal(20 )7-Arg8-Trp9-Lys10)NH2; a sub-nanomolar agonist at MC1R and MC5R, and a highly effective antagonist at MC3R and MC4R [17]. In the last few years, the NDP-aMSH, MTII and SHU9119 peptides have been frequently used as research tools in numerous in vitro and in vivo experiments designed to elucidate the roles of the melanocortin receptors in biological processes [8,10,14,15,18,19,22,23,28]. For example, they have been indispensable in recent intensive studies on MC4R, the receptor implicated in regulation of food intake and energy metabolism in rodents and humans. These studies led to an understanding that compounds active at MC4R might be useful in the treatment of eating disorders (obesity, cachexia, anorexia and others) [1–4,8–10,16–19,21,22,28,30]. In parallel with the receptor examination, structure-function studies on aMSH of the last several years have focused, almost exclusively, on the design of MC4R selective peptides [15]. Only sporadic efforts have been made to secure ligands specific for the other melanocortin receptors [12,14,23]. While some progress has been reported on the understanding of a biological role of the melanocortin receptors 1 and 3 in humans, the role of MC5R remains elusive [8,10,18,28,29]. Yet the widespread peripheral distribution of MC5R in mammals suggests this receptor’s involvement in many actions of the melanocortin peptides. There are indications that in rodents MC5R may affect some inflammatory and anxiolytic events, production of pheromone and lipids in the exocrine glands, and secretion of luteinizing hormone, however human pharmacological validation is not yet available for these proposed functions of MC5R [9,20,26,27,31]. Our recent structure-function studies on aMSH were directed towards syntheses and in vitro pharmacological evaluation of several cyclic peptides which are highly potent and selective hMC5R agonists [5]. These compounds are analogs of MTII and SHU9119 with a modified lactam ring, in positions of His6, Phe7 and Arg8. The most interesting compound is OBP-MTII, the Ac-Nle4-cyclo(Asp5-Oic6-D-4,40 Bip7-Pip8-Trp9-Lys10)-NH2 peptide (Oic: octahydroindole-2COOH, 4,40 -Bip: 4,40 -biphenylalanine, Pip: pipecolic acid), which is a high potency and affinity agonist at hMC5R (IC50 = 0.95 nM, EC50 = 0.99 nM) (5). This analog is more than 5000-fold selective versus melanocortin receptors 1b, 3 and 4 and therefore nearly inactive at these receptors at micromolar concentration. In the present study, similar alterations to the ‘‘essential core’’ of melanocortins have been applied to the sequence of NDP-aMSH. The new linear peptide thus obtained: Nle4,Oic6,D-4,40 -Bip7,Pip8-aMSH, shortly NODBP-aMSH, displayed a sub-nanomolar agonist potency at hMC5R (IC50 = 0.7 nM, EC50 = 0.4 nM) and was more than 3200-fold selective versus the human MC 1b, 3 and 4 receptors. Subsequent evaluation of several truncated analogs of NODBP-aMSH identified a penta-peptide which effectively

1022

peptides 28 (2007) 1020–1028

binds to and activates hMC5R at 1 nM concentration and which shows enhanced selectivity with respect to the melanocortin receptors 1b, 3 and 4. Interestingly, this compound lacks Trp8 in its structure, a residue which was believed to be a crucial component of an ‘‘essential core’’ of the melanocortin peptides.

of different doses of the selected antagonist as described in Ref. [2]. The antagonists were preincubated with the receptor expressing cells for 10 min prior to the addition of aMSH, and the assay was finished as described above.

3. 2.

Materials and methods

2.1.

Peptide synthesis, purification and characterization

Elongation of peptidyl chains on p-methylbenzhydrylamine resin (431A ABI peptide synthesizer), deprotection and cleavage of peptides from a resin with HF, and purification of the crude products by high-pressure liquid chromatography were performed as previously described in detail [2]. A standard gradient system of 10–100% buffer B in 30 min (G1) was used for analysis; buffer A was 0.1% trifluoroacetic acid in water and buffer B was 0.1% trifluoroacetic acid in acetonitrile. The second gradient system used for analysis was 0–100% buffer B in 30 min (G2); buffer A was 0.1% trifluoroacetic acid in water and buffer B was 0.1% trifluoroacetic acid in methanol. The chromatographically homogenous compounds were analyzed by electrospray mass spectrometry (Hewlett Packard series 1100 MSD spectrometer).

2.2.

Competitive binding assays

Binding activity of compounds was measured using membranes from Chinese hamster ovary (CHO) cells expressing the cloned melanocortin receptors. Binding reactions contained membranes, 200 pM [125I]NDP-aMSH (New England Nuclear Corp.), and increasing concentrations of unlabelled test compounds from 0.05 to 20,000 nM. Reactions were incubated for 1.5 h and then filtered as described previously [2]. Binding data were analyzed using GraphPad curve-fitting software. Active peptides were evaluated in 3 or more independent experiments.

2.3.

cAMP assays

Agonist activities of all compounds were measured using Chinese hamster ovary (CHO) cells expressing the cloned melanocortin receptors (see Ref. [2] for details). Cells were detached from tissue culture flasks, collected by 5 min centrifugation, and resuspended in Earle’s Balanced Salt solution (Life Technologies, Gaithersburg, MD) with addition of 10 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM glutamine, and 1 mg/ml bovine serum albumin. Compounds from 0.003 to 5000 nM concentration together with 0.6 mM IBMX were incubated at room temperature with dissociated cells for 40 min and lysed with 0.1 M HCl to terminate the assay (SMP 001J) or with Perkin-Elmer detection buffer (SMP-004). cAMP was quantitated by Perkin-Elmer Life Sciences (NEN) (Boston, MA) SMP-001J or SMP-004 Flashplate cAMP assay. Activation by compounds was compared to the maximum response to aMSH. Active peptides were evaluated in three or more independent experiments. For assay of functional antagonism, the aMSH dose response curves were performed in the absence and presence

Results

Analogs of aMSH listed in Tables 1 and 2 were prepared by solid phase syntheses as previously described (see Ref. [2] and the Experimental Section.) They were evaluated for their binding affinities to the human melanocortin receptors 1b, 3, 4 and 5 in the competitive binding assays using the radiolabeled ligand [125I]-NDP-aMSH and for their agonist potency in cAMP assays employing the CHO cells expressing these receptors. The human melanocortin 1b receptor (hMC1bR) possesses pharmacological properties indistinguishable from those of its isoform, human melanocortin receptor 1a (hMC1aR) [24]. Compounds from Tables 1 and 2 were not evaluated for their binding affinities and functional potencies at hMC2R. Also, functional antagonism was determined only for compound 5. Binding and functional data collected in our laboratory for the best-known melanocortin receptor ligands: aMSH, NDPaMSH, MTII and SHU9119, compounds 1–4, have been compiled in Table 1. This table also contains data for several analogs of NDP-aMSH, peptides 5–15, which were the subject of the present study. They were linear, 13-amino acid long peptides with unusual substituents in the His6-D-Phe7-Arg8Trp9 segment of NDP-aMSH. In the structure of compound 5, D-Phe in position 7 was replaced with D-Nal(20 ); a substitution known to yield peptides which act as antagonists at MC3R and MC4R, but agonists at MC1R and MC5R. The new analog Nle4, D-Nal(20 )7-aMSH, designated NDN-aMSH, displayed binding affinity similar to that of SHU9119 for all melanocortin receptors studied. Its functional activity was similar to that of SHU9119 as well: it was a highly effective antagonist at hMC3R (IC50 = 0.15 nM, Kb = 0.5 nM) and hMC4R (IC50 = 0.29 nM, Kb = 0.95 nM), a highly potent agonist at hMC5R (IC50 = 0.07 nM, EC50 = 0.09 nM), and a partial agonist at hMC1bR (IC50 = 0.12 nM, EC50 = 0.23 nM, 67% maximum activation). Previously we have defined alterations to the lactam ring of MTII/SHU9119 which account for high agonist potency and selectivity at hMC5R: substitutions of His6 with Oic, D-Phe7 with D-4,40 -Bip and Arg8 with Pip [5]. An analog of MTII encompassing these modifications, the Oic6,D-4,40 Bip7,Pip8MTII peptide, shortly OBP-MTII, compound 6, was a potent hMC5R agonist which was largely inactive at human MC 1b, 3 and 4 receptors [5]. In the present study, an analog of NDPaMSH with the same alterations to the 6-9 segment (the ‘‘essential core’’ of the melanocortin peptide) was measured to also be a potent and selective ligand for hMC5R, peptide 7. This was a sub-nanomolar agonist at hMC5R (IC50 = 0.7 nM, EC50 = 0.4 nM), more than 3200-fold selective with respect to the other melanocortin receptors. To evaluate involvement of the side chains in position 4, 6 and 8 of peptide 7 in interactions with hMC5R, compounds 8– 11 were prepared and evaluated. In the structures of these peptides, D-4,40 -Bip in position 7 was retained, but the residues in the above mentioned positions were replaced with the

Table 1 – Analogs of aMSH Ac-Ser1-Tyr-Ser-Met4-Glu-His6-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13-NH2 No.

Compound

Binding assay#

cAMP assay##

IC50(nM)

1 2

3 4 5

7

8 9 10 11

12 13 14

15

Nle4,Oic6,D-4, 40 Bip7,Tic 8 Nle4,Oic6,DNal(20 )7,Pip 8 Nle4,Oic6,D-4, 40 Bip7,Pip8aMSH-OH desAc,Nle4, Oic6,D-4, 40 Bip7,Pip8 -aMSH-OH

EC50(nM)

MC1bR

hMC3R

hMC4R

hMC5R

1b/5

3/5

4/5

MC1bR

3.9  0.9 0.39  0.01

19  2 0.78  0.02

19  2 1.6  0.03

120  19 0.27  0.01

0.033 1.4

0.16 2.8

0.16 5.9

3.4  0.1 0.87  0.04

0.36  0.1 0.62  0.11 0.12  0.01

3.1  0.94 0.23  0.04 0.15  0.02

0.18  0.05 0.07  0.01 0.29  0.02

1.2 + 0.15 0.65  0.01 0.073  0.02

0.3 1 1.6

2.6 0.35 2.1

0.8 0.1 4

0.32  0.3 0.36  0.02 (54%) 0.23  0.05 (67%)

1.1  0.06 5% @ 1 mM 3 % @ 2.5 mM

>5000

>5000

>5000

0.95  0.21

>5000

>5000

>5000

18% @ 5 mM

5% @ 2 mM

40% @ 2 mM

0.99  0.13

2400  410

4000  600

2500  300

0.74  0.21

3240

5400

3380

20% @ 5 mM

14% @ 5 mM

36% @ 5 mM

0.41  0.05

0.13  0.01 0.11  0.07 15  2.2

0.35  0.1 0.23  0.03 1200  100

0.47  0.16 0.41  0.003 39% @ 2 mM

0.082  0.02 0.083  0.03 6.3  0.26

1.6 1.3 2.4

4.3 2.8 190

5.7 5

1.2  0.6 0.51  0.1 (70%) 6.8  1.8 (34%)

0.44  0.08 (60%) 0.25  0.08 (60%) 560  180 (45%)

0.64  0.22 (70%) 1.2 + 0.4 (60%) 2% @ 1 mM

0.25  0.02 0.27  0.09 3.3  0.69

0.41  0.09

0.19  0.02

0.44  0.03

0.058  0.02

7.1

3.3

7.6

1.4  0.6 (59%)

0.54  0.01 (43%)

4% @ 2.5 mM

0.11  0.01

940  240

2300  610

1300  280

3.8  1.1

245

605

340

550  88 (29%)

2000  410

2% @ 10 mM

3  0.33

9% @ 1.25 mM

38% @ 1.3 mM

320  14

1.2  0.1

265

2% @ 0.625 mM

47% @ 10 mM

>6000

>8000

4.3  0.45

>1400

>1860

36% @ 10 mM

2900  500

>5000

2.7  0.1

1070

>1850

1600  88 (29%)

>1500 (21%)

hMC3R 1.1  0.05 0.29  0.006

hMC4R 1.9  0.08 0.58  0.01

0.26  0.01 0% @ 1 mM 0% @ 2.5 mM

hMC5R 16  0.5 0.37  0.006

2.3  0 06 0.094  0.003 0.088  0.09

0% @ 0.625 mM

0% @ 0.625 mM

2.4  0.32

2% @ 5 mM

9% @ 5 mM

6.3  0.59

1% @ 2.5 mM

1% @ 2.5 mM

3.6  0.49

peptides 28 (2007) 1020–1028

6

aMSH (Nle4,D-Phe7)aMSH [NDPaMSH] MTII SHU9119 (Nle4,D-Nal7)aMSH [NDN-aMSH] (Oic6,dBip7,Pip8)MTII [OBP-MTII] (Nle4,Oic6,D-4, 40 Bip7,Pip8)-aMSH [NODBP-aMSH] D-4,40 Bip 7 Nle4,D-4,40 Bip 7 Nle4,D-4,40 Bip7, Pip 8 Nle4,Oic6,D-4, 40 Bip 7

Selectivity

Concentration of peptide at 50% specific binding or, the percentage of inhibition (relative to [125I]-NDP-aMSH) observed at a given peptide concentration (mM). # refers to binding assay, ## refers to cAMP assay.

1023

1024

Table 2 – Truncated analogs of NODBP-aMSH (compound 7) Ac-Ser1-Tyr-Ser-Nle4-Glu-Oic6-D-4,40 Bip7-Pip8-Trp-Gly-Lys-Pro-Val13-NH2 No.

Compound

Binding assay#

cAMP Assay##

IC50(nM)

16

17

18

19

21

22

23

24

25

26 27

4

5

Ac-Ser -Nle -Glu Oic6-D-4,40 Bip7Pip8-Trp9-NH2 Ac-Nle4-Glu5Oic6-D-4,40 Bip7Pip8-Trp9-NH2 Ac-Glu5-Oic6D-4,40 Bip7-Pip8Trp9-NH2 Ac-Oic6-D-4, 40 Bip7-Pip8Trp9-NH2 Ac-Oic6-D-4, 40 Bip7-Pip8Trp9-Gly10-NH2 Ac-Oic6-D-4, 40 Bip7-Pip8Trp9-Gly10Lys11-NH2 Ac-Glu5-Oic6D-4,40 Bip7Pip8-Trp9Gly10-NH2 Ac-Nle4-Glu5Oic6-D-4,40 Bip7Pip8-Trp9Gly10-NH2 Ac-Nle4-Glu5Oic6-D-4, 40 Bip7-Pip8-Trp9Gly10-Lys11-NH2 Ac-Nle4-Glu5Oic6-D-4, 40 Bip7-Pip8-NH2 Ac-Glu5-Oic6-D-4, 40 Bip7-Pip8-NH2 Ac-Nle4-Glu5Oic6-D-4, 40 Bip7-NH2

MC1bR

hMC3R

hMC4R

hMC5R

MC1bR

hMC3R

hMC4R

>5000

>5000

>4000

1.9  0.52

1300  200 (22%)

4% @ 10 mM

2% @ 5 mM

3.8  0.61

3400  110

>8000

2000  470

3.7  0.084

>1000 (29%)

4% @ 5 mM

0% @ 5 mM

5.5  0.57

12% @ 10 mM

46% @ 10 mM

>2500

130  15

1% @ 5 mM

>2500

1% @ 5 mM

92  11

45% @ 20 mM

>10000

3200  900

450  75

8

2% @ 5 mM

2% @ 5 mM

20% @ 10 mM

1200  98 (62%)

4000  890

2100  140

670  130

95  3

7

11% @ 10 mM

2% @ 10 mM

3% @ 10 mM

210  21 (36%)

33% @ 10 mM

>8000

>4000

88  2.9

2% @ 10 mM

2% @ 5 mM

0% @ 5 mM

120  30

18% @ 10 mM

23% @ 10 mM

32% @ 10 mM

160  34

2% @ 5 mM

2% @ 5 mM

0% @ 5 mM

210  41

2800  620

>5000

2800 + 400

8.9  0.98

315

325

>2500 (23%)

4% @ 5 mM

1% @ 5 mM

13  16

2800  190

3700  570

2000 + 150

2.9  0.23

965

1270

690

14% @ 1.8 mM

11% @ 1.8 mM

0% @ 2.5 mM

1.2  0.15

870  22

760  15

2100  190

0.85  0.15

1020

890

2470

190  46 (38%)

820  62 (26%)

2% @ 5 mM

0.67  0.12

5% @ 1.25 mM

21% @ 1.25 mM

7% @ 1.25 mM

150  33

0% @ 0.625 mM

0% @ 0.625 mM

0% @ 0.625 mM

2% @ 10 mM

5% @ 10 mM

7% @ 10 mM

0% @ 5 mM

0% @ 5 mM

0% @ 5 mM

>7000

1b/5

920

42

3/5

EC50(nM)

>2160

22

4/5

540

hMC5R

120 + 19 >2500 (48%)

Concentration of peptide at 50% maximum cAMP accumulation and/or the percentage of cAMP accumulation (relative to [125I]-NDP-aMSH) observed at the designated mM concentration. Data are the average of 3 or more independent determination together with the standard error of the mean. #, ## see Table 1.

peptides 28 (2007) 1020–1028

20

3

Selectivity

peptides 28 (2007) 1020–1028

equivalent ones present in NDP-aMSH. These peptide hybrids, of compound 7 and NDP-aMSH, were potent but not selective hMC5R agonists. Similarly to NDP-aMSH, but unlike compound 7, peptides 8, 9 and 11 displayed high binding affinity for the melanocortin receptors 1b, 3 and 4. They were either partial or full agonists at MC receptors 1b, 3 and 4, with one exception – peptide 11 was not able to activate hMC4R even at micromolar concentrations. Analog 10 was about 10-fold less potent at hMC5R than compound 7; it was a partial agonist at hMC1b, 3R and practically inactive at hMC4R at micromolar concentrations. In an attempt to lower conformational flexibility of compound 7, an aromatic, bulky analog of Pip, tetrahydroquinoline-3-carboxylic acid (Tic) was incorporated in place of Pip8. The resultant compound 12 was a slightly less potent and selective hMC5R agonist than the parent peptide 7. Unlike the Pip8 peptide, the Tic8 analog 12 was able to form stable complexes with the other MC receptors and was a full, albeit weak agonist at hMC3R and a partial agonist at hMC1bR; it was therefore a compound of a lesser interest to us. Similarly, replacement of D-4,40 Bip7 in peptide 7 with D-Nal(20 ), peptide 13, resulted in a highly potent hMC5R agonist but of lower selectivity versus hMC4R (about 260-fold). In peptides 14 and 15, the C-terminal amide group of 7 was replaced with the carboxyl group. Additionally in peptide 15, the N-terminal acetyl group was omitted. These alterations slightly affected agonist potency of the new peptides at hMC5R; they were about 5- to 10-fold less potent than compound 7. In order to find the smallest fragment of 7 which retained high agonist potency and selectivity for hMC5R, truncated analogs of this peptide were synthesized and tested in the binding and functional assays. Compounds 16–24, listed in Table 2, encompassed the modified ‘‘essential core’’ Oic6-D4,40 Bip7-Pip8-Trp9, which was extended by one, two or three additional residues at either N-terminal, C-terminal or both ends. The new shortened analogs of 7 were not able to activate the human MC 1b, 3 and 4 receptors at micromolar concentrations but were agonists at hMC5R (EC50 = 1 nM to 1200 nM). For example, peptide 16 lacking the N-terminal Ser1Tyr2 segment and the C-terminal Gly10-Lys11-Pro12-Val13 fragment was a 3.8 nM agonist at hMC5R of high receptor subtype selectivity (>2300-fold). A still shorter peptide, compound 17, in which Ser3 was omitted as well, Ac-Nle4Glu5-Oic6-D-4,40 Bip7-Pip8-Trp9-NH2, was approximately 5-fold weaker hMC5R agonist than the parent peptide and was less selective with respect to other melanocortin receptors, about 500–2000-fold. However, omission of the Nle4 residue from a peptide structure as in 18, Ac-Glu5-Oic6-4,40 Bip7-Pip8-Trp9NH2, yielded a compound of a substantially lower potency at hMC5R, about 250-fold. Also, the tetrapeptide Ac-Oic6-4,40 Bip7Pip8-Trp9-NH2, compound 19, which encompassed only the modified ‘‘essential core’’ of 7 was a weak, partial agonist at hMC5R (54% activation at 2.5 mM) and inactive at the other melanocortin receptors. In the structures of peptides 20 and 21, the 6-9 segment of 7 was extended at the C-terminal end by Gly10 and Gly10-Lys11, respectively. These peptides were moderately potent, full hMC5R agonists with EC50 = 210 and 120 nM, respectively. Similarly moderate potency at hMC5R was displayed by the

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hexapeptide Ac-Glu5-Oic6-4,40 Bip7-Pip8-Trp9-Gly10-NH2, compound 22 (EC50 = 210 nM), in which the ‘‘essential core’’ of 7 was extended by one residue at the N-terminus (Glu5) and one residue at the C-terminus (Gly10.) However, subsequent incorporation of Nle4 at the N-terminus, as in the larger peptides 23 and 24, significantly increased these peptides hMC5R agonist potency and selectivity (EC50 = 13 and 1 nM, respectively.) The observed pharmacological properties of compounds 22-24 at hMC5R further support our previous observation [3] that the residues 4 and 5 of aMSH and MTII are crucial for agonist potency at this receptor. The high agonist potency and selectivity of the hexapeptide 17 at hMC5R prompted us to test its shorter analog in which the Nle4-Glu5 segment remained intact but C-terminal Trp9 was absent. Rather unexpectedly, pentapeptide 25, Ac-Nle4Glu5-Oic6-D-4,40 Bip7-Pip8-NH2, was also a high potency agonist at hMC5R (IC50 = 0.9 nM, EC50 = 0.7 nM) and showed partial agonist activity at hMC1bR and hMC3R. Although quite potent at hMC5R, compound 25 was less selective than peptide 7 with respect to the other melanocortin receptors. Further omission of one residue from either the N-terminal (Nle4) or C-terminal (Pip8) end of compound 25 resulted in tetra-peptides which were weak hMC5R agonists and nearly inactive at other melanocortin receptors studied, peptides 26 and 27.

4.

Discussion and conclusions

Since its discovery more than two decades ago, the NDP-aMSH peptide has frequently been utilized instead of the less potent endogenous hormone aMSH in various studies on the melanocortin system [21]. For example, in our work described here we used the iodinated derivative of NDP-aMSH, but not of aMSH, in competitive binding assays. However in the last several years, the cyclic peptide derived from NDP-aMSH, the MTII compound of an enhanced enzymatic stability has been applied almost exclusively for various in vivo studies [1,15,22,30]. Yet, for a great number of in vitro experiments, the linear NDP-aMSH peptide has remained a ligand-ofchoice. The best known analog of MTII is the SHU9119 peptide in which D-Phe in position 7 was replaced with D-Nal(20 ). This alteration to the ‘‘essential core’’ of melanocortin peptides, His6-Phe7-Arg8-Trp9, is recognized as crucial to the design of peptidic MC3/4R antagonists. While SHU9119 has captured attention of the research community almost immediately upon its description [17], a corresponding linear analog of aMSH with D-Nal(20 ) in position 7 and Nle in position 4 has gained little interest. However, when tested in our binding and functional assays, the Nle4,D-Nal(20 )7-aMSH peptide showed binding affinity similar to that of SHU9119 for all human melanocortin receptors studied, and also was an effective antagonist at hMC3R and hMC4R (Kb = 0.5 and 0.95 nM, respectively.) We designated this peptide NDN-aMSH. Due to the growing interest in compounds that are MC4R antagonists, and potentially useful for the treatment of cachexia, anorexia and other similar disorders of nutrient imbalance, NDN-aMSH, with structure and solubility properties similar to those of the natural hormone, aMSH, may find

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broader applications as a convenient research tool complimentary to SHU9119. Recently, we have reported synthesis of another analog of MTII which is a potent and selective agonist at hMC5R (Ref. [5], compound 6 in Table 1.) In the lactam ring of this peptide which we designated OBP-MTII, the basic His6-D-Phe7-Arg8 segment has been replaced with the hydrophobic Oic6-D4,40 Bip7-Pip8 cluster. In the present study, we showed that a linear analog of aMSH with the same hydrophobic residues in position 6-8 was an equally potent and selective hMC5R agonist, peptide 7, Ac-Ser1-Tyr-Ser-Nle-Glu-Oic6-D-4,40 Bip7Pip8-Trp9-Gly-Lys-Pro-Val13-NH2. Similarly to NDP-aMSH and NDN-aMSH, we labeled this analog NODBP-aMSH. The 6-8 segment (Oic6-D-4,40 Bip7-Pip8) of NODBP-aMSH also appears to be crucial for high agonist potency and selectivity at hMC5R, because even a single alteration to this part of the structure, such as incorporation of His in position 6 (peptide 10) or Arg in positions 8 (peptide 11), resulted in a loss of hMC5R selectivity. For the detailed structure-function studies on the Oic6-D4,40 Bip7-Pip8 segment in OBP-MTII see Ref. [5].

In the structure of NODBP-aMSH, compound 7, two basic residues His6 and Arg8 were replaced with hydrophobic, secondary amino acids (analogs of Pro: Oic and Pip.) These conformationally constrained residues, apparently, did not affect biologically active conformations of the lactam ring which are favorable to molecular recognition at hMC5R but destabilize those required for interactions with hMC1b,3,4R. Absence of ionic interactions between the side chains in position 6 and 8 of NODBP-aMSH and the receptors, and/or the steric constraints in the lactam ring introduced by Oic6 and Pip8, may additionally weaken ligand-receptor complexes at the counter selected receptors. When His or Arg was restored in position 6 or 8, the new analogs 10 and 11 bind effectively to all melanocortin receptors studied. Replacement of D-4,40 Bip7 in compound 7 with D-Nal(20 ) led to a potent hMC5R agonist, albeit less selective. Omission of the N-terminal acetyl group from peptide 7 and/or replacement of the C-terminal amide with the carboxyl group only slightly alters pharmacological properties of this peptide, indicating that like in the other full length derivatives of aMSH, both the N-terminus and C-terminus may not necessary be involved in receptor interactions but perhaps face outside environment. The NODBP-aMSH peptide, compound 7, similarly to NDPaMSH, may become a useful research tool for studies on hMC5R. However, this rather lengthy linear peptide is not particularly amendable to structure-functions studies focused on the development of low molecular weight peptides and

peptide-mimetics while a tetrapeptide encompassing its ‘‘essential core’’ may be. Yet, the Ac-Oic6-D-4,40 Bip7-Pip8Trp9-NH2 peptide, compound 19, was found to be only a partial micromolar agonist at hMC5R, practically inactive at the human MC 1b, 3 and 4 receptors. This data contrasted with the pharmacological properties of the tetra-peptides which encompass the modified ‘‘essential core’’ of NDP-aMSH, the Ac-His6-D-Phe7-Arg8-Trp9-NH2 peptide, and of NDN-aMSH, the Ac-His6-D-Nal(20 )7-Arg8-Trp9-NH2 peptide. Like NDPaMSH, the Ac-His6-D-Phe7-Arg8-Trp9-NH2 analog was a panagonist at hMC1b,3-5R, although a weak one; and Ac-His6-DNal(20 )7-Arg8-Trp9-NH2, similar to NDN-aMSH, was a modest agonist at hMC1b/5R and an antagonist at hMC3/4R (data not included.) Still, the inability of tetra-peptide 19 to activate fully hMC5R was consistent with our previous observation [3] that the residues 4 and 5 of the melanocortin peptides (absent in the structure of 19) are important for agonism at hMC5R. Separately, we anticipated that given the high agonist potency and selectivity for hMC5R of the linear NODBP-aMSH and cyclic OBP-MTII peptides (compound 6), the ‘‘open chain’’ analog of the latest, the linear (4-10)NODBP-aMSH peptide with Nle4 in its structure, Ac-Nle4-Glu5-Oic6-D-4,40 Bip7-Pip8Trp9-Gly10-NH2, compound 23, might also have similar properties at hMC5R. Indeed, in our functional assays peptide 23 was only a 13-fold weaker hMC5R agonist (IC50 = 7.5 nM, EC50 = 13 nM) than OBP-MTII (compound 6), and showed high receptor subtype selectivity. Furthermore, analogs of 23 in which Nle4 was omitted from their structures, compounds 1822, were substantially less potent at hMC5R. Their pharmacological properties thus again underlined the crucial role of a residue in position 4 in molecular recognition at hMC5R [3]. In contrast, omission from the structure of 23 the residues which are C-terminal to the ‘‘essential core’’ had rather negligible impact on potency and selectivity at hMC5R, peptides 16 and 17. From this portion of our study on truncated analogs of NODBP-aMSH, the Ac-Nle4-Glu5-Oic6-D-4,40 Bip7-Pip8-Trp9NH2 peptide, compound 17, emerged as the smallest linear peptide of selectivity and agonist potency at hMC5R similar to those of the parent peptide 7. Additionally, the residues 4-9, Nle4-Glu5-Oic6-D-4,40 Bip7-Pip8-Trp9, have been shown to constitute an ‘‘essential core’’ which is indispensable for high selectivity and agonist potency at hMC5R. This ‘‘core’’ consists of two segments: Nle4-Glu5 and Oic6-D-4,40 Bip7-Pip8-Trp9; the second occupies positions previously recognized as critical for interaction of the melanocortin peptides with the melanocortin receptors. In the hMC5R agonists discussed herein, 3 out of 4 residues of the 6-9 cluster are new, unusual, hydrophobic substituents and only Trp9 of the ‘‘original core’’ (His6-Phe7Arg8-Trp9) remains unchanged. To probe the role of Trp9 in the interactions with hMC5R, we evaluated an analog of 17 lacking Trp9 in its structure, Ac-Nle4-Glu5-Oic6-D-4,40 Bip7-Pip8-NH2, compound 25. This shorter peptide displayed a rather unanticipated high agonist potency at hMC5R (IC50 = 0.9 nM, EC50 = 0.7 nM). It is likely that the unusual residues in positions 6-8 of 25 (Oic6-D-4,40 Bip7-Pip8) stabilize different peptide conformations and interact differently with the melanocortin receptors than the residues His6-Phe7-Arg8 from the ‘‘unmodified core’’ [16]. The altered receptor-ligand recognition forces may consequently render the Trp9 residue redundant.

peptides 28 (2007) 1020–1028

An attempt to even further shorten the structure of 25 by omission of the Pip8 residue led to compound 27, Ac-Nle4Glu5-Oic6-D-4,40 Bip7-NH2, which was nearly inactive at hMC5R and the other melanocortin receptors. Therefore, we concluded that the Nle4-Glu5-Oic6-D-4,40 Bip7-Pip8 segment is critical for high agonist potency at hMC5R, while the Cterminal Trp9 residue is required for high receptor subtype selectivity. Although somewhat less selective than the full length hMC5R agonist 7, pentapeptide Ac-Nle4-Glu5-Oic6-D4,40 Bip7-Pip8-NH2, agonist 25, may serve as a lead compound for further structure-function studies aimed at the development of small MC5R ligands with improved pharmacological properties. Results of our studies in that direction will be described in a separate manuscript. Peptide 25 and other analogs of aMSH reported in this study should be further evaluated for their functional potency at and selectivity for hMC2R. In summary: our study has showed that an analog of NDPaMSH with the Oic6-D-4,40 Bip7-Pip8 segment in its structure is a potent and selective hMC5R agonist. This Nle4,Oic6,D4,40 Bip7,Pip8-aMSH peptide, shortly NODBP-aMSH, and its iodinated derivative may be useful research tools for the evaluation of the physiological role of hMC5R in humans and other species. Additionally we found that the truncated analog of NODBPaMSH which encompasses residues 4-8, Ac-Nle4-Glu5-Oic6-D4,40 Bip7-Pip8-NH2, activated hMC5R as effectively as the fulllength parent compound. This suggests that, unlike for the other reported synthetic and natural melanocortin peptides, the Trp9 residue is not required for agonism at hMC5R by the compounds presented in this analysis.

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