Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2’)-Orn-NH₂] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2’)-Arg-NH₂]

Abstract

Discovery of pan-antagonist ligands for the melanocortin receptors will help identify the physiological activities controlled by these receptors. The previously reported MC3R/MC4R antagonist Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH₂ was identified herein, for the first time, to possess MC1R and MC5R antagonist activity. Further structure-activity relationship studies probing the second and fourth positions were performed towards the goal of identifying potent melanocortin antagonists. Of the twenty-one tetrapeptides synthesized, thirteen possessed MC1R, MC3R, MC4R, and MC5R antagonist activity. Three tetrapeptides were more than 10-fold selective for the mMC1R, including 8 (LTT1-44, Ac-DPhe(pI)-DArg-Nal(2’)-Arg-NH₂) that possessed 80 nM mMC1R antagonist potency and was at least 40-fold selective over the mMC3R, mMC4R, and mMC5R. Nine tetrapeptides were selective for the mMC4R, including 14 (SSM1-8, Ac-DPhe(pI)-Arg-Nal(2’)-Orn-NH₂) with an mMC4R antagonist potency of 1.6 nM. This compound was administered IT into mice, resulting in a dose-dependent increase in food intake and demonstrating the in vivo utility of this compound series.

Graphical Abstract:

Introduction

The melanocortin receptors are G protein-coupled receptors (GPCRs) that are involved in many different biological pathways, including skin pigmentation,^1-2 steroidogenesis,² and energy homeostasis.^3-9 The five receptors identified to date are stimulated by agonists derived from the proopiomelanocortin (POMC) gene transcript and increase intracellular cAMP levels. The melanocortin system also possesses two endogenous antagonists, agouti^10-11 and agouti-related protein (AGRP).^12-14 Due to the myriad of functions controlled by the melanocortin receptors, the development of tool antagonist compounds that can individually or simultaneously block the MC1R, MC3R, MC4R, and MC5R may be beneficial in identifying the roles of these receptors in vivo or as potential therapeutic leads. As one example, central administration of MC4R antagonist ligands is well recognized to result in an increased feeding response.^{6, 15-18} Development of MC4R antagonist therapeutics may be useful for treating states of negative energy balance such as cachexia, anorexia, and failure to thrive in infants.

Naturally occurring agouti and AGRP are a logical starting scaffold for further antagonist development. While both antagonists possess an Arg-Phe-Phe tripeptide motif that is critical for binding and functional activity,^19-20 these ligands have different profiles at the melanocortin receptors. Recombinant mouse (m) agouti was reported to antagonize α-MSH stimulation of the mMC1R and human (h)MC4R, but not the rat (r)MC3R or mMC5R.²¹ A subsequent report observed human agouti antagonized α-MSH stimulation of the hMC1R, hMC3R, hMC4R, and hMC5R, with decreased potency at the hMC3R and hMC5R.²² In contrast, AGRP has been reported to interact with the MC3R and MC4R, but not the MC1R.^12-13 These data suggest that although a common tripeptide sequence is required for activity, the additional amino acids of agouti and AGRP impart the observed receptor selectivity. The large active forms of endogenous agouti [ASP(23-132)]^23-25 and AGRP [AGRP(83-132)]²⁶ complicate their use as lead scaffolds. A synthetic C-terminal domain of agouti (Agouti-YY, 53 residues) has been demonstrated to antagonize the hMC1R, hMC3R, and hMC4R,²⁷ while a 34 residue minimal fragment of AGRP [AGRP(87-120), C105A] can antagonize the hMC3R and hMC4R.²⁸ Further truncation to the active loop of AGRP (Arg-Phe-Phe-Asn-Ala-Phe) cyclized head-to-tail through a DPro-Pro motif has led to nanomolar potent MC3R and MC4R antagonist macrocycles following further structure-activity relationship (SAR) studies.^29-32

In addition to the naturally occurring antagonists, extensive SAR studies over decades have been performed on the endogenous melanocortin agonist ligands as starting templates. The POMC-derived agonists share a His-Phe-Arg-Trp tetrapeptide sequence. Inverting the stereochemistry of the Phe to DPhe results in more potent ligands, resulting in the common His-DPhe-Arg-Trp agonist sequence present in three FDA approved melanocortin peptide drugs.^33-35 Pioneering work by Hruby et al. identified that substituting the DPhe in the MTII template (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ with bulky aromatic substitutions [DPhe(pI) in SHU8914 or DNal(2’) in SHU9119] resulted in ligands with antagonist activity at the MC3R and MC4R.³⁶ The crystal structure of SHU9119 interacting with the MC4R indicates the DNal(2’) residue of SHU9119 interacts with Leu133 of the MC4R,³⁷ an MC4R residue previously identified to be important for antagonism.³⁸ The DNal(2’) of SHU9119 positions Leu133 in front of Trp258, inhibiting the movement of TM6 of the MC4R required for receptor activation.^39-41 This detailed molecular knowledge of the structural requirements for receptor signaling may be useful in the design of new ligands derived from known compounds.

While many MC3R and MC4R antagonists derived from endogenous agonist and antagonist ligands have been reported, fewer MC1R and MC5R antagonists have been functionally characterized. In part, this may be due to the historical approach used by classical pharmacologists that postulate that if a compound can displace a radiolabeled ligand and not induce a cAMP response, it is an antagonist without performing functional antagonist assays following a Schild paradigm.⁴² Due to this assumption, there are limited reports of functionally characterized MC1R and MC5R antagonists. Positional scanning libraries have been used to identify peptides and amino acids that alter α-MSH signaling in Xenopus laevis melanophores,⁴³ B16-F10 melanoma cells,⁴⁴ and human epidermal melanocytes⁴⁴ (all presumably expressing the MC1R). Although glycine amide derived from one of these studies⁴⁴ has been used to alter skin pigmentation in human trials,⁴⁵ the functional antagonist potencies of these ligands have not been reported. A series of α-MSH derivatives were found to be MC1R antagonists using the classic frog or lizard skin bioassays,^46-47 with the most potent peptide (Ac-Nle-Asp-Trp-DPhe-Nle-Trp-Lys-NH₂, pA₂ = 8.4 in the frog skin assay)⁴⁷ subsequently being used to explore MC1R inhibition and pain in mice.⁴⁸ A chimeric melanotropin-deltorphin analog (c[Gly-Cpg-DNal(2’)-Arg-Trp-Glu]-Val-Val-Gly-NH₂) was reported to be a competitive hMC1R antagonist (antagonist K_i = 53 nM).⁴⁹ A cyclic decapeptide derived from the active loop of AGRP (NH₂-Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dap]-Tyr-NH₂) was found to possess antagonist activity at the mMC1R (pA₂ = 5.92, antagonist K_i = 1200 nM),⁵⁰ which restored wild-type coloration in K5-noggin mice when administered intracutaneously.⁵¹ Although NDP-MSH, an MC1R agonist that increases pigmentation, has been approved to treat erythropoietic protoporphyria,³³ there remains an unmet need in the development of skin lightening agents, which may be derived from MC1R antagonists.

Similar to the MC1R, relatively few ligands have been functionally assayed as antagonists at the MC5R. Derivatives of SHU9119 were found to possess antagonist activity at the mMC5R.⁵² The most potent of these compounds (Ac-Nle-c[Asp-(1-Me)His-DNal(2’)-Arg-DNal(2’)-Lys]-NH₂) possessed an pA₂ value of 7.2 (antagonist K_i = 63 nM) at the mMC5R.⁵² Similar antagonist potency at the MC5R (pA₂ = 7.3) was reported from an AGRP-derived macrocyclic octapeptide scaffold (c[Pro-Arg-Phe-Phe-Dap-Ala-Phe-DPro]).⁵³ An alkylthioaryl bridged cyclic SHU9119 derivative was found to possess antagonist activity (pA₂ = 8.3, antagonist K_i = 5 nM) at the hMC5R,⁵⁴ similar to the antagonist potency (pA₂ = 8.7, antagonist K_i = 2 nM) found at the hMC5R for a cyclic γ-MSH analog (c[Nle-Val-DNal(2’)-Arg-Trp-Glu]-NH₂),⁵⁵ though the receptor constructs assayed in the γ-MSH analog may have been incorrect.⁵⁶ Common to these reported MC5R antagonists is a larger, cyclic structure. Due to the potential involvement of the MC5R in muscle glucose uptake⁵⁷ and acne (as reviewed),⁵⁸ the development of short, linear MC5R antagonist ligands may lead to probe compounds that can be used to explore the in vivo functions of the MC5R.

Few compounds have been assayed for functional antagonism at the different melanocortin receptors, perhaps due to the increased cost of running Schild antagonist assays (multiple concentrations of a purported antagonist with a concentration-response of a known agonist) compared to agonist or binding assays. Recombinant human agouti dose-dependently shifted the potency of α-MSH to stimulate the hMC1R, hMC3R, hMC4R, hMC5R,²² although recombinant mouse agouti was observed not to alter α-MSH potency at the rMC3R and the mMC5R (up to 0.7 and 100 nM concentrations, respectively).²¹ The HS024 ligand displaced [I¹²⁵]NDP-MSH at nanomolar to sub-nanomolar concentrations, did not result in accumulation of cAMP at concentrations up to 10 μM, and prevented α-MSH induced cAMP production at 100 nM concentrations of HS024 at the hMC1R, hMC3R, hMC4R, and hMC5R, suggesting the compound may be an antagonist at these receptors.⁵⁹ Subsequently, another group observed HS024 to possess full agonist activity at the hMC5R and rMC5R.⁶⁰ With these conflicting reports, there remains an unmet need for the development of a pan-melanocortin antagonist tool compounds that can simultaneously block the different melanocortin ligands.

Due to the unmet need for MC1R, MC5R, and pan-melanocortin antagonist ligands, herein a previously reported MC3R and MC4R antagonist tetrapeptide [Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH₂]⁶¹ with minimal MC1R and MC5R agonist activities was explored for potential MC1R and MC5R antagonism. As this tetrapeptide was observed to possess antagonist activity at the MC1R, MC3R, MC4R, and MC5R, an additional 20 novel tetrapeptides were synthesized to explore the second and fourth position Arg residues of the lead compound towards the goal of developing more potent, selective antagonists for the melanocortin receptors. The charge and stereochemistry of both positions were examined utilizing a series of basic, acidic, and uncharged residues (Figure 1) to test the structural requirements for antagonist potency and selectivity. Due to the observed nanomolar MC4R antagonist potency for several compounds herein, one tetrapeptide was centrally administered in mice to probe potential in vivo effects on appetite and validate this scaffold as a potential lead for treating disease states of negative energy balance.

Figure 1:

Structures of the substituted amino acids in this study. The amino acids are grouped by side chain type: basic (blue), uncharged (yellow), and acidic (red).

Results & Discussion

All peptides in this study were synthesized manually using standard microwave-assisted Fmoc techniques.^62-63 Each peptide possessed a C-terminal amidate functionality and were acetylated at the N-terminal. Following sidechain deprotection and cleavage from resin, peptides were purified by RP-HPLC. Tetrapeptides were at least 95% pure by analytical RP-HPLC (λ = 214 nm) in two solvent systems (Table 1) and possessed the correct molecular mass as determined by ESI-MS (University of Minnesota Mass Spectrometry Lab). The peptides were assessed for biological activity using the “AlphaScreen” cAMP kit with HEK293 cells stably expressing the mMC1R, mMC3R, mMC4R, or mMC5R, as previously described.^64-66 Since the MC2R is only stimulated by ACTH and not other endogenously derived POMC peptides,⁶⁷ it was not examined in the current study. Compounds were assayed for agonist activity by themselves or for antagonist activity in the presence of the synthetic melanocortin agonist NDP-MSH using a Schild antagonist experimental paradigm.⁴² Tetrapeptides that did not possess agonist or antagonist activity in two independent experiments were considered inactive at the concentrations assayed and were not further studied. Active compounds were studied in at least three independent experiments. Due to the inherent experimental error associated with these types of assays, we considered compounds that were within a 3-fold potency range to be equipotent. Since the “AlphaScreen” cAMP assay is a loss-of-signal assay, in which higher concentrations of compound result in lower assay signal, the data was normalized to baseline and maximal NDP-MSH signal for illustrative purposes, as previously described.^65-66

Table 1:

Characterization of tetrapeptides synthesized in this study.^a

Peptide	Compound ID	Sequence	k’ (MeCN)	k’ (MeOH)	M (cal)	M+H (obs)	purity
1	LTT1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH2	7.5	12.3	841.8	842.6	>99%
2	LTT1-24	Ac-DPhe(pI)-His-Nal(2’)-Arg-NH2	7.5	12.0	822.7	823.7	>99%
3	LTT1-16	Ac-DPhe(pI)-Lys-Nal(2’)-Arg-NH2	7.4	12.2	813.7	814.6	>99%
4	LTT1-28	Ac-DPhe(pI)-Orn-Nal(2’)-Arg-NH2	7.5	12.0	799.7	800.7	>99%
5	LTT1-32	Ac-DPhe(pI)-Dab-Nal(2’)-Arg-NH2	7.7	12.5	785.7	786.4	>99%
6	LTT1-36	Ac-DPhe(pI)-Dap-Nal(2’)-Arg-NH2	8.0	12.9	771.7	772.6	>98%
7	LTT1-40	Ac-DPhe(pI)-hArg-Nal(2’)-Arg-NH2	7.5	11.8	855.8	856.7	>99%
8	LTT1-44	Ac-DPhe(pI)-DArg-Nal(2’)-Arg-NH2	7.2	11.6	841.8	842.6	>98%
9	LTT1-52	Ac-DPhe(pI)-DLys-Nal(2’)-Arg-NH2	7.1	11.1	813.7	814.6	>99%
10	LTT1-48-4	Ac-DPhe(pI)-Cit-Nal(2’)-Arg-NH2	7.6	12.0	842.7	843.6	>99%
11	LTT1-56-2	Ac-DPhe(pI)-Glu-Nal(2’)-Arg-NH2	8.2	13.1	814.7	815.7	>98%
12	SSM1-6	Ac-DPhe(pI)-Arg-Nal(2’)-His-NH2	7.3	12.0	822.7	823.4	>96%
13	SSM1-3P1	Ac-DPhe(pI)-Arg-Nal(2’)-Lys-NH2	7.3	12.0	813.7	814.8	>98%
14	SSM1-8	Ac-DPhe(pI)-Arg-Nal(2’)-Orn-NH2	7.3	11.9	799.7	800.4	>98%
15	SSM1-10	Ac-DPhe(pI)-Arg-Nal(2’)-Dab-NH2	7.4	12.1	785.7	786.3	>99%
16	SSM1-12	Ac-DPhe(pI)-Arg-Nal(2’)-Dap-NH2	7.1	11.5	771.7	772.3	>99%
17	SSM1-14	Ac-DPhe(pI)-Arg-Nal(2’)-hArg-NH2	7.5	12.0	855.8	856.4	>97%
18	SSM1-16	Ac-DPhe(pI)-Arg-Nal(2’)-DArg-NH2	7.1	11.6	841.8	842.4	>99%
19	SSM1-18	Ac-DPhe(pI)-Arg-Nal(2’)-DLys-NH2	7.0	11.7	813.7	814.6	>98%
20	SSM1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Cit-NH2	7.3	11.9	842.7	843.6	>99%
21	JLB1-3	Ac-DPhe(pI)-Arg-Nal(2’)-Glu-NH2	8.0	12.9	814.7	815.4	>99%

^aHPLC k’ = [(peptide retention time – solvent retention time)/solvent retention time] in acetonitrile (MeCN; 10% acetonitrile in 0.1% trifluoroacetic acid/water and a gradient to 90% acetonitrile over 35 min) or methanol (MeOH; 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 peptide purity was determined by HPLC at a wavelength of 214 nm.

The lead tetrapeptide 1 [Ac-DNal(2’)-Arg-Nal(2’)-Arg-NH₂] was previously reported to minimally stimulate the MC1R and MC4R (25% activation at 100 μM concentrations)⁶¹ and possessed antagonist activity at the mMC3R and mMC4R (pA₂ = 7.8 and 9.0, respectively).⁶¹ It was resynthesized in this study (1, LTT1-20), and was found to possess similar activation at the mMC1R and mMC4R at 100 μM concentrations (35% and 25%, respectively; Table 2 and Figure 2) and antagonist activity at the mMC3R and mMC4R (pA₂ values of 7.5 and 8.9, respectively; Table 3 and Figure 2) compared to the prior report. Due to the minimal agonist response of 1 (LTT1-20) at the mMC1R and mMC5R, this ligand was assayed for antagonist activity at these two receptors for the first time herein (Figure 2). Compound 1 (LTT1-20) possessed antagonist activity at the mMC1R and mMC5R (pA₂ = 7.7 and 6.6, respectively; Table 3 and Figure 2), demonstrating this tetrapeptide is an antagonist at all four melanocortin receptors, with nanomolar potencies at each receptor (antagonist K_i = 20, 32, 1.3, and 250 nM at the mMC1R, mMC3R, mMC4R, and mMC5R, respectively).

Table 2:

Tetrapeptide agonist pharmacology at the mouse melanocortin receptors.^a

Peptide	Compound ID	Sequence	EC50 (nM)
			mMC1R	mMC3R	mMC4R	mMC5R
	NDP-MSH		0.010±0.001	0.079±0.008	0.72±0.08	0.19±0.008
1	LTT1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH2	35% @ 100 μM	>100,000	25% @ 100 μM	>100,000
2	LTT1-24	Ac-DPhe(pI)-His-Nal(2’)-Arg-NH2	20% @ 100 μM	>100,000	>100,000	>100,000
3	LTT1-16	Ac-DPhe(pI)-Lys-Nal(2’)-Arg-NH2	20% @ 100 μM	>100,000	>100,000	>100,000
4	LTT1-28	Ac-DPhe(pI)-Orn-Nal(2’)-Arg-NH2	20% @ 100 μM	>100,000	>100,000	>100,000
5	LTT1-32	Ac-DPhe(pI)-Dab-Nal(2’)-Arg-NH2	>100,000	>100,000	>100,000	>100,000
6	LTT1-36	Ac-DPhe(pI)-Dap-Nal(2’)-Arg-NH2	>100,000	>100,000	>100,000	>100,000
7	LTT1-40	Ac-DPhe(pI)-hArg-Nal(2’)-Arg-NH2	25% @ 100 μM	>100,000	>100,000	>100,000
8	LTT1-44	Ac-DPhe(pI)-DArg-Nal(2’)-Arg-NH2	25% @ 100 μM	>100,000	>100,000	>100,000
9	LTT1-52	Ac-DPhe(pI)-DLys-Nal(2’)-Arg-NH2	>100,000	>100,000	>100,000	>100,000
10	LTT1-48-4	Ac-DPhe(pI)-Cit-Nal(2’)-Arg-NH2	40% @ 100 μM	>100,000	>100,000	>100,000
11	LTT1-56-2	Ac-DPhe(pI)-Glu-Nal(2’)-Arg-NH2	>100,000	>100,000	>100,000	>100,000
12	SSM1-6	Ac-DPhe(pI)-Arg-Nal(2’)-His-NH2	Partial Agonist 400±100 55%NDP	35% @ 100 μM	>100,000	>100,000
13	SSM1-3P1	Ac-DPhe(pI)-Arg-Nal(2’)-Lys-NH2	30% @ 100 μM	>100,000	>100,000	>100,000
14	SSM1-8	Ac-DPhe(pI)-Arg-Nal(2’)-Orn-NH2	Partial Agonist 3000±1000 40%NDP	>100,000	>100,000	>100,000
15	SSM1-10	Ac-DPhe(pI)-Arg-Nal(2’)-Dab-NH2	Partial Agonist 1100±600 55%NDP	>100,000	>100,000	>100,000
16	SSM1-12	Ac-DPhe(pI)-Arg-Nal(2’)-Dap-NH2	30% @ 100 μM	>100,000	>100,000	>100,000
17	SSM1-14	Ac-DPhe(pI)-Arg-Nal(2’)-hArg-NH2	40% @ 100 μM	>100,000	>100,000	>100,000
18	SSM1-16	Ac-DPhe(pI)-Arg-Nal(2’)-DArg-NH2	>100,000	>100,000	>100,000	>100,000
19	SSM1-18	Ac-DPhe(pI)-Arg-Nal(2’)-DLys-NH2	>100,000	>100,000	>100,000	>100,000
20	SSM1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Cit-NH2	>100,000	>100,000	>100,000	>100,000
21	JLB1-3	Ac-DPhe(pI)-Arg-Nal(2’)-Glu-NH2	>100,000	>100,000	>100,000	>100,000

^aThe indicated errors represent the standard error of the mean from at least three independent experiments. A percentage denotes the percent maximal stimulatory response (compared to NDP-MSH) observed at 100 μM, but not enough stimulation was observed to determine an EC₅₀ value. The use of >100,000 indicates that the compound was examined but lack agonist activity at concentrations up to 100 μM in at least two independent experiments. Tetrapeptides labeled partial agonists were observed to possess partial agonist activity, with the apparent EC₅₀ values and percentage of receptor activation relative to NDP-MSH.

Figure 2:

Illustration of the antagonist pharmacology of 1 (LTT1-20) at the mMC1R, mMC3R, mMC4R, and mMC5R. Due to the increased potency at the mMC4R, a different set of concentrations were used at this receptor as indicated in the figure. Antagonist K_i values were calculated from pA₂ values using the relationship [pA₂= −Log(K_i)].

Table 3:

Tetrapeptide antagonist pharmacology at the mouse melanocortin receptors.^a

Peptide	Compound ID	Sequence	mMC1R		mMC3R		mMC4R		mMC5R
			pA2	Ki (nM)	pA2	Ki (nM)	pA2	Ki (nM)	pA2	Ki (nM)
	SHU9119		NA		9.2±0.2	0.63	9.2±0.2	0.63	NA
1	LTT1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH2	7.7±0.1	20	7.5±0.2	32	8.9±0.2	1.3	6.6±0.1	250
2	LTT1-24	Ac-DPhe(pI)-His-Nal(2’)-Arg-NH2	6.2±0.2	630	5.7±0.2	2000	7.0±0.2	100	<5.5
3	LTT1-16	Ac-DPhe(pI)-Lys-Nal(2’)-Arg-NH2	6.5±0.2	320	6.7±0.1	200	8.1±0.1	8	6.1±0.1	800
4	LTT1-28	Ac-DPhe(pI)-Orn-Nal(2’)-Arg-NH2	6.7±0.1	200	<5.5		7.0±0.2	100	<5.5
5	LTT1-32	Ac-DPhe(pI)-Dab-Nal(2’)-Arg-NH2	7.2±0.1	63	<5.5		6.6±0.2	250	<5.5
6	LTT1-36	Ac-DPhe(pI)-Dap-Nal(2’)-Arg-NH2	7.0±0.3	100	<5.5		5.8±0.2	1600	<5.5
7	LTT1-40	Ac-DPhe(pI)-hArg-Nal(2’)-Arg-NH2	6.5±0.1	320	7.4±0.1	40	8.9±0.2	1.3	6.7±0.2	200
8	LTT1-44	Ac-DPhe(pI)-DArg-Nal(2’)-Arg-NH2	7.1±0.1	80	<5.5		5.5	3200	<5.5
9	LTT1-52	Ac-DPhe(pI)-DLys-Nal(2’)-Arg-NH2	6.2±0.8	630	<5.5		<5.5		<5.5
10	LTT1-48-4	Ac-DPhe(pI)-Cit-Nal(2’)-Arg-NH2	6.2±0.2	630	5.8±0.3	1600	6.5±0.3	320	<5.5
11	LTT1-56-2	Ac-DPhe(pI)-Glu-Nal(2’)-Arg-NH2	<5.5		<5.5		<5.5		<5.5
12	SSM1-6	Ac-DPhe(pI)-Arg-Nal(2’)-His-NH2	6.6±0.1	250	7.0±0.1	100	7.8±0.3	16	5.9±0.2	1300
13	SSM1-3P1	Ac-DPhe(pI)-Arg-Nal(2’)-Lys-NH2	6.7±0.1	200	7.4±0.2	40	8.2±0.2	6.3	6.3±0.2	500
14	SSM1-8	Ac-DPhe(pI)-Arg-Nal(2’)-Orn-NH2	6.4±0.3	400	7.4±0.1	40	8.8±0.1	1.6	6.4±0.2	400
15	SSM1-10	Ac-DPhe(pI)-Arg-Nal(2’)-Dab-NH2	6.5±0.2	320	7.8±0.1	16	8.8±0.1	1.6	6.7±0.2	200
16	SSM1-12	Ac-DPhe(pI)-Arg-Nal(2’)-Dap-NH2	6.6±0.2	250	7.3±0.4	50	8.1±0.1	8	6.3±0.2	500
17	SSM1-14	Ac-DPhe(pI)-Arg-Nal(2’)-hArg-NH2	7.3±0.2	50	7.7±0.5	20	8.2±0.2	6.3	6.0±0.2	1000
18	SSM1-16	Ac-DPhe(pI)-Arg-Nal(2’)-DArg-NH2	6.7±0.3	200	6.5±0.3	320	7.7±0.1	20	5.8±0.2	1600
19	SSM1-18	Ac-DPhe(pI)-Arg-Nal(2’)-DLys-NH2	6.2±0.2	630	6.5±0.3	320	7.7±0.2	20	5.9±0.2	1300
20	SSM1-20	Ac-DPhe(pI)-Arg-Nal(2’)-Cit-NH2	5.9±0.2	1300	6.8±0.3	160	8.5±0.2	3.2	6.1±0.1	800
21	JLB1-3	Ac-DPhe(pI)-Arg-Nal(2’)-Glu-NH2	5.9±0.3	1300	6.6±0.1	250	7.7±0.3	20	5.7±0.2	2000

^aThe indicated error represents the standard error of the mean determined from at least three independent experiments. Antagonist pA₂ values were determined using a Schild analysis and the agonist NDP-MSH. The use of <5.5 indicates that no antagonist potency was observed in the highest concentration range assayed (10,000, 5,000, 1,000, and 500 nM) in at least two independent assays. Antagonist K_i values were calculated from pA₂ values using the relationship [pA₂= -Log(K_i)]. NA denotes that SHU9119 was not assayed for antagonist activity at the mMC1R or mMC5R as it is a full agonist at these receptors.

Due to the observed nanomolar antagonist potency of 1 (LTT1-20) at the melanocortin receptors, a series of amino acids (Figure 1) were substituted at the second or fourth position Arg residues towards the goal of developing more potent and/or selective antagonist ligands. The amino acids His and Lys were incorporated to explore the effects of substituting other positively charged L-amino acid sidechains. To probe sidechain length, a series of residues possessing an amine extended by a varying of methylene units (1, 2, 3, or 4 for Dap, Dab, Orn, or Lys, respectively) were incorporated, as was adding a methylene unit in the Arg side chain resulting in the hArg residue. Incorporation of DArg and DLys examined the stereochemical requirements with a basic charge. The importance of the charge was examined by incorporating the neutral Cit residue (a bioiostere of Arg that replaces a nitrogen atom with an oxygen) and by incorporating a negatively charged Glu residue.

Substitutions at the Arg² Position:

Ten amino acids were substituted at the second position of the scaffold, resulting in tetrapeptides 2 – 11. Comparing the antagonist pharmacology for these ten compounds at the mMC4R, one substitution at the second position (hArg, 7, LTT1-40) resulted in equipotent antagonist activity compared to tetrapeptide 1 (LTT1-20; pA₂ = 8.9 for both; Table 3). This can be visualized by looking at a plot of the different substitutions at the second position by receptor subtype (Figure 3A), with equal height bars for Arg and hArg at the mMC4R. Substitution of other basic amino acids with L-stereochemistry (His, 2, LTT1-24; Lys, 3, LTT1-16; Orn, 4, LTT1-28; Dab, 5, LTT1-32; Dap, 6, LTT1-36) resulted in compounds with 6- to 1230-fold decreased antagonist potency at the mMC4R compared to lead 1 (LTT1-20). Sequentially, shortening a sidechain possessing a terminal amine by one methylene unit from Lys (3, LTT1-16), to Orn (4, LTT1-28), Dab (5, LTT1-32), and Dap (6, LTT1-36) serially decreased the pA₂ values observed at the mMC4R (Figure 3A), indicating a longer sidechain resulted in a more potent MC4R antagonist. Incorporating basic D-amino acids [8 (LTT1-44) and 9 (LTT1-52)] resulted in no observed antagonist activity at the concentrations assayed at the mMC4R, as did incorporating an acidic Glu residue (11, LTT1-56-2). Substituting the uncharged Cit (10, LTT1-48-4) bioisotere at the second position decreased antagonist potency 240-fold relative to the lead compound 1 (LTT1-20). These data suggest that the steric size and charge of a guanidyl group at the second position are important for potent mMC4R antagonism in this tetrapeptide scaffold, as the most potent ligands in this set possessed an Arg or hArg amino acid. No compounds substituted in the second position possessed agonist activity at the mMC4R (Table 2).

Figure 3:

Graphical illustration summarizing the antagonist potencies (pA₂) of tetrapeptides substituted at the (A) second and (B) fourth positions at the mMC1R, mMC3R, mMC4R, and mMC5R. A higher pA₂ value represents a more potent compound.

The same two residue substitutions in the second position that were most potent at the mMC4R (hArg, 7, LTT1-40 and Lys, 3, LTT1-16) were also the most potent mMC3R antagonist ligands (with pA₂ = 7.4 and 6.7, respectively) in this set of 10 compounds. The hArg substitution (7, LTT1-40) was an equipotent antagonist at the mMC3R compared to 1 (LTT1-20), while the Lys-substituted 3 (LTT1-16) decreased potency 6-fold at this receptor. Two other substitutions (Cit, 10, LTT1-48-4 and His, 2, LTT1-24) resulted in ligands with measurable mMC3R antagonist activity (pA₂ = 5.8 and 5.7, respectively). Other basic (Orn, 4, LTT1-28; Dab, 5, LTT1-32; Dap, 6, LTT1-36), D-stereochemistry (DArg, 8, LTT1-44; DLys, 9, LTT1-52) and acidic (Glu, 11, LTT1-56-2) substitutions at the second position resulted in no mMC3R antagonist activity at the concentrations assayed. Similar to the mMC4R, this set of compounds also did not result in agonist activity at the mMC3R.

The hArg (7, LTT1-40) and Lys (3, LTT1-16) substitutions at the second position resulted in antagonist pA₂ values of 6.7 and 6.1 respectively at the mMC5R. These substitutions were equipotent to the lead compound 1 (LTT1-20, pA₂ = 6.6), and were also the most potent second position substitutions at the mMC3R and mMC4R. None of the other substitutions in the second position resulted in antagonist activity at the mMC5R at the concentrations assayed, and no substitutions resulted in measurable agonist activity up to 100 μM concentrations.

In contrast to the mMC5R, most of the second position substitutions resulted in compounds with measurable antagonist activity at the mMC1R (Table 3 and Figure 3). Three substitutions (Dab, 5, LTT1-32; DArg, 8, LTT1-44; Dap, 6, LTT1-36) possessed 3- to 5-fold decreased antagonist potencies relative to the lead ligand 1 (LTT1-20) at the mMC1R. Incorporation of the remaining basic residues or the neutral Cit (10, LTT1-48-4) residue decreased antagonist potencies 10- to 30-fold relative to 1 (LTT1-20), whereas incorporation of the basic Glu amino acid (11, LTT1-56-2) resulted in no antagonist activity at the concentrations assayed. Several of these substitutions partially activated the mMC1R at 100 μM concentrations, resulting in 20-40% of the maximal NDP-MSH signal.

The work presented herein expands upon the previously reported substitutions of the second position with the tetrapeptide lead scaffold. Previously, an Arg or DNal(2’) were substituted at the second position, resulting in the tetrapeptides Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH₂ (same as lead 1, LTT1-20 in the present experiment) and Ac-DPhe(pI)-DNal(2’)-Nal(2’)-Arg-NH₂.⁶¹ The tetrapeptide possessing an Arg in the second position was a more potent mMC3R and mMC4R antagonist (pA₂ values of 7.8 and 9.0, respectively)⁶¹ compared to the DNal(2’) substituted compound (pA₂ values of <5.5 and 5.8),⁶¹ indicating a basic charge in the second position was important for mMC3R and mMC4R antagonist potency. The SAR presented herein demonstrates that amino acids possessing L-stereochemistry and a basic guanidyl group in the second position of this tetrapeptide scaffold (Arg, 1, LTT1-20 and hArg, 7, LTT1-40) results in the most potent mMC3R and mMC4R antagonist ligands. While three residues at the second position resulted in antagonist activity at the mMC5R (Arg, 1, LTT1-20; hArg, 7, LTT1-40; Lys, 3, LTT1-16), all but one substitution resulted in antagonist activity at the mMC1R, indicating that more substitutions are amenable for mMC1R antagonist activity in the scaffold. Substitutions in the second position resulted in 2 additional pan-melanocortin antagonists at the mMC1R, mMC3R, mMC4R, and mMC5R (3, LTT1-16 and 7, LTT1-40, in addition to 1, LTT1-20), as well as yielding a control compound possessing an acidic Glu residue (11, LTT1-56-2) that did not possess antagonist activity at the receptors assayed.

Substitutions at the Arg⁴ Position:

In addition to the second position, the fourth position Arg residue was substituted with a set of amino acids in order to develop potent and/or selective antagonist ligands, resulting in the ten tetrapeptides 12 - 21. Three substitutions (Orn, 14, SSM1-8; Dab, 15, SSM1-10; Cit, 20, SSM1-20) were found to result in equipotent antagonist activity compared to the lead ligand 1 (LTT1-20) at the mMC4R (Table 3, Figure 3B). Other substitutions containing an L-amino acid with a primary amine (Lys, 13, SSM1-3P1; Dap, 16, SSM1-12) or guanidyl (hArg, 17, SSM1-14) groups decreased potency 5- to 6-fold at the mMC4R compared to 1 (LTT1-20). Incorporation of an imidazole (His, 12, SSM1-6), D-stereochemistry (DArg, 18, SSM1-16; DLys, 19, SSM1-18), or acidic residues (Glu, 21, JLB1-3) further decreased potency 12- to 15-fold relative to 1 (LTT1-20). All substitutions resulted in nanomolar antagonist potencies at the mMC4R, while none of these substitutions resulted in mMC4R agonist activity.

At the mMC3R, basic residues with an amine (Lys, 13, SSM1-3P1; Orn, 14, SSM1-8; Dab, 15, SSM1-10; Dap, 12, SSM1-12) or a guanidyl (hArg, 17, SSM1-14) group in the fourth position were found to be equipotent antagonists compared to the lead tetrapeptide 1 (LTT1-20). Incorporation of a basic His (12, SSM1-6), uncharged Cit (20, SSM1-20), or acidic Glu (21, JLB1-3) decreased antagonist potency 3-, 5-, and 8-fold, respectively, at the mMC3R compared to 1 (LTT1-20), while substituting basic amino acids with D-stereochemistry (DArg, 18, SSM1-16; DLys, 19, SSM1-18) decreased mMC3R antagonist potency 10-fold. The tetrapeptide incorporating a His residue in the fourth position (12, SSM1-6) was the only ligand from the library that partially activated the mMC3R, resulting in 30% the maximal NDP-MSH signal at 100 μM concentrations.

Residues that possessed a primary amine (Lys, 13, SSM1-3P1; Orn, 14, SSM1-8; Dab, 15, SSM1-10; Dap, 12, SSM1-12) that were substituted in the fourth position were found to be equipotent mMC5R antagonists compared to 1 (LTT1-20). Tetrapeptides incorporating the uncharged Cit (20, SSM1-20) or basic hArg (17, SSM1-14) residues in the fourth position possessed 3- and 4-fold decreased antagonist potency at the mMC5R compared to 1 (LTT1-20). Compounds with His (12, SSM1-6), DArg (18, SSM1-16), DLys (19, SSM1-18), and Glu (21, JLB1-3) at the fourth position were 5- to 8-fold less potent antagonists at the mMC5R compared to 1 (LTT1-20). None of the fourth position substitutions resulted in measurable agonist activity at the mMC5R.

The guanidyl-containing hArg tetrapeptide (17, SSM1-14) was the only fourth position substitution that resulted in equipotent antagonist potency at the mMC1R compared to 1 (LTT1-20). Substituting a Lys (13, SSM1-3P1) or DArg (18, SSM1-16) resulted in ligands with 10-fold decreased antagonist potency at the mMC1R compared to 1 (LTT1-20). Other L-stereochemistry basic substitutions (His, 12, SSM1-6; Orn, 14, SSM1-8; Dab, 15, SSM1-10; Dap, 16, SSM1-12) decreased potency 10- to 20-fold relative to 1 (LTT1-20) at the mMC1R, while incorporating DLys (19, SSM1-18), Cit (20, SSM1-20), or Glu (21, JLB1-3) further decreased mMC1R potency 30- to 65-fold. Compounds possessing a Lys (13, SSM1-3P1), Dap (16, SSM1-12), and hArg (17, SSM1-14) partially activated the mMC1R at 100 μM concentrations (30-40% the maximal NDP-MSH signal), similar to 1 (LTT1-20). Three compounds possessed partial agonist pharmacology at the mMC1R (Figure 4), with His (12, SSM1-6), Orn (14, SSM1-8) or Dab (15, SSM1-10) substitutions at the fourth position. These compounds possessed EC₅₀ values of 400, 3,000, and 1,000 nM, respectively, and activated the mMC1R at 40-55% of the maximal NDP-MSH signal.

Figure 4:

Illustration of the partial agonist pharmacology of 12 (SSM1-6), 14 (SSM1-8), and 15 (SSM1-10) at the mMC1R with the control full agonist NDP-MSH.

Previously reported substitutions at the fourth position for this tetrapeptide scaffold have included a DLys (same as 19, SSM1-18 in the present study), DNal(2’), and Arg (same as 1, LTT1-20 in the present study) residues.⁶¹ The reported pA₂ values at the mMC3R and mMC4R for Ac-DPhe(pI)-Arg-Nal(2’)-DNal(2’)-NH₂ (<5.5 and 6.7),⁶¹ Ac-DPhe(pI)-Arg-Nal(2’)-DLys-NH₂ (6.8 and 8.0),⁶¹ and Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH₂ (7.8 and 9.0)⁶¹ indicated that having a basic in the fourth position increased mMC3R and mMC4R antagonist potency. This work identifies that residues at the fourth position possessing a basic amine or guanidyl sidechain group result in potent mMC3R antagonists in this scaffold, while having a basic amine, guanidyl, or Cit substitution result in MC4R antagonist activity with sub-10 nM potency. All of the fourth position substitutions incorporated were MC5R antagonists, which were within a 10-fold potency range of the lead tetrapeptide 1 (LTT1-20). Every substitution at the fourth position also resulted in MC1R antagonist activity; therefore all of fourth position substitutions assayed resulted in pan-melanocortin antagonists at the mMC1R, mCM3R, mMC4R, and mMC5R.

Second versus Fourth Position Substitutions:

Differences in the melanocortin receptor activity profiles of the substitutions at the second and fourth positions were observed. In general, substitutions at the second position resulted in compounds that were inactive at different receptor subtypes, while incorporation of the same residues at the fourth position maintained activity at the receptors assayed. This can be visualized by comparing Figure 3A and 3B, where many substitutions were inactive at the mMC3R and mMC5R in Figure 3A, while antagonist activity was observed for all substitutions in Figure 3B. This can further be examined by graphing the pA₂ values for the different substitutions at each receptor for both positions (Figure 5). While antagonist activity is observed at the mMC1R (Figure 5A) for substitutions at both positions, several substitutions at the second position do not result in measurable mMC3R (Figure 5B) and mMC5R (Figure 5D) antagonist activity. Furthermore, there is a much broader range of mMC4R antagonist potencies for the second position substitutions as compared to the fourth position (bar size of the blue and red, respectively, Figure 5C). All of the data suggest that the second position has a stricter requirement for a basic guanidyl group for potent MC3R, MC4R, and MC5R antagonist activity. The fourth position appears more amendable to substitutions to maintain antagonist potency at the melanocortin receptors.

Figure 5:

Graphical illustration comparing the antagonist potencies (pA₂) with the same amino acid substitutions at the second and fourth positions at the (A) mMC1R, (B) mMC3R, (C) mMC4R, and (D) mMC5R. A higher pA₂ value represents a more potent compound.

Receptor Selectivity:

Of the compounds assayed, none of them were found to be selective (10-fold) antagonists for the mMC3R or mMC5R (Table 3, Figure 3). Nine tetrapeptides were at least 10-fold selective for the mMC4R over the other receptors: 1 (LTT1-20), 3 (LTT1-16), 7 (LTT1-40), 14 (SSM1-8), 15 (SSM1-10), 18 (SSM1-16), 19 (SSM1-18), 20 (SSM1-20), and 21 (JLB1-3). Three compounds were at least 10-fold selective for the mMC1R over the other receptors. The mMC1R receptor selectivity can be visualized in Figure 3A, where the blue mMC1R bar is higher than any other receptor for Dap (6, LTT1-36), DArg (8, LTT1-44), and DLys (9, LTT1-52). While the lead tetrapeptide 1 (LTT1-20) is the most potent mMC1R antagonist (antagonist K_i = 20 nM), it is more potent at the mMC4R (antagonist K_i = 1.3 nM) and equipotent at the mMC3R (antagonist K_i = 32 nM). The tetrapeptide substituted with DArg at the second position (8, LTT1-44) is 4-fold less potent compared to 1 (LTT1-20) at the mMC1R (antagonist K_i = 80 nM), but is at least 40-fold selective for the mMC1R over the other melanocortin receptors assayed (Figure 6), and may be a useful lead compound in further SAR studies to identify mMC1R selective ligands that may be important for regulating pigmentation.

Figure 6:

Illustration of the antagonist pharmacology of antagonist pharmacology of 8 (LTT1-44) at the mMC1R, mMC3R, mMC4R, and mMC5R. Antagonist K_i values were calculated from pA₂ values using the relationship [pA₂= -Log(K_i)].

In Vivo Feeding Study:

Nine of the tetrapeptides in the present study were at least 10-fold selective antagonists for the mMC4R over the remaining melanocortin receptors (Table 1). The MC4R has been linked to appetite and body weight. An obese phenotype is observed when the MC4R is knocked out in mice.⁷ Upon central administration in mice, MC4R agonists decrease and antagonist ligands increase food intake.^{6, 15, 68} To probe the potential effects on food intake, tetrapeptide 14 (SSM1-8; Figure 7) was selected as a representative compound to examine the potential in vivo effects of this tetrapeptide series. The greater than 10-fold selectivity for 14 (SSM1-8) for the MC4R would support a conclusion that any observed effects on feeding would most likely be mediated by the MC4R. This compound was dosed via intrathecal (IT) administration directly into the spinal canal, as previously reported for other melanocortin ligands.^{15, 68} The IT route was selected to permit direct CNS administration of the tetrapeptide to assess physiological effects of the compound at the target tissue as a proof of concept study. The FDA approval of the melanocortin peptide agonist setmelanotide (Imcivree^™)³⁵ for weight loss in patients with select genetic conditions as a daily subcutaneous injection suggest that peptide melanocortin drugs may be optimized for alternate administration routes for clinical use. Food intake following 10 nmol/mouse and 15 nmol/mouse doses were compared to vehicle control. Both male and female mice were used in approximately equal amounts per treatment group in this study. Since no statistical differences were observed between sexes, male and female data were collapsed into the different treatment groups for data analysis.

Figure 7:

Illustration of the antagonist pharmacology of 14 (SSM1-8) at the mMC1R, mMC3R, mMC4R, and mMC5R. Due to the increased potency at the mMC4R, a different set of concentrations were used at this receptor as indicated in the figure. Antagonist K_i values were calculated from pA₂ values using the relationship [pA₂= -Log(K_i)].

Compared to vehicle treated mice, the 10 nmol/mouse dose significantly increased cumulative food intake at 4 and 6 h (Figures 8, S1). Mice treated with the 15 nmol/mouse dose had significantly increased food intake at 2, 4, and 6 h, as compared to vehicle control, and had a relative increase compared to the 10 nmol/mouse dose at 2, 4, and 6 h (Figures 8, S1), indicating a compound-dose response pharmacological effect. There was no significant difference in cumulative food intake at 24 h post treatment for either treatment group, as compared to vehicle treated mice (data not shown). Body weights before (t = 0) and post (t = 24 and 48 h) treatment were not statistically significantly different from vehicle treated control mice, or at any of the treatment doses relative to each other at each of the respective time points (Figure S2). This pilot study indicates that the tetrapeptide scaffold is sufficiently potent to induce a measurable in vivo feeding response, and may be useful in the development of therapeutic agents that can increase appetite.

Figure 8:

Cumulative food intake following intrathecal (IT) administration of vehicle control or 14 (SSM1-8) directly into the spinal column and central nervous system in wild type mice (C57Bl6). Both male and female mice were examined in approximately equal numbers. No statistically significant difference between sex was observed for cumulative food intake relative to vehicle control values, so the sexes were collapsed into compound treatment groups (n ≥ 8 per treatment group). Data is shown as mean ± SEM. Data were analyzed using the PRISM program (v9; GraphPad Inc.) by a two-way ANOVA with a simple effects within rows multiple comparison relative to vehicle control treated mice. *p< 0.05, ***p<0.001.

Conclusion

This study examines the second and fourth position Arg residues in the previously reported mMC3R and MC4R antagonist tetrapeptide Ac-DPhe(pI)-Arg-Nal(2’)-Arg-NH₂, discovered herein to possess mMC1R and mMC5R antagonist activity. Of the twenty-one synthesized compounds, thirteen were found to possess pan-antagonist activity at the MC1R, MC3R, MC4R, and MC5R (with varying selectivity profiles), identifying this compound series as potential probes for assessing the antagonist activity of these melanocortin ligands. Three tetrapeptides were found to be more than 10-fold selective for the mMC1R, including 8 (LTT1-44), with an antagonist K_i = 80 nM at the mMC1R and at least 40-fold selectivity for the mMC1R over the mMC3R, mMC4R, and mMC5R. An additional nine tetrapeptides were found to be selective for the mMC4R. Tetrapeptides 14 (SSM1-8) was 25-fold selective for the mMC4R and was selected as a representative compound to investigate this tetrapeptide series effects on food intake in vivo. A dose-dependent increased food intake response was observed, indicating these tetrapeptide scaffolds may be potential leads for negative energy balance disease states such as cachexia, anorexia, and failure to thrive.

Experimental

Tetrapeptide Synthesis and Purification:

Unless otherwise mentioned, amino acids, the Rink-amide MBHA resin, and coupling reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Vivitide. The Fmoc-DArg(Pbf)-OH, Fmoc-Orn(Boc)-OH and Fmoc-Dap(Boc)-OH amino acids were purchased from Bachem. The Fmoc-DPhe(pI) amino acid was purchased from Alfa Aesar. Dichloromethane (DCM), methanol (MeOH), acetonitrile (MeCN), dimethylformamide (DMF), and anhydrous diethyl ether were purchased from Fisher (Fair Lawn, NJ). Trifluoroacetic acid (TFA), dimethyl sulfoxide (DMSO), piperidine, triisopropylsilane (TIS), thioanisole, and N,N-diisopropylethylamine (DIEA) were purchased from Sigma-Aldrich (St. Louis, MO). All reagents and chemicals were ACS grade or better and were used without further purification. The control peptide SHU9119 was synthesized in a prior report.⁶⁹

Tetrapeptides were synthesized using standard N-α-fluorenylmethoxycarbonyl (Fmoc) methodologies^62-63 on a manual microwave synthesizer (Discover SPS; CEM, Matthews, NC), as previously reported.^{61, 65, 70} The resin was swelled in DCM before iterative deprotection and amino acid coupling steps were used to assemble the tetrapeptides. The Fmoc deprotection step used a two-stage deprotection strategy with 20% piperidine in DMF (1 x 2 min at room temperature, followed by 1 x 4 min using microwave irradiation to 75 °C and 30W). Microwave coupling reactions were carried out at 75 °C and 30W for 5 min (10 min for coupling Arg residues), except for coupling His, which utilized a lower temperature (50 °C for 5 min). Most coupling reactions utilized 3.1 eq of the amino acid, 3 eq HBTU, and 5 eq of DIEA. For coupling Arg, hArg, and DArg, higher equivalents were employed (5.1 eq Arg, 5 eq HBTU, and 7.1 eq DIEA). In all cases, reaction progress was monitored using a ninhydrin assay,⁷¹ and deprotection and coupling reactions were repeated if necessary. Following the removal of the terminal Fmoc group, tetrapeptides were acetylated by adding a 3:1 acetic anhydride:pyridine solution and mixing for 30 min at room temperature. The resin was then washed with DMF, DCM, and MeOH. Tetrapeptides were cleaved from resin and sidechain deprotected using a 91:3:3:3 mixture of TFA:H₂O:TIS:thioanisole for 2 h at room temperature, and then precipitated using ice-cold diethyl ether. Crude peptides were pelleted on a Sorvall Legend XTR centrifuge (4,000 rpm, 4 °C, 4 min) and dried overnight in a vacuum desiccator.

The crude tetrapeptides were purified on a C18 RP-HPLC semipreparative column (Vydac 218TP1010, 1.0 cm x 25 cm) using a Shimadzu system equipped with a UV detector. Peptides were at least 95% pure (λ = 214 nm) as ascertained by analytical RP-HPLC (Vydac 218TP104, 0.46 cm x 25 cm) on a Shimadzu system equipped with a PDA detector in two solvent systems (MeCN and MeOH) and possessed the correct average molecule mass by ESI-MS (Table 1; Bruker BioTOF II ESI/TOF-MS; LeClaire-Dow Instrumentation Facility, University of Minnesota).

cAMP “AlphaScreen” Bioassay:

The purified tetrapeptides were dissolved in DMSO at a stock concentration of 10⁻² M (NDP-MSH in H₂O at a stock concentration of 10⁻⁴ M), and assayed using HEK293 cells stably expressing the mouse MC1R, MC3R, MC4R, and MC5R and the “AlphaScreen” cAMP bioassay kit (PerkinElmer) according to the manufacturer’s instructions and as previously described.^{64, 66, 72}

Briefly, cells 70–90% confluent were dislodged with Versene (Gibco) at 37 °C and plated 10,000 cells/well in a 384-well plate (Optiplate) with 10 μL of freshly prepared stimulation buffer (1 x HBSS, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA, pH = 7.4) with 0.5 μg of anti-cAMP acceptor beads per well. The cells were stimulated with the addition of 5 μL of stimulation buffer containing peptide (concentrations from 10⁻⁴ to 10⁻¹³ M, determined by ligand potency) or forskolin (10⁻⁴ M) and incubated in the dark at room temperature for 2 h.

Following stimulation, streptavidin donor beads (0.5 μg) and biotinylated-cAMP (0.62 μmol) were added to the wells in a green light environment with 10 μL of lysis buffer (5 mM HEPES, 0.3% Tween-20, 0.1% BSA, pH = 7.4) and the plates were incubated in the dark at room temperature for an additional 2 h. Plates were read on a Enspire (PerkinElmer) Alpha-plate reader using a pre-normalized assay protocol (set by the manufacturer).

Schild Analysis:

Compounds that did not possess agonist activity were evaluated for antagonist activity using a Schild paradigm.⁴² Briefly, a 7-point concentration response curve of NDP-MSH was assayed by itself and in the presence of 4 different concentrations of a peptide ligand (starting concentrations of 10,000, 5,000, 1,000, and 500 nM, and adjusted to lower concentrations for more potent compounds). Peptides that shifted the curves in a rightward concentration-dependent manner (higher concentrations of test peptide resulted in higher observed EC₅₀ values) were considered antagonists. For each compound demonstrating antagonist activity, a linear regression was performed after plotting the each concentration according to the following parameters: -Log[Concentration (M)] (x-axis) versus the Log[((EC₅₀ NDP + Antagonist at that Concentration)/(EC₅₀ NDP alone)) −1] (y-axis). The point at which the linear regression crosses the x-axis in this plot is defined as the pA₂ value.

Data Analysis:

The pA₂ and EC₅₀ values represent the mean of at least three independent experiments performed in duplicate replicates. Compounds that were not active in two independent experiments at the concentrations assayed (>100,000 nM for agonist assays; 10,000, 5,000, 1,000, and 500 nM for antagonist assays) were not furthered examined. The pA₂ and EC₅₀ values and associated standard errors (SEM) were determined by fitting the data to a nonlinear least-squares analysis using the PRISM program (version 4.0, GraphPad Inc). The peptides were assayed as TFA salts and not corrected for peptide content.

Animals:

This study was conducted in accordance with the guidelines set up by the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota. Wildtype (WT) male and female mice with a mixed 129/Sv×C57BL/6J background derived from an in house breeding colony were used throughout this experiment as previously reported in literature.^{15, 17, 65, 68} Mice were housed in a temperature-controlled room (23°-25°C) and maintained on a reversed 12-h light/dark cycle (lights off at 11:00am). Mice had ad libitum access to normal chow (Harlan Teklad 2018 Diet: 18.6% crude protein, 6.2% crude fat, 3.5% crude fiber, with energy density of 3.1kcal/g) and water.

Study design:

All intrathecal (IT)^{15, 68, 73} administration experiments were a crossover design and standard chow was provided ab libitum. Mice were housed in standard polycarbonate conventional cages provided by the University of Minnesota’s Research Animal Resources. Compound 14 (SSM1-8) was dissolved to a stock solution of 4 nmols/μL in 2% DMSO:98% H₂O. The day of compound administration, an aliquot of stock solution was diluted with autoclaved water to concentrations of 10 nmol or 15 nmol in 5 μL 1% DMSO and 1.5% DMSO, respectively. Desired experimental dose (5 μL) or vehicle control (5 μL) was administered via IT injection two hours before lights out (t =0 h). Food intake was manually measured at T = 0, 2, 4, 6, 24, 48, and 72 hours post-injection using a standard laboratory scale balance (Sartorius Quintix 512-1s, Bohemia, New York). This paradigm measures nocturnal food consumption of free feeding mice. It was chosen because it causes minimal disruption of normal feeding patterns and homeostasis, yet is sensitive to subtle changes that can be masked in fasting paradigms.⁷⁴ It is also a sensitive way to measure the effects of compounds that acutely result in a full feeling which might only reduce the size of an initial meal (e.g. two hour time point measurements), but no other.⁷⁴ Mice were given 6-7 days recovery between treatments to reestablish pretreatment body weight and feeding patterns. Male and female mice were used in approximately equal numbers (n ≥ 4 for each sex). No statistical difference between sex was observed for cumulative intake relative to vehicle control, so the sexes were collapsed into compound treatment groups (n ≥ 8 per treatment group). Data was analyzed using the PRISM program (v9.0; GraphPad Inc.) by a two-way ANOVA with a simple effects within rows multiple comparison relative to vehicle control treated mice in order to compare individual doses to vehicle control administration at each time point. Statistical significance is considered if p<0.05.

Abbreviations Used:

ACTH

adrenocorticotropic hormone

AGRP

agouti-related protein

cAMP

3',5'-cyclic adenosine monophosphate

DIEA

N,N-diisopropylamine

ESI-MS

electrospray ionization mass spectrometry

Fmoc

fluorenylmethoxycarbonyl

GPCR

G protein-coupled receptor

HBSS

Hanks’ balanced salt solution

HBTU

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

IBMX

isobutylmethylxanthine

IT

intrathecal

MBHA

methylbenzhydrylamine

MC1R

melanocortin 1 receptor

MC2R

melanocortin 2 receptor

MC3R

melanocortin 3 receptor

MC4R

melanocortin 4 receptor

MC5R

melanocortin 5 receptor

MeCN

acetonitrile

MeOH

methanol

MSH

melanocyte-stimulating hormone

NDP-MSH

4-norleucine, 7-D-phenylalanine-α-melanocyte-stimulating hormone (melanotan I)

DNPe

array detector

POMC

pro-opiomelanocortin

RP-HPLC

reversed-phase high-pressure liquid chromatography

SAR

structure-activity relationship

SEM

standard error of the mean

TIS

triisopropylsilane