The melanocortin receptor (MCR) family is a member of the G-protein coupled receptor (GPCR) superfamily that stimulates the adenylate cyclase signal transduction pathway and contains five melanocortin receptor subtypes (MC1–5R). (1-6) The melanocortin system includes the endogenous agonists α-, β-, and γ-melanocortin stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) which are derived from posttranslational modifications of the POMC gene. (7, 8) Two endogenous antagonists [agouti related protein (AGRP) (9) and agouti (10)] have been discovered to antagonize the centrally expressed MC3R and MC4Rs. (11) All the endogenous agonists share a common His-Phe-Arg-Trp (6–9; α-MSH numbering) pharmacophore domain in their primary amino acid sequence. (12-14) This tetrapeptide sequence is postulated to be important for melanocortin receptor molecular recognition and ligand-induced receptor activation. (15-18) The MC1R, expressed in melanocytes, is involved in the regulation of skin and hair pigmentation. (1, 2, 19) The MC2R is stimulated only by the ACTH agonist and is expressed in the adrenal cortex to regulate steroidogenesis. (1) The MC3R is expressed in the gut, placenta, heart, and brain. The MC3R has been reported to be involved in metabolism and energy homeostasis, via mechanism(s) remaining to be characterized. (3, 20-22) The MC4R is expressed primarily in the brain and regulates feeding behavior, energy homeostasis, and sexual function. (5, 23-25) The MC5R is expressed in a wide variety of tissues, both centrally and peripherally, and is involved in exocrine gland function in mice. (6, 26, 27) The mouse MC3R and MC4R are expressed in the brain and have 60% similarity in primary amino acid sequence, but possess distinct ligand pharmacological profiles. Central activation of the MC3R and MC4R is postulated to mediate the effects of the melanocortin pathway on energy homeostasis as both the MC3R knockout (KO) (21, 22) and MC4R KO mice show alterations in energy balance. (25) Intracerebroventricular (ICV) administration of the synthetic melanocortin agonist (MTII) decreased food intake, while both the endogenous AGRP antagonist and the synthetic SHU9119 antagonist increased food intake. (23, 28)
Single nucleotide polymorphisms (SNPs) of the hMC4R have been identified in human patients with severe obesity. Farooqi et al. investigated children with severe obesity (under the age of 10) and found that ∼6% of these early onset childhood obesity patients possessed hMC4R single nucleotide polymorphisms (SNPs). (29, 30) To date, greater than 100 SNPs have been reported in human patients. Seventy of these SNPs have been characterized in vitro (cell culture) in attempts to identify the putative underlying molecular mechanistic defects resulting from the amino acid change(s) in the hMC4R. (31-33) In 2011, the MC3R was postulated to be involved in the regulation of food intake by using a mixed pharmacology ligand that possessed partial agonist/antagonist activity at the mMC3R and full agonist activity at the mMC4R. (28) Intracerebroventricular administration of this compound in the wild type, MC3RKO, and MC4RKO mice resulted in decreased food intake. (28) These, and other genetic and pharmacological studies (involving both animals and humans), support the role of the central melanocortins in the regulation of satiety and energy homeostasis. Selective ligands are needed to understand underlying molecular mechanism(s) by which the MC3R and the MC4R regulate energy balance. The discovery and use of these molecular probes to identify and characterize physiological functions associated with the MCR system are still being sought after.
In continuous efforts to identify potent, selective, and enzymatically stable melanocortin ligands, we have incorporated a bioactive small molecule heterocycle into a peptide in attempts to overcome the inherent problems associated with the endogenous peptides that may limit their consideration for drug development. The strategy applied modifies the peptide backbone resulting in changes in intra- and/or intermolecular interactions postulated to contribute to melanocortin receptor preferred “bioactive” conformation(s) while providing increased protease stability. We have previously reported systematic exploration of the reverse turn mimetic template studied herein with the His 6, Phe 7, and Arg 8 residue domain modifications (α-MSH numbering) that resulted in the identification of a potent agonist ligand at the mMC4R (AMW610). (34) However, this compound was only ∼5-fold more selective for the mMC4R versus the mMC3R.
The aromatic indole side chain of the Trp amino acid, a constituent of the melanocortin agonist pharmacophore His 6-Phe 7-Arg 8-Trp 9 (α-MSH numbering), has been postulated to play a key role in ligand–receptor interactions. (13, 14, 35, 36) Substitution of the Trp residue by an Ala amino acid in various peptide templates was reported to reduce agonist potency at the melanocortin receptor subtypes, particularly at the MC3R. (37-39) Therefore, we hypothesized that the modifications of this Trp residue side chain, in the template presented herein, may lead to potent and/or selective analogues at the melanocortin receptors. It has also been postulated that a peptide-heterocyclic scaffold could lead to conformationally constrained ligands that might be more stable for in vitro and in vivo studies as compared to their linear endogenous counterpart(s). The aim of this study was to (i) elucidate the role of the Trp amino acid side chain in the peptide-heterocyclic scaffold in attempts to obtain potent and selective ligands and (ii) study the scope of this novel template on serum stability over the endogenous α-MSH peptide ligand. Peptides, in general, can be promising therapeutic agents by having a number of advantages over small molecules, in terms of specificity and affinity for targets. However, general endogenous peptide low protease stability often traditionally limits their further consideration in drug development. Standard approaches to make peptides less susceptible to serum proteases, for example, using modifications with unnatural amino acids, d-amino acids, β-amino acids, cyclization of peptide chain at the termini, modifications of the peptide backbone, and so forth, have been well established. These approaches often make the peptide structure more constrained and therefore less prone to protease degradation in serum. In the present study, we compared the compound containing a heterocyclic-peptide backbone modification and a disulfide bridge at the N- and C-terminus, which can restrict a peptide’s backbone conformation and modify their stability in serum. In addition, in vivo experiments were performed to assess the physiological effect on food intake of this new chemical probe. Central administration of AST3-88 decreased a cumulative feeding response in rats 48 h post-ICV treatments. Herein, we report the design, functional, and structural characterization of a potent and selective MC4R full agonist and present the studies of proteolytic stability and in vivo properties of this template for the first time. Taken together, this study provides new information on the structural prerequisites for potent, selective, and increased serum stable melanocortin ligands.
The development of MC3R and MC4R selective ligands has the therapeutic potential for the treatment of body weight and feeding related disorders. A synergistic role of the MC3R in conjunction with the MC4R for feeding and energy homeostasis are emerging. (28, 40) Thus, ligands and molecular probes that can discriminate in a subtype specific manner (agonist and/or antagonist), and that can be used to probe the physiological roles of melanocortin receptor subtypes are still needed in the field. Toward this goal, a number of small molecules and peptide ligands have been explored and reported in the literature by a plethora of academic and industrial research laboratories. Our strategy in this study is to combine the use of a bioactive small molecule and incorporate it into a potent cyclic peptide template in attempts to gain further insight into melanocortin receptor molecular recognition, selectivity, and increase protease stability. We report herein the AST3-88 melanocortin agonist that possesses a selective MC4R agonist profile over the MC3R (>50-fold), enhanced serum stability, and results in decreased food intake in vivo.
The compound AST3-88, and its stereoisomer AMW6103, were synthesized using previously reported method(s) and is summarized in Scheme 1. (34, 41) To probe the importance of the heterocyclic stereochemistry within the ring moiety, the Cys α-carbons (marked with * in Figure 1) in both AST3-88 (l-configuration) and AMW6103 (d-configuration) were designed and synthesized. The Fmoc-Trp(Boc)-aldehyde was synthesized from the corresponding acid via Fmoc-Trp(Boc)-amide and introduced via a reductive amination method to the resin bound peptide chain. The thioether ring was formed on the resin by mild on-bead cyclization method. (34, 41) After completion of the synthesis, the ligand was cleavage and the crude peptide was dissolved in a 20% DMSO/water solution and stirred at room temperature to form the disulfide-bridge. The peptides AST3-88 and AMW6103 possessed the correct molecular weights as determined by mass spectrometry. The purity of these peptides (>95%) was assessed by analytical RP-HPLC in two diverse solvent systems (data provided in the experimental section).
Scheme 1. a
Scheme a Reagents and conditions: (a) 20% piperidine/DMF; (b) 3 equiv Fmoc-Asn(Trt)-OH, 3 equiv BOP or HBTU, 6 equiv DIEA, DMF; (c) Fmoc-Trp(Boc)-aldehyde, NaBH 3 CN, AcOH, DMF; (d) Fmoc-Cys(SBu _t_)-OH (R or S), BOP or HBTU, DIEA, DMF; (e) (ClCH 2 CO)2 O, NEM, DCM; (f) Bu 3 P/H 2 O/THF; (g) NEM, DMF, heat 55–60 °C; (h) TFA/TIS/EDT/H 2 O 91:3:3:3; (i) 20% DMSO/H 2 O, rt.
Figure 1. (A) Illustration of the compounds (i) heterocycle, (ii) AMW610, (iii) AST3-88, and (iv) AMW6103. The stereochemistry of the thioether ring is represented by an asterisk (*). Compounds AST3-88 and AMW610 contain l-Cys, and compound AMW6103 contains a d-Cys at this position. The side chain of His is depicted in green, DPhe in orange, Arg in blue, the heterocycle moiety in pink, and the disulfide bridge is circled in yellow. (B) Amino acid sequences of key endogenous and synthetic melanocortin peptides.
The compounds were tested for agonist functional activity at the mouse MC1R, MC3R, MC4R, and MC5R using a 96-well cAMP based β-galactosidase reporter gene bioassay (Table 1). (42) The NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH 2) peptide (43) is one of the standard melanocortin agonists used in the study of melanocortin receptors and was included herein as a reference control and as an internal control for maximal ligand efficacy (100%). The pharmacology of the heterocyclic moiety alone and peptides AMW3-130 and AMW610 (Figure 1) were also included for pharmacological comparisons of the different but related templates. The AMW3-130 molecule was designed as a chimeric cyclic peptide template that incorporated the AGRP based antagonist scaffold that upon substitution of the AGRP Arg-Phe-Phe pharmacophore residues with the agonist His-DPhe-Arg-Trp pharmacophore resulted in the conversion of a weak antagonist into a potent sub-nanometer agonist that was able to functionally rescue polymorphic hMC4R SNPs. (33) In 2002, the heterocyclic moiety (44) (Figure 1) was reported to possess nanomolar MCR functional agonist activity and initial studies were performed incorporating this moiety into the AMW3-130 peptide template. (34, 41) The first study identified the AMW610 template (Figure 1) as possessing the most potent nanomolar MCR full agonist functionality of the SAR at the time. (34) Biophysical experiments, similar to the ones reported herein, were performed in attempts to correlate SAR and identify solution-phase structural differences between the peptides containing different orientations of the heterocycle. (8, 34) Thus, for structural and functional comparative purposes, the AMW3-130 and AMW610 ligands have been included herein.
At the mMC1R and mMC3R, AST3-88 resulted in an equipotent compound, within the 3-fold inherent experimental error, as compared to AMW610. At the mMC4R, AST3-88 resulted in a ∼5 nM full agonist that is 18-fold more potent at this receptor than AMW610. At the mMC5R, AST3-88 resulted in 6-fold increased agonist potency as compared to AMW610. The AMW6103 ligand, possessing the D-configuration at the Cys (6) residue of the heterocyclic ring (Figure 1), result
