Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation.

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

Structural studies on Hgr3 orphan receptor ligand prolactin-releasing peptide.

Prolactin-releasing peptides (PrRPs) are two novel bioactive peptides of 20 and 31 residues, dubbed respectively PrRP20 and PrRP31, isolated from bovine hypothalamic tissues as ligands of the orphan seven-transmembrane domain receptor Hgr3. The first biological activity identified for these peptides was the release of prolactin. Recent data on biological activities of PrRPs as well as on the localization of their receptors in numerous central nervous system sites suggested new potential actions of PrRPs in the regulation of the central nervous system and the possibility of identifying an alternative central role for these peptides. We describe here the synthesis and the structural characterization of the peptide PrRP20 by CD and NMR spectroscopies. A 3D model was built on the basis of the NMR data collected in a water/sodium dodecyl sulfate mixture. This system provides an amphipatic medium able to mimic the cell membrane. The main structural feature of the PrRP20 is an alpha-helical secondary structure spanning the 10 C-terminal residues. The conformational properties of PrRP20 are discussed in considering the sequence similarity observed between the Hgr3 and the neuropeptide Y (NPY) receptors. This similarity, together with the data showing a number of biological activities common to PrRP and NPY peptides, leads us to formulate the hypothesis that similar structural elements could exist in the ligands as well. In fact, PrRP20 and NPY are well aligned in the C-terminal portion, where they share an amphipatic alpha-helical secondary structure. Interestingly, the homology between the two sequences involves residues crucial for NPY biological activity. The conformational characterization of PrRP20 and the comparison with NPY are a valuable starting point for the rational design of subsequent SAR studies aimed at identifying PrRP analogues acting as either agonists or antagonists at the Hgr3 receptor. Such PrRP analogues could be useful receptorial tools able to clarify the multiple biological functions hypothesized for the PrRP receptor in the central nervous system.

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor.

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.

Binding sites for melanin-concentrating hormone (MCH) in brain synaptosomes and membranes from peripheral tissues identified with highly tritiated MCH.

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in the brain of all vertebrate species. In chromatophores of teleost fishes it induces pigment granule aggregation. In mammals, however, its physiological function is not yet clear. Attempts to identify the site(s) of its action by binding analysis failed because radioiodinated MCH with the natural sequence was devoid of biological activity. We have now synthesized an analogue of rat/human MCH, [Pra4,8,12,19]-MCH, containing four L-propargylglycine (Pra) residues in positions 4, 8, 12, and 19 for catalytic tritiation to norvaline ([3H4]Nva) residues, each of which containing four tritium atoms. The resulting [3H]-MCH ([(3H4)Nva4,8,12,19]-MCH) had a specific radioactivity of approx. 12,200 GBq/mmol (330 Ci/mmol) and retained a biological activity of 10% as compared to rat/human MCH when tested in the carp scale assay. A series of qualitative binding studies performed with rat crude membranes from brain and peripheal tissues as well as with rat brain synaptosomes using the [3H]-MCH radioligand provided the first evidence for the presence of MCH receptors in mammalian tissues. The data showed that specific binding is present in the hypothalamus, hippocampus and in the adrenal gland while none was detected in the brain cortex or spleen. Owing to the tendency of [3H]-MCH to non-specific binding to tissue, glass and plastic surfaces, a saturation binding analysis with this radioligand was not possible.

Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay.

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.

The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK.

We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide [PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version] to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. [125I]PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited [125I]PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.