PT-141 (bremelanotide) is a synthetic cyclic heptapeptide analogue of α-melanocyte-stimulating hormone (α-MSH). It is an agonist at four of the five melanocortin receptors, with MC4R and MC3R the subtypes of most interest, and it is used as a reference ligand in receptor binding, cAMP accumulation and reporter assays on recombinant cell lines. PT-141 from NuVion is supplied as a laboratory chemical for in vitro research use only.
Key facts
| Type | Synthetic cyclic peptide, melanocortin receptor agonist |
| Amino acid count | 7 |
| Sequence | Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH |
| Molecular formula | C50H68N14O10 |
| Molecular weight | 1025.2 g/mol |
| CAS number | 189691-06-3 |
| Synonyms | Bremelanotide, PT141 |
| Supplied form | Lyophilised powder in a sealed vial |
Structure and chemistry
The peptide is built around His-Phe-Arg-Trp, the tetrapeptide that all melanocortins use to engage their receptors and that corresponds to residues 6 to 9 of α-MSH. Four changes turn that core into bremelanotide. Norleucine replaces the methionine at position 4, which removes an oxidation-prone thioether without changing side chain length. D-phenylalanine replaces L-phenylalanine at position 7, which protects the neighbouring bonds from proteases and raises receptor potency. The side chains of an aspartate at position 5 and a lysine at position 10 are joined by a lactam bridge, which holds the pharmacophore in the β-turn that the receptors recognise. The N-terminus is acetylated.
Bremelanotide differs from Melanotan 2 only at the C-terminus. Melanotan 2 is the C-terminal amide and bremelanotide the free carboxylic acid, a change that raises the mass by about 1 Da, adds one negative charge at neutral pH and leaves the receptor profile largely unchanged. Because the aspartate and lysine side chains are consumed in the lactam, the only free charged groups are the arginine guanidinium, the C-terminal carboxylate and the partly protonated histidine, so the net charge at pH 7.4 is close to zero.
The cyclic backbone, the acetylated N-terminus and the D-residue together make the peptide resistant to exopeptidases and to most endopeptidases, so it persists in serum-containing medium far longer than linear α-MSH. Tryptophan and histidine are the residues most sensitive to oxidation and to light, so solutions are kept in the dark. The tryptophan also gives the peptide a usable absorbance at 280 nm and intrinsic fluorescence, which makes quantification straightforward compared with aromatic-free peptides.
Mechanism of action
The five melanocortin receptors are class A G protein-coupled receptors. MC2R responds only to ACTH and does not bind bremelanotide. At the other four, bremelanotide is an agonist with a rank order of potency of MC1R, MC4R, MC3R and then MC5R in cAMP assays on recombinant cell lines, and MC1R and MC4R are activated at low nanomolar concentrations. The profile is non-selective and close to that of Melanotan 2, its C-terminal amide.
Canonical signalling runs through Gαs. Receptor activation stimulates adenylyl cyclase, raises intracellular cAMP, activates protein kinase A and phosphorylates the transcription factor CREB. MC4R has additional outputs that depend on the cell background: coupling to Gαq/11 with a calcium response, ERK1/2 phosphorylation, β-arrestin recruitment followed by internalisation, and, in a G protein-independent manner shown in heterologous expression systems, closure of the inward-rectifier potassium channel Kir7.1. Which output dominates depends on receptor density and on accessory proteins such as MRAP2, which is why assay reports state the cell line and expression level.
The melanocortin system also has endogenous antagonists. Agouti-related protein (AgRP) is an inverse agonist and competitive antagonist at MC3R and MC4R, and agouti signalling protein (ASIP) plays the same role at MC1R. Bremelanotide responses in cAMP assays are shifted rightward by these proteins and by synthetic antagonists such as SHU9119, which makes it a convenient agonist against which to characterise new antagonists and allosteric modulators. At MC1R in cultured melanocytes, the same cAMP signal raises expression of the transcription factor MITF and the activity of tyrosinase, the standard readout for MC1R agonism in cell culture.
Research applications
- Radioligand displacement binding at recombinant MC1R, MC3R, MC4R and MC5R in HEK293 or CHO cell membranes, typically against iodinated NDP-α-MSH
- cAMP accumulation assays (HTRF, ELISA) and CRE-luciferase reporter assays for concentration-response curves and EC50 values
- β-arrestin recruitment and receptor internalisation assays for biased signalling comparisons with α-MSH and Melanotan 2
- Electrophysiology of Kir7.1 coupling in MC4R-expressing cells
- Antagonist and allosteric modulator characterisation using AgRP, ASIP and SHU9119 as reference competitors
- LC-MS reference standard and a model cyclic lactam peptide for studies of D-amino acid and cyclisation effects on protease resistance
PT-141 sits in the Reproductive Signalling category alongside the other G protein-coupled receptor ligands used in signalling assays.
Handling in the laboratory
The lyophilised powder is reconstituted with bacteriostatic water. Run the water down the inside of the vial and swirl gently; do not vortex, since the peptide is amphipathic and foams. The reconstitution calculator gives the concentration that a chosen water volume produces from the vial content. For binding and cAMP assays the stock is diluted serially into assay buffer on the day, and low-binding tubes and tips are used because cyclic hydrophobic peptides adsorb to plastic at nanomolar concentrations.
Keep the sealed vial dry, away from light and refrigerated as described in the product documentation. Once reconstituted, keep the solution refrigerated, protected from light and used within the period stated on the documentation, and aliquot to avoid repeated freeze-thaw cycles. The compound is characterised by RP-HPLC for purity and by mass spectrometry for identity. On electrospray the singly charged ion is observed near m/z 1026, and the 1 Da difference from Melanotan 2 is resolved by a high-resolution instrument, which is the check to run if there is any doubt about which of the two analogues is in the vial.
Testing and supply from NuVion
NuVion has PT-141 independently tested by Janoshik Analytical. Most batches are tested, and the Certificate of Analysis for a tested batch is published on the product page, reporting purity by RP-HPLC and identity by mass spectrometry. Manufacture is GMP-audited, the peptide is supplied lyophilised in sealed vials, and orders are dispatched from within Australia. Certificates for other compounds are collected in the Certificate of Analysis library.
Related compounds
The closest relative is Melanotan 2, the C-terminal amide of the same cyclic sequence, which is the usual comparator in melanocortin receptor assays. Kisspeptin, a KISS1R agonist signalling through Gαq, and Oxytocin, an OXTR agonist, are the other G protein-coupled receptor ligands in the category.
Frequently asked questions
What is PT-141 used for in research?
PT-141 is used as a reference agonist for melanocortin receptor pharmacology: radioligand binding, cAMP accumulation, reporter gene, β-arrestin recruitment and internalisation assays on cells expressing MC1R, MC3R, MC4R or MC5R. It is also used as a model cyclic lactam peptide in stability and mass spectrometry method development.
How does PT-141 differ from Melanotan 2?
The two share the same cyclic sequence, Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]. Melanotan 2 carries a C-terminal amide and PT-141 a C-terminal carboxylic acid. The mass differs by about 1 Da, the acid carries one extra negative charge, and the receptor selectivity profile is similar across the four melanocortin receptors that bind them.
How is PT-141 supplied and stored?
NuVion supplies PT-141 as a lyophilised powder in a sealed vial. Store the sealed vial dry, away from light and refrigerated per the product documentation. After reconstitution with bacteriostatic water, keep the solution refrigerated and protected from light, and use it within the period given on the documentation.
Is PT-141 a therapeutic good in Australia?
No. PT-141 from NuVion is a laboratory chemical for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the TGA. It is not for human or veterinary use.
Research use only. This product is a laboratory chemical supplied for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.
