Melanotan II: Melanocortin Receptor Research

Melanotan II is a synthetic cyclic heptapeptide, Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2, designed at the University of Arizona in the 1980s as a conformationally constrained analog of the melanocortin hormone α-MSH. It is a nonselective agonist across the melanocortin receptors MC1R, MC3R, MC4R and MC5R, which signal through Gs and cyclic AMP. In the United States it has never held an approved application, and FDA documented that position in writing in 2007.

Three things define the compound: the 23-membered lactam ring that holds its active core in a fixed shape, the receptor family it is nonselective across, and a regulatory record that sets it apart from the one approved α-MSH analog. This profile covers each, with identity data checked against PubChem and the primary literature.

Melanotan 2 at a glance

The formula and mass above describe the free base. Synthetic peptides of this type are isolated as acetate salts and carry residual water, so the mass of powder in a vial is larger than the mass of peptide it contains. The certificate for a lot states the counter-ion and the peptide content; how to read a peptide certificate of analysis explains where both appear.

From α-MSH to a cyclic lactam

α-MSH is one of the peptides cut from pro-opiomelanocortin. It has 13 residues, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, and structure-activity work through the 1970s and 1980s reduced it to a message sequence, His-Phe-Arg-Trp, with two substitutions that raised potency: norleucine for methionine at position 4, and the D-isomer of phenylalanine at position 7.

In 1989 Al-Obeidi, Castrucci, Hadley and Hruby reported a series built on that core. They prepared seven cyclic lactam analogs of α-MSH(4-13) and α-MSH(4-10), varying the bridging residues (Glu or Asp at position 5; Lys, Orn, Dab or Dpr at position 10) and closing the ring either in solution or on the solid-phase support. Scored against α-MSH in the lizard (Anolis carolinensis) skin melanophore bioassay of the period, the Asp5/Lys10 analogs were the most potent of the set, and the paper reports that rings of 23 members were about 100-fold more potent than α-MSH while larger or smaller rings were less so. The α-MSH(4-10) member of that series, Ac-[Nle4,Asp5,D-Phe7,Lys10]α-MSH(4-10)-NH2, is Melanotan II.

Two design choices in that structure still matter in the laboratory. Norleucine removes the oxidation-prone methionine of the parent hormone. The side-chain-to-side-chain lactam leaves a normal N-terminus and C-terminus, so the peptide is cyclic through a branch rather than head to tail, and it still ends in an amide. That amide is the only difference between Melanotan II and PT-141 (bremelanotide), which carries a free acid in the same position.

The melanocortin receptor family

Five melanocortin receptors are known. All are class A (rhodopsin-like) G protein-coupled receptors, and all couple canonically to Gs, adenylyl cyclase and a rise in intracellular cyclic AMP. Four respond to α-MSH; MC2R responds to ACTH alone.

The family is unusual in having endogenous antagonists as well as agonists: agouti signaling protein at MC1R, and agouti-related protein at MC3R and MC4R. MC1R is the receptor on melanocytes that governs the switch between eumelanin and pheomelanin synthesis, which is why the earliest assays for this whole series were pigment-cell assays in amphibian and reptile skin.

Melanotan II is a broad agonist across the four α-MSH-responsive subtypes. For assay design, that has consequences:

• One receptor per system. A readout from a cell line carrying more than one melanocortin receptor cannot be assigned to a subtype. Heterologous expression of a single receptor in HEK293 or CHO cells is the standard arrangement.
• Cyclic AMP is the first readout. Because every subtype couples to Gs, cyclic AMP accumulation or a cyclic AMP reporter is the usual way to build a concentration-response curve.
• A matched antagonist exists. Hruby and colleagues showed in 1995 that replacing D-Phe7 in this exact scaffold with the bulkier D-2'-naphthylalanine gives SHU9119, an antagonist at MC4R (pA2 9.3) and MC3R (pA2 8.3) with minimal agonist activity, while remaining a full agonist at MC1R and MC5R. Because it shares the Melanotan II ring, it is the natural pharmacological control.

Three α-MSH analogs, side by side

Melanotan II is often confused with the linear analog sold under the name Melanotan I, which is the same molecule as the approved drug afamelanotide, and with PT-141. The differences are structural and easy to tabulate.

Afamelanotide is the [Nle4,D-Phe7] substitution applied to the full 13-residue chain without cyclization, which is why its formula is so much larger. The Scenesse label describes it as a melanocortin 1 receptor agonist. Anhydrolabs lists that compound as Melanotan I; it is a research reagent, not the approved implant.

Research systems the compound appears in

The literature on this scaffold falls into three groups, and each uses the peptide for a different purpose.

The first is the pigment-cell bioassay that the compound was born in. Al-Obeidi and colleagues scored their cyclic series in isolated frog (Rana pipiens) and lizard skin preparations, and reported that the same analogs ranked differently in the two species, which they read as evidence for distinct melanocyte receptors. Those assays predate the cloning of the receptors and are read today as historical structure-activity data rather than as subtype assignments.

The second is recombinant receptor pharmacology. Once MC1R through MC5R had been cloned in the early 1990s, the Arizona group and others re-tested the same peptides on cloned human and mouse receptors expressed in cultured cells, which is how the agonist and antagonist profiles quoted above were established. This is the format most current work uses: one receptor per cell line, cyclic AMP or a β-arrestin recruitment readout, and a reference agonist such as [Nle4,D-Phe7]α-MSH on the same plate.

The third is structural and computational. The 1989 design itself came out of molecular-dynamics modeling of α-MSH, and the constrained ring was the test of that model; later work on melanocortin receptor structure continues to use rigid analogs as probes of the binding pocket, because a cyclic peptide samples far fewer conformations than a linear one of the same length.

A practical note for any of the three: because a synthetic cyclic peptide can contain a linear precursor or a side-product with the same mass, a bioassay run without an identity check on the lot is measuring the vial, not the molecule.

Regulatory status in the United States

Melanotan II has no approved application in the United States, and the agency's position is on the public record. A 2016 FDA debarment order (81 FR 79501, Docket No. FDA-2015-N-4169) recounts the history: on or about August 30, 2007 the agency sent a warning letter to Melanocorp, Inc. stating that, based on statements on the company's website, Melanotan II "constituted a new drug under the FD&C Act that could not be introduced or delivered for introduction into interstate commerce without an FDA approved application." A November 29, 2007 letter reiterated that the agency considered it an unapproved new drug, and a December 28, 2007 letter stated that unapproved new drugs do not qualify for export. The company's president was later convicted of two counts of conspiracy and permanently debarred.

That record is about marketing a compound for use in people. It does not restrict the use of Melanotan II as a laboratory reagent, and it is the reason a supplier's framing matters. Anhydrolabs supplies this compound for in-vitro research only, not for human or veterinary diagnosis, treatment, or consumption; the terms are set out on the research use page.

Handling Melanotan II in the laboratory

Anhydrolabs supplies Melanotan II as a lyophilized powder in vacuum-sealed vials, and in 10-vial kits. The handling points below follow from the structure rather than from any application.

• Storage. Keep sealed vials cold, dry and dark; −20 °C is the usual long-term condition for a lyophilized peptide, and colder for multi-year holding. Bring a vial to room temperature before opening so that moisture does not condense onto the powder. How to store peptides covers the reasoning.
• Stock solutions. The peptide is soluble in water and in dilute acetic acid. Prepare a concentrated stock, split it into single-use aliquots and freeze them so that no aliquot is thawed twice; the method is in how to reconstitute peptides.
• Oxidation and light. With methionine replaced by norleucine, the remaining liability is Trp9, which is sensitive to light and to dissolved oxygen. Amber vials, minimal headspace and short handling times all help.
• Concentration checks. The single tryptophan gives useful absorbance at 280 nm, so a spectrophotometer reading can be compared against the nominal concentration of a stock, allowing for salt and water content.
• Adsorption. Cationic, partly hydrophobic peptides bind to glass and to some plastics at low concentration. Low-binding tubes and a carrier protein in dilute working solutions are common precautions.
• Identity, not just purity. Melanotan II and PT-141 differ by about 0.98 Da, less than the spread of the isotope envelope, so a low-resolution mass measurement can confuse them. High-resolution mass spectrometry and retention time against a reference standard settle identity; a purity figure alone does not.