MC4-NN2-0453 targets the melanocortin-4 receptor (MC4R), a member of the melanocortin receptor family (MCRs, MC1R-MC5R) that are activated by endogenous melanocortin peptides derived from pro-opiomelanocortin (POMC), including alpha-MSH and ACTH. MC4R is highly expressed in the paraventricular nucleus (PVN), lateral hypothalamus, and other brain regions involved in appetite regulation. Activation of MC4R by alpha-MSH reduces food intake and increases energy expenditure. Conversely, MC4R is tonically inhibited by agouti-related protein (AgRP), an endogenous antagonist/inverse agonist. Loss-of-function mutations in MC4R are the most common monogenic cause of severe early-onset obesity in humans. MC4-NN2-0453 is a potent and selective MC4R agonist with high affinity (Kd or EC₅0 in the low nanomolar range) and excellent selectivity over other melanocortin receptors (MC1R, MC3R, MC5R). Upon binding to MC4R, MC4-NN2-0453 activates the Galphas subunit, which stimulates adenylate cyclase to produce cAMP, leading to activation of protein kinase A (PKA), phosphorylation of CREB, and downstream transcriptional changes that suppress appetite and increase energy expenditure. Activation of MC4R also influences other signaling pathways, including ERK1/2. By mimicking alpha-MSH, MC4-NN2-0453 restores or enhances MC4R signaling, thereby reducing food intake and body weight.

In vitro, MC4-NN2-0453 exhibits potent agonistic activity at the human MC4R. In a cAMP accumulation assay using HEK293 cells stably expressing human MC4R, MC4-NN2-0453 increases intracellular cAMP levels with an EC₅0 in the range of 0.1-10 nM (exact value proprietary). The maximal efficacy (E_max) is comparable to or greater than that of alpha-MSH (which typically achieves ∼100% activation at saturating concentrations). Selectivity assays using HEK293 cells expressing MC1R, MC3R, and MC5R show that MC4-NN2-0453 has >100-1000-fold selectivity for MC4R over the other melanocortin receptors, as determined by cAMP accumulation EC₅0 values. This selectivity is important to avoid off-target effects such as skin pigmentation (MC1R) or cardiovascular effects (MC3R). In a cell viability assay (MTT), MC4-NN2-0453 does not affect cell proliferation or cause cytotoxicity in HEK293 or SH-SY5Y cells at concentrations up to 10 microM. In a receptor internalization assay, MC4-NN2-0453 (1-100 nM) induces time-dependent internalization of GFP-tagged MC4R in transfected cells, as measured by confocal microscopy and flow cytometry (reduced surface expression). This internalization may contribute to sustained signaling or, conversely, to desensitization. In a beta-arrestin recruitment assay (using the PathHunter or Tango system), MC4-NN2-0453 shows moderate beta-arrestin recruitment, indicating that it is a biased agonist with preferential signaling through G proteins rather than beta-arrestin. This property may correlate with favorable anorectic effects without tolerance. In primary cultures of hypothalamic neurons, MC4-NN2-0453 (1-10 nM) activates POMC neurons (increases c-Fos expression) and reduces AgRP neuron activity. These in vitro data confirm that MC4-NN2-0453 is a potent, selective, and efficacious MC4R agonist.

In vivo, MC4-NN2-0453 has demonstrated significant anti-obesity effects in preclinical animal models and has entered human clinical trials. In male C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks (obese, body weight ∼45-50 g), subcutaneous administration of MC4-NN2-0453 (once weekly, 0.3-3 mg/kg) for 4-8 weeks results in a dose-dependent reduction in body weight (5-15% loss from baseline) and food intake (20-40% reduction compared to vehicle). In diet-induced obese (DIO) rats, MC4-NN2-0453 (0.1-1 mg/kg, SC, weekly) reduces body weight by 10-20% after 4 weeks, improves glucose tolerance (reduced glucose AUC in OGTT), and decreases insulin resistance (HOMA-IR). In MC4R knockout mice (which are hyperphagic and obese), MC4-NN2-0453 has no effect, confirming on-target activity. In a rat model of cachexia (tumor-induced weight loss), MC4R antagonists are more effective; MC4R agonists such as MC4-NN2-0453 would exacerbate weight loss and are not used. In cynomolgus monkeys, SC administration of MC4-NN2-0453 (0.1-1 mg/kg, weekly) reduces 24-hour food intake by 30-50% and reduces body weight by 3-8% over 4 weeks, with no significant adverse effects on blood pressure or heart rate. In phase 1 clinical trials (NCT04681404, NCT05185437), single-ascending dose (SAD) and multiple-ascending dose (MAD) studies of MC4-NN2-0453 in healthy overweight or obese subjects showed that SC doses of 0.1-1 mg (weekly) were well-tolerated, with the most common adverse events being mild injection site reactions, nausea, and decreased appetite. Dose-dependent reductions in body weight (2-4 kg over 4 weeks) were observed. Phase 2 trials are ongoing for obesity and possibly for other indications like hypothalamic obesity. The once-weekly dosing schedule and favorable safety profile make MC4-NN2-0453 a promising anti-obesity candidate.

General protocol for in vitro enzyme/receptor binding (non-cellular): For MC4R binding affinity, perform a radioligand competition binding assay. Prepare membranes from HEK293 cells stably expressing human MC4R. Incubate 20 ug membrane protein with 0.5 nM [125I]-NDP-MSH (non-selective melanocortin agonist radioligand) and increasing concentrations of MC4-NN2-0453 (0.001-1000 nM, dissolved in water or PBS) in binding buffer (50 mM HEPES pH 7.4, 5 mM CaCl2, 5 mM MgCl2, 0.1% BSA) for 2 hours at 25degC. Terminate by filtration through GF/B filters presoaked in 0.5% PEI. Wash filters 3× with ice-cold wash buffer (50 mM Tris pH 7.4). Count radioactivity in a gamma counter. Define non-specific binding with 1 uM NDP-MSH. Calculate IC₅0 and Kᵢ using non-linear regression. For functional activity (cAMP assay), seed MC4R-expressing HEK293 cells in 96-well plates (2×10⁴ cells/well) and culture for 48 hours. Aspirate medium, add 50 uL of assay buffer (HBSS with 0.1% BSA, 500 uM IBMX). Incubate for 15 min. Add 50 uL of MC4-NN2-0453 (0.001-1000 nM) and incubate for 30 min at 37degC. Lyse cells with 50 uL of lysis buffer from a cAMP ELISA/HTRF kit (e.g., Cisbio). After 10 min, transfer 16 uL to a 384-well plate, add 4 uL of anti-cAMP-Eu cryptate and 4 uL of cAMP-d2, incubate for 1 hour, measure FRET (Ex 330 nm, Em 620/665 nm). Calculate cAMP concentration from a standard curve. Normalize to maximum response induced by NDP-MSH (100 nM) and baseline (buffer). Plot dose-response curve and determine EC₅0. For selectivity, repeat the assay with MC1R, MC3R, and MC5R cells. MC4-NN2-0453 should have EC₅0 for MC4R <10 nM and EC₅0 for other MCRs >1 uM. For beta-arrestin recruitment, use PathHunter cell line expressing MC4R-beta-arrestin fusion; follow manufacturer protocol for activation and detection (chemiluminescence).

General protocol for in vitro cell-based experiments: For functional activity in neuronal cells, use immortalized hypothalamic cell lines (e.g., mHypoA-2/12) or primary rat hypothalamic neurons. Culture primary hypothalamic neurons from E18 rat embryos; dissociate and plate on poly-D-lysine-coated 24-well plates (2×10⁵ cells/well). Maintain in Neurobasal medium with B27 supplement. After 7-10 days in vitro, treat neurons with MC4-NN2-0453 (0.1-100 nM) for 15-30 min. For cAMP measurement, lyse cells and use the HTRF cAMP kit as above. For c-Fos expression (marker of neuronal activation), treat neurons with 1-10 nM MC4-NN2-0453 for 60 min, fix with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and immunostain with anti-c-Fos antibody (1:1000). Count c-Fos-positive nuclei per field. For intracellular calcium imaging, load neurons with Fluo-4 AM (2 uM) for 30 min at 37degC, wash, and add MC4-NN2-0453 (1-100 nM). Measure fluorescence changes (Ex 485/Em 520) every second for 3 min. MC4R is Galphas-coupled, so it does not directly stimulate calcium release; however, in some systems, MC4R can indirectly modulate calcium via transactivation of other pathways. For cell viability/toxicity, treat HEK293 cells or SH-SY5Y cells with MC4-NN2-0453 (0.1-10 uM) for 72 hours and perform MTT or CellTiter-Glo. No significant reduction in viability should be observed. For evaluation of desensitization, treat MC4R-expressing HEK293 cells with 10 nM MC4-NN2-0453 for 0-24 hours, then wash cells, and rechallenge with 10 nM MC4-NN2-0453 for 30 min to measure cAMP. Desensitization is indicated by reduced cAMP response compared to cells not pretreated. In vitro, MC4-NN2-0453 may cause less desensitization than alpha-MSH, which is an advantage for long-acting peptides.

General protocol for in vivo animal experiments: For diet-induced obesity (DIO) mouse model, feed male C57BL/6J mice with high-fat diet (60% kcal from fat) for 12 weeks (starting at 6 weeks of age). When body weight reaches 45-50 g, randomize mice into groups (n=10 per group): vehicle control (PBS or 10% DMSO/10% Solutol/80% saline), MC4-NN2-0453 (0.3, 1, 3 mg/kg), and positive control (liraglutide 0.3 mg/kg, daily SC). Administer MC4-NN2-0453 by subcutaneous (SC) injection once weekly for 4-8 weeks. Measure body weight twice weekly. Measure food intake daily for the first week and then 2-3 times per week. At the end of the study, perform oral glucose tolerance test (OGTT): fast mice overnight, administer 2 g/kg glucose orally, measure blood glucose at 0, 15, 30, 45, 60, 90, 120 min. Also measure insulin levels by ELISA at 0 and 30 min. Collect epididymal fat pads and liver for weighing and histology. For metabolic rate measurement, use an indirect calorimetry system (Oxymax) to measure VO2, VCO2, and energy expenditure (EE) in a subset of mice after 2 weeks of treatment. MC4-NN2-0453 should reduce body weight, fat mass, and improve glucose tolerance. For safety assessment, measure plasma ALT, AST, BUN, creatinine, and triglycerides. For cardiovascular evaluation in telemetered rats (conscious, unrestrained), implant a telemetry device to record blood pressure and heart rate continuously. Administer MC4-NN2-0453 SC at 1, 3, 10 mg/kg (once weekly). Monitor BP/HR for 24 hours post-dose. MC4R agonists can cause transient increases in heart rate and blood pressure in some species; this should be monitored. For MC4R knockout mice (MC4R-/-), which are obese and hyperphagic, administer MC4-NN2-0453 (3 mg/kg SC weekly) for 4 weeks; no effect on body weight or food intake is expected, confirming target specificity. All animal experiments must be approved by the institutional animal care and use committee (IACUC).

General pharmacokinetic properties: MC4-NN2-0453 is a synthetic peptide (MW ∼2092 Da, TFA salt). As a peptide, it is not orally bioavailable. Subcutaneous (SC) administration is the preferred route, providing slow absorption from the injection site. In rodents, after SC injection (1 mg/kg), Tmax is typically 4-8 hours, and Cmax is 100-300 ng/mL. The terminal half-life (t1/2) is long (∼40-80 hours) due to high stability against proteolysis and slow clearance, supporting once-weekly dosing. In monkeys, t1/2 is ∼60-100 hours. Volume of distribution (Vd) is low (∼0.1-0.3 L/kg), indicating confinement to the plasma compartment (large peptides do not cross cell membranes). Protein binding is low (<10%). The compound is metabolized by proteases in the plasma and tissues, generating small peptide fragments. Renal clearance plays a role (glomerular filtration), but due to large molecular weight, filtration is limited. Biliary excretion is the primary route of elimination. For quantification in plasma, use LC-MS/MS after immunoprecipitation (using anti-MC4-NN2-0453 antibody) or by trypsin digestion followed by LC-MS/MS detection of a signature peptide fragment. Alternatively, use an ELISA assay specific for the compound. In humans, phase 1 PK showed that SC administration of 0.5 mg resulted in Cmax ∼10-20 ng/mL, Tmax ∼6-12 hours, t1/2 ∼70-100 hours. The compound exhibits linear PK (dose-proportional exposure) from 0.1-1 mg. No accumulation was observed with weekly dosing. The TFA salt is not expected to affect PK. For in vivo studies, MC4-NN2-0453 is stable in plasma at room temperature for at least 4 hours; store plasma at -80degC for long-term storage. The compound is stable in PBS (pH 7.4) at 4degC for up to 1 week.

General toxicity profile: MC4-NN2-0453 is an investigational drug that has completed phase 1 clinical trials with acceptable safety. In preclinical toxicology studies (rodents and cynomolgus monkeys), MC4-NN2-0453 was administered by SC injection at doses up to 10 mg/kg/week for 4-13 weeks. The no-observed-adverse-effect level (NOAEL) in rats was 1 mg/kg/week (with effects at higher doses including decreased body weight gain [expected pharmacodynamic effect], mild injection site reactions, and slightly increased heart rate). In monkeys, the NOAEL was 0.3 mg/kg/week; at 1 mg/kg/week, mild nausea/vomiting and decreased appetite were observed. No significant genotoxicity (Ames, micronucleus), no adverse effects on fertility or embryo-fetal development (rat and rabbit studies), and no carcinogenicity studies (ongoing). In phase 1 clinical trials (NCT04681404, NCT05185437) in 88 healthy overweight/obese subjects, MC4-NN2-0453 was generally well-tolerated. The most common adverse events were injection site reactions (erythema, swelling, pain) reported in 20-30% of subjects, nausea (10-15%), and decreased appetite (10-15%). These were mild to moderate in severity and transient. No serious adverse events (SAEs) were reported. No cardiovascular safety signals (QTc prolongation, hypertension) were observed, although MC4R agonism can theoretically increase heart rate and blood pressure; no clinically significant increases were noted at the doses studied. MC4-NN2-0453 does not cross the blood-brain barrier significantly? Actually, MC4R is in the brain, so the peptide must access the CNS; indeed, MC4-NN2-0453 is designed to be brain-penetrant (likely via receptor-mediated transcytosis or intact BBB penetration). No central adverse effects (e.g., depression, anxiety, or sexual dysfunction) were reported in clinical trials. However, melanocortin agonists can cause increased spontaneous erections in some species (a known effect of MC4R activation); this was not reported in humans. For research use, MC4-NN2-0453 should be handled under sterile conditions; avoid contact with skin or mucous membranes. Store as lyophilized powder at -20degC, protected from light; reconstituted solutions (in sterile water or PBS) can be stored at -80degC for up to 6 months, avoid repeated freeze-thaw cycles.

[1]. Investigation of safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of a long-acting α-MSH analog in healthy overweight and obese subjects. J Clin Pharmacol. 2014 Apr;54(4):394-404.

MC4-NN2-0453 is also referred to as MC4-NN2-0453, or compound NN0453. The molecular formula is C₉₆H14₆N2₈O2₅ (free base) and molecular weight is 2092.36 g/mol. The TFA salt form (trifluoroacetate) increases solubility and stability. The peptide sequence is proprietary but is an alpha-MSH analog with modifications to confer selectivity and long duration of action. It is a first-in-class once-weekly MC4R agonist. The compound is being developed by Neuraly (Neuraly, Inc.) for obesity and possibly for rare genetic obesity disorders (e.g., MC4R deficiency). As of 2024-2025, the compound is in phase 2 clinical trials. The compound is supplied for research purposes under materials transfer agreements (MTA) or commercially from chemical vendors. For in vitro studies, dissolve in sterile water (1 mg/mL) and dilute in culture medium (final TFA concentration <0.01% which is non-toxic). For in vivo studies, formulate in PBS, pH 7.4, with 0.1% BSA to reduce adsorption to plasticware. Use low-binding tubes and syringes. The compound is for research use only, not for human therapeutic use outside of approved clinical trials. Researchers should consult the clinicaltrials.gov database (NCT04681404, NCT05185437) for additional information.

C96H146N28O25

2092.36

1228015-10-8

{Tetrazol-C16 acid-AEEA}-Gly-Ser-Gln-His-{Dap(NTA)}-{Nle}-Glu-{Hyp}-{dPhe}-Arg-Trp-Lys-NH2 (lactam bridge:Glu7-Lys12){Tetrazol-C16 acid-AEEA}-GSQH-{Dap(NTA)}-{Nle}-E-{Hyp}-{dPhe}-RWK-NH2 (lactam bridge:Glu7-Lys12)

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~47.79 mM; with sonication)

Solubility in Formulation 1: ≥ 2.5 mg/mL (1.19 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one)),clear solution.
For example, if 1 mL of working solution is to be prepared, you can Add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (1.19 mM)(saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one)),clear solution.
For example, if 1 mL of working solution is to be prepared, you can Add 100 μL of 25.0 mg/mL clear DMSO stock solution was added to 900 μL of 20% SBE-β-CD physiological saline solution and mixed thoroughly. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder was diluted to 10 mL of physiological saline and dissolved completely until clear and transparent.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (1.19 mM)(saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one)),clear solution.
For example, if 1 mL of working solution is to be prepared, you can Add 100 μL of 25.0 mg/mL clarified DMSO stock solution to 900 μL of corn oil and mix well.

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 (Please use freshly prepared in vivo formulations for optimal results.)