p38 MAPK; NLRP1
The primary target of Muramyl Dipeptide is NOD2 (nucleotide-binding oligomerization domain-containing 2), an intracellular pattern recognition receptor. MDP binds to NOD2 to induce NF-κB signaling and the production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. NOD2 is a protein associated with susceptibility to Crohn's disease and responds to bacterial preparations of lipopolysaccharides and peptidoglycans. MDP also activates NLRP3, further enhancing the immune response. The stereochemical configuration (L-Ala-D-isoGln) is essential for NOD2 agonist activity.

MDP directly augments osteoblast differentiation and bone-forming gene expression by Runx2 activation. MDP has no direct effect, but it indirectly inhibits osteoclast differentiation by lowering the RANKL/OPG ratio. The MDP receptor, Nod2, is expressed more frequently when MDP is present, and Nod2-deficient individuals do not experience MDP-induced bone formation or osteoblast activation. [1]
MDP binds to NOD2 and induces NF-κB signaling, leading to the production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. It has an EC50 of 46-148 nM in HEK-Blue hNOD2 cells. MDP induces polarization of proinflammatory Ly6Chigh into Ly6Clow patrolling monocytes. The compound demonstrates NOD2 agonist activity with stereochemical specificity, where the L,L-diastereomer is more than 1,000-fold less active. MDP also activates NLRP3 inflammasome, further enhancing immune responses.

Muramyl dipeptide (MDP)-treated mice show increased bone and mineral density due to enhanced bone formation. Surprisingly, pre- or post-treatment with MDP reduces bone loss in mouse models of RANKL-induced osteoporosis.[1]
MDP has immunoadjuvant activity in vivo, enhancing immune responses to antigens. It is a breakdown product of bacterial cell wall peptidoglycan and activates macrophages. MDP causes arthritis and stimulates cellular and humoral immunity. The compound's immunomodulatory effects make it useful as an adjuvant in vaccine studies and cancer immunotherapy research. The aqueous formulation provides transient receptor engagement ideal for temporal dissection of trained immunity.

Enzyme-linked immunosorbent assay[1]
The levels of OPG and RANKL in culture supernatant and bone marrow extracellular fluid were measured using Quantikine enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.

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Real-time reverse transcriptase–polymerase chain reaction[1]
The mRNA expression of ALP, BSP, Runx2, Nod2, and GAPDH in osteoblasts was determined by using real-time reverse transcriptase–polymerase chain reaction (real-time RT-PCR) as described.28 The sequences of each primer are as follows: ALP: forward 5′-CCAACTCTTTTGTGCCAGAGA-3′ and reverse 5′-GGCTACATTGGTGTTGAGCTTTT-3′; BSP, forward 5′-GAATGCTGTGTCCTCTGAAG-3′ and reverse 5′-AATCCTCGCTCTCTGCATGG-3′; Runx2: forward 5′-AACGATCTGAGATTTGTGGGC-3′ and reverse 5′-CCTGCGTGGGATTTCTTGGTT-3′; Nod2: forward 5′-CCTGGTACGTGCCCAAAGTAG-3′ and reverse 5′-GCCAAGTAGAAAGCGGCAAA-3′; and GAPDH: forward 5′-AGGTCGGTGTGAACCGGATTTG-3′ and reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
No specific non-cell assay protocol is available for MDP. For NOD2 binding studies, standard cell-free assays include surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) using purified recombinant NOD2 protein and MDP. These methods measure binding affinity and kinetics between the ligand and receptor. Alternatively, competitive binding assays using radiolabeled MDP could be employed to determine binding parameters. The stereochemical specificity of the NOD2-MDP interaction can be verified using the inactive L,L-diastereomer as a control.

In 48-well plates with 2×104 cells/400 l per well, MC3T3-E1 cells, BMSCs, or primary osteoblast precursors from mouse calvaria are plated before being incubated with osteoblast induction medium in the absence or presence of MDP. Every two days, half of the medium is replaced with brand-new osteoblast induction medium. At days 6 and 12, the cells are stained with alizarin red S to detect bone mineralization and with ALP to detect osteoblast differentiation.
No specific cell-based assay protocol is available for MDP. For NOD2 activity, standard assays use HEK293 cells transfected with NOD2 and an NF-κB luciferase reporter. Cells are treated with MDP for 4-6 hours, and luciferase activity is measured to quantify NOD2 activation. The EC50 for MDP in this system is 46-148 nM. Other readouts include cytokine production (e.g., IL-8, TNF-α) measured by ELISA, and NLRP3 inflammasome activation assessed by IL-1β or caspase-1 cleavage. The inactive L,L-diastereomer serves as a negative control.

Five-week-old C57BL/6 mice, one-day-old neonatal mice, B6.129S1-Nod2tm1Flv/J mice
1.25 mg/kg
IP

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Micro–computed tomography[1]
Mice acclimated for 1 week were intraperitoneally administered with 200 µL of 1.25 mg/kg MDP (n = 5) or 200 µL PBS (n = 5) at days 0 and 4. At day 7 after initial administration, the femurs were removed from mice and fixed in 10% formalin. The bones were scanned using X-ray micro–computed tomography (μCT) at 70 kV, 142 mA, 10 W, 0.5 mm Aluminium filter, and 7 µm per pixel scan resolution. The μCT images were reconstructed by the SkyScan NRecon program and analyzed using the SkyScan Dataviewer 1.3.2 and SkyScan CT analyzer software version 1.8.1.5. Three-dimensional (3D) images were created by SkyScan CT volume version 2.0. To quantitatively analyze the trabecular bone, 144 slides starting from 144 sections above the distal growth plate were selected and the region of interest of trabecular bone was defined. The trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated from voxel-based 3D reconstruction of μCT images.

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Calcein double labeling and calculation of mineral apposition rate[1]
Mice were intraperitoneally administered with 20 mg/kg of calcein 1 day before the first administration of MDP at 1.25 mg/kg or PBS at days 1 and 5. The mice were intraperitoneally administered with 20 mg/kg of calcein again 1 day after the last MDP administration. At day 9 after the first injection with MDP, the femurs were fixed and embedded in methyl methacrylate. The resin blocks were sectioned and the calcein-labeled sections were observed using confocal microscopy . The mineral apposition rate (MAR) is the distance between the midpoints of two labels. Mineralizing surface/bone surface (MS/BS), MAR, and bone formation rate (BFR) were quantified using the OsteoMeasure software
No specific animal protocol is available for MDP. MDP is used as an immunoadjuvant in vaccine studies. Standard in vivo protocols involve co-administration of MDP with antigens in mice, followed by measurement of antibody titers and immune cell activation. MDP-induced arthritis models can be used to study inflammatory mechanisms. For cancer immunotherapy studies, MDP may be combined with tumor antigens or checkpoint inhibitors to enhance anti-tumor immune responses. Dosing, route of administration, and treatment schedules vary depending on the experimental objectives.

No detailed pharmacokinetic data is publicly available for MDP. MDP has a molecular weight of 492.5 and formula C19H32N4O11. As a glycopeptide, it would have limited oral bioavailability and is typically administered parenterally. The compound is soluble in DMSO and should be stored dry, dark, and at 0-4°C for short term or -20°C for long term. The aqueous formulation provides transient receptor engagement, suggesting rapid clearance or metabolism. Comprehensive pharmacokinetic studies would be required for therapeutic applications.

No detailed toxicology data is publicly available for MDP. As a bacterial cell wall component and immunostimulant, MDP can induce inflammatory responses. Toxicological evaluation would be required for therapeutic development. The compound's ability to induce arthritis in animal models highlights the potential for inflammatory side effects if not properly controlled. However, when used as an adjuvant, the benefits of enhanced immune responses may outweigh the risks at appropriate doses. The compound is for research use only.

[1]. Muramyl Dipeptide, a Shared Structural Motif of Peptidoglycans, Is a Novel Inducer of Bone Formation through Induction of Runx2. J Bone Miner Res. 2017 Jul;32(7):1455-1468.

[2]. Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation. Cell Death Differ. 2015 Oct;22(10):1676-86.

Muramyl dipeptide is a glycopeptide with the chemical name N-propionyl-L-alanyl-D-α-glutamine, in which the pro-R hydrogen of the propionyl group is replaced by an oxygen atom at the 3-position of 2-acetamido-2-deoxy-β-D-glucopyranose. It is a component of peptidoglycan in Gram-positive and Gram-negative bacteria and can be used as an immune adjuvant. It is the conjugate acid of N-acetyl-β-D-muramyl-L-alanyl-D-isoglutamine (1-). Muramyl dipeptide is a natural component of bacterial cell walls and has the ability to activate macrophages. It is a peptidoglycan immune adjuvant, originally isolated from bacterial cell wall fragments; it also has pyrogenic effects and may cause arthritis; it can stimulate humoral and cellular immunity.
Muramyl Dipeptide is also known as N-Acetylmuramyl-L-alanyl-D-isoglutamine. It has the molecular formula C19H32N4O11 and molecular weight 492.5 g/mol. The SMILES notation is CC(C(=O)NC(CCC(=O)O)C(=O)N)NC(=O)C(C)OC(C(C=O)NC(=O)C)C(C(CO)O)O. It appears as a solid powder and is soluble in DMSO. The compound should be stored dry, dark, and at 0-4°C for short term or -20°C for long term. MDP is the minimal immunologically active glycopeptide substructure of bacterial cell wall peptidoglycan. No approved drug status has been identified.

C19H34N4O10

492.47800

492.206

C, 47.69; H, 7.16; N, 11.71; O, 33.44

53678-77-6

53678-77-6

451714

N-Acetylmuramyl-L-alanyl-D-isoglutamine

White to off-white solid powder

1.5±0.1 g/cm3

1023.8±65.0 °C at 760 mmHg

572.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.578

-2.97

8

11

12

34

765

8

C[C@H](NC([C@H](O[C@@H]1[C@@H](NC(C)=O)C(O[C@@H]([C@H]1O)CO)O)C)=O)C(N[C@@H](C(N)=O)CCC(O)=O)=O

BSOQXXWZTUDTEL-QAQREVAFSA-N

InChI=1S/C19H32N4O11/c1-7(17(30)23-10(16(20)29)4-5-12(26)27)21-18(31)8(2)33-15-13(22-9(3)25)19(32)34-11(6-24)14(15)28/h7-8,10-11,13-15,19,24,28,32H,4-6H2,1-3H3,(H2,20,29)(H,21,31)(H,22,25)(H,23,30)(H,26,27)/t7-,8+,10+,11+,13+,14+,15+,19?/m0/s1

(4R)-4-[[(2S)-2-[[(2R)-2-[(3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoyl]amino]propanoyl]amino]-5-amino-5-oxopentanoic acid

Adjuvant Peptide; Muramyl Dipeptide; N-Acetylmuramyl-L-alanyl-D-isoglutamine; Adjuvant Peptide; 53678-77-6; N-Acetylmuramyl-L-alanyl-D-isoglutamine; CHEMBL1779325; (4R)-4-[[(2S)-2-[[(2R)-2-[(3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoyl]amino]propanoyl]amino]-5-amino-5-oxopentanoic acid; (4R)-4-[[(2S)-2-[[(2R)-2-[(2S,3R,4R,5S,6R)-3-Acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoyl]amino]propanoyl]amino]-5-amino-5-oxopentanoic acid; MFCD00077638; Ac-muramyl-Ala-D-Glu-NH2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 98~260 mg/mL (199~527.9 mM)
Ethanol: ~98 mg/mL (~199 mM)

Solubility in Formulation 1: ≥ 6.5 mg/mL (13.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 65.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
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: ≥ 6.5 mg/mL (13.20 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 65.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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: ≥ 6.5 mg/mL (13.20 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 65.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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