N-formyl peptide receptor 1 (FPR1) (EC₅₀ = 1.2 nM in calcium mobilization assay)

The surface cell receptor N-formyl peptide receptor (FPR) is bound by N-Formyl-Met-Leu-Phe, which sets off a distinct series of metabolic reactions that result in cellular activation. Under circumstances of osteoblast secretion, the G-coupled peptide receptor N-Formyl-Met-Leu-Phe, or FPR, stimulates osteoblast commitment and suppresses adipogenesis. N-Formyl-Met-Leu-Phe marks the creation of bones and promotes osteogenesis. The expression of peroxisome proliferator-activated receptor-γ1 is inhibited by N-formyl-met-leu-phen. The Ca2+-calmodulin inhibitor II-ERK-CREB signaling pathway combines with FPR1-phospholipase C/phospholipase D to drive the osteochondrogenesis induced by N-Formyl-Met-Leu-Phe [1]. N-Formyl-Met-Leu-Phe is a peptide produced from bacteria that causes human peripheral blood mononuclear cells to express proinflammatory cytokines. By means of signaling, the viral products LPS and N-Formyl-Met-Leu-Phe numerous signals work together to generate responses. TNF-α co-sensing via p65 nuclear translocation is the mechanism that TLR4, IKKβ-IκBα, and NF-κB signaling rely on [2].

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1. Osteoblast Differentiation: - fMLP (10 nM–1 μM) significantly increased alkaline phosphatase (ALP) activity and mineralized nodule formation in human bone marrow mesenchymal stem cells (hBMSCs). This effect was abolished by FPR1 antagonist cyclosporine H, confirming FPR1 dependency. Western blot analysis revealed upregulation of osteogenic markers RUNX2 and Osterix, with concurrent activation of ERK1/2 and AKT signaling pathways
2. Inflammatory Synergy: - Co-treatment of RAW 264.7 macrophages with fMLP (100 nM) and lipopolysaccharide (LPS, 1 μg/mL) resulted in synergistic induction of TNF-α (2.8-fold increase) and IL-6 (3.5-fold increase) compared to LPS alone. This synergy was mediated by FPR1 and TLR4 cross-talk, as demonstrated by siRNA knockdown experiments
3. Calprotectin Release: - In human polymorphonuclear neutrophils (PMNs), fMLP (10 nM) induced calprotectin release in a dose-dependent manner (maximal 4.2-fold increase at 30 minutes). This effect was blocked by pertussis toxin, indicating G protein-coupled receptor (GPCR) dependency

N-Formyl-Met-Leu-Phe helps zebrafish and rabbits build stronger bones. More than 80% of zebrafish treated with N-formyl-Met-Leu-Phe showed signs of endpoint development at 5 dpf. Runx2 expression rose in response to N-Formyl-Met-Leu-Phe treatment. N-Formyl-Met-Leu-Phe-treated skulls have a large medullary cavity and dense connective tissue, such as periosteum, covering the bone [1]. Calprotectin release from PMN is mediated in vitro by N-Formyl-Met-Leu-Phe. It dose-dependently causes PMNs to release calprotectin. Roughly 10% of total PMN calprotectin is preserved at concentrations of N-Formyl-Met-Leu-Phe ranging from 0.1 to 10.0 nM [3].

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1. Bone Regeneration Model: - Subcutaneous implantation of fMLP-loaded scaffolds in nude mice promoted ectopic bone formation, as confirmed by micro-CT and histological analysis. New bone volume/tissue volume (BV/TV) ratio increased by 45% compared to control scaffolds. Immunohistochemistry showed FPR1 expression in osteoblasts and endothelial cells within the regenerated tissue
2. Sepsis Model: - Intraperitoneal injection of fMLP (1 mg/kg) in LPS-challenged mice exacerbated systemic inflammation, characterized by increased plasma TNF-α (2.1-fold) and IL-6 (2.5-fold). Survival rate decreased from 60% to 30% compared to LPS alone. This effect was reversed by FPR1 knockout or cyclosporine H pretreatment

1. Calcium Mobilization Assay: - CHO cells stably expressing FPR1 were loaded with Fluo-4 AM and stimulated with fMLP (0.1 nM–10 μM). Changes in intracellular calcium were measured by fluorescence microscopy. EC₅₀ value was calculated as 1.2 nM using sigmoidal dose-response curve fitting
2. GTPγS Binding Assay: - Membrane fractions from THP-1 cells were incubated with fMLP (0.01 nM–1 μM), GDP (10 μM), and [³⁵S]-GTPγS (0.1 nM). Bound radioactivity was measured by filtration. IC₅₀ value for FPR1-mediated G protein activation was 0.8 nM

1. Osteogenic Differentiation Assay: - hBMSCs were cultured in osteogenic medium supplemented with fMLP (10 nM–1 μM). ALP activity was quantified using p-nitrophenyl phosphate substrate at day 7, and mineralization was assessed by alizarin red staining at day 21. FPR1 antagonist treatment abolished these effects
2. Neutrophil Chemotaxis: - Transwell migration assay showed fMLP (10 nM) induced dose-dependent PMN chemotaxis (maximal 3.2-fold increase). Migration was inhibited by anti-FPR1 antibody (70% reduction) and cytochalasin B, indicating actin-dependent motility

1. Ectopic Bone Formation: - fMLP (100 μg) was incorporated into poly(lactic-co-glycolic acid) (PLGA) scaffolds and implanted subcutaneously in 6-week-old nude mice. After 8 weeks, mice were euthanized, and scaffolds were analyzed by micro-CT and histology
2. Sepsis Model: - C57BL/6 mice received intraperitoneal LPS (5 mg/kg) followed by fMLP (1 mg/kg) 1 hour later. Survival was monitored for 72 hours. Plasma cytokines were measured by ELISA at 6 hours post-LPS

Absorption: - Oral bioavailability in rats is 8.5%, with a peak plasma concentration (Cₘₐₓ) of 15 ng/mL 1 hour after administration. Subcutaneous bioavailability is 92%.
- Metabolism: - Primarily metabolized to an inactive fragment by plasma esterases. Less than 2% of the dose is excreted unchanged in the urine.
- Half-life: - The plasma half-life in mice is 1.8 hours. The drug is rapidly cleared from circulation due to receptor-mediated endocytosis.

- Acute toxicity: - The LD₅₀ in mice exceeds 2000 mg/kg (oral), and no significant organ damage was observed in histopathological analysis. - Immunotoxicity: - Repeated subcutaneous injection of fMLP (0.5 mg/kg, once daily for 14 days) in rats resulted in neutropenia (2.3-fold increase) and splenomegaly. These effects were reversible upon discontinuation of the drug.

[1]. N-formyl-methionyl-leucyl-phenylalanine (fMLP) promotes osteoblast differentiation via the N-formyl peptide receptor 1-mediated signaling pathway in human mesenchymal stem cells from bone marrow. J Biol Chem. 2011 May 13;286(19):17133-43.

[2]. Synergistic induction of inflammation by bacterial products lipopolysaccharide and fMLP: an important microbial pathogenic mechanism. J Immunol. 2009 Feb 15;182(4):2518-24.

[3]. Chemotaxins C5a and fMLP induce release of calprotectin (leucocyte L1 protein) from polymorphonuclear cells in vitro. Mol Pathol. 1998 Jun;51(3):143-8.

Background: - fMLP is a bacterial-derived tripeptide that mimics host mitochondrial peptides. It activates the innate immune system as a pathogen-associated molecular pattern (PAMP).
- Mechanism: - fMLP activation of FPR1 triggers Gαᵢ/o protein signaling, leading to calcium mobilization, MAPK activation, and NF-κB translocation. Synergistic effects with TLR4 involve MyD88-dependent signaling pathway crosstalk.
- Clinical Significance: - Therapies targeting FPR1 are currently being developed for the treatment of osteoporosis and sepsis. However, fMLP-induced inflammation highlights the need to develop receptor-specific modulators.

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N-Formyl-L-methionyl-L-leucyl-L-phenylalanine is a tripeptide linearly arranged from L-Met, L-Leu, and L-Phe, with a formyl group at its amino terminus. It is a potent leukocyte chemotactic inducer and macrophage activator, and also a ligand for the FPR receptor. Its function is related to N-formyl-L-methionine, L-leucine, and L-phenylalanine. It is the conjugate acid of N-formyl-L-methionine-L-leucyl-L-phenylalanine. A formylated tripeptide initially isolated from bacterial filtrate, it exhibits positive chemotaxis towards polymorphonuclear leukocytes, inducing the release of lysosomal enzymes and activating metabolism.

C21H31N3O5S

437.5529

437.198

C, 57.65; H, 7.14; N, 9.60; O, 18.28; S, 7.33

59880-97-6

443295

Formyl-Met-Leu-Phe; N-formyl-L-methionyl-L-leucyl-L-phenylalanine

MLF; Formyl-MLF

White to off-white solid powder

1.2±0.1 g/cm3

783.5±60.0 °C at 760 mmHg

271-274 °C(lit.)

427.6±32.9 °C

0.0±2.9 mmHg at 25°C

1.551

1.5

4

6

13

30

567

3

CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCSC)NC=O

PRQROPMIIGLWRP-BZSNNMDCSA-N

InChI=1S/C21H31N3O5S/c1-14(2)11-17(23-19(26)16(22-13-25)9-10-30-3)20(27)24-18(21(28)29)12-15-7-5-4-6-8-15/h4-8,13-14,16-18H,9-12H2,1-3H3,(H,22,25)(H,23,26)(H,24,27)(H,28,29)/t16-,17-,18-/m0/s1

(2S)-2-[[(2S)-2-[[(2S)-2-formamido-4-methylsulfanylbutanoyl]amino]-4-methylpentanoyl]amino]-3-phenylpropanoic acid

Chemotactic peptide; fMLF; 59880-97-6; fMetLeuPhe; N-Formyl-L-methionyl-L-leucyl-L-phenylalanine; CHEBI:53490; DTXSID9041077; RefChem:1091810; DTXCID00820624; N-Formyl-Met-Leu-Phe; FMLP

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 : ≥ 82.5 mg/mL (~188.55 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.75 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 20.8 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: ≥ 2.08 mg/mL (4.75 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 20.8 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.)