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| Targets |
CXCL4-CCL5[1]
The primary molecular target of Cyclic MKEY TFA is the heterodimerization interface of CXCL4 and CCL5. By binding to the surface of CXCL4 (PF4) or CCL5 (RANTES), it sterically prevents the two chemokines from forming the functional CXCL4-CCL5 heterodimer, which is known to promote monocyte adhesion to endothelium and induce inflammatory signaling in vascular cells (endothelial cells, smooth muscle cells, and macrophages). The compound does not inhibit the individual chemokines alone; rather, it specifically disrupts their synergistic interaction. |
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| ln Vitro |
In in vitro assays, Cyclic MKEY TFA inhibits the CXCL4-CCL5 heterodimer-dependent chemotaxis of monocytes (e.g., THP-1 cells, primary human monocytes). In a transwell migration assay, the heterodimer (50-100 ng/mL each) induces potent monocyte migration; co-incubation with Cyclic MKEY TFA (0.1-10 microM) reduces migration back to basal levels. The peptide also blocks heterodimer-induced Ca2+ flux and ERK1/2 phosphorylation in target cells, confirming that the signaling pathway is dependent on the chemokine heterodimer. There is no direct inhibition of CCL5 or CXCL4 alone.
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| ln Vivo |
Cyclic MKEY TFA has been evaluated in several in vivo disease models. In the ApoE-/- (atherosclerosis-prone) mouse model, administration of the cyclic peptide (5-10 mg/kg, i.p. twice weekly for 8-12 weeks) significantly reduced the formation of atherosclerotic plaques (quantified by Oil Red O or Sudan IV staining of the aorta). The protective effect is associated with decreased macrophage infiltration and inflammatory cytokine levels (TNF-alpha, IL-6, MCP-1). In a mouse model of aortic aneurysm (Angiotensin II infusion), Cyclic MKEY TFA reduces aortic dilatation and prevents aortic rupture. Furthermore, in a mouse model of ischemic stroke (middle cerebral artery occlusion, MCAO), Cyclic MKEY TFA (administered i.p. 10 mg/kg, one hour post-occlusion) reduces infarct volume and improves neurological scores, by blocking the influx of inflammatory cells into the brain.
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| Enzyme Assay |
The binding of Cyclic MKEY TFA to CXCL4 or CCL5 is determined by surface plasmon resonance (SPR). Immobilized CXCL4 or CCL5 on a CM5 sensor chip is exposed to increasing concentrations (0.1-100 microM) of the cyclic peptide. The real-time binding kinetics (association rate ka, dissociation rate kd) and equilibrium dissociation constant (KD) are calculated. Typically, the affinity (KD) is in the low micromolar range (e.g., 1-5 microM). Alternatively, a fluorescence anisotropy competition assay is performed: labeled CXCL4-CCL5 heterodimer is incubated with increasing concentrations of unlabeled Cyclic MKEY TFA; the decrease in anisotropy reflects competitive displacement.
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| Cell Assay |
The ability of Cyclic MKEY TFA to block heterodimer-induced chemotaxis is assessed using a 24-well transwell plate (5 microm pore size). THP-1 monocytes or freshly isolated human peripheral blood mononuclear cells (PBMCs) are serum-starved for 4 hours. Lower chambers are filled with medium containing CXCL4 (50-100 ng/mL) + CCL5 (50-100 ng/mL) in the presence or absence of Cyclic MKEY TFA (0.1-10 microM). Upper chambers receive 2 × 10⁵ cells in 100 microL of serum-free medium. Cells are allowed to migrate for 2-4 hours at 37degC in a 5% CO2 incubator. Migrated cells in the lower chamber are collected and counted by flow cytometry (counting events for a fixed time) or using a CyQuant cell proliferation assay kit (fluorescence measurement). Percent migration inhibition is calculated relative to the positive control (heterodimer only). Intracellular Ca2+ flux: cells are loaded with Fluo-4 AM (2 microM, 30 min at 37degC) and then stimulated with the CXCL4-CCL5 heterodimer (50 ng/mL each) with or without Cyclic MKEY TFA (10 microM). Fluorescence is recorded on a plate reader (excitation 488 nm, emission 525 nm) for 200 seconds. Cyclic MKEY TFA blocks the rapid peak in Ca2+ response.
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| Animal Protocol |
For in vivo efficacy in the MCAO stroke model: Adult male C57BL/6 mice (8-10 weeks, 20-25 g) are subjected to transient (60 minutes) middle cerebral artery occlusion via the intraluminal filament technique under isoflurane anesthesia. Recirculation is confirmed by laser Doppler flowmetry. At 1 hour and 6 hours post-reperfusion, Cyclic MKEY TFA (10 mg/kg) or vehicle control (saline) is administered intraperitoneally (i.p.). At 72 hours after ischemia, mice are euthanized. Brains are removed, sliced into 2 mm coronal sections, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to quantify infarct volume (unstained white areas). Neurological deficits are assessed using a standardized 5-point scale (0: no deficit, 4: severe deficit). Immunohistochemistry with anti-CD68 (macrophage) or anti-MPO (neutrophil) antibodies is performed to evaluate inflammatory cell infiltration. For the atherosclerosis model: Female ApoE-/- mice (6-8 weeks old) are fed a high-fat diet (21% fat, 0.15% cholesterol) for 12 weeks. Cyclic MKEY TFA (5 mg/kg, i.p., three times per week) or PBS vehicle is administered for 12 weeks. Aortas are collected and stained en face with Oil Red O. The percentage of plaque area (lesion area/total aortic area) is quantified.
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| ADME/Pharmacokinetics |
As a synthetic cyclic peptide (approx. molecular weight 1000-1500 Da), Cyclic MKEY TFA is rapidly cleared from circulation. In mice, the plasma half-life after i.p. or i.v. injection is estimated to be 15-60 minutes. The cyclic structure provides significantly improved stability against proteases compared to linear peptides, but renal filtration (glomerular filtration) remains the primary elimination route due to the small size. The TFA counterion is present to improve solubility in aqueous buffers and saline. For in vivo studies, the compound is typically dissolved in sterile PBS (1-5 mg/mL) and administered i.p. (allows slower absorption) to prolong exposure compared to i.v. injection. No significant plasma protein binding has been reported.
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| Toxicity/Toxicokinetics |
Cyclic MKEY TFA exhibits low acute toxicity in mice. In typical efficacy studies using doses of 5-20 mg/kg (i.p. or i.v.), no significant adverse effects (body weight loss >15%, gross behavioral abnormalities, morbidity) are observed compared to vehicle controls. No hepatotoxicity (elevated ALT/AST) or nephrotoxicity (elevated BUN/creatinine) has been reported at these doses. The peptide is not known to be mutagenic or clastogenic. However, as a research chemical, standard precautions (PPE: lab coat, gloves, safety glasses) should be used. Avoid direct inhalation of aerosolized powder. Long-term toxicity studies have not been formally reported but given the moderate dose levels and short duration of studies (2-12 weeks), it is considered relatively safe for research purposes.
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| References |
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| Additional Infomation |
Cyclic MKEY TFA is a synthetic cyclic peptide derived from the sequences of CXCL4 (PF4) and CCL5 (RANTES). The “MKEY” sequence refers to a conserved motif in chemokine proteins that is responsible for heterodimer formation. The cyclic form (via a thioether or amide cyclization, depending on the synthetic method) locks the peptide into a conformation that mimics the heterodimerization interface. By blocking CXCL4-CCL5 heterodimerization, the compound is a first-in-class anti-inflammatory agent that does not inhibit chemokine monomers (which are essential for normal immune surveillance). This compound is a valuable tool for studying chemokine synergy and is being explored preclinically as a potential therapy for chronic inflammatory diseases (atherosclerosis, abdominal aortic aneurysm) and acute neuroinflammation (stroke). Cyclic MKEY TFA is for research use only and is not an approved drug for human use.
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| Molecular Formula |
C115H175F3N28O36S2
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| Molecular Weight |
2644.89
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| Related CAS # |
Cyclic MKEY
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.3781 mL | 1.8904 mL | 3.7809 mL | |
| 5 mM | 0.0756 mL | 0.3781 mL | 0.7562 mL | |
| 10 mM | 0.0378 mL | 0.1890 mL | 0.3781 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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