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| Targets |
The primary target of CCX777 is the atypical chemokine receptor 3 (ACKR3/CXCR7). ACKR3 is a member of the chemokine receptor family but is classified as "atypical" because it does not couple to G proteins to activate classical signaling pathways that lead to cell migration. Instead, ACKR3 functions as a scavenger receptor, binding and internalizing chemokines (particularly CXCL12 and CXCL11) to regulate their extracellular levels. It also signals through β-arrestins, which can lead to distinct downstream effects. CCX777 is a partial agonist that specifically promotes the recruitment of β-arrestin-2 to ACKR3. This means it induces a conformational change in the receptor that facilitates β-arrestin-2 binding, but it does so with less efficacy than a full agonist. By selectively modulating the β-arrestin pathway, CCX777 allows researchers to study the functional consequences of β-arrestin-mediated ACKR3 signaling in isolation from G protein signaling. This is particularly important because the role of ACKR3 in various diseases, including cancer, is often attributed to its β-arrestin-dependent functions, such as cell survival, proliferation, and metastasis. CCX777's partial agonism provides a nuanced tool to probe the specific contributions of this pathway.
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| ln Vitro |
In vitro studies have focused on characterizing CCX777's ability to modulate ACKR3 signaling. The primary activity measured is its capacity to promote the recruitment of β-arrestin-2 to ACKR3. This is typically assessed using cell-based assays designed to detect protein-protein interactions, such as Bioluminescence Resonance Energy Transfer (BRET) or enzyme fragment complementation (e.g., PathHunter®). In these assays, ACKR3 and β-arrestin-2 are tagged with complementary fragments of a reporter enzyme or with a BRET donor/acceptor pair. Upon treatment with CCX777, the recruitment of β-arrestin-2 to the receptor brings the fragments into proximity, generating a measurable signal. As a partial agonist, CCX777 elicits a submaximal response compared to a full agonist. This property is crucial for understanding its mechanism of action. CCX777 is also used to study the downstream consequences of β-arrestin-2 recruitment, such as receptor internalization and the activation of downstream signaling cascades like ERK/MAPK. These in vitro studies are essential for defining CCX777's pharmacological profile and for validating its use as a tool to study ACKR3 biology.
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| ln Vivo |
In vivo activity data for CCX777 is limited, as it is primarily a research tool used in vitro. However, its role as a modulator of ACKR3 suggests potential in vivo applications in studying cancer and inflammation. ACKR3 is overexpressed in various cancers and is involved in tumor growth, metastasis, and angiogenesis. By modulating ACKR3 signaling, CCX777 could be used in animal models to study the role of this receptor in tumor progression. For example, it could be administered to mice bearing xenograft tumors to assess its effect on tumor growth and metastasis. However, specific in vivo protocols, such as dosing regimens, routes of administration, and pharmacokinetic data, are not detailed in standard product descriptions. The compound is soluble in DMSO at 50 mg/mL, which can be used to formulate it for in vivo administration. Its stability is guaranteed for at least two years when stored at -20°C. Overall, while the in vivo potential of CCX777 is acknowledged, its primary use remains in vitro for mechanistic studies of ACKR3 signaling.
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| Enzyme Assay |
The in vitro receptor binding and functional assays for CCX777 are designed to measure its interaction with ACKR3 and its effect on β-arrestin-2 recruitment. A standard assay is the β-arrestin recruitment assay, which is a cell-based, functional assay that measures the ability of a compound to promote the interaction between a receptor and β-arrestin. In this assay, cells are engineered to express ACKR3 fused to a ProLink™ tag (a small peptide fragment of β-galactosidase) and β-arrestin-2 fused to the larger fragment of the enzyme (EA). Upon receptor activation and β-arrestin-2 recruitment, the two enzyme fragments complement each other to form an active β-galactosidase, which cleaves a chemiluminescent substrate to produce a measurable signal. CCX777 is added to the cells at various concentrations, and the resulting signal is measured. As a partial agonist, CCX777 produces a signal that is lower than the maximum achieved by a full agonist. The data is used to generate a concentration-response curve to determine its potency and efficacy. Another method is the BRET assay, where ACKR3 is tagged with a Renilla luciferase (donor) and β-arrestin-2 with a fluorescent protein (acceptor). Upon recruitment, energy transfer occurs, and the BRET ratio is measured. These assays provide quantitative data on CCX777's activity as a β-arrestin-2 partial agonist for ACKR3.
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| Cell Assay |
In vitro cell-based assays for CCX777 are used to study its functional consequences on ACKR3-expressing cells. These assays go beyond measuring β-arrestin recruitment to assess downstream signaling and cellular outcomes. A common approach is to measure the phosphorylation of downstream kinases such as ERK1/2, which is a known pathway activated by ACKR3. Cells expressing ACKR3 are treated with CCX777, and the level of phosphorylated ERK (p-ERK) is measured by Western blotting or ELISA. This confirms that the β-arrestin recruitment leads to functional signaling. Another cell-based assay is the assessment of receptor internalization. ACKR3 is known to internalize upon activation. Cells expressing a fluorescently tagged ACKR3 can be treated with CCX777, and the receptor's localization can be visualized using confocal microscopy. The degree of internalization can be quantified by measuring the fluorescence in the cytoplasm versus the plasma membrane. Additionally, cell proliferation or migration assays can be performed to study the functional effects of ACKR3 modulation in cancer cells. In these assays, cancer cell lines that express ACKR3 are treated with CCX777, and their proliferation rate or migratory ability is measured. These cell-based assays are crucial for understanding the biological significance of CCX777's partial agonist activity on ACKR3.
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| Animal Protocol |
Specific in vivo animal protocols for CCX777 are not detailed in standard product descriptions. As a research compound, its in vivo use would be determined by the specific research question being addressed. A typical protocol for studying a compound like CCX777 in vivo would involve its administration to animal models of disease, such as xenograft models for cancer. In such a study, immunodeficient mice would be implanted with human cancer cells that express ACKR3. Once tumors are established, the mice would be randomized into treatment groups. CCX777 would be formulated for injection, likely using a vehicle that includes DMSO (in which it is soluble at 50 mg/mL), PEG, and saline. The compound would be administered via intraperitoneal (i.p.) or intravenous (i.v.) injection at a predetermined dose and schedule. Tumor growth would be monitored by caliper measurements. At the end of the study, tumors would be harvested for analysis of signaling pathways and cell proliferation markers. Other in vivo models could include inflammation models, given the role of chemokine receptors in immune cell trafficking. However, because CCX777 is a partial agonist of β-arrestin-2 recruitment, the specific outcomes would be highly dependent on the model and the role of ACKR3 in that context. The compound's stability for at least two years at -20°C is a key consideration for long-term studies.
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for CCX777 is not provided in standard product descriptions. As a research compound, its PK profile would need to be determined experimentally. Some basic properties can be inferred from its chemical structure. CCX777 has a molecular weight of 593.79 g/mol and a molecular formula of C31H43N7O3S, indicating it is a relatively large, lipophilic molecule with a significant number of heteroatoms. It is soluble in DMSO at 50 mg/mL, but its aqueous solubility is not specified. For in vivo administration, it would need to be formulated with co-solvents and surfactants. Its logP value is not listed, but its structure suggests it may have moderate to high lipophilicity, which could lead to extensive plasma protein binding and a long half-life. For storage, it is recommended to keep the compound at -20°C, where it is stable for at least two years. It is shipped with ice packs to maintain its stability during transit. A comprehensive PK study would involve administering CCX777 to rodents via both intravenous and oral routes, collecting blood samples at various time points, and analyzing plasma concentrations using LC-MS/MS to determine key parameters such as half-life, clearance, volume of distribution, and oral bioavailability. Such data is essential for any in vivo study to ensure proper dosing and interpretation of results.
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| Toxicity/Toxicokinetics |
Toxicological data for CCX777 is not provided in standard product descriptions. As a research compound intended for in vitro use, its toxicity profile has not been extensively characterized. The compound is classified as a small-molecule partial agonist of ACKR3, and its effects are expected to be mediated through its interaction with this specific receptor. The safety of CCX777 would depend on the role of ACKR3 in normal tissues and the potential for off-target effects. ACKR3 is expressed in various tissues, including the brain, heart, and immune cells, and its modulation could have physiological consequences. For example, because ACKR3 is a scavenger receptor for CXCL12, its partial agonism could affect the levels of this important chemokine, potentially impacting stem cell mobilization or immune cell trafficking. However, no specific toxicological data, such as LD50, organ toxicity, or genotoxicity, is available. When handling CCX777, standard laboratory safety precautions should be followed, including the use of personal protective equipment (PPE) and working in a well-ventilated area. Any in vivo use would require prior toxicological assessment to determine a safe and effective dose range.
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| References | |
| Additional Infomation |
CCX777 is a research compound and is not approved for any clinical use. It is a small-molecule partial agonist that promotes the recruitment of β-arrestin-2 to the atypical chemokine receptor 3 (ACKR3/CXCR7). Its mechanism of action is unique in that it specifically targets the β-arrestin pathway of ACKR3, allowing researchers to study the non-canonical signaling of this receptor in isolation from G protein-dependent pathways. ACKR3 is an important receptor in cancer biology, as it is overexpressed in many tumors and is involved in cell survival, proliferation, and metastasis. CCX777 is therefore a valuable tool for studying the role of β-arrestin-mediated ACKR3 signaling in cancer. It is also used to study the receptor's role in inflammation and stem cell biology. The compound is available for research purposes only and is not intended for human or animal therapeutic use. Its high purity (>98% by HPLC) and defined chemical properties make it a reliable reagent for mechanistic studies. CCX777 is typically stored at -20°C and is stable for at least two years, ensuring its utility in long-term research projects.
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| Molecular Formula |
C31H43N7O3S
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| Molecular Weight |
593.783225297928
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| Exact Mass |
593.314
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| CAS # |
1226686-36-7
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| PubChem CID |
59366167
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.9
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
42
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| Complexity |
845
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(N2CCOCC2)=NC(=C1)C(CC(NCCN1CCCC1)=O)N1CCN(C2=C(C=CC3=CC=CN=C23)OC)CCC1
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| InChi Key |
PUPJAMOHYJWOKK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H43N7O3S/c1-40-27-8-7-24-6-4-9-33-29(24)30(27)37-14-5-13-36(16-17-37)26(22-28(39)32-10-15-35-11-2-3-12-35)25-23-42-31(34-25)38-18-20-41-21-19-38/h4,6-9,23,26H,2-3,5,10-22H2,1H3,(H,32,39)
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| Chemical Name |
3-[4-(7-methoxyquinolin-8-yl)-1,4-diazepan-1-yl]-3-(2-morpholin-4-yl-1,3-thiazol-4-yl)-N-(2-pyrrolidin-1-ylethyl)propanamide
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| Synonyms |
CCX 777CCX777 CCX-777
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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) |
DMSO : ~50 mg/mL (~84.21 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.21 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.21 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6841 mL | 8.4206 mL | 16.8413 mL | |
| 5 mM | 0.3368 mL | 1.6841 mL | 3.3683 mL | |
| 10 mM | 0.1684 mL | 0.8421 mL | 1.6841 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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