An antagonist of CCR2
INCB3344 is a novel potent and selective antagonist of CCR2 receptor, which possesses an IC 50 of 10 nM for CCL2. [1]
CCR2 is a chemokine receptor mainly expressed on monocytes which acts as the key receptor in mediating their tissue influx in the context of immune-based inflammation. CCR2 is a G protein-coupled receptor (GPCR), whose ligands include the chemokines MCP family, including CCL2, CCL7, CCL8. These ligands bind CCR2 receptor with high affinity and elicit a chemotactic signal which leads to directed migration of the receptor-bearing cells. CCL2 has been shown to be relevant in high concentrations in various inflammatory lesions, implicating this chemokine as a physiologically important chemotactic signal for monocytes. [1]
Characterization of the pharmacological activity of INCB3344 was first evaluated by testing its ability to inhibit CCL2 binding to CCR2 in a whole cell binding assay using a murine monocyte cell line, WEHI-274.1 and 125I-labeled mCCL2 as a tracer. The binding IC 50 of INCB3344 in this assay was determined to be 10±5 nM, and inhibition greater than 90% binding was observed at a concentration of 90nM . The chemotaxis inhibitory activity of different concentrations of INCB3344 was evaluated using 30nM mCCL2 as the agonist. The result showed a similar potency to the binding assay. Selectivity of INCB3344 was evaluated against a panel of GPCRs including several human chemokine receptors using radioligand binding assays. Results from these studies demonstrate at least 100-fold selectivity of INCB3344 against all of the receptors tested. [1]
INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse delayed-type hypersensitivity model. The histopathological analysis of tissues from the delayed-type hypersensitivity model illustrates that inhibitory activity of CCR2 leads to a substantial reduction in tissue inflammation. [1]
Reference:
[1].? Brodmerkel C M, Huber R, Covington M, et al. Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344[J]. The Journal of Immunology, 2005, 175(8): 5370-5378.
| Kinase experiment [1]: | |
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Binding assays |
WEHI 274.1 (murine monocytic cell line) cells were used in a whole cell binding assay. Cells (5×105) inRPMI 1640, 0.1% BSA, 20 mM HEPES were added to various concentrations of INCB3344 in RPMI 1640 followed immediately by the addition of 150 pM 125I-labeled mCCL2 (mouse CCL2(JE)) and incubated for 30 min at room temperature (RT). For the nonspecific control, 0.3 μM mCCL2 was added in place of INCB3344. Cells were then harvested through 1.2-μM polyvinylidene difluoride filters, the filters were airdried, and binding was determined by counting in a gamma counter. Antagonist activity is reported as the inhibitor concentration required for IC50 of specific binding. Specific binding is defined as the total binding minus the nonspecific binding and typically represents 97% of the total binding. |
| Cell experiment [1]: | |
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Cell lines |
WEHI-274.1 cells |
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Preparation method |
Dissolved in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months. |
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Reaction Conditions |
0.3-300 nM; 6 min. |
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Applications |
In WEHI-274.1 cells, INCB3344 inhibits monocyte chemotaxis with IC50 value of 10 nM using 30 nM mCCL2 as the agonist. INCB3344 blocks ERK phosphorylation in response to mCCL2 with IC50 value of 3-10 nM, which is mediated by CCR2 signaling. |
| Animal experiment [1]: | |
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Animal models |
Female BALB/c mice (20 g) received i.p. injection of thioglycolate. |
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Dosage form |
30, 60, or 100 mg/kg BID; 48 h; administrated orally. |
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Applications |
INCB3344 dose-dependently reduces total cell number in the lavage fluid and inhibits monocyte influx by 36%, 55% and 73% at 30 mg/kg, 60 mg/kg and 100 mg/kg, respectively. |
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Other notes |
Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal. |
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参考文献: [1]. Brodmerkel CM, Huber R, Covington M, et al. Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344[J]. The Journal of Immunology, 2005, 175(8): 5370-5378. | |
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