CCR2-RA-[R]
(Synonyms: (5R)-4-乙酰基-1-(4-氯-2-氟苯基)-5-环己基-1,5-二氢-3-羟基-2H-吡咯-2-酮) 目录号 : GC31714
CCR2-RA -[R]能以非竞争性结合的方式抑制CCR2，主要是通过阻断激活相关的构象变化和G蛋白结合界面的形成。
![CCR2-RA-[R] Chemical Structure](/media/struct/GC3/GC31714.png "CCR2-RA-[R] Chemical Structure")
Cas No.:512177-83-2
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Kinase experiment [1]: | |
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Preparation Method |
For dissociation experiments, the membranes were first incubated with 3 nM CCR2-RA-[R]CCR2-RA-[R] for 90 minutes. Dissociation was initiated by the addition of 10 mM of JNJ-27141491 at different time points. |
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Reaction Conditions |
3 nM [3H]CCR2-RA-[R] for 90 minutes |
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Applications |
CCR2-RA-[R] had IC50 values of 103 + 18. Saturation binding experiments yielded a Kp of 5.8 + 0.2 nM with a Bmax of 9.7 + 0.2 pmol/mg. |
| Cell experiment [1]: | |
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Cell lines |
U2OS-CCR2 cells |
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Preparation Method |
For the antagonist assays, cells were first preincubated for 30 minutes with increasing concentrations of antagonist(including CCR2-RA-[R]) or vehicle control that was added in 5 ul of compound solution (final concentration of 0.25% dimethylsulfoxide).Subsequently, cells were stimulated with an ECso concentration (3 nM) of CCL2. |
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Reaction Conditions |
Increasing concentrations of the antagonists(including CCR2-RA-[R] were added 30 minutes before agonist stimulation to determine their IC 50 value.( No specific concentration) |
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Applications |
INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA-[R] showed a noncompetitive or allosteric mode of inhibition. |
| Animal experiment [2]: | |
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Animal models |
Adult female Sprague-Dawley rats (150-200 g) |
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Preparation Method |
CCR2-RA-[R] were given a one-time intraperitoneal (i.p.) injection one hour prior to behavioral testing. |
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Dosage form |
10 mg/kg CCR2-RA-[R] for 1 time |
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Applications |
CCR2-RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia |
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References: [1]. Zweemer AJ, Nederpelt I, ,et,al. Multiple binding sites for small-molecule antagonists at the CC chemokine receptor 2. Mol Pharmacol. 2013 Oct;84(4):551-61. doi: 10.1124/mol.113.086850. Epub 2013 Jul 22. PMID: 23877010. | |
CCR2-RA -[R] can inhibit CCR2 in a non-competitive binding manner, mainly by blocking activation-related conformational changes and the formation of G-protein binding interfaces[1,2]. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII[3].
CCR2-RA-[R] had IC50 values of 103 nM.Saturation binding experiments yielded a Kp of 5.8 + 0.2 nM with a Bmax of 9.7 + 0.2 pmol/mg.CCR2-RA-[R] clearly showed noncompetitive antagonism for CCR2 with respect to CCL2, as indicated by a decrease in CCL2's efficacy in the presence of increasing concentrations of CCR2RA-[R][1].
Focal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia[4]. CCR2i CCR2-RA-[R] (MCP-1 receptor inhibitor) were administered via intraperitoneal injection. With or without PDX, treatment with CCR2-RA-[R] reduced the number of neutrophils and recruited macrophages, indicating that the basic function of PDX was disappeared after using CCR2 inhibitors(CCR2-RA-[R])[5]. CCR2 RA-[R] enhances the response to ¨?PD-1 by promoting the counts of progenitor Tex[6]. Compared to the model group, the fluorescence intensity of spleen was markedly decreased by Pirfenidone and CCR2-RA-[R] Moreover, Compared to the model group, the fluorescence intensity of liver metastasis foci was dramatically reduced by Pirfenidone and CCR2-RA-[R][7].
References:
[1]. Zweemer AJ, Nederpelt I,et,al. Multiple binding sites for small-molecule antagonists at the CC chemokine receptor 2. Mol Pharmacol. 2013 Oct;84(4):551-61. doi: 10.1124/mol.113.086850. Epub 2013 Jul 22. PMID: 23877010.
[2]. Zheng Y, Qin L, ,et,al.Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature. 2016 Dec 15;540(7633):458-461. doi: 10.1038/nature20605. Epub 2016 Dec 7. PMID: 27926736; PMCID: PMC5159191.
[3]. Zweemer AJ, Bunnik J, ,et,al.Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2. Mol Pharmacol. 2014 Oct;86(4):358-68. doi: 10.1124/mol.114.093328. Epub 2014 Jul 14. PMID: 25024169.
[4]. Bhangoo S, Ren D, ,et,al. Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors. Mol Pain. 2007 Dec 12;3:38. doi: 10.1186/1744-8069-3-38. PMID: 18076762; PMCID: PMC2228278.
[5]. Ye Y, Zhang HW, et,al. PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury. J Cell Mol Med. 2020 Sep;24(18):10604-10614. doi: 10.1111/jcmm.15679. Epub 2020 Jul 31. PMID: 32735065; PMCID: PMC7521295.
[6]. Yihua Xu, Hao Wang, et,al. DFB Suppresses Obesity-Driven CRC Via Restricting Progenitor to Terminally Exhausted T Cell Differentiation, 19 October 2021, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-952538/v1]
[7]. Chen C, Yao X, et,al. Dahuang Zhechong Pill suppresses colorectal cancer liver metastasis via ameliorating exosomal CCL2 primed pre-metastatic niche. J Ethnopharmacol. 2019 Jun 28;238:111878. doi: 10.1016/j.jep.2019.111878. Epub 2019 Apr 13. PMID: 30986521.
CCR2-RA -[R]能以非竞争性结合的方式抑制CCR2，主要是通过阻断激活相关的构象变化和G蛋白结合界面的形成[1,2] .对于 CCR2-RA-[R]，发现最重要的结合残基是 NPxxYx(5,6)F 基序的高度保守的酪氨酸 Y(7.53) 和苯丙氨酸 F(8.50)，以及 V(6.36) TM-VI 底部和螺旋-VIII 中的 K(8.49)[3].
CCR2-RA-[R] 的 IC50 值为 103 nM。饱和结合实验产生的 Kp 为 5.8 + 0.2 nM，Bmax 为 9.7 + 0.2 pmol/mg。CCR2-RA-[R] 清楚地显示出非竞争性拮抗作用CCR2 相对于 CCL2 的变化，如在 CCR2RA-[R][1] 浓度增加的情况下 CCL2 的功效降低所示。
局灶性神经脱髓鞘增加了神经损伤同侧和对侧后爪在术后第 3 天和 POD28 之间对机械刺激的行为反射反应。 CCR2 RA-[R] 对神经损伤大鼠的治疗产生了触觉痛觉过敏的立体特异性双侧逆转 [4]。 CCR2i CCR2-RA-[R]（MCP-1 受体抑制剂）通过腹膜内注射给药。有或没有 PDX，CCR2-RA-[R] 治疗减少了中性粒细胞的数量并募集了巨噬细胞，表明在使用 CCR2 抑制剂（CCR2-RA-[R]）后 PDX 的基本功能消失了[5 ]。 CCR2 RA-[R] 通过促进祖细胞 Tex[6] 的计数来增强对 ¨»PD-1 的反应。与模型组相比，吡非尼酮和CCR2-RA-[R]脾脏荧光强度显着降低；此外，与模型组相比，吡非尼酮和CCR2-RA-[R]肝转移灶荧光强度显着降低。 R][7].
| Cas No. | 512177-83-2 | SDF | |
| 别名 | (5R)-4-乙酰基-1-(4-氯-2-氟苯基)-5-环己基-1,5-二氢-3-羟基-2H-吡咯-2-酮 | ||
| Canonical SMILES | O=C1C(O)=C(C(C)=O)[C@@H](C2CCCCC2)N1C(C(F)=C3)=CC=C3Cl | ||
| 分子式 | C18H19ClFNO3 | 分子量 | 351.8 |
| 溶解度 | DMSO : ≥ 125 mg/mL (355.32 mM);Water : < 0.1 mg/mL (insoluble) | 储存条件 | Store at -20°C |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.8425 mL | 14.2126 mL | 28.4252 mL |
| 5 mM | 0.5685 mL | 2.8425 mL | 5.685 mL |
| 10 mM | 0.2843 mL | 1.4213 mL | 2.8425 mL |
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动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
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| % DMSO % % Tween 80 % saline | ||||||||||
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2.
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Revealing the Positive Binding Cooperativity Mechanism between the Orthosteric and the Allosteric Antagonists of CCR2 by Metadynamics and Gaussian Accelerated Molecular Dynamics Simulations
ACS Chem Neurosci2020 Feb 19;11(4):628-637.PMID: 31968162DOI: 10.1021/acschemneuro.9b00630
CC chemokine receptor 2 (CCR2) and its endogenous CC chemokine ligands are associated with numerous inflammatory, neurodegenerative diseases, and cancer. CCR2 is becoming an attractive target in the treatment of autoimmune disease and neurodegenerative diseases. The orthosteric antagonist BMS-681 and allosteric antagonist CCR2-RA-[R] of CCR2 show positive binding cooperativity. We performed well-tempered metadynamics simulations and Gaussian accelerated MD simulations to reveal the influence of the orthosteric antagonist on the unbinding of allosteric antagonist of CCR2. We revealed different unbinding pathways of CCR2-RA-[R] in binary complex CCR2-VT5 and ternary complex CCR2-73R-VT5. The different unbinding pathways of CCR2-RA-[R] are due to the conformational dynamics of TM6. We obtained the significant conformational differences of the intracellular side of TM6 upon CCR2 binding to different ligands by GaMD simulation. The conformational dynamics of TM6 are consistent with the unbinding pathway analysis. GaMD simulations indicate that BMS-681 binding restricts the bend of intracellular side of TM6 by stabilizing the extracellular sides of TM6 and TM7. The charged residues Arg2065.43 of TM5 and Glu2917.39 of TM7 play key roles in stabling TM7 and TM6. TM6 and TM7 are crucial components in the orthosteric and allosteric binding sites. Our results illustrate the conformational details about the effect of the orthosteric antagonist on the allosteric antagonist of CCR2. The conformational dynamics of CCR2 upon binding to different ligands can provide a rational basis for development of allosteric ligands of CCR2.
Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists
Nature2016 Dec 15;540(7633):458-461.PMID: 27926736DOI: 10.1038/nature20605
CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.
Multiple binding sites for small-molecule antagonists at the CC chemokine receptor 2
Mol Pharmacol2013 Oct;84(4):551-61.PMID: 23877010DOI: 10.1124/mol.113.086850
The chemokine receptor CCR2 is a G protein-coupled receptor that is activated primarily by the endogenous CC chemokine ligand 2 (CCL2). Many different small-molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all these antagonists lack clinical efficacy, and therefore a better understanding of their mechanism of action is warranted. In this study, we examined the pharmacological properties of small-molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist (125)I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 [N-(2-(((3S,4S)-1-((1r,4S)-4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide] and CCR2-RA, were radiolabeled to study the binding site in greater detail. We discovered that [(3)H]INCB3344 and [(3)H]CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we examined CCR2 inhibition in multiple functional assays, including a novel label-free whole-cell assay. INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA showed a noncompetitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified into two groups with different binding sites and thereby different modes of inhibition. We have provided further insights in CCR2 antagonism, and these insights are important for the development of novel CCR2 inhibitors.
Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2
Mol Pharmacol2014 Oct;86(4):358-68.PMID: 25024169DOI: 10.1124/mol.114.093328
The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.
Dahuang Zhechong Pill suppresses colorectal cancer liver metastasis via ameliorating exosomal CCL2 primed pre-metastatic niche
J Ethnopharmacol2019 Jun 28;238:111878.PMID: 30986521DOI: 10.1016/j.jep.2019.111878
Ethnopharmacological relevance: Dahuang Zhechong Pill (DZP) is a classical formula from "Synopsis of Prescriptions of the Golden Chamber". It has been used for treatment of abdominal masses (including tumorous diseases) for thousands of years.
Aim of the study: Our previous work showed that DZP suppresses CCl-4 induced hepatic fibrosis by downregulating the expression of interleukin-13. We aimed to test if DZP suppresses the metastasis of colorectal cancer (CRC) by ameliorating the fibrosis status of the future metastatic organ.
Materials and methods: Liver metastasis was observed by injection of MC38-EGFP cells with stably expressing enhanced green fuorescence protein beneath the splenic capsule of C57BL/6J mice. MC38-EGFP-derived exosomes were analyzed by Label-free comparative proteomics. mRNA expression was determined by Quantitative PCR. Protein expression was determined by immunohistochemistry, immunofuorescence and Western blot. Collagen deposition was determined by Masson staining. All data were statistically analyzed using SPSS.
Results: DZP drastically reduced the metastatic tumor number and fluorescence intensity in a splenic liver metastasis model. It also lowered the expression of mature TGF-¦± and decreased the fibronectin contents & collagen deposition. Exosome proteomics showed that the upregualted CC chemokine ligand-2 (CCL2) was repressed by DZP treatment. Importantly, DZP markedly lowered the expression of CCL2 and its receptor CCR2 in the liver. Exosomal CCL2 activated macrophage recruitment and shifted the M1/M2 paradigm to a M2 phenotype. DZP reduced the macrophage infiltration and attenuated the M2 polarizaion in tumor-bearing mice liver. It decreased the F4/80 positive areas and specifically reduced the ratio of CCR2+ positive macrophage. Anti-fibrosis and inhibition of CCR2 suppress the growth and metastasis of CRC.
Conclusions: DZP inhibits the liver metastasis of CRC by suppressing CCL2 mediated M2-skewing paradigm and ameliorating the pro-fibrotic microenvironment.