Wan Yuntao, Jakway James P, Qiu Hongchen, Shah Himanshu, Garlisi Charles G, Tian Fang, Ting Pauline, Hesk David, Egan R W, Billah M Motasim, Umland Shelby P
Department of Allergy, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Eur J Pharmacol. 2002 Dec 5;456(1-3):1-10. doi: 10.1016/s0014-2999(02)02621-3.
Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.
对CC趋化因子受体3（CCR3）的研究一直局限于使用放射性标记的激动剂趋化因子。一种小分子CCR3拮抗剂，2-[(6-氨基-2-苯并噻唑基)硫代]-N-[1-[(3,4-二氯苯基)甲基]-4-哌啶基]乙酰胺，半羽（I），被氚标记并用于药理学研究。半羽（I）对人CCR3转染细胞和嗜酸性粒细胞的解离常数（K(d)）分别为5.0±0.4和4.3±1.8 nM，并且非竞争性抑制[125I]嗜酸性粒细胞趋化因子结合以及嗜酸性粒细胞趋化因子诱导的[35S]鸟苷-5'-O-(3-硫代三磷酸)（[35S]GTPγS）结合。在嗜酸性粒细胞或转染细胞中，[125I]嗜酸性粒细胞趋化因子：[3H]半羽（I）结合位点的比例分别为35%或13%，尽管两种结合位点在转染细胞中均有过表达。在降低的NaCl和GDP浓度下，通过基础和嗜酸性粒细胞趋化因子诱导的[35S]GTPγS结合的变化证明，CCR3在CCR3转染细胞中自发地与G蛋白偶联。因此，半羽（I）被鉴定为反向激动剂。相比之下，CCL18和I-TAC（干扰素诱导的T细胞α趋化因子）是中性拮抗剂，抑制嗜酸性粒细胞趋化因子诱导的[35S]GTPγS结合，对CCR3与G蛋白的基础偶联影响最小。嗜酸性粒细胞趋化因子、嗜酸性粒细胞趋化因子-2和单核细胞趋化蛋白（MCP）-4是诱导[35S]GTPγS结合的完全激动剂；嗜酸性粒细胞趋化因子-3、MCP-3、调节激活正常T细胞表达和分泌的因子（RANTES）、病毒巨噬细胞炎性蛋白-I（vMIP-I，卡波西肉瘤相关疱疹病毒巨噬细胞炎性蛋白-）和vMIP-II是部分激动剂，表明这是一种定量激动剂效力的灵敏方法。
