Young P W, Buckle D R, Cantello B C, Chapman H, Clapham J C, Coyle P J, Haigh D, Hindley R M, Holder J C, Kallender H, Latter A J, Lawrie K W, Mossakowska D, Murphy G J, Roxbee Cox L, Smith S A
SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK.
J Pharmacol Exp Ther. 1998 Feb;284(2):751-9.
A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.
一种放射性碘化配体，[125I]SB - 236636 [（S）-（-）3 - [4 - [2 - [N - （2 - 苯并恶唑基）-N - 甲基氨基]乙氧基] - 3 - [125I]碘苯基] - 2 - 乙氧基丙酸]，被研发出来，它对过氧化物酶体增殖物激活受体（PPARγ）的γ亚型具有特异性。[125I]SB - 236636与全长人重组PPARγ1以及含有人类PPARγ1配体结合域的谷胱甘肽S - 转移酶（GST）融合蛋白具有高亲和力结合（KD = 70 nM）。使用这种配体，我们对来自大鼠、小鼠和人类的脂肪来源细胞中的结合位点进行了表征。在竞争实验中，强效抗高血糖药物罗格列酮（BRL - 49653）与完整脂肪细胞中的位点具有高亲和力结合（大鼠、3T3 - L1和人类脂肪细胞的IC50分别为12、4和9 nM）。其他噻唑烷二酮类药物对PPARγ1配体结合域的结合亲和力（IC50）与在脂肪细胞中测定的结果相当，并反映了这些药物作为3T3 - L1脂肪细胞中葡萄糖转运刺激剂和体内抗高血糖药物的效力顺序：罗格列酮>吡格列酮>曲格列酮。[125I]SB - 236636结合的竞争具有立体选择性，因为α - 三氟乙氧基丙酸胰岛素增敏剂的（S） - 对映体SB - 219994在重组人PPARγ1上的IC50值比（R） - 对映体SB - 219993低770倍。SB - 219994在完整脂肪细胞中也表现出更高的结合亲和力，这反映了其作为抗糖尿病药物的效力高100倍。结果强烈表明，完整脂肪细胞中[125I]SB - 236636的高亲和力结合位点是PPARγ，并且啮齿动物和人类脂肪细胞中胰岛素增敏剂结合的药理学非常相似，而且可以预测体内抗高血糖活性。
