Herdon H J, Godfrey F M, Chan W N
Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, CM19 5AW, Essex, UK.
Neuropharmacology. 2000 Sep;39(12):2457-63. doi: 10.1016/s0028-3908(00)00071-x.
SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [(3)H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [(125)I]-labelled form of SB-217644 (trans-6-acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [(125)I]-SB-217644 shows a similar binding profile to that of [(3)H]-SB-204269, with a maximum specific binding capacity (B(max)) of 286+/-12 fmol/mg protein, but has twenty-fold higher affinity (K(d) value 1.7+/-0.1 nM). The high affinity and high specific activity of [(125)I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [(125)I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K(d) value of 2.7+/-0.3 nM and a B(max) value of 55+/-11 fmol/mg protein, with a 7.3+/-0.3% yield of solubilised binding sites. [(125)I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [(125)I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [(125)I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.
SB - 204269（反式 - （+） - 6 - 乙酰基 - 4S - （4 - 氟苯甲酰氨基） - 3,4 - 二氢 - 2,2 - 二甲基 - 2H - 苯并[b]吡喃 - 3R - 醇）在一系列动物癫痫模型中显示出抗惊厥活性，具有高治疗指数且无副作用。我们之前报道了大鼠前脑中[(3)H] - SB - 204269的一个新型结合位点的特性，该位点具有与其他已知抗惊厥作用位点无关的独特特征。我们现在描述使用SB - 204269的类似物[(125)I]标记的SB - 217644（反式 - 6 - 乙酰基 - 4S - （3 - 碘苯甲酰氨基） - 3,4 - 二氢 - 2,2 - 二甲基 - 2H - 苯并[b]吡喃 - 3R - 醇）来研究这个新型结合位点。在大鼠前脑细胞膜中，[(125)I] - SB - 217644显示出与[(3)H] - SB - 204269相似的结合特征，最大特异性结合容量（B(max)）为286±12 fmol/mg蛋白质，但亲和力高二十倍（K(d)值为1.7±0.1 nM）。[(125)I] - SB - 217644的高亲和力和高比活性使其可用于检测和表征结合位点的去污剂增溶形式。[(125)I] - SB - 217644与胆酸盐增溶的大鼠小脑的特异性结合显示K(d)值为2.7±0.3 nM，B(max)值为55±11 fmol/mg蛋白质，增溶结合位点的产率为7.3±0.3%。[(125)I] - SB - 217644还用于全细胞结合测定，以研究一系列细胞系中新型结合位点的特性。发现大鼠脑神经元和神经胶质原代培养物以及几种中枢神经系统相关细胞系中[(125)I] - SB - 217644的特异性结合水平与大鼠前脑细胞膜中的相似。这个新型结合位点的增溶，以及使用[(125)I] - SB217644在增溶组织和全细胞中对其进行定量和表征的能力，将有助于进一步研究以最终确定SB - 204269的分子靶点。
