Milligan G, Kellett E, Dacquet C, Dubreuil V, Jacoby E, Millan M J, Lavielle G, Spedding M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, G12 8QQ, Glasgow, UK.
Neuropharmacology. 2001 Mar;40(3):334-44. doi: 10.1016/s0028-3908(00)00162-3.
S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.
S 14506在化学结构上与5-HT(1A)受体反向激动剂螺哌隆相关，但S 14506在体外和体内均表现为这些受体已知的最有效激动剂之一。在海马体膜中，[(3)H]-S 14506（K(d)=0.79±0.2 nM；B(max)=400±32 fmol/mg蛋白质）与5-HT(1A)受体的特异性结合类似于拮抗剂，因为它会被鸟苷-5'-O-(3-硫代三磷酸)（GppNHp）增强，而GppNHp会减少经典激动剂[(3)H]-8-羟基二丙胺基四氢萘（[(3)H]-8-OH-DPAT）（K(d)=1.5±0.5 nM；B(max)=303±20 fmol/mg蛋白质）的结合。锰、镁和钙会减少[(3)H]-S 14506与5-HT(1A)受体的结合，而[(3)H]-8-OH-DPAT的结合会增加。此外，钠会显著减少[(3)H]-8-OH-DPAT的结合，而不影响[(3)H]-S 14506的结合。[(3)H]-S 14506也以高亲和力与稳定表达于中国仓鼠卵巢（CHO）细胞膜中的人5-HT(1A)受体结合（K(d)=0.13±0.05 nM；B(max)=2.99±0.60 pmol/mg蛋白质）：B(max)是[(3)H]-8-OH-DPAT的两倍。GppNHp强烈降低[(3)H]-8-OH-DPAT的结合，但几乎不改变[(3)H]-S 14506的结合；钙、镁和锰对CHO细胞中[(3)H]-S 14506的结合影响很小。拮抗剂（WAY 100635、WAY 100135）和反向激动剂（螺哌隆和米氮平）以高亲和力取代[(3)H]-S 14506的结合，希尔斜率接近1，而激动剂（5-羟色胺和5-羧色胺）显示出低亲和力和低希尔斜率：部分激动剂（丁螺环酮、伊沙匹隆）表现出中间特性。在人5-HT(1A)受体与G(ialpha1)的融合蛋白中，该化合物能有效增加高亲和力GTP酶，其希尔斜率比5-羟色胺更陡，这可能表明存在正协同性。在这些实验中，S 14506的最大反应与5-羟色胺相当，表明它是一种完全激动剂。在分子建模研究中，使用5-HT(1A)受体的三位点模型，S 14506跨越5-羟色胺识别位点和假定可激活受体与G蛋白相互作用的“精氨酸开关”（DRY微结构域）。因此，有可能在G蛋白偶联受体上合成与反向激动剂在结构上相关但具有高效激动剂活性且表现出一些拮抗剂/反向激动剂结合特征的配体。
