Marchant J S, Beecroft M D, Riley A M, Jenkins D J, Marwood R D, Taylor C W, Potter B V
Department of Pharmacology, Tennis Court Road, University of Cambridge, Cambridge, CB2 1QJ U.K.
Biochemistry. 1997 Oct 21;36(42):12780-90. doi: 10.1021/bi971397v.
The glyconucleotides adenophostin A and B are the most potent known agonists at type 1 inositol trisphosphate [Ins(1,4,5)P3] receptors, although their stuctures differ markedly from that of Ins(1,4,5)P3. Equilibrium competition binding with [3H]Ins(1,4,5)P3 and unidirectional 45Ca2+ flux measurements were used to examine the effects of adenophostin A in hepatocytes, which express predominantly type 2 Ins(1,4,5)P3 receptors. Both Ins(1,4,5)P3 (Kd = 8.65 +/- 0.98 nM) and adenophostin A (Kd = 0.87 +/- 0.20 nM) bound to a single class of [3H]Ins(1,4,5)P3-binding site and each fully mobilized the same intracellular Ca2+ pool; although, adenophostin A (EC50 = 10.9 +/- 0.7 nM) was more potent than Ins(1,4,5)P3 (EC50 = 153 +/- 11 nM). Working on the assumption that it is the phosphorylated glucose component of the adenophostins that mimics the critical features of Ins(1,4,5)P3, we synthesized various phosphorylated disaccharide analogs containing this structure. The novel disaccharide-based analogs, sucrose 3,4,3'-trisphosphate [Sucr(3,4,3')P3], alpha,alpha'-trehalose 3,4,3',4'-tetrakisphosphate [Trehal(3,4,3',4')P4], alpha,alpha'-trehalose 2,4,3', 4'-tetrakisphosphate [Trehal(2,4,3',4')P4], and methyl 3-O-(alpha-d-glucopyranosyl)-beta-d-ribofuranoside 2,3', 4'-trisphosphate [Rib(2,3',4')P3], were all able to mobilize the same intracellular Ca2+ pool as Ins(1,4,5)P3 and adenophostin A; although, none was as potent as adenophostin A. The rank order of potency of the analogs, adenophostin A > Ins(1,4,5)P3 approximately Rib(2,3',4')P3 > Trehal(2,4,3',4')P4 > Glc(2',3,4)P3 approximately Trehal(3,4,3',4')P4 > Sucr(3,4,3')P3, was the same in radioligand binding and functional assays of hepatic Ins(1,4,5)P3 receptors. Both Rib(2,3',4')P3, which was as potent as Ins(1,4,5)P3, and Trehal(2,4,3',4')P4 bound with significantly higher affinity ( approximately 27 and approximately 3-fold, respectively) than the only active carbohydrate agonist of Ins(1,4,5)P3 receptors previously examined [Glc(2',3,4)P3]. We conclude that phosphorylated disaccharides provide novel means of developing high-affinity ligands of Ins(1,4,5)P3 receptors.
糖核苷酸腺嘌呤宿主素A和B是已知对1型肌醇三磷酸[Ins(1,4,5)P3]受体最有效的激动剂，尽管它们的结构与Ins(1,4,5)P3明显不同。利用与[3H]Ins(1,4,5)P3的平衡竞争结合和单向45Ca2+通量测量来研究腺嘌呤宿主素A对主要表达2型Ins(1,4,5)P3受体的肝细胞的影响。Ins(1,4,5)P3（Kd = 8.65 +/- 0.98 nM）和腺嘌呤宿主素A（Kd = 0.87 +/- 0.20 nM）都与一类[3H]Ins(1,4,5)P3结合位点结合，并且各自完全动员相同的细胞内Ca2+池；尽管如此，腺嘌呤宿主素A（EC50 = 10.9 +/- 0.7 nM）比Ins(1,4,5)P3（EC50 = 153 +/- 11 nM）更有效。基于腺嘌呤宿主素的磷酸化葡萄糖成分模拟Ins(1,4,5)P3的关键特征这一假设，我们合成了各种含有该结构的磷酸化二糖类似物。新型的基于二糖的类似物，蔗糖3,4,3'-三磷酸[Sucr(3,4,3')P3]、α,α'-海藻糖3,4,3',4'-四磷酸[Trehal(3,4,3',4')P4]、α,α'-海藻糖2,4,3',4'-四磷酸[Trehal(2,4,3',4')P4]以及甲基3-O-(α-D-吡喃葡萄糖基)-β-D-呋喃核糖苷2,3',4'-三磷酸[Rib(2,3',4')P3]，都能够像Ins(1,4,5)P3和腺嘌呤宿主素A一样动员相同的细胞内Ca2+池；尽管如此，没有一种像腺嘌呤宿主素A那样有效。在肝脏Ins(1,4,5)P3受体的放射性配体结合和功能测定中，类似物的效力顺序为：腺嘌呤宿主素A > Ins(1,4,5)P3 ≈ Rib(2,3',4')P3 > Trehal(2,4,3',4')P4 > Glc(2',3,4)P3 ≈ Trehal(3,4,3',4')P4 > Sucr(3,4,3')P3。与之前研究过的Ins(1,4,5)P3受体唯一的活性碳水化合物激动剂[Glc(2',3,4)P3]相比，效力与Ins(1,4,5)P3相当的Rib(2,3',4')P3和Trehal(2,4,3',4')P4的结合亲和力分别显著更高（分别约为27倍和约3倍）。我们得出结论，磷酸化二糖为开发Ins(1,4,5)P3受体的高亲和力配体提供了新方法。
