Disaccharide polyphosphates based upon adenophostin A activate hepatic D-myo-inositol 1,4,5-trisphosphate receptors

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 (K(d) = 8.65 ± 0.98 nM) and adenophostin A (K(d) = 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], α,α'-trehalose 3,4,3',4'- tetrakisphosphate [Trehal-(3,4,3',4')P₄], α,α'-trehalose 2,4,3',4'- tetrakisphosphate [Trehal(2,4,3',4')P₄], and methyl 3-O-(α-D- glucopyranosyl)-β-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 ≃ 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, 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 (~27 and ~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.