Acyclophostin: a ribose-modified analog of adenophostin A with high affinity for inositol 1,4,5-trisphosphate receptors and pH-dependent efficacy.

Adenophostin A is the most potent known agonist of D-myo-inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptors. Equilibrium competition binding studies with 3H-Ins(1,4,5)P3 showed that the interaction of a totally synthetic adenophostin A with both hepatic and cerebellar Ins(1,4,5)P3 receptors was indistinguishable from that of the natural product. At pH 8.3, a synthetic analog of adenophostin A (which we named acyclophostin), in which most elements of the ribose ring have been removed, bound with substantially higher affinity (Kd = 2.76 +/- 0.26 nM) than Ins(1,4,5)P3 (Kd = 7.96 +/- 1.02 nM) to the 3H-Ins(1,4,5)P3-binding sites of hepatic membranes. At pH 7, acyclophostin (EC50 = 209 +/- 12 nM) and Ins(1,4,5)P3 (EC50 = 153 +/- 11 nM) stimulated 45Ca++ release to the same maximal extent and from the same intracellular stores of permeabilized hepatocytes. Comparison of the affinities of a range of Ins(1,4,5)P3 and adenophostin analogs with their abilities to stimulate Ca++ release revealed that although all other agonists had similar EC50/Kd ratios, that for acyclophostin was significantly higher. Similar results were obtained with cerebellar membranes, which express almost entirely type 1 InsP3 receptors. When the radioligand binding and functional assays of hepatocytes were performed under identical conditions, the higher EC50/Kd ratio for acyclophostin was retained at pH 8.3, but it was similar to that for Ins(1,4,5)P3 when the assays were performed at pH 7. To directly assess whether acyclophostin was a partial agonist of hepatic Ins(1,4,5)P3 receptors, the kinetics of 45Ca++ efflux from permeabilized hepatocytes was measured with a temporal resolution of 80 ms using rapid superfusion. At pH 7, the kinetics of 45Ca++ release, including the maximal rate of release, evoked by maximal concentrations of acyclophostin or Ins(1,4,5)P3 were indistinguishable. At pH 8.3, however, the maximal rate of 45Ca++ release evoked by a supramaximal concentration of acyclophostin was only 69 +/- 7% of that evoked by Ins(1,4,5)P3. We conclude that acyclophostin is the highest affinity ribose-modified analog of adenophostin so far synthesized, that at high pH it is a partial agonist of inositol trisphosphate receptors, and that it may provide a structure from which to develop high-affinity antagonists of inositol trisphosphate receptors.