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-myoinositol 1,4,5- trisphosphate [Ins(1,4,5)P₃] receptors. Equilibrium competition binding studies with ³H-Ins(114,5)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 (K(d) = 2.76 ± 0.26 nM)than Ins(1,4,5)P₃ (K(d): 7.96 ± 1.02 nM) to the ³H-lns(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/K(d) 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/K(d) 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 acyciophostin or Ins(1,4,5)P₃ were indistinguishable. At pH 8.3, however, the maximal rate of ⁴⁵Ca⁺⁺ relase 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 ribbose-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.