Acyclophostin: A Ribose-Modified Analog of Adenophostin A with High Affinity for Inositol 1,4,5-Trisphosphate Receptors and pH-Dependent Efficacy

Abstract

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.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…At the purified InsP 3 R1, adenophostin B is about 10 times more potent than InsP 3 (EC 50 values: 11 nM versus 100 nM) and exhibits a positive cooperativity in binding that is not observed with InsP 3 (Hirota et al, 1995). Adenophostin A is also about a 10-fold more potent Ca 2ϩ releaser than InsP 3 in permeabilized hepatocytes (Marchant et al, 1997b;Beecroft et al, 1999) that have predominance of InsP 3 R2 versus InsP 3 R1 (Ͼ80% versus Ͻ20%) (Wojcikiewicz, 1995;De Smedt et al, 1997).…”

“…They are full InsP 3 R agonists in rat cerebellar microsomes (which express InsP 3 R1 almost exclusively) (Wojcikiewicz, 1995) with potencies about 100 times higher than InsP 3 potency (EC 50 values: adenophostin A ϭ 1.4 nM, adenophostin B ϭ 1.5 nM, and InsP 3 ϭ 170 nM). In binding data, adenophostin A has an affinity about seven times higher than InsP 3 in rat cerebellar microsomes (K D values: 0.91 nM versus 6.75 nM) (Marchant et al, 1997a) and about five times higher in hepatic membranes (Beecroft et al, 1999). Consistent with this, the displacement of [ 3 H]InsP 3 by adenophostin has an IC 50 value about 20 to 50 times lower than InsP 3 in rat cerebellar microsomes (Takahashi et al, 1994;Murphy et al, 1997) and about 10 times lower than in porcine cerebellar microsomes (Shuto et al, 1998).…”

Pharmacology of inositol trisphosphate receptors

“…Since the synthesis of adenophostins and their analogues involves fewer steps than that of chiral inositol phosphates, adenophostins provide an alternative approach to develop high-affinity selective ligands for the IP 3 R [4,34,94,121,183].…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…AdA, a fungal glyconucleotide metabolite (448), and its many analogs (1,23,51,102,290,388,397,398,420,444,465) were discovered as agonists of the InsP 3 R. Although their molecular structures are significantly different from those of InsP 3 and its analogs (198), they activate the channel by interacting with the InsP 3 binding site (157). AdA binds InsP 3 R with substantially higher affinity and is significantly more potent in stimulating InsP 3 Rmediated Ca 2ϩ release than its natural agonist InsP 3 .…”

“…Adenophostin A (AdA), a fungal glyconucleotide metabolite (448), and its analogs (23,290,420) are potent agonists of the InsP 3 R. Although their molecular structures are significantly different from those of InsP 3 and its analogs (198), they activate the channel by interactions with the InsP 3 binding site (157). Molecular docking of AdA into the core domain crystal structure was consistent with experimental structure-activity relationships and provided some possible clues to the mechanisms involved in the high affinity of AdA for the InsP 3 R (397).…”