Vol. 55, Issue 1, 109-117, January 1999

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

Mike D. Beecroft, Jonathan S. Marchant, Andrew M. Riley, Nicole C. R. Van Straten, Gijs A. Van der Marel, Jacques H. Van Boom, Barry V. L. Potter, and Colin W. Taylor

Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, England (M.D.B., J.S.M., C.W.T.); Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, England (A.M.R., B.V.L.P.); and Leiden Institute of Chemistry, Gorleaus Laboratories, University of Leiden, 2300 RA Leiden, the Netherlands (N.C.R.V.S., G.A.V.d.M., J.H.V.B.)

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 (K_d = 2.76 ± 0.26 nM) than Ins(1,4,5)P₃ (K_d = 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/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 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.