Abstract

Adenophostin A is the most potent known agonist of inositol 1,4,5-trisphosphate (InsP₃) receptors. Ca²⁺release from permeabilized hepatocytes was 9.9 ± 1.6-fold more sensitive to adenophostin A (EC₅₀, 14.7 ± 2.4 nM) than to InsP₃ (145 ± 10 nM), consistent with the greater affinity of adenophostin A for hepatic InsP₃receptors (K _d = 0.48 ± 0.06 and 3.09 ± 0.33 nM, respectively). Here, we systematically modify the structures of the glucose, ribose, and adenine moieties of adenophostin A and use Ca²⁺ release and binding assays to define their contributions to high-affinity binding. Progressive trimming of the adenine of adenophostin A reduced potency, but it fell below that of InsP₃ only after complete removal of the adenine. Even after substantial modifications of the adenine (to uracil or even unrelated aromatic rings, retaining the β-orientation), the analogs were more potent than InsP₃. The only analog with an α-ribosyl linkage had massively decreased potency. The 2′-phosphate on the ribose ring of adenophostin A was essential and optimally active when present on a five-membered ring in a position stereochemically equivalent to its location in adenophostin A.Xylo-adenophostin, where xylose replaces the glucose ring of adenophostin A, was only slightly less potent than adenophostin A, whereas manno-adenophostin (mannose replacing glucose) had similar potency to InsP₃. These results are consistent with the relatively minor role of the 3-hydroxyl of InsP₃ (the equivalent is absent fromxylo-adenophostin) and greater role of the equatorial 6-hydroxyl (the equivalent is axial inmanno-adenophostin). This is the first comprehensive analysis of all the key structural elements of adenophostin A, and it provides a working model for the design of related high-affinity ligands of InsP₃ receptors.