Molecular recognition at the myo-inositol 1,4,5-trisphosphate receptor. 3- Position substituted myo-inositol 1,4,5-trisphosphate analogues reveal the binding and Ca²⁺ release requirements for high affinity interaction with the myo-inositol 1,4,5-trisphosphate receptor

Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) analogues equatorially substituted at the 3-position have been synthesized to probe the structure-activity relationship of the Ins(1,4,5)P₃-receptor subsite adjacent to the native 3-hydroxy (3-OH) of Ins(1,4,5)P₃. This study was prompted, in part, by our observation that myo-inositol 1,3,4,5- tetrakisphosphate (Ins(1,3,4,5)P₄), the 3-position phosphorylated product of Ins(1,4,5)P₃ was a full agonist at the Ca²⁺-mobilizing Ins(1,4,5)P₃ receptor of SH-SY5Y cells (Wilcox, R. A., Challiss, R. A. J., Liu, C., Potter, B. V. I., and Nahorski, S. R. (1993) Mol. Pharmacol. 44, 810-817). The 3-position Ins(1,4,5)P₃ analogues were equatorially substituted with groups spanning the steric range between the 3-OH of Ins(1,4,5)P₃ and the 3- phosphate of Ins(1,3,4,5)P₄; in order of increasing 3-position steric bulk these were: 3-fluoro-, 3-chloro-, 3-amino-, 3-bromo-, 3-methoxy-, and 3- phosphorothioate-Ins(1,4,5)P₃. The analogues were assessed at the specific Ins(1,4,5)P₃ binding-site of bovine adrenal cortex and for Ca²⁺ mobilizing activity in saponin-permeabilized SH-SY5Y human neuroblastoma cells. A correlation was observed between increasing molecular volume of the 3- position substituent and respective decreases in both affinity and Ca²⁺ mobilizing efficacy. Further analysis of the data also revealed that Ins(1,4,5)P₃ analogues with equatorial 3-OH, 3-phosphate, and 3- phosphorothioate substituents interacted more favorably with Ins(1,4,5)P₃ recognition sites than would be predicted by purely steric considerations. In contrast, 3-C-trifluoromethyl-Ins(1,4,5)P₃ (which is axially substituted, but retains the native 3-OH of Ins(1,4,5)P₃) interacted with Ins(1,4,5)P₃ recognition sites with virtually the same potency as Ins(1,4,5)P₃, indicating that the binding pocket of the Ins(1,4,5)P₃-receptor was not sterically restrictive with respect to axially oriented 3-position substituents. We conclude that the Ins(1,4,5)P₃ receptor has favorable non- covalent binding interactions with the equatorial 3-position substituents of Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ and that these interactions significantly ameliorate the steric constraints of the Ins(1,4,5)P₃ receptor binding pocket.