MOLECULAR RECOGNITION AT THE MYOINOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR- 3-POSITION SUBSTITUTED MYOINOSITOL 1,4,5-TRISPHOSPHATE ANALOGS REVEAL THE BINDING AND CA2-AFFINITY INTERACTION WITH THE MYOINOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR( RELEASE REQUIREMENTS FOR HIGH)

Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3] analo gues equatorially substituted at the and position have been synthesize d to probe the structure-activity relationship of the Ins(1,4,5)P-3-re ceptor subsite adjacent to the native 3-hydroxy (3-OH) of Ins(1,4,5)P- 3. This study was prompted, in part, by our observation that myo-inosi tol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P-4), the 3-position phosph orylated product of Ins(1,4,5)P-3 was a full agonist at the Ca2+-mobil izing Ins(1,4,5)P-3 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)P-3 analogues were equatorially substituted with groups spanning the steric range between the 3-OH of Ins(1,4,5)P-3 and the 3-phosphate of Ins(1,3,4,5)P-4; in order of increasing 3-position steric bulk these were: 3-fluoro, 3-chl oro-, 3-amino-, 3-bromo-, 3-methoxy-, and 3-phosphorothioate-Ins(1,4,5 )P-3. The analogues were assessed at the specific Ins(1,4,5)P-3 bindin g site of bovine adrenal cortex and for Ca2+ mobilizing activity in sa ponin-permeabilized SH-SY5Y human neuroblastoma cells. A correlation w as observed between increasing molecular volume of the 3-position subs tituent and respective decreases in both affinity and Ca2+ mobilizing efficacy. Further analysis of the data also revealed that Ins(1,4,5)P- 3 analogues with equatorial 3-OH, 3-phosphate, and 3-phosphorothioate substituents interacted more favorably with Ins(1,4,5)P-3 recognition sites than would be predicted by purely steric considerations. In cont rast, 3-C-trifluoromethyl-Ins(1,4,5)P-3 (which is axially substituted, but retains the native 3-OH of Ins(1,4,5)P-3) interacted with Ins(1,4 ,5)P-3 recognition sites with virtually the same potency as Ins(1,4,5) P-3, indicating that the binding pocket of the Ins(1,4,5)P-3-receptor was not sterically restrictive with respect to axially oriented 3-posi tion substituents. We conclude that the Ins(1,4,5)P-3 receptor has fav orable non-covalent binding interactions with the equatorial 3-positio n substituents of Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 and that these int eractions significantly ameliorate the steric constraints of the Ins(1 ,4,5)P-3 receptor binding pocket.