Defining the minimal structural requirements for partial agonism at the type I myo-inositol 1,4,5-triphosphate receptor

The novel synthetic analogues D-3-fluoro-myo-inositol 1,5-bisphosphate-4-phosphorothioate, [3F-Ins(1,5)P₂-4PS], D-3-fluoro-myo-inositol 1,4-bisphosphate-5-phosphorothioate [3F-Ins(1,4)P₂-5PS], and D-3-fluoro-myo-inositol 1-phosphate-4,5-bisphosphorothioate [3F-Ins(1)P-(4,5)PS₂] were utilised to define the structure-activity relationships which could produce partial agonism at the Ca²⁺ mobilising myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptor. Based on prior structure-activity data we hypothesised that tile minimal structural requirements for Ins(1,4,5)P₃ receptor partial agonism, were phosphorothioate substitution of the crucial vicinal 4,5-bisphosphate pair accompanied by another structural perturbation, such fluorination of 3-position of the myo-inositol ring. All the analogues fully displaced [³H]]Ins(1,4,5)P₃ from a single Ins(1,4,5)P₃ binding site in pig cerebellar membranes [3F-Ins(1,5)P₂-4PS (IC₅₀ = 26 nM), 3F-Ins(1,4)P₂-5PS (IC₅₀ = 80 nM) and 3F-Ins(1)P-(4,5)PS₂ (IC₅₀ = 109 nM) cf. Ins(1,4,5)P₃ (IC₅₀ = 11 nM)]. In contrast, 3F-Ins(1,5)P₂-4PS (IC₅₀ = 424 nM) and 3F-Ins(1,4)P₂-5PS (IC₅₀ = 3579 nM) were weak full agonists at the Ca²⁺ mobilising Ins(1,4,5)P₃ receptor of permeabilised SH-SY5Y neuroblastoma cells, being respectively 4- and 36-fold less potent than Ins(1,4,5)P₃ (EC₅₀ = 99 nM). While 3F-Ins(1)P-(4,5)PS₂ (EC₅₀ = 11345 nM) was a partial agonist releasing only 64.3 ± 1.9% of the Ins(1,4,5)P₃-sensitive intracellular Ca²⁺ pools. 3F-Ins(1)P-(4,5)PS₂ was unique among the Ins(1,4,5)P₃ receptor partial agonists so far identified in having it relatively high affinity for the Ins(1,4,5)P₃ binding site, accompanied by a significant loss of intrinsic activity for Ca²⁺ mobilisation. This improved affinity was probably due to the retention of the 1-position phosphate, which enhances interaction with the Ins(1,4,5)P₃ receptor. 3F-Ins(1)P-(4,5)PS₂ may be an important lead compound for the development of efficient Ins(1,4,5)P₃ receptor antagonists.