IP3 Receptor Ligand Research Articles

Effects of Linker Flexibility and Conformational Changes of IP3 Receptor on Split Luciferase Complementation Assay.

Background: IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.Objectives: Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1–604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and Methods: In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.Results: As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.Conclusions: It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Medicinal Chemical Studies Based on the Theoretical Design of Bioactive Compounds

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.

A synthetic cyclitol-nucleoside conjugate polyphosphate is a highly potent second messenger mimic.

Reactions that form sec-sec ethers are well known, but few lead to compounds with dense functionality around the O-linkage. Replacement of the α-glucopyranosyl unit of adenophostin A, a potent d-myo-inositol 1,4,5-trisphosphate (IP3R) agonist, with a d-chiro-inositol surrogate acting substantially as a pseudosugar, leads to "d-chiro-inositol adenophostin". At its core, this cyclitol-nucleoside trisphosphate comprises a nucleoside sugar linked via an axial d-chiro-inositol 1-hydroxyl-adenosine 3'-ribose ether linkage. A divergent synthesis of d-chiro-inositol adenophostin has been achieved. Key features of the synthetic strategy to produce a triol for phosphorylation include a new selective mono-tosylation of racemic 1,2:4,5-di-O-isopropylidene-myo-inositol using tosyl imidazole; subsequent conversion of the product into separable camphanate ester derivatives, one leading to a chiral myo-inositol triflate used as a synthetic building block and the other to l-5-O-methyl-myo-inositol [l-(+)-bornesitol] to assign the absolute configuration; the nucleophilic coupling of an alkoxide of a ribose pent-4-ene orthoester unit with a structurally rigid chiral myo-inositol triflate derivative, representing the first sec-sec ether formation between a cyclitol and ribose. Reaction of the coupled product with a silylated nucleobase completes the assembly of the core structure. Further protecting group manipulation, mixed O- and N-phosphorylation, and subsequent removal of all protecting groups in a single step achieves the final product, avoiding a separate N6 protection/deprotection strategy. d-chiro-Inositol adenophostin evoked Ca2+ release through IP3Rs at lower concentrations than adenophostin A, hitherto the most potent known agonist of IP3Rs.

A fluorescence-based method for evaluating inositol 1,4,5-trisphosphate receptor ligands: Determination of subtype selectivity and partial agonist effects

Inositol 1,4,5-trisphosphate (IP3) receptors consist of three subtypes: IP3R1, IP3R2, and IP3R3. Although numerous IP3 receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP3 receptor ligands using FRET-based IP3 biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP3 or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP3 in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP3R1, as evidenced by the modified selectivity following replacement of the 5′-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5′-deoxy-5′-phenyladenophostin A possessed a partial agonistic effect on IP3R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP3R ligands, including potency, efficacy, and subtype selectivity.

アデノフォスチンA の構造展開に基づくIP3受容体リガンドの設計と合成：細胞内Ca2+動員シグナル伝達系機能分子の開発を目指して

アデノフォスチンA の構造展開に基づくIP3受容体リガンドの設計と合成：細胞内Ca2+動員シグナル伝達系機能分子の開発を目指して

Synthesis of Adenophostin A Analogues Conjugating an Aromatic Group at the 5‘-Position as Potent IP3 Receptor Ligands

Previous structure-activity relationship studies of adenophostin A, a potent IP(3) receptor agonist, led us to design the novel adenophostin A analogues 5a-c, conjugating an aromatic group at the 5'-position to develop useful IP(3) receptor ligands. The common key intermediate, a D-ribosyl alpha-D-glucoside 10alpha, was stereoselectively synthesized by a glycosidation with the 1-sulfinylglucoside donor 11, which was conformationally restricted by a 3,4-O-cyclic diketal protecting group. After introduction of an aromatic group at the 5-position of the ribose moiety, an adenine base was stereoselectively introduced at the anomeric beta-position to form 7a-c, where the tetra-O-i-butyryl donors 9a-c were significantly more effective than the corresponding O-acetyl donor. Thus, the target compounds 5a-c were synthesized via phosphorylation of the 2', 3' ', and 4' '-hydroxyls. The potencies of compounds 5a-c for Ca(2+) release were shown to be indistinguishable from that of adenophostin A, indicating that bulky substitutions at the 5'-position of adenophostin A are well-tolerated in the receptor binding. This biological activity of 5a-c can be rationalized by molecular modeling using the ligand binding domain of the IP(3) receptor.

Synthesis and biological activity of adenophostin derivatives as IP3 receptor ligands.

The three derivatives of adenophostin A, which were the C-glycosidic analogs 3a and 3b, and the 5'-phenyl analog 4, were designed and synthesized as efficient IP3 receptor ligands.

ChemInform Abstract: Synthesis of 3,7-Anhydro-D-glycero-D-ido-octitol 1,5,6-Trisphosphate as an IP3 Receptor Ligand Using a Radical Cyclization Reaction with a Vinylsilyl Tether as the Key Step. Conformational Restriction Strategy Using Steric Repulsion B

3,7-Anhydro-d-glycero-d-ido-octitol 1,5,6-trisphosphate (5) was designed as a novel IP3-receptor ligand having a C-glycosidic structure and was synthesized via a radical cyclization reaction with a temporary connecting vinylsilyl tether as the key step. The phenyl 2-O-dimethylvinylsilyl-3,4,6-tri-O-benzyl-1-seleno-β-d-glucopyranoside (7), in the usual 4C1-conformation, was successively treated with Bu3SnH/AIBN and under Tamao oxidation conditions to give a mixture of five C-glycosidic products. On the other hand, similar successive treatment of the corresponding 3,4-di-O-TBS-protected substrates 13 and 24, which were in an unusual 1C4-conformaion due to the steric repulsion between the bulky silyl protecting groups, gave the desired 1α-C-glycosides 18 and 25, respectively, as the major products. Thus, the course of the radical cyclization was effectively controlled by a change in the conformation of the pyranose ring into a 1C4-form due to steric repulsion between the adjacent bulky TBS-protecting groups ...

Inositol 1,4,5-trisphosphate and adenophostin analogues induce responses in turtle olfactory sensory neurons.

Using the whole-cell mode of the patch-clamp technique, we recorded inward currents in response to inositol-1,4,5-trisphosphate (IP3) and adenophostin analogues in turtle olfactory sensory neurons. Dialysis of IP3 into the neurons induced inward currents with an increase in membrane conductance in a dose-dependent manner under the voltage-clamp conditions (holding potential -70 mV). The application of Ca2+-free Ringer solution to neurons previously dialysed with IP3 induced inward currents that were reversibly inhibited by application of Na+, Ca2+-free Ringer solution, normal Ringer solution or 10 microM ruthenium red. Dialysis of the adenophostin analogues, novel IP3 receptor ligands, also induced inward currents with an increase in membrane conductance. The magnitude of the responses to the adenophostin analogues varied among these derivatives. The application of Ca2+-free Ringer solution to neurons previously dialysed with the adenophostin analogues induced inward currents that were inhibited by the application of normal Ringer solution. The reversal potential of inward currents induced by an adenophostin analogue was similar to that induced by IP3, suggesting that inward currents induced by the adenophostin analogue were generated by a similar ionic mechanism to that induced by IP3. The present study demonstrated that IP3-mediated transduction pathways exist in turtle olfactory receptor neurons and that adenophostin analogues act as agonists of IP3.

Synthesis of Adenophostin Analogues Lacking the Adenine Moiety as Novel Potent IP3 Receptor Ligands: Some Structural Requirements for the Significant Activity of Adenophostin A

1-O-Tetrahydrofuranyl-α-d-glucopyranose derivatives 5−8 were designed and synthesized as novel IP3 receptor ligands. The glycosidation reactions between fluoroglycosyl donor 23 and tetrahydrofuran derivatives 11−14 as glycosyl acceptors selectively gave the corresponding α-glycosides, which were converted into the target compounds 5−8 via the introduction of phosphate groups using the phosphoramidite method. Among these compounds, 1-O-tetrahydrofuranyl-α-d-glucopyranose trisphosphate derivatives 5 and 8 significantly inhibited the binding of [3H] IP3 to IP3 receptor from porcine cerebella, with IC50 values of 25 and 27 nM, respectively, which were comparable to the affinity of IP3 itself.