Stephen J. Mills

Biphenyl 2,3′,4,5′,6-pentakisphosphate, a novel inositol polyphosphate surrogate, modulates Ca2+ responses in rat hepatocytes

Benzene polyphosphates containing phosphate groups on one ring are Ins(1,4,5)P3 5‐phosphatase inhibitors when evaluated against type‐I Ins(1,4,5)P3 5‐phosphatase. A novel biphenyl derivative, biphenyl 2,3′,4,5′,6‐pentakisphosphate, with five phosphate groups on two rings was synthesized: It inhibited the activity of two inositol 5‐phosphatases: type I and SHIP2 with Ins(1,3,4,5)P4 as substrate. The inhibition was competitive with respect to the substrate. IC50 value measured in rat hepatocytes, which contains the native Ins(1,4,5)P3 5‐phosphatase, was in the micromolar range at 1.0 μM Ins(1,4,5)P3 as substrate. Biphenyl 2,3′,4,5′,6‐pentakisphosphate did not affect the activity of Ins(1,4,5)P3 3‐kinase A in the 5‐100 μM range. Surprisingly, experimental evidence supports an effect of biphenyl 2,3′,4,5′,6‐pentakisphosphate at the level of the Ins(1,4,5)P3 receptor. Finally, when injected into rat hepatocytes, the analog affected the frequency of Ca2+ oscillations in a positive or negative way depending on its concentration. At very high concentrations of the analog, Ca2+ oscillations were even suppressed. These data were interpreted as a dual effect of the biphenyl 2,3′,4,5′,6‐pentakisphosphate on cytosolic [Ca2+] increases: an activation effect through an increase in Ins(1,4,5)P3 level via Ins(1,4,5)P3 5‐phosphatase inhibition and an inhibitory effect, which was exerted directly on the Ins(1,4,5)P3 receptor. Thus, our data show for the first time that the frequency of Ca2+ oscillations in response to a Ca2+‐mobilizing agonist can be controlled by inhibitors of type‐I Ins(1,4,5)P3 5‐phosphatase.—Vandeput, F., Combettes, L., Mills, S. J., Backers, K., Wohlkönig, A., Parys, J. B., De Smedt, H., Missiaen, L., Dupont, G., Potter, B. V. L., Erneux, C. Biphenyl 2,3′,4,5′,6‐pentakisphosphate, a novel inositol polyphosphate surrogate, modulates Ca2+ responses in rat hepatocytes. FASEB J. 21, 1481–1491 (2007)

A synthetic polyphosphoinositide headgroup surrogate in complex with SHIP2 provides a rationale for drug discovery

Phosphoinositides regulate many cellular processes, and cellular levels are controlled by kinases and phosphatases. SHIP2 (SH2 (Src homology 2)-domain-containing inositol-phosphatase-2) plays a critical role in phosphoinositide signaling, cleaving the 5-phosphate from phosphatidylinositol 3,4,5-trisphosphate. SHIP2 is thought to be involved in type-2 diabetes and obesity, conditions that could therefore be open to pharmacological modulation of the enzyme. However, rational design of SHIP2 inhibitors has been limited by the absence of a high-resolution structure. Here, we present a 2.1 Å resolution crystal structure of the phosphatase domain of SHIP2 bound to the synthetic ligand biphenyl 2,3′,4,5′,6-pentakisphosphate (BiPh(2,3′,4,5′,6)P5). BiPh(2,3′,4,5′,6)P5 is not a SHIP2 substrate but inhibits Ins(1,3,4,5)P4 hydrolysis with an IC50 of 24.8 ± 3.0 μM, (Km for Ins(1,3,4,5)P4 is 215 ± 28 μM). Molecular dynamics simulations suggest that when BiPh(2,3′,4,5′,6)P5 binds to SHIP2, a flexible loop folds over and encloses the ligand. Compounds targeting such a closed conformation might therefore deliver SHIP2-specific drugs.

Synthesis of myo-inositol 1,2,4,5-tetrakisphosphate, a Ca2+-mobilising tetrakisphosphate with a potency similar to myo-inositol 1,4,5-trisphosphate

Abstract The synthesis of myo-inositol 1,2,4,5-tetrakisphosphate from inositol is described; this tetrakisphosphate is a highly potent Ca2+-mobilising agonist at the Ins(1,4,5)P3 receptor.

Benzene polyphosphates as tools for cell signalling: inhibition of inositol 1,4,5-trisphosphate 5-phosphatase and interaction with the PH domain of protein kinase Balpha.

Novel benzene polyphosphates were synthesised as inositol polyphosphate mimics and evaluated against type‐I inositol 1,4,5‐trisphosphate 5‐phosphatase, which only binds soluble inositol polyphosphates, and against the PH domain of protein kinase Bα (PKBα), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5‐phosphatase inhibitor is benzene 1,2,4‐trisphosphate (2, IC50 of 14 μM), a potential mimic of D‐myo‐inositol 1,4,5‐trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P3 5‐phosphatase inhibitor is benzene 1,2,4,5‐tetrakisphosphate, with an IC50 of 4 μM. Biphenyl 2,3′,4,5′,6‐pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P3 5‐phosphatase (IC50 of 1 μM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P3 5‐phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBα with apparent higher affinity than to type I Ins(1,4,5)P3 5‐phosphatase. The most potent ligand for the PKBα PH domain, measured by inhibition of biotinylated diC8‐PtdIns(3,4)P2 binding, is biphenyl 2,3′,4,5′,6‐pentakisphosphate (4, Ki=27 nm). The approximately 80‐fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation–π interaction. These new molecular tools will be of potential use in structural and cell signalling studies.

3-Hydroxybenzene 1,2,4-Trisphosphate, a Novel Second Messenger Mimic and unusual Substrate for Type-I myo-Inositol 1,4,5-Trisphosphate 5-Phosphatase: Synthesis and Physicochemistry

3‐Hydroxybenzene 1,2,4‐trisphosphate 4 is a new myo‐inositol 1,4,5‐trisphosphate analogue based on the core structure of benzene 1,2,4‐trisphosphate 2 with an additional hydroxyl group at position‐3, and is the first noninositol based compound to be a substrate for inositol 1,4,5‐trisphosphate 5‐phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the 31P NMR spectrum displayed monophasic behaviour in which phosphate‐1 and phosphate‐2 behaved independently in most of the studied pH range. For compound 4, phosphate‐2 and phosphate‐4 interacted with the 3‐OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co‐operativity between these groups. Phosphate‐1 and phosphate‐2 strongly interacted with each other and phosphate‐4 experienced repulsion because of the interaction of the 3‐OH group. Benzene 1,2,4‐trisphosphate 2 is resistant to inositol 1,4,5‐trisphosphate type I 5‐phosphatase catalysed dephosphorylation. However, surprisingly, 3‐hydroxybenzene 1,2,4‐trisphosphate 4 was dephosphorylated by this 5‐phosphatase to give the symmetrical 2,3‐dihydroxybenzene 1,4‐bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at −15 °C, and 1H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3‐OH group in compound 4 confirms a critical role for the 6‐OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.

Myo-inositol 1,4,6-trisphosphate: A new synthetic Ca2+-mobilising inositol phosphate

Abstract The synthesis of myo -inositol 1,4,6-trisphosphate from myo -inositol is described; this novel trisphosphate is a potent Ca 2+ -mobilising agonist at the Ins(1,4,5)P 3 receptor and is derived from structure-activity considerations of myo -inositol 1,3,4,6-tetrakisphosphate.

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3‐kinase and type I Ins(1,4,5)P3 5‐phosphatase. The catalytic domain of Ins(1,4,5)P3 3‐kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C‐terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3‐kinase A in Escherichia coli as a His‐ and S‐tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D‐2‐deoxy‐Ins(1,4,5)P3 was devised. D‐2‐Deoxy‐Ins(1,4,5)P3 and D‐3‐deoxy‐Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D‐2‐deoxy‐Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3‐kinase. Therefore, the axial 2‐hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3‐hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3‐hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.

Myo-inositol 1,4,6-trisphosphorothioate and myo-inositol 1,3,4-trisphosphorothioate: New synthetic Ca2+-mobilising partial agonists at the inositol 1,4,5-trisphosphate receptor

Abstract Syntheses of myo -inositol 1,4,6-trisphosphorothioate and 1,3,4-trisphosphorothioate from myo -inositol are described; these trisphosphorothioates, derived from structure-activity considerations of myo -inositol 1,3,4,6-tetrakisphosphate, are low intrinsic activity partial agonists at the platelet Ins(1,4,5)P 3 receptor.

Synthesis of the enantiomers of myo-inositol 1,2,4,5-tetrakisphosphate, a regioisomer of myo-inositol 1,3,4,5-tetrakisphosphate

Routes for the synthesis of racemic myo-inositol 1,2,4,5-tetrakisphosphate DL-Ins(1,2,4,5)P4 5ab and the chiral antipodes D- and L-myo-inositol 1,2,4,5-tetrakisphosphate 5a and 5b, respectively, are described. For the synthesis of racemate 5ab, 3,6-di-O-benzoyl-1,2:4,5-di-O -isopropylidene-myo-inositol 7ab is prepared in two steps from myo-inositol. The ketals are hydrolysed under acidic conditions to give DL-1,4-di-O-benzoyl- myo-inositol 8ab. Phosphitylation of compounds 8ab using chloro(diethoxy)phosphine in the presence of base, followed by oxidation and a three-step deprotection strategy, gives DL-Ins(1,2,4,5)P4 5ab.The chiral tetrakisphosphates 5a and 5b are synthesized using a different route. The 4,5-isopropylidene group of DL-3,6-di-O -benzyl-1,2:4,5-di-O-isopropylidene-myo -inositol 13ab are selectively removed under mild acidic conditions to give diol 14ab. p-Methoxybenzylation at the 4,5-positions followed by acid hydrolysis of the cis-isopropylidene ketal affords cis-diol 16ab. Selective coupling of (S)-(+)-O -acetylmandelic acid with diol 16ab at the equatorial hydroxy group provides two diastereoisomers 18 and 19, which are separated by chromatography. Basic hydrolysis of the individual diastereoisomers provides the enantiomers 16a and 16b. Acidic hydrolysis gives D- and L-3,6-di-O-benzyl- myo-inositol 20a and 20b, respectively. Phosphitylation and oxidation of tetraols 20a and 20b gives the fully blocked derivatives, which are deprotected to give tetrakisphosphates 5a and 5b, respectively. The absolute configuration of compound 20a is established by a chemical method. DL-1,2:4,5-Di-O -isopropylidene-myo-inositol 12ab is coupled to (S)-(+)-O -acetylmandelic acid to give a mixture of bis-esters 26 and 27 and crystallisation of the mixture of diastereoisomers affords pure isomer 27. Basic hydrolysis gives the pure enantiomer 12a (for which the absolute configuration is known) and benzylation followed by acid hydrolysis gives tetraol 20a with the same physical properties as compound 20a prepared by a different route described previously. D-Ins(1,2,4,5)P4 5a is a potent mobiliser of intracellular Ca2+ ions in permeabilised platelets, while L-Ins(1,2,4,5)P4 5b is inactive.

Synthesis of potent Ins(1,4,5)P3 5-phosphatase inhibitors by modification of myo-inositol 1,3,4,6-tetrakisphosphate

Three myo-inositol tetrakisphosphate analogues were synthesised based upon myo-inositol 1,3,4,6-tetrakisphosphate: 2,5-di-O-methyl myo-inositol-1,3,4,6-tetrakisphosphate 19 and its phosphorothioate derivative 22, together with myo-inositol 1,3,4,6 tetrakisphosphorothioate 25. These compounds were prepared by phosphitylating 2,5-di-O-methyl-myo-inositol and 2,5-di-O-benzyl-myo-inositol followed by oxidation with t-butylhydroperoxide or sulfoxidation at room temperature using sulfur in a mixed solvent of DMF and pyridine. Sulfoxidation was complete within 15 min; however, without DMF, the reaction was much slower, and required overnight. When evaluated against Ins(1,4,5)P(3) 5-phosphatase, 3-kinase and for Ca(2+) release at the Ins(1,4,5)P(3) receptor, only weak activity was observed for Ca(2+) release. 22 and 25 are potent 5-phosphatase inhibitors and 25 is a moderate inhibitor of 3-kinase. Thus, we have synthesised potent enzyme inhibitors, which do not mobilise Ca(2+) and devised conditions for quick, clean and inexpensive sulfoxidation of inositol polyphosphite intermediates.