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Published as doi: 10.1096/fj.06-7691com.
 (The FASEB Journal. 2007;21:1481-1491.)
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Biphenyl 2,3',4,5',6-pentakisphosphate, a novel inositol polyphosphate surrogate, modulates Ca2+ responses in rat hepatocytes

Fabrice Vandeput*, Laurent Combettes{dagger}, Stephen J. Mills{ddagger}, Katrien Backers*, Alexandre Wohlkönig§, Jan B. Parys||, Humbert De Smedt||, Ludwig Missiaen||, Geneviève Dupont, Barry V. L. Potter{ddagger} and Christophe Erneux*,1

* Institut de Recherche Interdisciplinaire (IRIBHM), Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium;

{dagger} INSERM UMR-S757, Université de Paris-Sud, Orsay, France;

{ddagger} Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom;

§ CNRS-UMR8161, Institut de biologie de Lille, France; and

|| Laboratorium voor Fysiologie, Dept. Molecular Cell Biology, KULeuven, Campus Gasthuisberg, Leuven, Belgium; and

ULB, Faculté des Sciences, Belgium

1Correspondence: Institut de Recherche Interdisciplinaire (IRIBHM), Université Libre de Bruxelles, Campus Erasme, Bldg. C, 808 Route de Lennik, 1070 Brussels, Belgium. E-mail: cerneux{at}ulb.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


Key Words: inositol 5-phosphatase • SHIP2 • Ins(1,4,5)P3 receptor • Ca2+ signaling • benzene polyphosphates


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DISTINCT FORMS OF inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates, that is, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), or phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (1 , 2) . In mammalian cells, this family contains a series of distinct genes and splice variants (2 , 3) . All inositol polyphosphate 5-phosphatases share an inositol 5-phosphatase catalytic domain and various protein modules probably responsible for specific cell localization or recruitment (SH2 domain, proline-rich sequences, prenylation sites). Type-I and the SH2-domain-containing inositol 5-phosphatase 2 (SHIP2) are two members of this family of proteins involved in Ca2+ (4 , 5) and insulin signaling, respectively, as well as the control of obesity (6 , 7) .

We initiated this study in order to identify novel inositol 5-phosphatase inhibitors as molecular tools for studies using intact cells. The availability of phosphatase inhibitors of this family of enzymes could facilitate a comparison of their catalytic domains. It will also allow us to initiate intact-cell studies with suitable phosphate-masked derivatives by modulating phosphatase activities, as previously shown for the various isoforms of cyclic nucleotide phosphodiesterases (8) , for example. This would allow a better understanding of their individual functions in the control of intracellular levels of inositol phosphates and of phosphoinositides.

On the basis of modeling studies, we hypothesized that Ins(1,4,5)P3 5-phosphatase activity plays an important role in the control of the frequency of Ca2+ oscillations in response to Ca2+-mobilizing agonists (9) . This hypothesis could be tested using the model of connected hepatocytes by injecting the phosphatase into one cell. Connected hepatocytes display quasi-identical Ca2+ oscillations in response to stimulation by noradrenaline or vasopressin (10) . Therefore, the noninjected cell provides the control situation, allowing for a direct visualization of the effect of 5-phosphatase (11) .

In the present study, one of our aims was to inject an inositol 5-phosphatase inhibitor, which might increase the intracellular level of Ins(1,4,5)P3, producing a new steady-state level of Ins(1,4,5)P3 that could control the frequency of Ca2+ oscillations.

Benzene polyphosphates are polyanionic compounds that can accommodate phosphate groups around a six-membered planar ring with a similar, but more rigid phosphate regiochemistry to a natural inositol phosphate and can potentially interact with inositol polyphosphate-binding proteins. Additionally, the nature of the benzene ring may encourage the formation of new intermolecular ligand-protein interactions such as {pi}–cation interactions that cannot be observed in inositol polyphosphates. These criteria make the benzene ring an interesting replacement for the conformationally mobile myo-inositol ring. Benzene 1,2,4-trisphosphate (Bz(1,2,4)P3) was the first simple benzene polyphosphate to be modeled on the Ins(1,4,5)P3 structure and has an arrangement of phosphate groups similar to 2,3,6-trideoxy Ins(1,4,5)P3, a weak agonist for Ca2+ release at the Ins(1,4,5)P3 receptor. Poitras et al. found that Bz(1,2,4)P3 interacted with the Ins(1,4,5)P3 receptor of adrenal-cortex microsomes, albeit with a 10,000 fold weaker binding affinity than Ins(1,4,5)P3 (12) . Bz(1,2,4)P3 did not release Ca2+, but weakly inhibited Ca2+ release in a dose-dependent way. We recently prepared a novel series of benzene polyphosphates that inhibit type-I Ins(1,4,5)P3 5-phosphatase with different potency. Interestingly, most of the analogs were not dephosphorylated, one exception being 3-hydroxybenzene 1,2,4-trisphosphate ((OH) Bz(1,2,4)P3), which was a very efficient substrate of type-I 5-phosphatase (13) .

In this study, we report the synthesis of a related, but highly novel, derivative with five phosphate groups on two rings (a biphenyl moiety) biphenyl 2,3',4,5',6-pentakisphosphate (biphenyl(2,3',4,5',6)P5, Fig. 1 ). It inhibits the activity of two inositol 5-phosphatases: type-I Ins(1,4,5)P3 5-phosphatase and SHIP2. It is the most potent inhibitor of the current series. Surprisingly, we found that it also binds to the Ins(1,4,5)P3 receptor type 1 ((IP)3R1) and inhibits Ins(1,4,5)P3-induced Ca2+ release in permeabilized cells. When injected into intact hepatocytes, it was able to affect both positively and negatively the frequency of Ca2+ oscillations depending on its concentration and on the sensitivity of the cell.


Figure 1
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  		Figure 1. Structures of biphenyl-2,4,6,3',5'-pentaol (1), 2,3',4,5',6-pentakis(diethylphosphoryloxy)-biphenyl (2), and biphenyl 2,3',4,5',6-pentakisphosphate (3). The scheme of the synthesis is: i) Diisopropylethylamine, Cl-P(OEt)2, CH2Cl2, (reverse addition), then mCPBA, (76%); ii) CH2Cl2 TMSBr, then purification on Q-Sepharose ion exchange resin, triethylammonium bicarbonate 0->2.0 M (91.5%).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The synthesis of Ins(1,3,4,5)P4 (14) , as well as the synthesis of three- and four-phosphorylated analogs, has been reported ((13) and unpublished results). CompeteTM (Protease inhibitor cocktail) was from Roche Diagnostics (Mannheim, Germany). ProBondTM Nickel-Chelating Resin was purchased from Invitrogen (Carlsbad, CA). Hyperfilm-MP, enhanced chemoluminescence (ECL®), Western blot analysis reagents, and HiLoad Superdex 75 prep grade were from Amersham. [3H]Ins(1,4,5)P3 (22Ci/mmol) and [3H] Ins(1,3,4,5)P4 (22 Ci/mmol) were from DuPont NEN-PerkinElmer. Dowex 1-X8 (formate form) was from Bio-Rad Laboratories (Hercules, CA). A bacterial construct encoding the catalytic domain of Ins(1,4,5)P3 3-kinase A was expressed and purified as reported in Poinas et al. (15) . The cloning and expression of human brain type-I InsP3 5-phosphatase were carried out as described previously (16) . A glutathione S-transferase (GST)-SHIP2 construct encoding the catalytic domain of SHIP2 was expressed as previously reported (17) . R23–11 cells used in this study are triple Ins(1,4,5)P3-receptor-knockout cells derived from chicken DT40 chicken B lymphoma cells, which are stably transfected with mouse IP3R1 (18) .

Synthesis of biphenyl-2,3',4,5',6-pentakisphosphate
2,3',4,5',6-pentakis(diethylphosphoryloxy)-biphenyl (2)
A mixture of dry CH2Cl2 (10 ml) and diethyl chlorophosphite (1.0 ml, 7.0 mmol) and dry N,N-diisopropylethylamine (2.1 ml, 12 mmol) was stirred at room temperature. Biphenyl 2,4,6,3',5'-pentanol (1) (234 mg, 1.0 mmol) was slowly added to the mixture, and ultrasound was used to dissolve the solid. As the compound dissolved, the solution became a deeper yellow color, which was cooled using dry ice and stirred for 30 min. 3-Chloroperoxybenzoic acid (2.0 g, 11.58 mmol) dissolved in CH2Cl2 (25 ml) was added quickly, and the solution was stirred for a further 30 min. The mixture was purified by flash chromatography [EtOAc then EtOAc–EtOH (5:1)], Rf = 0.34, EtOAc–EtOH (5:1), to give the pure title compound (2) as a yellow oil 699 mg (76%). 1H NMR (270 MHz, CDCl3) 1.16–1.22 (12 H, m, 2 x ArOPO(OCH2CH3)2), 1.31–1.39 (18 H, m, 3 x ArOPO(OCH2CH3)2), 3.88–3.96 (8 H, m, 2 x ArOPO(OCH2CH3)2), 4.16–4.28 (12 H, m, 3 x ArOPO(OCH2CH3)2), 7.02–7.03 (2 H, m, Ar), 7.16–7.18 (1 H, m, Ar) 7.20–7.21 (2 H, m, Ar); 31P NMR (162 MHz, CDCl3) –6.80 (2 P, 2 x ArOPO(OCH2CH3)2), –6.97 (1 P, ArOPO(OCH2CH3)2), –7.38 (2 P, 2 x ArOPO(OCH2CH3)2); (MS, FAB+) Calcd for C32H56O20P5 [M + H]+ 915.2053. Found 915.2066. Calcd for C32H55O20P5 C, 42.02, H, 6.06. Found C, 41.6, H, 5.98.

Biphenyl 2,3',4,5',6-pentakisphosphate (3)
Compound (2) (227 mg, 248 µmoles), was dissolved in dry CH2Cl2 (10 ml). Bromotrimethylsilane (2.0 ml, 15.15 mmol) was added, and the solution was stirred for 22 h at room temperature. The solvents were evaporated off, and the remaining syrup was stirred in MeOH (10 ml) for 30 min. Final purification of compound (3) was accomplished over Q-Sepharose Fast Flow using a linear gradient of 0->2.0 M TEAB buffer. The title compound eluted at a buffer concentration of 1.3–2.0 M and produced (3) , a glassy triethylammonium salt, 227 µmoles (91.5%). 1H NMR (270 MHz, D2O) 6.87 (1 H, m, Ar), 6.96 (2 H, m, Ar), 7.05 (2 H, m, Ar); 31P NMR (109 MHz, D2O) –1.33 (1 P, ArOPO32–), –1.76 (2 P, 2 x ArOPO32–), –2.66 (2 P, 2 x ArOPO32–); (MS, FAB–) 633.1, Calcd for C12H14O20P5 [M - H]– 632.8772. Found 632.8783.

Preparation of a lysate of rat hepatocytes
Hepatocytes (107 cells) were lyzed in 1 ml of ice-cold lysis buffer containing 10 mM Tris pH 8.0, 0.15 M KCl, 0.5% Nonidet P-40, 2 mM 2-mercaptoethanol, 0.1 M NaF, protease and phosphatase inhibitors (5 µM leupeptin, 0.1 mM pefabloc, 2.5 µM okadaic acid, 1 mM Na3VO4, 20 mM EDTA).

Preparation of hepatocytes for [Ca2+]i measurements
Isolated rat hepatocytes were prepared from fed female Wistar rats by limited collagenase digestion of rat liver, as described previously (11) . Under these conditions, ~20% of the cells were associated by two (doublet) or three (triplet) and were distinguished from aggregates of nonconnected cells in conventional light microscopy by screening for dilated bile canaliculi, indicators of maintained functional polarity. After isolation, rat hepatocytes were maintained (5x105 cells/ml) at 4°C in Williams’ medium E supplemented with 10% fetal calf serum, penicillin (100,000 U/ml), and streptomycin (100 µg/ml). Cell viability, assessed by trypan blue exclusion, remained greater than 96% for 4–5 h.

Microinjection
Microinjection was performed using an Eppendorf microinjector (5242), as described previously (10) . Micropipettes with an internal tip diameter of 0.5 µm (Femtotips, Eppendorf) were filled with biphenyl(2,3',4,5',6)P5 at the indicated concentration together with 5 mM Fura-2 in a buffer solution containing 100 mM KCl, 20 mM NaCl, 10 mM HEPES adjusted to pH 7.1. After microinjection, cells were allowed to recover for at least 10 min. The success of microinjection was assessed by monitoring the morphology of the cells before and after manipulation and checking the ability of the cell to retain injected Fura-2 and low [Ca2+]i.

Determination of [Ca2+]i changes in hepatocytes
Ca2+ imaging was carried out as described previously (11) . Briefly, the excitation light was supplied by a high-pressure xenon arc lamp (75 W), and the excitation wavelengths were selected using 340- and 380-nm filters (10-nm bandwidth) mounted in a processor-controlled rotating filter wheel (Sutter, Novato, CA) between the UV lamp and the microscope. Fluorescence images were collected by a CCD camera (Princeton Instruments, Evry, France), digitized and integrated in real time by an image processor (Metafluor, Princeton Instruments).

Malachite green phosphatase assay
The enzyme activity of the 5-phosphatases was measured with a phosphate-release assay using an acidic malachite green dye. Di-C8 phosphoinositides, inositol phosphates or analogs were diluted in 30-µl assay buffer (50 mM HEPES (pH 7.4), 2 mM MgCl2, 1 mg/ml BSA). The phosphatase reaction was initiated by adding the enzyme diluted in assay buffer (15 µl) to the substrates, and samples were incubated at 37°C. After 7 min, reactions were stopped by the addition of 15 µl 0.1 M EDTA. 75 µl of malachite green reagent was added to 50 µl of the reaction solution. Samples were allowed to stand for 10 min for color development before measuring absorbance at 650 nm. Inorganic-phosphate release was quantified by comparison to a standard curve of KH2PO4 in dH2O.

Ins(1,4,5)P3/Ins(1,3,4,5)P4 phosphatase assay
Inositol phosphatase activity was assayed at 37°C, using [3H]Ins(1,4,5)P3 or [3H]Ins(1,3,4,5)P4 in 50 mM HEPES (pH 7.4), 2 mM MgCl2, 1 mg/ml BSA, and the appropriate enzyme dilution in a final volume of 50 µl. The assay was initiated by adding the substrate, stopped after 7 min by addition of 1 ml ice-cold 0.4 M ammonium formate/0.1 M formic acid or 0.7 M ammonium formate/0.1 M formic acid (for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphatase assay, respectively), and the resulting solution was immediately applied to Dowex columns. [3H]Ins(1,4)P2, the product of [3H]Ins(1,4,5)P3 5-phosphatase, and [3H]Ins(1,3,4)P3, the product of [3H]Ins(1,3,4,5)P4 5-phosphatase, were separated on 0.5 ml and 1 ml Dowex columns, respectively. [3H]Ins(1,4,5)P3 3-kinase assay was performed as described before (19) .

[3H]Ins(1,4,5)P3 binding
The [3H]Ins(1,4,5)P3 binding assay was performed on microsomes of Sf9 cells overexpressing IP3R1 by a rapid filtration method, exactly as described previously (20) . Incubation (0.15 mg protein/sample) was performed on ice at pH 7.4 in the presence of 7 nM [3H]Ins(1,4,5)P3. In control samples, the specific binding amounted to 97 ± 1% of the total binding.

Ca2+-release studies in permeabilized R23–11 cells
Cell pellets of R23–11 cells stably expressing IP3R1 were resuspended in intracellular medium (120 mM KCl, 30 mM HEPES, pH 7.4, 1 mM MgCl2, 1 mM ATP, 25 mM phosphocreatine, 50 U of creatine kinase and 5 µM Fluo-3) and transferred to a 4-ml fluorescence quartz cuvette thermostatically maintained at 37°C. Cell density was 5 x 107 cells/ml. Mild treatment of the cells with digitonin (50 µM) disrupted the plasma membrane. The Fluo-3 fluorescence ({lambda}ex=503 nm and {lambda}em=530 nm) was measured with an Aminco–Bowman Series 2 spectrometer (Spectronic Unicam, Rochester, NY). A23187 (8 µM) was added at the end of each experiment to measure the total releasable Ca2+. The fluorescence signal F was calibrated by first adding 0.5 mM Ca2+ (Fmax) and then adding 5 mM EGTA (Fmin). The free [Ca2+] was calculated using the equation:

Formula 1(1)

Description of the mathematical model
We used a previously published mathematical model to describe Ca2+ dynamics and Ins(1,4,5)P3 metabolism (9) . Given the predominant role played by 5-phosphatase with respect to 3-kinase in Ins(1,4,5)P3 metabolism, Ins(1,3,4,5)P4 was not considered. The model was modified to take into account the inhibitions of both the Ins(1,4,5)P3 receptor and the 5-phosphatase by biphenyl(2,3',4,5',6)P5. Thus, the fraction of activatable channels (Eq. 1 in (9) ) became

Formula 1
where [BP] stands for the concentration of biphenyl(2,3',4,5',6)P5, and KBP is the inhibition constant of the InsP3R by biphenyl(2,3',4,5',6)P5. The rate of 5-phosphatase (eq. 3 in (9) ) became

Formula 1
where KIBP represents the inhibition constant of the 5-phosphatase by biphenyl(2,3',4,5',6)P5.

Parameter values were the same as reported earlier in Fig. 2 of (9) , except for k1 = 14.4606 s–1, k_ = 0.217 s–1, Kact = 0.6 µM and KIP= 0.2 µM. Moreover, KBP= 7 µM and KIBP= 0.5 µM. These latter values were arbitrary, the only constraint being the fact that 5-phosphatase had a higher affinity for biphenyl(2,3',4,5',6)P5 than the Ins(1,4,5)P3 receptor. In Fig. 8 , the values for biphenyl(2,3',4,5',6)P5 concentrations are 0, 1.5, 25, and 40 µM from top to bottom. A low stimulus corresponded to a rate of phospholipase C activity (VPLC) equal to 0.03 µMs–1, and a high stimulation to 0.3 µMs–1.


Figure 2
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  		Figure 2. Overlay of the structures of two inositol phosphatase inhibitors Ins(1,3,4,5)P4 and biphenyl(2,3',4,5',6)P5. Ins(1,3,4,5)P4 (in purple) and biphenyl(2,3',4,5',6)P5 (in gray) were built in Sybyl7.1 (Tripos associates). Charges were calculated by the Gasteiger-Huckel method, and each ligand was optimized to convergence using the MMFF94s method. Atoms of phosphate-1, phosphate-3, and phosphate-5 of Ins(1,3,4,5)P4 were overlaid on the phosphate-4, phosphate-2, and phosphate-6 of the biphenyl compound using the fit atoms command in Sybyl7.1. This gave the best fit root mean square deviation of 1.32 Å between equivalent atoms.


Figure 3
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  		Figure 3. Effect of biphenyl(2,3',4,5',6)P5 on recombinant enzymes and crude rat hepatocyte lysate. Effect of 1–100 µM of biphenyl(2,3',4,5',6)P5 on recombinant enzymes at 10 µM Ins(1,3,4,5)P4 as substrate (top panel). Effect of 0.5–10 µM of biphenyl(2,3',4,5',6)P5 on rat hepatocyte lysate at 1 µM Ins(1,4,5)P3 (bottom panel). The data are representative of three different experiments and are means of triplicates ± SD.


Figure 4
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  		Figure 4. The effect of benzene polyphosphate analogs on recombinant Ins(1,4,5)P3 3-kinase A. Effect of 0–100 µM of the various analogs on Ins(1,4,5)P3 3-kinase activity at 1 µM Ins(1,4,5)P3 substrate level. Data are expressed as a percentage of control value (activity in the presence of water) ± SD.


Figure 5
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  		Figure 5. Effect of various concentrations of analogs on [3H]Ins(1,4,5)P3 binding. [3H]Ins(1,4,5)P3 binding on microsomes of Sf9 cells overexpressing IP3R1 was performed in the presence of various concentrations of the indicated compounds. Values are expressed as a percentage of the binding observed in control conditions and are presented as means ± SEM.


Figure 6
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  		Figure 6. Ca2+ release in permeabilized R23–11 cells expressing IP3R1. Ca2+ release was measured as described in Materials and Methods. Biphenyl(2,3',4,5',6)P5 was added 1 min before Ins(1,4,5)P3. The inhibitory effect of biphenyl(2,3',4,5',6)P5 on Ca2+ release induced by 100 and 300 nM Ins(1,4,5)P3 was reproduced in three independent experiments.


Figure 7
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  		Figure 7. Ca2+ signals in response to 0.1–10 µM noradrenaline in hepatocytes. One cell of the doublet (lower trace in each panel) was microinjected with Fura-2 and 10 µM (A) and 300 µM (B and C) biphenyl(2,3',4,5',6)P5 in the injection pipette. Hepatocytes doublets were challenged with noradrenaline (Nor, 0.1, 1, or 10 µM) for the time shown by the horizontal bar. Results are representative of a large number of injected cells as indicated in the text.


Figure 8
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  		Figure 8. Numerical simulations of Ca2+ oscillations in response to agonist stimulation at different concentrations of biphenyl(2,3',4,5',6)P5 injected to the cell. The different panels show simulated Ca2+ oscillations at two different levels of stimulation in the absence (upper panel) or in the presence of biphenyl(2,3',4,5',6)P5 (three lower panels, increasing concentrations as indicated). Traces have been obtained with a classical inhibition term for type I inositol 5-phosphatase and Ins(1,4,5)P3 receptor by biphenyl(2,3',4,5',6)P5. A critical assumption of the model is that the 5-phosphatase has a higher affinity for the analog than the Ins(1,4,5)P3 receptor. See Materials and Methods for a description of the equations and parameter values. The data were obtained at one- and at a ten-fold higher concentration of agonist as indicated by the horizontal line.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biphenyl(2,3',4,5',6)P5 is a potent inhibitor of two inositol 5-phosphatases
In a screening of several tris- and tetrakis-phosphorylated benzene derivatives, we observed that they were potent inhibitors of recombinant type-I Ins(1,4,5)P3 5-phosphatase (13) . The inhibition was very much influenced by the number and position of the phosphate groups on the benzene ring. To establish the specificity of these new inhibitors of inositol 5-phosphatase, we compared two members of this family of enzymes: type-I inositol 5-phosphatase and SHIP2. Type-I inositol 5-phosphatase controls the degradation of Ins(1,4,5)P3 (4) , and SHIP2 has been shown to control PtdIns(3,4,5)P3 levels both in vitro (21) and in vivo (22 , 23) . Both enzymes could use Ins(1,3,4,5)P4 as substrate, which was therefore used in comparative studies (Table 1 ). Ins(1,4,5)P3 was not a substrate of SHIP2, and the related truncated lipid analog di-C8 PtdIns(3,4,5)P3 was not a substrate of type-I inositol 5-phosphatase (Table 1) .


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  		Table 1. Substrate specificity of type-I inositol 5-phosphatase and SHIP2 (assayed by malachite green phosphatase assay)

In this study, in addition to the various regioisomeric tetraphosphorylated analogs on one benzene ring, a novel pentakisphosphate derivative with phosphates spread over two benzene rings was also evaluated (Table 2 ). It appeared to be a rather potent inhibitor of the two inositol 5-phosphatases with IC50-values in the low micromolar range using 10 µM Ins(1,3,4,5)P4 as a substrate; it was also a better inhibitor compared to the tetrakis-phosphorylated analogs on one benzene ring for both enzymes (Table 2) . The mechanism of 5-phosphatase inhibition appears to be competitive with respect to the substrate (data not shown). When SHIP2 was compared to type-I Ins(1,4,5)P3 5-phosphatase, biphenyl(2,3',4,5',6)P5 was a better inhibitor of SHIP2 (IC50 = 1.8 µM) compared to type I Ins(1,4,5)P3 5-phosphatase (IC50 = 7.9 µM, Fig. 3 ). The biphenyl molecule also inhibited the crude Ins(1,4,5)P3 5-phosphatase in a lysate of rat hepatocytes (IC50 = 1 µM at 1 µM Ins(1,4,5)P3, Fig. 3 ). When incubated in the presence of recombinant type-I Ins(1,4,5)P3 5-phosphatase or SHIP2, none of the benzene tetrakisphosphate molecules nor biphenyl(2,3',4,5',6)P5 at 100 µM was dephosphorylated, as measured in a phosphate-release assay (the assays were run at different concentrations of enzyme and Ins(1,3,4,5)P4 used as positive control, was very significantly dephosphorylated as expected from our radiolabeled assay resolved on Dowex columns, data not shown).


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  		Table 2. Inhibitory potency of benzene polyphosphate analogs

Biphenyl(2,3',4,5',6)P5 is not an inhibitor of Ins(1,4,5)P3 3-kinase A
To further characterize the properties of biphenyl(2,3',4,5',6)P5, we tested its effect on two different proteins that use Ins(1,4,5)P3 either as substrate or ligand: Ins(1,4,5)P3 3-kinase and the IP3RI. In the study of Poitras et al., benzene 1,2,4-trisphosphate was an inhibitor of the activity of Ins(1,4,5)P3 3-kinase of bovine adrenal cortex (IC50 = 0.4 µM at 6 µM Ins(1,4,5)P3). This result prompted us to evaluate the effect of the biphenyl analog on recombinant Ins(1,4,5)P3 3-kinase A, the major isoenzyme expressed in brain (24) . Our data in this study were compared with several inositol phosphate analogs, some of which being potent inhibitors, such as D-2-deoxy Ins(1,4,5)P3 and D-3-deoxy Ins(1,4,6)P3 (15) . At concentrations up to 30 µM of biphenyl(2,3',4,5',6)P5, Bz(1,2,3,4)P4, Bz(1,2,3,5)P4 or Bz(1,2,4,5)P4, the inositol 3-kinase activity was not affected (Fig. 4 ). Significant inhibition of activity only started at 100 µM. We conclude that biphenyl(2,3',4,5',6)P5 and the tetrakis-phosphorylated one-ring analogs are rather poor inhibitors of the Ins(1,4,5)P3 3-kinase A.

Direct effect of biphenyl(2,3',4,5',6)P5 on IP3R1
It was reported that several inositol 5-phosphatase inhibitors that are Ins(1,3,4,5)P4 derivatives are also able to inhibit the binding of Ins(1,4,5)P3 to IP3R1 (25) . A series of tri- and tetra-phosphorylated analogs was tested on [3H]Ins(1,4,5)P3 binding on microsomes of Sf9 cells overexpressing IP3R1: biphenyl(2,3',4,5',6)P5 was clearly the most potent inhibitor of Ins(1,4,5)P3 binding (50% displacement being observed at 1.4 µM). Bz(1,2,4)P3 and Bz(1,2,3,4)P4 were not capable of displacing bound [3H]Ins(1,4,5)P3 in the 1–10 µM range (Fig. 5 ). The displacement of [3H]Ins(1,4,5)P3 binding by the benzene tetrakisphosphates was also studied at a fixed concentration of 10 µM (Table 3 ). The most potent inhibitor of the tetra-phosphorylated analogs was Bz(1,2,4,5)P4 (47% of the control at 10 µM), which was also the most potent inhibitor of type-I inositol 5-phosphatase activity. In the same experiment, 10 µM biphenyl(2,3',4,5',6)P5 decreased the binding of [3H]Ins(1,4,5)P3 to 14% of its control value (Table 3) . Our data indicate that the number and position of the phosphate groups on the benzene ring influence the recognition pattern of phosphorylated benzene molecules at the IP3R1.


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  		Table 3. [3H]Ins(1,4,5)P3 binding in control conditions and in the presence of the indicated compounds (10 µM)

Ins(1,3,4,5)P4, a well characterized inhibitor of type-I inositol 5-phosphatase, is also an inhibitor of [3H]Ins(1,4,5)P3 binding (50% displacement at 4.8 µM, Fig. 5 ).

Biphenyl(2,3',4,5',6)P5 is an inhibitor of Ins(1,4,5)P3-induced Ca2+ release
As biphenyl(2,3',4,5',6)P5 competed with Ins(1,4,5)P3 for the binding to IP3R1, we examined its functional effect on Ins(1,4,5)P3-induced Ca2+ release. An ideal model for this analysis are the permeabilized triple Ins(1,4,5)P3-receptor-knockout R23–11 cells expressing IP3R1 (18) . Biphenyl(2,3',4,5',6)P5 inhibited the Ins(1,4,5)P3 5-phosphatase activity in lysates of the same cells also with the same potency compared to recombinant Ins(1,4,5)P3 5-phosphatase (data not shown). The addition of Ins(1,4,5)P3 at concentrations of 50, 100, or 150 nM, induced Ca2+ release from the nonmitochondrial intracellular stores as previously reported (18) . This Ca2+ release was inhibited by 1 or 4 µM biphenyl(2,3',4,5',6)P5, added 1 min before 100 or 300 nM Ins(1,4,5)P3 (Fig. 6 ). A23187 was added at the end of the experiment to evaluate the total amount of releasable Ca2+. Therefore, the interaction between the analog and the IP3R1 is functionally relevant in this cell model since at higher concentrations, it overrules the effect of the analog on Ins(1,4,5)P3 5-phosphatase, which is also present.

Dual effect of biphenyl(2,3',4,5',6)P5 on Ca2+ oscillations in hepatocytes
On the basis of the experimentally determined kinetic parameters of the Ins(1,4,5)P3 5-phosphatase and 3-kinase, it was predicted that the 5-phosphatase primarily controls the levels of Ins(1,4,5)P3 and, thus the occurrence and frequency of Ca2+ oscillations (9) . We have used the model of doublets of hepatocytes that are tightly coupled by gap junctions. Coordination of Ca2+ oscillations among connected hepatocytes is fully dependent on Ins(1,4,5)P3 diffusion through gap junctions. It was previously shown that Ca2+ signals in the two cells were different when active 5-phosphatase (as compared to inactive enzyme) had been injected into one cell of the doublet at low (0.1 µM) and maximal (10 µM) noradrenaline concentrations (26) . In response to 0.1 µM noradrenaline, [Ca2+]i remained at a low basal level in the phosphatase-injected cell and oscillated at low frequency in the adjacent cell (26) . This was interpreted by a reduction in the concentration of Ins(1,4,5)P3 in the injected cells and by an intermediate concentration in the noninjected cell. We therefore compared the Ca2+ oscillations between the hepatocyte doublets, which had been injected with biphenyl(2,3',4,5',6)P5. These experiments were designed at two different concentrations of the analog (10 µM and 300 µM) in the injection pipette. We assumed the intracellular concentration of the benzene polyphosphate inside the cell to be at least 10-fold lower. At 10 µM (injected concentration), we observed two different responses: either an increase or a decrease in the frequency of Ca2+ oscillations. An increase in the frequency of the oscillations is shown in Fig. 7 A at 0.1–1 µM noradrenaline (the injected cell being the bottom trace and is compared to the upper trace). For 17 doublets in which one cell was injected, 4 cells showed an increase and 4 cells a decrease in the frequency of Ca2+ oscillations; 9 cells showed no effect. At 300 µM of the biphenyl analog in the injection pipette, the Ca2+ response in the injected cell was inhibited either partially (depending on the agonist concentration) or completely (Fig. 7B, C , respectively). At 1 µM noradrenaline, low-frequency Ca2+ oscillations were detected in the injected cell, whereas high-frequency oscillations were observed in the adjacent cells, suggesting that the biphenyl compound was mainly active in the injected cells (Fig. 7B at 1 µM noradrenaline). In 21 doublets from which one cell was injected, 11 cells showed a partial inhibition and only responded at 1.0 µM noradrenaline, whereas 10 cells showed a total inhibition at 0.1–10 µM noradrenaline (as shown in Fig. 7C ).

Simulation of the behavior of biphenyl(2,3',4,5',6)P5 in intact hepatocytes
Using a model for Ca2+ oscillations and the different parameters of both the synthesis and metabolism of Ins(1,4,5)P3, we previously simulated the presence of Ca2+ oscillations in agonist-stimulated cells. It was suggested that the phosphatase primarily controls the levels of Ins(1,4,5)P3 and thereby the occurrence and frequency of Ca2+ oscillations (9) . We used the same model and introduced biphenyl(2,3',4,5',6)P5 as a competitive inhibitor at both the level of the 5-phosphatase and at the Ins(1,4,5)P3 binding site of the Ins(1,4,5)P3 receptor. We introduced a lower dissociation constant for the phosphatase as compared to the binding to the Ins(1,4,5)P3 receptor. When a low concentration of the analog was present inside the cells, the model predicted an increase in the frequency of Ca2+ oscillations in response to a stimulus (1.5 µM as compared to control cell in Fig. 8 ). At higher concentrations, the Ca2+ response was totally lost. Ca2+ oscillations were recovered when the concentration of the stimulus was 10-fold higher (Fig. 8 at 25 µM of biphenyl(2,3',4,5',6)P5). The fact that in our experimental observations, the frequency of Ca2+ oscillations could either decrease or increase at low concentration of biphenyl(2,3',4,5',6)P5, depending on the injected cell, was interpreted as the consequence of its interaction at two proteins: First, on Ins(1,4,5)P3 5-phosphatase, thereby increasing the intracellular levels of Ins(1,4,5)P3 and second, on the Ins(1,4,5)P3 receptor itself by an inhibitory effect on Ins(1,4,5)P3 binding. This dual effect depended on the dissociation constant of the analog with respect to the 5-phosphatase as compared to the Ins(1,4,5)P3 receptor. In this framework, at high concentrations of biphenyl(2,3',4,5',6)P5, the concentration of Ins(1,4,5)P3 in the cell would be high (because of 5-phosphatase inhibition), but this would not allow any significant [Ca2+]i increase, because the receptor would also be inhibited. Our simulation data suggest that in the cell, biphenyl(2,3',4,5',6)P5 had a higher apparent affinity for the phosphatase as compared to the Ins(1,4,5)P3 receptor. Indeed, if the affinity for the Ins(1,4,5)P3 receptor would be higher than for the phosphatase, an increase in the frequency of Ca2+ oscillations at low agonist concentrations (as shown in Fig. 7A ) could never be observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inositol and lipid phosphatases can act at the 5-, 4-, and 3-position of the inositol ring, generating either inactive compounds or novel second messengers (27) . Several soluble inositol phosphates and phosphoinositides are dephosphorylated at the 5-position by the large family of inositol 5-phosphatases, which in mammalian cells consist of 10 different enzymes (1) . Their primary structures show the presence of a catalytic domain and a series of protein interaction motives which target the enzymes to different parts of the cell. Inositol 5-phosphatase inhibitors could be useful tools in order to evaluate the role of individual enzymes in any cell model and compare their relative activities and eventual redundancy in cells. Type-I inositol 5-phosphatase appears to be a membranous enzyme as shown initially in liver or brain tissue (28 , 29) . Cloning studies have shown that it is the shorter enzyme in the mammalian inositol 5-phosphatase family (412 amino acids) and that it contains a C-terminal prenylation site (30) . It controls the levels of Ins(1,4,5)P3 and the occurrence of Ca2+ oscillations in intact cells. A mutation of the prenylation site also abolished its effect on [Ca2+]i (4) .

We looked for inositol 5-phosphatase inhibitors using human type-I inositol 5-phosphatase produced in bacteria. Initial studies of Poitras et al. using a relatively crude enzyme have suggested that benzene polyphosphate analogs could be of interest in that respect (12) . A wider study of benzene polyphosphate analogs containing two, three, and four phosphate groups, show that they may act as type-I inositol 5-phosphatase inhibitors (13) . They were not substrates at 100 µM, indicating that if they interact at the active site, they do not completely adopt the structure of Ins(1,4,5)P3 or Ins(1,3,4,5)P4, the two natural substrates of that enzyme. One exception is 3-hydroxybenzene 1,2,4-trisphosphate, which was dephosphorylated by type-I Ins(1,4,5)P3 5-phosphatase to 2,3-dihydroxybenzene 1,4-bisphosphate (13) .

Surprisingly, we show here that the most potent inhibitor was biphenyl(2,3',4,5',6)P5. Compared to bisphosphoglycerate, the first type-I inositol 5-phosphatase inhibitor identified (Ki of approx. 0.35 mM with Ins(1,4,5)P3 as substrate (31) ), biphenyl(2,3',4,5',6)P5 is much more potent, having an IC50-value in the low micromolar range, and was not a substrate using the phosphate release assay when tested at 100 µM. A possible explanation for the potency of biphenyl(2,3',4,5',6)P5 is that the phosphate groups of this molecule can nearly overlay with the phosphate groups of Ins(1,3,4,5)P4, (Fig. 2) a very potent inhibitor of type I inositol 5-phosphatase. However, in contrast to this molecule, it is not a substrate.

This result prompted us to consider whether biphenyl(2,3',4,5',6)P5 could also inhibit the activity of the lipid phosphatase SHIP2. Because this enzyme was shown to accept Ins(1,3,4,5)P4 as a substrate, we used that substrate in our comparative studies. Although we clearly showed that Ins(1,4,5)P3 was not a substrate of SHIP2, the biphenyl analog was an inhibitor of SHIP2 and was even more potent compared to its effect on type-I enzyme. The rank order of potency (biphenyl > Bz(1,2,4,5)P4) was conserved between the two enzymes, suggesting that they share some conserved structural characteristics. In another study, although not tested as an inhibitor, Ins(1,2,3,4,5)P5 was reported to be a good substrate of SHIP2 (32) . Therefore, highly phosphorylated molecules such as biphenyl(2,3',4,5',6)P5 could be very well tolerated at the active site of SHIP2. We have observed that assay conditions could largely influence the lipid phosphatase activity of SHIP2: the presence of phosphatidylserine vesicles stimulated the activity of SHIP2 with PtdIns(3,4,5)P3 di-C8 as substrate, whereas type-I Ins(1,4,5)P3 5-phosphatase was not sensitive to the presence of this anionic lipid (17) . The inhibitory effect of biphenyl(2,3',4,5',6)P5 was also seen with PtdIns(3,4,5)P3 di-C8 as substrate but only in the absence of phosphatidylserine. In the presence of the anionic lipid at 200 µM, the inhibitory effect was no longer observed (data not shown). Therefore, although we have established that biphenyl(2,3',4,5',6)P5 shows an affinity for SHIP2, the potency of inhibition of its natural lipid substrate may still rely on assay conditions. A high-throughput assay for SHIP2 of a 91,060-member compound library resulted in the identification of SHIP2 inhibitors, although no chemical structures were presented in this study (33) . A comparison of the best inhibitors of that study with biphenyl(2,3',4,5',6)P5 will be interesting and may provide a route to more potent novel inhibitors.

The biphenyl analog was not an inhibitor of the Ins(1,4,5)P3 3-kinase A produced in bacteria. Because Ins(1,4,5)P3 3-kinase A is very specific in the recognition of Ins(1,4,5)P3 and tolerates only very few modifications of its structure, thus, this result was not unexpected (15 , 34) . Data in the study of Poitras et al. indicated that benzene 1,2,4-trisphosphate competitively blocked Ins(1,4,5)P3 binding to adrenal cortex microsomes with a half-maximal concentration at 34 µM (12) . Our conditions for measuring [3H]Ins(1,4,5)P3 binding have the advantage of using a single Ins(1,4,5)P3 receptor subtype, and the assay was performed at pH of 7.4, which is more physiologically relevant than in many other studies. Among the phosphorylated benzene analogs we have tested, biphenyl(2,3',4,5',6)P5 was by far the most potent competitor of [3H]Ins(1,4,5)P3 binding on IP3R1. We suggest that the displacement is not just electrostatic: competition of [3H] Ins(1,4,5)P3 binding by nonradioactive Ins(1,4,5)P3 occurs at much lower concentrations with a 50% displacement around 50 nM and a nearly complete displacement observed at 100 nM (20) . Moreover, when the tris-phosphorylated benzene regioisomers were compared, Bz(1,2,4,5)P4 was always more efficient as compared to Bz(1,2,3,4)P4 or Bz(1,2,3,5)P4 (Table 3) . Given the fact that Ins(1,3,4,5)P4, a potent type-I 5-phosphatase inhibitor, also inhibited the binding of Ins(1,4,5)P3 to the receptor (25) , this would indicate that the receptor and the phosphatase must share some common structural requirements. It was suggested in another study that Ins(1,3,4,5)P4 acted at two steps: at low concentrations, it facilitates store-operated Ca2+ influx by inhibition of Ins(1,4,5)P3 5-phosphatase (25) . At higher concentrations, it was as an inhibitor of Ins(1,4,5)P3 receptors, enabling Ins(1,3,4,5)P4 to act as a potent bimodal regulator of cellular sensitivity to Ins(1,4,5)P3. Thus, it provides both facilitatory and inhibitory feedback on Ca2+ signaling (25) .

High concentrations of biphenyl(2,3',4,5',6)P5 were shown to inhibit the Ca2+ response in two different cell models: in B cells overexpressing the IP3R1 and in hepatocytes. This is supported by the fact that it inhibits the binding of Ins(1,4,5)P3 to IP3R1, thereby preventing any Ca2+ response. The most important feature of our study is the observation that biphenyl(2,3',4,5',6)P5 may positively or negatively affect the frequency of Ca2+ oscillations stimulated by noradrenaline. Because we injected the analog into hepatocytes, the result is probably dependent on its effective concentration that is experienced by the Ins(1,4,5)P3 receptor. It affects the phosphatase activity in a very restricted window of concentration of both agonist and analog. In our injections, this has implied the use of 10 µM of biphenyl(2,3',4,5',6)P5 in the injection pipette. The data presented here highlight two important properties of a type-I phosphatase inhibitor such as Ins(1,3,4,5)P4 and the biphenyl polyphosphate molecule: 1) at high concentrations, it inhibits the receptor and therefore the Ca2+ response and 2) at low concentrations, it provides a window where it increases the level of Ins(1,4,5)P3 producing a new steady-state level of this important molecule. This is essentially the result of its interaction with the 5-phosphatase and the enzymatic properties of that enzyme compared to those of the Ins(1,4,5)P3 3-kinase. Consequently, this increase in Ins(1,4,5)P3 concentration activates the frequency of Ca2+ oscillations. This interpretation is supported by our model of Ca2+ oscillations, taking into account the fact that the dissociation constant is lower for the Ins(1,4,5)P3 5-phosphatase than for the Ins(1,4,5)P3 receptor (Fig. 8) .

In conclusion, our data suggest that biphenyl(2,3',4,5',6)P5 could be an interesting core molecule in order to control intracellular levels of Ins(1,4,5)P3 and its effect in Ins(1,4,5)P3-induced Ca2+ release. At low concentrations, it inhibits the Ins(1,4,5)P3 5-phosphatase activity and participates in the generation of a new steady-state level of Ins(1,4,5)P3. At high concentration, it primarily acts by inhibiting the binding of Ins(1,4,5)P3 to its receptor and, by this mechanism, the Ca2+ response. The phosphatase appears to be a key enzyme in the control of Ca2+ oscillations, and further study will be needed to investigate the physiological consequences of this control in hepatocytes and other cells. The close functional relationship between the phosphatase and the Ins(1,4,5)P3 receptor converging in the control of Ca2+ oscillations is consistent with the fact that in yeast, it is another pathway that is used in the metabolism of Ins(1,4,5)P3: an inositol phosphate multikinase, controls the level of Ins(1,4,5)P3 and the synthesis of inositol pentakisphosphate (35) . Yeast also does not have an Ins(1,4,5)P3 receptor nor an Ins(1,4,5)P3 5-phosphatase (36) .

Biphenyl(2,3',4,5',6)P5 could be a lead molecule in the synthesis of cell-permeable Ins(1,4,5)P3 5-phosphatase inhibitors that could be exploited as biological tools. It is achiral and easier to synthesize than a chiral inositol polyphosphate or related derivative. Furthermore, the synthesis of the masked lipophilic phosphate derivative which can be used without microinjection should be easy to accomplish since only the phosphate groups have to be protected. These compounds may also inhibit other isoenzymes within the inositol 5-phosphatase family. They could be evaluated as competitors with respect to the Ins(1,4,5)P3 receptor, and as we have shown here, could positively or negatively affect the frequency of Ca2+ oscillations in response to a Ca2+-mobilizing agonist.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Fonds de la Recherche Scientifique Médicale, and by a grant from the FRFC-IM Communauté Française de Belgique and Wellcome Trust Programme Grant support (060554 to BVLP). This work was executed in the framework of research network IAPV-05 (Belgium Science Policy). We thank Mrs. Colette Moreau and Mr. Tomas Luyten for their technical help and advice, Dr. Andrew Riley for helpful discussions and Dr. M. Trusselle for the molecular modeling overlay. We also thank Dr. T. Kurosaki (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) for the R23–11 cell line.

Received for publication November 15, 2006. Accepted for publication December 14, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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