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SHORT COMMUNICATION

The Phosphoinositide-Specific Phospholipase C Inhibitor U73122 (1-(6-((17ß-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) Spontaneously Forms Conjugates with Common Components of Cell Culture Medium

Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Belmont, Surrey, United Kingdom (N.E.W., W.J.C., R.R., Y.M.N., W.A., P.W.S., S.A.E., F.I.R.); and Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom (N.P.J., M.K.)

(Received December 21, 2006; accepted March 28, 2007)

	Abstract

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Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca²⁺ release from inositol 1,4,5-triphosphate-sensitive stores. U73122 [GenBank] (1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 [GenBank] has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 [GenBank] to common components of cell culture medium, namely L-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 [GenBank] on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM L-glutamine) was 150, 60, 32, 30, and 18 min, respectively. However, U73122 [GenBank] was not recoverable from medium supplemented with 0.5% BSA. U73122 [GenBank] underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 [GenBank] were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca²⁺ release in a human epidermoid carcinoma cell line (A431) by U73122 [GenBank] was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 [GenBank] to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.

U73122 (1-(6-((17ß-3-methoxyestr-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione) is firmly established as the archetypal inhibitor of PLC. Recently, the lack of selectivity of PLC inhibition by U73122 [GenBank] has been raised: U73122 [GenBank] was shown to possess estrogenic activity (Cenni and Picard, 1999) and to inhibit a range of unrelated proteins including 5-lipoxygenase (Feisst et al., 2005), histamine H1 receptor (Hughes et al., 2000), adenosine A₁ receptor (Walker et al., 1998), and plasma membrane Ca²⁺ channels (Berven and Barritt, 1995; Pulcinelli et al., 1998). U73122 [GenBank] is also more potent at inhibiting the mobilization of Ca²⁺ and plasma membrane Ca²⁺ channels compared with PLC inhibition, as reported by several authors (Berven and Barritt, 1995; Grierson and Meldolesi, 1995; Wang, 1996).

This study identifies the conjugates of U73122 [GenBank] formed on incubation with standard cell culture media. The inactive analog of U73122 [GenBank] , U73343 [GenBank] , in which the maleimide is substituted with a nonreactive succinimide, does not form these conjugates. We also show that the presence of BSA in the medium of a Ca²⁺ release assay abolishes the inhibitory potency of U73122. [GenBank] It is proposed that the availability of U73122 [GenBank] to interact with PLC in an intact cell system will be severely hampered through conjugation of the maleimide group to nonspecific biological nucleophiles, and therefore, inhibitory activity observed in cell-based assay cannot be assumed to occur via PLC.

	Materials and Methods

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Materials. U73122 [GenBank] (Fig. 1A) and U73343 [GenBank] (Fig. 1B) were obtained from Calbiochem (Merck, Nottingham, UK). Compounds were prepared as 5 mM solutions in DMSO and stored at –80°C until required. Standards were checked for degradation products by LC/MS immediately before use. Glutathione, L-glutamine, BSA (lipid-free), DMSO, MCDB131 medium, DMEM (without L-glutamine and phenol red), HBSS, complete medium, EGF, and phosphate-buffered saline (PBS) tablets were obtained from Sigma-Aldrich (Poole, UK). Fisher Scientific (Loughborough, UK) supplied HPLC-grade solvents.

View larger version (21K): [in this window] [in a new window]   FIG. 1. A, U73122. B, the inactive succinimide-containing analog U73343. C, inhibition (IC50) of PLC by U73122 determined using Phospholipid FlashPlates (PerkinElmer Life and Analytical Sciences, Boston, MA), in the absence (; r2 0.98 , Hill slope 2.6) and presence (; r2 0.67 , Hill slope 1.3) of DTT (1 mM). D, disappearance of U73122 (5 µM) after incubation (37°C) with MCDB131 without BSA (), DMEM supplemented with 2 mM L-glutamine (), complete medium (gray inverted triangle), HBSS containing 2 mM glutamine (gray square), or PBS () and stability of U73343 (5 µM) after incubation (37°C) with MCDB131 without BSA ().

PLC Inhibition in Vitro. Inhibition of PLC by U73122 [GenBank] in the presence or absence of dithiothreitol (DTT; 1 mM) was assessed as described previously (Bembenek et al., 2003; Katan et al., 2003). In brief, after addition of enzyme, the rate of tritiated PIP₂ hydrolysis was monitored in the presence or absence of U73122 [GenBank] (0.5–100 µM) with or without DTT (1 mM). Human PLC₂ used for this assay was sourced from insect (Sf9) cells expressing full-length PLC₂ containing a C-terminal His₆ tag. Experimental details for the expression of PLC₂ protein and its subsequent purification have been described previously (Rodriguez et al., 2001).

Ca²⁺ Release Assay in A431 Cells. U73122 [GenBank] was incubated with A431 (American Type Culture Collection via LGC Promochem, Middlesex, UK), an epidermoid carcinoma cell line overexpressing epidermal GFR (Bembenek et al., 2003; Katan et al., 2003). A431 cells cultured in DMEM containing 2 mM glutamine were harvested using TrypLE Express (Invitrogen Corporation, Paisley, UK) and seeded at a density of 5 x 10⁴ cells per well in 100 µlina sterile flat, clear-bottomed, black-walled 96-well microtiter plate (Corning B.V. Life Sciences, Schiphol-Rijk, The Netherlands). Cells were incubated in a humidified incubator (5% CO₂ in air, 37°C) for 24 h before the experiment to allow cells to attach. Probenecid was prepared in 1 M NaOH (2.5 mM), diluted (1:1 v/v) with 20 mM HEPES in HBSS, and 200 µl was added to 10 ml of Calcium 3 dye (Molecular Devices Corp., Berkshire, UK). Medium was removed from the wells and replaced with 50 µl of Calcium 3 dye containing 2.5 µM probenecid and incubated for 60 min. During the incubation time, HBSS (50 µl) containing 10 µM U73122 [GenBank] , 10 µM U73122 [GenBank] , and 4 mM glutamine, 10 µM U73122 [GenBank] and 1% BSA (lipid-free), or 0.1% DMSO was added at 30, 45, 55, or 60 min. After incubation, cells were challenged with EGF (50 µl of 5 µg · ml^–1 in HBSS) and the Ca²⁺ release response was determined. Ca²⁺ release was measured at 37°C on a fluorometric imaging plate reader (Molecular Devices Corp.) with excitation 488 nm and emission filter 510 to 570 nm over a 2-min period immediately after EGF addition.

Incubation with Cell Culture Medium. U73122 [GenBank] or U73343 [GenBank] was incubated (37°C) with PBS (10 mM), glutathione (15 mg · ml^–1 in 10 mM PBS), L-glutamine (2 mM in HBSS), MCDB131 medium, MCDB131 supplemented with 0.5% BSA, complete medium, DMEM supplemented with 2 mM L-glutamine. Samples were incubated directly on the LC/MS autosampler (37°C) and aliquots (25 µl) taken sequentially for analysis over a total incubation period of 60 min.

Extraction of U73122 [GenBank] from Plasma. After addition of U73122 [GenBank] (1 µM) to mouse plasma protein, precipitation was effected by addition of 3 volume-equivalents of either methanol or acetonitrile to the plasma sample. After centrifugation (13,000g, 5 min, 4°C) the supernatant was analyzed by LC/MS. Liquid-liquid extraction using 10 volume-equivalents of either ethyl acetate or dichloromethane was also undertaken, followed by centrifugation (1000g,15 min, 4°C) and evaporation of the organic layer to dryness under N₂. Samples were reconstituted in mobile phase before analysis by LC/MS. The impact of pH on extraction efficiency was also assessed by addition of an equal volume of either 0.1 M HCl or 0.1 M NaOH to the plasma sample before liquid-liquid extraction.

LC/MS Analysis. Analysis was performed on a LCQ classic system [liquid chromatography quadrupole ion storage system (quistor)] from Thermo Scientific (Hemel Hempstead, Herfordshire, UK). The LC system consisted of a P400 pump. AS3000 autosampler, and SN400 system controller interface (Spectra Systems, Thermo Fisher Separations products), connected to the quistor. Data handling was achieved with Xcalibur (version 1.1). Chromatographic separation was facilitated on a Synergi 4µ Polar-RP 80A 50 x 3mm analytical column (Phenomenex, Macclesfield, Cheshire, UK) using a mobile phase with initial conditions of 90% formic acid (0.1%) and 10% methanol at a total flow rate of 1 ml · min^–1. A linear gradient with the methanol content rising to 90% from 0.5 to 6 min was initiated and held for a further 3.5 min. Detection of analytes was achieved using the following LCQ conditions: N₂ sheath gas 80, N₂ auxiliary gas 20, spray voltage 4.5 kV, capillary voltage 10 V, capillary temperature 250°C, and tube lens offset –10 V. The collision energy for MS² and MS³ analysis was set between –35 and –45 V. Concentrations of the compound were normalized using the peak area of the compound analyzed immediately after addition to the medium. Graphical representation of results was constructed using Prism (version 3.03; GraphPad Software Inc., San Diego, CA).

	Results

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In Vitro Inhibition of the PLC Enzyme by U73122. [GenBank] U73122 [GenBank] inhibited PLC₂ with an IC₅₀ value of 5.3 µM. Further analysis found that in the presence of 1 mM DTT, the inhibitory effect of U73122 [GenBank] was reduced substantially, having an IC₅₀ value of >100 µM (Fig. 1C). The inhibition of PLC by U73122 [GenBank] was in good agreement with other published data (Smith et al., 1990). The current assay was specifically designed for high throughput screening implementation and the results obtained using U73122 [GenBank] confirmed its reliability and validity to identify PLC inhibitors in a high throughput screening format.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Extracted ion chromatogram of the [M + H]+ ion of U73122 (m/z 465) (A), U73122 hydrolysis product (m/z 483) (B), U73122-glutathione conjugate (m/z 772) (C), and U73122-glutamine conjugate (m/z 611) (D).

Two products were detected on incubation of U73122 [GenBank] with PBS, one of which had the expected [M + H]⁺ ion of U73122 [GenBank] (m/z 465; Fig. 2A). The MS² spectrum of U73122 [GenBank] had fragmentation ions of m/z 173 (C₁₂H₁₃O^·+), m/z 197 (C₁₀H₁₇N₂O₂⁺), and m/z 269 (C₁₉H₂₅O^·+) (Fig. 3A). The second product had a [M + H]⁺ ion of m/z 483 (18 amu greater than U73122 [GenBank] ; Fig. 2B). The MS² spectrum of m/z 483 showed that hydrolysis occurred on the maleimide group (Fig. 3B). This phenomenon has been described previously for a maleimide functional group (Khan, 1984).

View larger version (32K): [in this window] [in a new window]   FIG. 3. Spectra and proposed fragmentation patterns for MS2 of U73122 (A), MS2 of U73122 hydrolysis product (B), MS3 of U73122-glutathione product (C), and MS3 of U73122-glutamine product (D).

From these results it was predicted that U73122 [GenBank] would undergo conjugation with thiol-containing compounds present in cell culture medium such as cysteine. However, U73122 [GenBank] -cysteine conjugates were not detected after incubation of U73122 [GenBank] with culture medium. The predominant [M + H]⁺ ion detected after incubation of U73122 [GenBank] with cell culture medium had a m/z of 611, which corresponded to the U73122 [GenBank] -glutamine conjugate (Fig. 2D). Incubation of U73122 [GenBank] with L-glutamine alone resulted in the formation of a species with an [M + H]⁺ ion and a retention time identical to that observed in the culture media. MS² analysis of the molecular ion revealed a spectrum with two product ions of m/z 465 and m/z 594 (data not shown). MS³ analysis showed that the m/z 465 ion gave the spectrum characteristic of U73122 [GenBank] itself, confirming that the molecular ion was a conjugate of U73122 [GenBank] (data not shown). The m/z 594 product ion of [M + H]⁺ ion 611 arose from loss of 17 Da and showed further loss of 28 Da. These neutral losses are likely to arise from successive losses of ammonia and CO from the amide group (Fig. 3D).

Inhibition of EGF-Stimulated Ca²⁺ Release from A431 Cells. The inhibition of an EGF-evoked release of Ca²⁺ by U73122 [GenBank] was measured in A431 cells and the effect of addition of glutamine and BSA on potency was assessed. U73122 [GenBank] readily formed a conjugate with glutamine and, therefore, it was anticipated that addition of glutamine would decrease the activity of the compound. However, glutamine did not appear to diminish the potency of U73122 [GenBank] inhibition of EGF-stimulated Ca²⁺ release (data not shown). BSA (0.5%) caused a marked reduction in the potency of U73122 [GenBank] that was dependent on the preincubation time of the cells with the compound and BSA (Fig. 4A). After 30 min of incubation of U73122 [GenBank] (5 µM) and 0.5% BSA with the A431 cells, no inhibition of EGF-elicited Ca²⁺ release was detected and the Ca²⁺ curve was identical to that without addition of U73122 [GenBank] .

View larger version (22K): [in this window] [in a new window]   FIG. 4. A, inhibition of EGF-stimulated Ca2+ release in A431 cells after coincubation of the cells with 5 µM U73122 and 0.5% BSA for 0, 5, 15, and 30 min (n = 3). B, Michael addition reaction of the conjugation of the maleimide of U73122 with biological nucleophiles (Nuc).

	Discussion

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U73122 is a weak inhibitor (IC₅₀ 5 µM) of PLC in the enzyme-based assay. The reactivity of the maleimide group is critical for this activity as its replacement with a succinimide (U73343 [GenBank] ), or coincubation with DTT, renders the compound inactive. However, the inherent reactivity of the maleimide functional group may prevent U73122 [GenBank] from interacting effectively with PLC in more complex cell-based assays. In A431 cells, U73122 [GenBank] was capable of inhibiting the Ca²⁺ release response after stimulation with EGF. Although inhibition of Ca²⁺ can be rationalized by PLC inhibition, there are no definitive data demonstrating that U73122 [GenBank] exerts this effect by directly interacting with PLC in a cellular assay. The supplementation of HBSS with 0.5% BSA caused a reduction in the inhibitory potency of U73122 [GenBank] even when preincubated for as little as 5 min with the cell line. This clearly demonstrates competition for U73122 [GenBank] between cellular proteins and BSA.

On incubation of U73122 [GenBank] with PBS, the compound was chemically unstable and readily underwent hydrolysis of the maleimide group, a reaction that has been previously characterized with maleimide itself (Khan, 1984). U73122 [GenBank] readily formed a conjugate with glutathione, a ubiquitous thiol source present in cells. The spectral data obtained for the U73122 [GenBank] -glutathione conjugate supports the proposition that conjugation occurs via the thiol of glutathione, showing neutral loss of 129 Da and a fragment ion of U73122 [GenBank] conjugated to a thiol group on the maleimide (Fig. 3C). Similarly, the conjugation of N-ethylmaleimide and maleimidosugars with the thiol of glutathione has been reported previously (Lee and Samuels, 1961; Shin et al., 2001).

Surprisingly, U73122 [GenBank] did not appear to form thiol conjugates when incubated with cysteine-rich media MCDB131 or DMEM. Conjugation of U73122 [GenBank] with L-glutamine was identified as the principal component in cell culture medium, and its formation was confirmed on incubation of U73122 [GenBank] with 2 mM L-glutamine in PBS. Glutamine contains three nucleophilic centers that can undergo conjugation with the maleimide moiety: an amide, amino, and carboxylic acid group. However, spectral data excluded the possibility of conjugation through the amide group. It anticipated that the glutamate would react via the more nucleophilic amino group (as depicted in Fig. 3D) rather than the carboxylate. Although this reaction cannot be confirmed based on the mass spectral data obtained, maleimide conjugation to glutamine via the amino group has been reported previously (Sharpless and Flavin, 1966). The lack of thiol conjugation in medium could result from L-glutamine having more nucleophilic characteristics under the experimental conditions, or the instability of thiol conjugates formed prevented their detection. Supplementation of cell culture medium with 0.5% BSA resulted in the instantaneous disappearance of U73122 [GenBank] , probably by covalent binding to nucleophilic centers present on BSA. It was reported that maleimide analogs of the anticancer drugs doxorubicin and carboplatin selectively conjugated to the cysteine-34 position of human serum albumin (Warnecke et al., 2004), and therefore, it is conceivable that U73122 [GenBank] conjugates to the equivalent cysteine residue in BSA. However, conjugation of U73122 [GenBank] to other nucleophilic centers cannot be discounted. These data clearly indicate that although the maleimide is reactive and capable of reacting with a number of nucleophilic species, there was a level of selectivity probably governed by chemical and steric constraints.

The maleimide group undergoes a Michael addition reaction with biological nucleophiles to form the respective conjugate (Fig. 4B). The electron-deficient double bond, activated by the two carbonyl groups present in the maleimide of U73122 [GenBank] , is absent in U73343 [GenBank] , and therefore, this type of reactivity is precluded for the inactive analog. The chemical reactivity of U73122 [GenBank] must raise questions about the ability of the compound to distribute effectively within a cell-based assay (or in vivo) and interact with the target enzyme. Alkylation by the reactive maleimide moiety of U73122 [GenBank] has previously been suggested to account for the nonspecific effects of U73122 [GenBank] (Horowitz et al., 2005). Although U73122 [GenBank] inhibited PLC-dependent processes in cells (measured by PIP₂ decrease/inositol 1,4,5-triphosphate increase and diacylglycerol production), it also had many non-PLC-mediated effects that could be mimicked by N-ethylmaleimide (NEM) (Horowitz et al., 2005). Pretreatment of cells with NEM prevented the additional effects of U73122 [GenBank] (or vice versa), indicating that NEM and U73122 [GenBank] were capable of alkylating the same nucleophilic groups (Horowitz et al., 2005).

In cell-based assays, therefore, U73122 [GenBank] will rapidly bind to exposed nucleophilic thiols and/or amines present either in macromolecules in the medium (e.g., serum or albumin) or on plasma membrane proteins limiting the availability of the compound to interact with PLC in intact cells. In the current study, the presence of glutamine in the Ca²⁺ assay did not interfere with the inhibition of EGF-stimulated calcium release by U73122 [GenBank] , even though it readily conjugated with glutamine. However, U73122 [GenBank] had a half-life of 60 min when incubated with glutamine in HBSS (Fig. 1D); thus, it appears that a sufficient concentration remained to elicit an effect in the cell assay. This is in contrast to the addition of BSA to the medium, where a rapid change in the potency of U73122 [GenBank] was observed, demonstrating the reactivity to BSA nucleophilic centers. Therefore, the availability of U73122 [GenBank] in cell-based assays that incorporate supplementation of culture medium with BSA or serum will be extremely short-lived. Caution is recommended in using U73122 [GenBank] to attribute PLC-dependent cellular events, because the inherent chemical reactivity of U73122 [GenBank] will have an impact on the experimental outcome in the presence of biological nucleophiles (i.e., any cell-based assay). Therefore, it is vitally important for the advancement of understanding the role of PLC in signal transduction pathways to identify a chemically stable PLC inhibitor to replace U73122 [GenBank] and/or to supplement the studies with inhibition of gene or protein expression (e.g., antisense oligonucleotides, small interfering RNA).

	Acknowledgments

 
We thank Sam Ranasinghe, University College London, for technical support.

	Footnotes

 
This work was supported by Cancer Research UK [CUK] Programme Grant C309/A8274.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.014498.

ABBREVIATIONS: PLC, phosphoinositide-specific phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; U73122 [GenBank] , 1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U73343 [GenBank] , 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; LC/MS, liquid chromatography-mass spectrometry; EGF, epidermal growth factor; DTT, dithiothreitol; NEM, N-ethylmaleimide; HBSS, Hanks' buffered saline solution; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium without phenol red and L-glutamine; A431, human epithelial carcinoma cell line; MCDB131, optimized basal nutrient medium; GFR, growth factor receptor.

Address correspondence to: Nicola Wilsher, DMPK, Cancer Research UK Centre for Cancer Therapeutics, 15 Cotswold Road, Institute of Cancer Research, Belmont, Surrey SM2 5NG. E-mail: nicola.wilsher{at}icr.ac.uk

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.014498v1 35/7/1017    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wilsher, N. E. Articles by Raynaud, F. I. Search for Related Content PubMed PubMed Citation Articles by Wilsher, N. E. Articles by Raynaud, F. I.