- Email: [email protected]
Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes
Biochemical and Biophysical Research Communications 415 (2011) 696–701
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes Alessia Grozio a,b, Enrico Millo a,b, Lucrezia Guida a,b, Tiziana Vigliarolo a,b, Marta Bellotti a,b, Annalisa Salis a,b, Chiara Fresia a,b, Laura Sturla a,b, Mirko Magnone a,b, Andrea Galatini c, Gianluca Damonte a,b, Antonio De Flora a,b, Santina Bruzzone a,b,d, Luca Bagnasco e, Elena Zocchi a,b,d,⇑ a
Department of Experimental Medicine (DIMES), Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova, Italy Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV 9, 16132 Genova, Italy Department of Chemistry and Industrial Chemistry, via Dodecaneso 31, 16146 Genova, Italy d Advanced Biotechnology Center (ABC), Largo Rosanna Benzi 10, 16132 Genova, Italy e Department of Internal Medicine, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy b c
a r t i c l e
i n f o
Article history: Received 27 October 2011 Available online 6 November 2011 Keywords: Abscisic acid antagonist Granulocytes Monocytes Inflammation LANCL2
a b s t r a c t The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E2 (PGE2) by human granulocytes, release of PGE2 and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of antiinflammatory agents. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The phytohormone abscisic acid (ABA) is involved in important functions in higher plants, including response to abiotic stress, regulation of seed germination, control of stomatal closure and of gene transcription [1]. ABA has recently been shown to stimulate several functions of human granulocytes (phagocytosis, reactive oxygen species and nitric oxide production, chemotaxis) through a signalling pathway sequentially involving a pertussis toxin-sensitive G-protein/receptor complex, activation of adenylate cyclase with cAMP overproduction, protein kinase A activation, ADP-ribosyl cyclase phosphorylation and consequent cADPR overproduction, leading to an increase of the [Ca2+]i [2]. The increase of intracellular ABA and its release by activated human granulocytes indicate that ABA is an endogenous hormone in humans [2]. ABA also stimulates the functional activity of human monocytes: nanomolar ABA evokes a Abbreviations: ABA, abscisic acid; LANCL2, Lanthionine synthetase component C-like protein 2; GST, glutathione S-transferase; PGE2, prostaglandin E2; MCP-1, monocyte chemoattractant protein 1; H 2 DCFDA, dichlorodihydrofluorescein diacetate. ⇑ Corresponding author at: DIMES, Viale Benedetto XV 1, 16132 Genova, Italy. Fax: +39 0 10 354415. E-mail address: [email protected] (E. Zocchi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.10.143
cADPR-mediated [Ca2+]i rise, leading to NF-jB translocation, increase of cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production, release of monocyte chemoattractant protein 1 (MCP-1) and of metalloprotease 9 (MMP-9), all events involved in atherogenesis [3]. Monocytes release ABA when they are exposed to thrombin-activated platelets, as occurs at the injured vascular endothelium, and ABA behaves as an autocrine and paracrine signal, stimulating monocyte migration and MCP-1 release and vascular smooth muscle cell (VSMC) migration and proliferation [3]. These results, and presence of ABA in human arterial plaques at a 10-fold higher concentration compared to normal arterial tissue [3], suggest that ABA may play a central role in the molecular cross-talk between platelets, monocytes and VSMC leading to the development of the atherosclerotic lesion. To study the role of ABA in cellular and animal models of inflammation and atherosclerosis, pharmacological tools through which the effect of ABA can be switched off are needed. Here we describe the functional characterization of a synthetic ABA analog, previously shown to partially inhibit ABA transport in plant cells [4]. This ABA analog is endowed with anti-inflammatory properties on human granulocytes and monocytes in vitro through its ability to compete with ABA for binding to cell membranes and to the recently identified human ABA receptor, LANCL2 [5].
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2. Materials and methods
2.6. Determination of intracellular cAMP levels
2.1. Synthesis of 20 a,30 a-dihydro-20 a, 30 a-epoxyabscisic acid (analog #10)
Granulocytes (1.5 107/ml) resuspended in HBSS with Ca2+ and Mg2+ were preincubated for 5 min at 25 °C with 10 lM cAMP phosphodiesterase inhibitor (Sigma). Duplicate 200 ll-aliquots of cells were incubated for 1 min at 25 °C without (control) or with 1 lM ABA, in the absence or presence of analog #10 (0.01 nM to 50 lM). The intracellular cAMP concentration ([cAMP]i) was determined by radioimmunoassay [2].
Synthesis of analog #10 was performed essentially as described in [6], with slight modifications. Briefly, (±) 2-cis, 4-trans abscisic acid (ABA) (30 mg, 0.113 mmol, in 1 ml methanol) was supplemented with 30% H2O2 (250 ll, 8.09 mmol) and 6 N NaOH (50 ll) at 0 °C. The mixture was stirred, protected from light, for 96 h at 0 °C and then diluted to 5 ml with H2O. After lowering the pH to 2.0 with 3 N HCl, the mixture was extracted 3 times with 3 ml of ethyl acetate. The organic layer was concentrated under reduced pressure. Analysis of the crude products was performed by HPLC– ESI-MS and analog #10 was purified by RP-HPLC. 2.2. Liquid chromatography-electrospray mass spectrometry (HPLC– ESI-MS) and NMR of analog #10 Analysis of the crude product was performed using an Agilent 1100 series LC/MSD ion trap instrument on an analytical Phenomenex C18 Luna column (4.6 250 mm), at a flow rate of 1 ml/min. The detection wavelength was 254 nm and MS and MS/MS spectra were acquired in negative ion mode (range m/z 50–300). The product was then purified by reverse phase high performance liquid chromatography (RP-HPLC) on a Shimadzu LC-9A preparative HPLC equipped with a Phenomenex C18 Luna column (21.20 250 mm), at a flow rate of 15 ml/min. The purity of analog #10 used in all experiments was P95%. Extracts of analog #10 from the stability experiments were injected into an Agilent 1100 capillary chromatographic system with an Alltech C18 Hypercarb column (1 100 mm) coupled to an Agilent 1100 Series LC/MSD Trap mass spectrometer, equipped with an orthogonal geometry electrospray and an ion trap analyzer, at a flow rate of 0.03 ml/min. The solvent program used for both analytical and preparative HPLC was a gradient starting with 15% solvent A for 5 min, linearly increasing to 70% solvent B in 30 min and up to 100% B in 10 min. Solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile.
2.7. Competition binding of analog #10 on human granulocytes Granulocytes (7 106 per determination) were incubated in triplicate for 60 min at 25 °C in 100 ll of HBSS with 10 nM [3H]ABA (50 Ci/mmol, GE Healthcare), in the presence of increasing concentrations of analog #10 (0.01 nM to 100 lM), with or without excess (1 mM) unlabeled ABA. Cells were then centrifuged (30 s at 16,000 g), the supernatants were discarded, cell pellets were washed once in ice-cold HBSS and the radioactivity was determined on a Packard b-counter. The specific binding was calculated as the difference between total binding and binding in the presence of excess unlabeled ABA. 2.8. Competition binding of analog #10 on human LANCL2 Recombinant LANCL2–GST was purified and bound to a GSHcoupled magnetic resin as described before [5]. The protein (approximately 10 lg) was resuspended in 50 ll of ABA-binding buffer (25 mM Tris–HCl pH 8, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% Triton-X 100) and incubated, in triplicate, for 60 min at 25 °C with 100 nM [3H]-ABA (20 Ci/mmol, Biotrend Radiochemicals, Köln, Germany), in the presence of increasing concentrations of analog #10 (0.01 nM to 100 lM). Magnetic beads were then washed once with 1.5 ml of ice-cold binding buffer and separated from the supernatant with a magnet particle concentrator. LANCL2 resin was resuspended in 200 ll water and 4.0 ml Ultima-Gold (Perkin Elmer, Milano, Italy) and the radioactivity was determined after 18 h with a Packard b-counter. The specific [3H]-ABA binding was calculated as the difference between total binding and nonspecific binding, obtained with excess unlabeled ABA (2.5 mM). 2.9. Chemotaxis
2.3. MS and MS/MS spectrum of analog #10 m/z = 279.1 [MH]: rel. int. of fragment ions, ESI-MS [MH], m/z 260.9 (25%), m/z 234.9 (75%), m/z 216.9 (100%), m/z 198.8 (9%), m/z 190.9 (13%), m/z 180.8 (6%), m/z 168.8 (8%). 2.4. NMR spectra of analog #10 1
H NMR: 300 Hz, DMSO-d6, T = 25 °C; d 0.79 [s, 3H, 80 ]; 0.96 [s, 3H, 90 ];1.27 [s, 3H, 70 ]; 1.93 (d, 1H, 50 , J = 15.4 Hz]; 1.98 [d, 3H, 6, J = 1.2 Hz]; 2.74 [d, 1H, 50 , J = 15.5 Hz]; 3.22 [s, 1H, 30 ]; 5.66–5.68 [m, 1H, 2]; 6.22 [dd, 1H, 5, J = 15.8, 0.5 Hz]; 7.81 [dd, 1H, 4, J = 15.8, 0.7 Hz]. 13 C NMR: 300 Hz, DMSO-d6, T = 25 °C; d 19.65 [70 ]; 20.93 [6]; 24.92 [80 ]; 26.02 [90 ]; 41.55 [60 ]; 47.28 [50 ]; 62.11 [30 ]; 66.71 [20 ]; 76.15 [10 ]; 118.12 [2]; 126.9 [4]; 138.18 [5]; 149.31 [3]; 166.95 [1]; 206.00 [40 ].
ABA (1 lM) and analog #10 (0.1 nM to 5.0 lM), alone or in combination, were added to the bottom wells of chemotaxis chambers. Granulocyte suspensions (25 ll at 10 106/ml in HBSS, PBS, and 0.5% albumin, 39:16:1) were placed on top of the filters (3 lmdiameter), the plates were incubated for 90 min at 37 °C and transmigrated cells were quantified as described in [2]. 2.10. Phagocytosis Granulocytes (1 106/100 ll HBSS) were preincubated for 10 min at 25 °C with 20 lM ABA, without or with analog #10 (0.5 nM to 1 lM). Fluorescent latex beads (Sigma, Catalog No. L5655) were then added (2.5 105 beads/100 ll): after 0, 15 and 30 min, 100 ll of each suspension was centrifuged at 200g for 10 s. Cell pellets were washed with 1 ml of ice-cold HBSS and resuspended in 350 ll of ice-cold HBSS. The fluorescence of 100 ll aliquots was determined in triplicate with a plate reader (Fluostar Optima, BMG Labtechnologies, Offenburg, Germany).
2.5. Isolation of human granulocytes and monocytes 2.11. Determination of ROS production Granulocytes, monocytes and peripheral blood mononuclear cells (PBMC) were isolated from buffy coats [2,3], prepared from freshly drawn blood of healthy human volunteers.
Granulocytes were loaded with the ROS-sensitive fluorescent probe dichlorodihydrofluorescein diacetate (H2DCFDA) as described
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Fig. 1. Analog #10 inhibits the [cAMP]i rise induced by ABA and competes with ABA for binding to human granulocytes and to recombinant LANCL2. (A) Granulocytes were incubated for 1 min without (control) or with 1 lM ABA, without or with analog #10. The percentage of inhibition of the [cAMP]i rise was calculated as the ratio between the [cAMP]i increase over control values in cells incubated with both ABA and analog #10, and the [cAMP]i increase over control values in cells incubated with ABA alone. Incubation of granulocytes with analog #10 alone did not modify the [cAMP]i as compared to control values, at any one of the concentrations tested. (B) Granulocytes were incubated with 10 nM [3H]ABA, without or with excess unlabeled ABA, in the presence of analog #10. The specific binding was analyzed by nonlinear regression using the software GraphPad Prism v5. The calculated Ki was 0.46 nM (R2 = 0.99). Results are the mean ± SD of seven experiments. (C) Recombinant LANCL2–GST protein was incubated with 100 nM [3H]ABA, without or with excess unlabeled ABA, in the presence of analog #10. The specific binding was analyzed by nonlinear regression using the software GraphPad Prism v5. The calculated Ki was 6.21 nM (R2 = 0.99). Results are the mean ± SD of three experiments.
in [2] and 100 ll-aliquots of cells (1 106/ml in HBSS) were incubated in 96-well plates with 1 lM ABA, with or without analog #10 (0.01 nM to 1 lM). Fluorescence was monitored every 10 s for 10 min with a fluorescence plate reader.
the extraction yield, and samples were processed as described in [2] for the extraction of ABA. Extracts were analyzed by HPLC– ESI-MS and the concentration of analog #10 was calculated from the HPLC peak area.
2.12. Quantification of PGE2 and MCP-1
2.14. Cytotoxicity
Monocytes (2.5 107/dish in RPMI with 20% autologous serum) and granulocytes (1 107/500 ll in RPMI with 10% fetal calf serum, FCS) were cultured for 6 h at 37 °C with 100 nM ABA, with or without analog #10 (0.1–1.0 lM). The PGE2 and MCP-1 concentrations in the medium were determined by ELISA (Cayman Chemical Co. and GE Healthcare, respectively) and normalized to the cell protein content.
Human granulocytes or PBMC (4 105/well) were incubated in 96-well plates in a humidified atmosphere at 37 °C with 5% CO2 in RPMI containing 10% FCS, with or without analog #10. After 24 h, the number of live cells was estimated with the CellTiter Cell Proliferation Assay (Promega).
2.13. Stability of analog #10
Results from structure–function studies of ABA analogs in plants suggest that the structural requirements for the phytohormonal functions of ABA are the stereochemistries at the C-10 , the C-70 , the 2-Z-enoic acid side chain and the C-40 carbonyl group [7,8]. We previously reported that ABA biotinylated at the C-40 keto group binds to granulocyte membranes and is antagonized by excess unmodified ABA [2]. Thus, the C-40 carbonyl group does not
Analog #10 (10 lM) was incubated at 37 °C in deionized water, or in HBSS with Ca2+ and Mg2+, or in human plasma, or in HBSS containing 5 107 granulocytes/ml. Aliquots of the incubations were withdrawn at time zero and after 2 and 24 h. [3H]-ABA (5000 cpm, 50 fmol) was added as internal standard, to estimate
3. Results and discussion
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Fig. 2. Analog #10 inhibits ABA-induced chemotaxis, phagocytosis, ROS and PGE2 production in human granulocytes. (A) Granulocytes were allowed to migrate toward a solution containing 1 lM ABA, without or with analog #10. Results are expressed as Chemotaxis Index (CI) = no of cells migrated toward the chemoattractant/no of cells migrated toward medium. Analog #10 alone did not induce cell migration at any one of the concentrations tested. (B) Granulocytes were preincubated with ABA, or with ABA and analog #10 and then allowed to phagocytose fluorescent beads for the times indicated. The fluorescence increase over time zero values is shown (n = 3). (C) Granulocytes loaded with the ROS-sensitive fluorescent probe H2DCFDA were incubated for 10 min with 1 lM ABA, with or without analog #10. The fluorescence increase over time zero values is shown (n = 3). D. Granulocytes were incubated for 6 h without (control) or with ABA 1 lM, alone or together with analog #10. PGE2 release is shown relative to control (n = 3). ⁄⁄⁄p < 0.001, ⁄⁄0.001 < p < 0.01, ⁄0.01 < p < 0.5, ns p > 0.5 compared to ABA, by one way Anova with Dunnett’s Multiple Comparison post test (n = 3).
appear to be essential for ABA binding to granulocytes. On the basis of this result and of the studies in plants, four regions of the ABA molecule were modified for structure–activity analysis on human granulocytes: the C-1 carboxyl group (which was replaced with ester or amide moieties or with carbon chains of different length), the C-20 and C-30 configuration (the double bond was removed through epoxidation), the C-40 carbonyl group (which was reduced or substituted with amino groups), and the ring saturation (the cyclohexene was substituted with an aromatic ring). One of the 22 compounds synthesized, 20 a,30 a-dihydro-20 a, 30 a-epoxyabscisic acid (analog #10) (Supplementary data Fig. S1), had been already produced as an intermediate for the synthesis of other ABA analogs [6] and later shown to behave as a competitive inhibitor of the saturable uptake of (+)-[3H]ABA by barley cells, with similar efficacy as ABA [4]. We synthesized analog #10 in racemic form, starting from (±)ABA because previous results had shown that (+)-ABA and ()-ABA were similarly effective at stimulating granulocyte functions [2]. Also in plants, both ABA enantiomers display biological activity
[9,10]. Epoxidation of (±)-ABA with H2O2 in an alkaline environment afforded a racemic mixture of one diastereoisomer only, because addition of the hydrogen peroxide occurs at the less hindered a-face, opposite to the side chain [6]. As activation of adenylate cyclase is among the first molecular events triggered by ABA in human granulocytes [2], the 22 synthetic ABA analogs were tested for their ability to inhibit the ABA-triggered increase of the [cAMP]i in these cells. The [cAMP]i increased by 35 ± 5% (n = 20) in granulocytes incubated for 1 min at 25 °C with 1 lM ABA as compared to untreated cells. Analog #10, with an EC50 of 0.7 ± 0.2 nM, was the most effective among the compounds tested at inhibiting the [cAMP]i increase (Fig. 1A). For competition binding experiments, intact human granulocytes were incubated for 60 min at 25 °C with 10 nM [3H]-ABA and increasing concentrations of analog #10 (Fig. 1B). The calculated IC50 value was 0.87 nM (95% confidence intervals: from 0.48 to 1.58 nM). This value was used to calculate the Ki by applying the Cheng–Prusoff equation, Ki = IC50/1 + ([ABA]/KDABA), and considering KDABA = 11 nM [2]. The Ki of analog #10 was
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Fig. 3. Analog #10 abrogates MCP-1 and PGE2 production induced by ABA in human monocytes. Monocytes were incubated for 6 h without (control) or with 100 nM ABA, alone or with 1 lM analog #10. Release of PGE2 (white bars) and MCP-1 (black bars) is shown relative to control (n = 3). ⁄⁄⁄p < 0.001, ⁄⁄0.001 < p < 0.01, ⁄ 0.01 < p < 0.5, ns p > 0.5 compared to ABA, by one way Anova with Dunnett’s Multiple Comparison post test (n = 3).
0.46 nM (95% confidence intervals: from 0.25 to 0.83 nM), i.e. approximately 20 times lower than the KD of ABA for the same binding sites. LANCL2–GST was previously shown to bind [3H]-ABA with an apparent KD value of 2.4 lM and ABA was displaced by both (+)ABA and ()-ABA [5]. In competition binding experiments, analog 10 showed a higher affinity constant for LANCL2 compared to
ABA: the calculated IC50 value was 6.56 nM (95% confidence intervals: from 4.62 to 9.32) (Fig. 1C). This value was used to calculate the Ki by applying the Cheng–Prusoff equation, Ki = IC50/1 + ([ABA]/ KDABA), and considering KD ABA = 2400 nM [5]. The Ki of analog #10 was 6.21 nM (95% confidence intervals: from 4.37 to 8.82). Next, we investigated whether analog #10 antagonized the stimulatory effect of exogenous ABA on chemotaxis, phagocytosis, ROS and PGE2 production by human granulocytes. Analog #10 inhibited cell migration induced by 1 lM ABA in a concentrationdependent manner, with an approximate IC50 of 1 nM (Fig. 2A). Pre-incubation of granulocytes with increasing concentrations of analog #10 also inhibited phagocytosis of fluorescent beads induced by 20 lM ABA, with an approximate IC50 at 30 min of 0.5 nM (Fig. 2B). Analog #10 also inhibited ROS production and PGE2 release induced by 1 lM ABA, in a concentration-dependent manner, with an approximate IC50 of 0.5 and 1 nM, respectively (Fig. 2C and D). Altogether, these results indicate that analog #10 abrogates all pro-inflammatory effects induced by exogenous ABA on human granulocytes at concentrations 10–200 times lower than ABA. Finally, 1 lM analog #10 abrogated the stimulatory effect of 100 nM ABA on the release of both PGE2 and MCP-1 in monocytes (Fig. 3). The approximate IC50 value of analog #10 on PGE2 and MCP-1 release induced by 100 nM ABA was 1 nM. The stability of analog #10 in solution was assayed in deionized water, HBSS, human plasma and in HBSS containing human granulocytes at a cell density 10 times higher than the one present in human blood. No significant degradation of analog #10 was observed upon incubation for up to 24 h at 37 °C in any of the different media tested (Fig. 4A). In the presence of granulocytes, the concentration of analog #10 decreased slightly over time, although approximately 80% of the initial concentration was still present after 24 h incubation (Fig. 4A). Finally, no significant decrease of cell viability compared to untreated controls was observed upon incubation of granulocytes or of PBMC for 24 h in the presence of up to 1 mM analog #10 (Fig. 4B). This concentration is 6 logs higher than the highest IC50 value observed for analog #10 on the ABA-triggered pro-inflammatory activities of granulocytes and monocytes.
Fig. 4. Analog #10 is stable in solution and non toxic to human granulocytes and PBMC. (A) Analog #10 (10 lM) was incubated at 37 °C in deionized water (H2O), or in HBSS with Ca2+ and Mg2+ (HBSS), or in human plasma, or in HBSS containing 5 107 granulocytes/ml (granulocytes). After 2 and 24 h the concentration of analog #10 was determined by HPLC-MS. Results are expressed as percentage of analog #10 relative to time zero (n = 3). (B) Granulocytes (white bars) or PBMC (black bars) (4 105/well) were incubated without (control) or with analog #10 for 24 h. Cell viability was determined with a colorimetric assay. The percentage of live cells relative to control is shown (n = 4).
A. Grozio et al. / Biochemical and Biophysical Research Communications 415 (2011) 696–701
In conclusion, here we show that analog #10 competes with ABA for binding to granulocyte membranes and to purified recombinant LANCL2 and inhibits the functional activation induced by exogenous ABA on human granulocytes and monocytes. Stability in solution and absence of cytotoxicity on granulocytes and on PBMC are important pre-requisites for testing analog #10 in animal models of inflammation, as a possible lead compound of a new family of anti-inflammatory drugs acting as ABA antagonists.
[2]
[3]
[4]
Acknowledgments This work was supported in part by the Italian Ministry of Education, University and Scientific Research, the University of Genova, the Liguria region, the Fondazione CARIGE, the Compagnia di S. Paolo and the European Union.
[5]
[6] [7]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.10.143. References [1] X. Xue-Xuan, S. Hong-Bo, M. Yuan-Yuan, X. Gang, S. Jun-Na, G. Dong-Gang, R. Cheng-Jiang, Biotechnological implications from abscisic acid roles in cold stress and leaf senescence as an important signal for improving plant
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sustainable survival under abiotic-stressed conditions, Crit. Rev. Biotechnol. 30 (2010) 222–230. S. Bruzzone, I. Moreschi, C. Usai, L. Guida, G. Damonte, A. Salis, S. Scarfì, E. Millo, A. De Flora, E. Zocchi, Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger, Proc. Natl. Acad. Sci. USA 104 (2007) 5759–5764. M. Magnone, S. Bruzzone, L. Guida, G. Damonte, E. Millo, S. Scarfì, C. Usai, D. Palombo, A. De Flora, E. Zocchi, Abscisic acid released by human monocytes activates monocytes and vascular smooth muscle cells responses involved in atherogenesis, J. Biol. Chem. 284 (2009) 17808–17818. M. Perras, P.A. Rose, E.W. Pass, K.B. Chatson, J.J. Balsevich, S.R. Abrams, Defining steric, electronic and conformational requirements of carrier-mediated uptake of abscisic acid in barley suspension culture cells, Phytochemistry 46 (1997) 215–222. L. Sturla, C. Fresia, L. Guida, A. Grozio, T. Vigliarolo, E. Mannino, E. Millo, L. Bagnasco, S. Bruzzone, A. De Flora, E. Zocchi, Binding of abscisic acid to human LANCL2, Biochem. Biophys. Res. Commun. (2011) doi: 10.1016/ j.bbrc.2011.10.079. Y. Todoroki, N. Hirai, H. Ohigashi, Synthesis, biological activity and metabolism of (S)-(+)-30 -fluoroabscisic acid, Tetrahedron 51 (1995) 6911–6926. G.C. Churchill, B. Ewan, M.J.T. Reaney, S.R. Abrams, L.W. Gusta, Structure– activity relationships of abscisic acid analogs based on the induction of freezing tolerance in Bromegrass (Bromus inermis Leyss) cell cultures, Plant Physiol. 100 (1992) 2024–2029. M.K. Walker-Simmons, M.J.T. Reaney, S.A. Quarrie, P. Perata, P. Vernieri, S.R. Abrams, Monoclonal antibody recognition of abscisic acid analogs, Plant Physiol. 95 (1991) 46–51. J.J. Balsevich, A.J. Cutler, N. Lamb, L.J. Friesen, E.U. Kurz, M.R. Perras, S.R. Abrams, Response of cultured maize cells to (+)-abscisic acid, ()-abscisic acid, and their metabolites, Plant Physiol. 106 (1994) 135–142. B.L. Lin, H.J. Wang, J.S. Wang, L.I. Zaharia, S.R. Abrams, Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-() and S-(+) isomers, J. Exp. Bot. 56 (2005) 2935–2948.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes Alessia Grozio a,b, Enrico Millo a,b, Lucrezia Guida a,b, Tiziana Vigliarolo a,b, Marta Bellotti a,b, Annalisa Salis a,b, Chiara Fresia a,b, Laura Sturla a,b, Mirko Magnone a,b, Andrea Galatini c, Gianluca Damonte a,b, Antonio De Flora a,b, Santina Bruzzone a,b,d, Luca Bagnasco e, Elena Zocchi a,b,d,⇑ a
Department of Experimental Medicine (DIMES), Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova, Italy Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV 9, 16132 Genova, Italy Department of Chemistry and Industrial Chemistry, via Dodecaneso 31, 16146 Genova, Italy d Advanced Biotechnology Center (ABC), Largo Rosanna Benzi 10, 16132 Genova, Italy e Department of Internal Medicine, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy b c
a r t i c l e
i n f o
Article history: Received 27 October 2011 Available online 6 November 2011 Keywords: Abscisic acid antagonist Granulocytes Monocytes Inflammation LANCL2
a b s t r a c t The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E2 (PGE2) by human granulocytes, release of PGE2 and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of antiinflammatory agents. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The phytohormone abscisic acid (ABA) is involved in important functions in higher plants, including response to abiotic stress, regulation of seed germination, control of stomatal closure and of gene transcription [1]. ABA has recently been shown to stimulate several functions of human granulocytes (phagocytosis, reactive oxygen species and nitric oxide production, chemotaxis) through a signalling pathway sequentially involving a pertussis toxin-sensitive G-protein/receptor complex, activation of adenylate cyclase with cAMP overproduction, protein kinase A activation, ADP-ribosyl cyclase phosphorylation and consequent cADPR overproduction, leading to an increase of the [Ca2+]i [2]. The increase of intracellular ABA and its release by activated human granulocytes indicate that ABA is an endogenous hormone in humans [2]. ABA also stimulates the functional activity of human monocytes: nanomolar ABA evokes a Abbreviations: ABA, abscisic acid; LANCL2, Lanthionine synthetase component C-like protein 2; GST, glutathione S-transferase; PGE2, prostaglandin E2; MCP-1, monocyte chemoattractant protein 1; H 2 DCFDA, dichlorodihydrofluorescein diacetate. ⇑ Corresponding author at: DIMES, Viale Benedetto XV 1, 16132 Genova, Italy. Fax: +39 0 10 354415. E-mail address: [email protected] (E. Zocchi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.10.143
cADPR-mediated [Ca2+]i rise, leading to NF-jB translocation, increase of cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production, release of monocyte chemoattractant protein 1 (MCP-1) and of metalloprotease 9 (MMP-9), all events involved in atherogenesis [3]. Monocytes release ABA when they are exposed to thrombin-activated platelets, as occurs at the injured vascular endothelium, and ABA behaves as an autocrine and paracrine signal, stimulating monocyte migration and MCP-1 release and vascular smooth muscle cell (VSMC) migration and proliferation [3]. These results, and presence of ABA in human arterial plaques at a 10-fold higher concentration compared to normal arterial tissue [3], suggest that ABA may play a central role in the molecular cross-talk between platelets, monocytes and VSMC leading to the development of the atherosclerotic lesion. To study the role of ABA in cellular and animal models of inflammation and atherosclerosis, pharmacological tools through which the effect of ABA can be switched off are needed. Here we describe the functional characterization of a synthetic ABA analog, previously shown to partially inhibit ABA transport in plant cells [4]. This ABA analog is endowed with anti-inflammatory properties on human granulocytes and monocytes in vitro through its ability to compete with ABA for binding to cell membranes and to the recently identified human ABA receptor, LANCL2 [5].
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2. Materials and methods
2.6. Determination of intracellular cAMP levels
2.1. Synthesis of 20 a,30 a-dihydro-20 a, 30 a-epoxyabscisic acid (analog #10)
Granulocytes (1.5 107/ml) resuspended in HBSS with Ca2+ and Mg2+ were preincubated for 5 min at 25 °C with 10 lM cAMP phosphodiesterase inhibitor (Sigma). Duplicate 200 ll-aliquots of cells were incubated for 1 min at 25 °C without (control) or with 1 lM ABA, in the absence or presence of analog #10 (0.01 nM to 50 lM). The intracellular cAMP concentration ([cAMP]i) was determined by radioimmunoassay [2].
Synthesis of analog #10 was performed essentially as described in [6], with slight modifications. Briefly, (±) 2-cis, 4-trans abscisic acid (ABA) (30 mg, 0.113 mmol, in 1 ml methanol) was supplemented with 30% H2O2 (250 ll, 8.09 mmol) and 6 N NaOH (50 ll) at 0 °C. The mixture was stirred, protected from light, for 96 h at 0 °C and then diluted to 5 ml with H2O. After lowering the pH to 2.0 with 3 N HCl, the mixture was extracted 3 times with 3 ml of ethyl acetate. The organic layer was concentrated under reduced pressure. Analysis of the crude products was performed by HPLC– ESI-MS and analog #10 was purified by RP-HPLC. 2.2. Liquid chromatography-electrospray mass spectrometry (HPLC– ESI-MS) and NMR of analog #10 Analysis of the crude product was performed using an Agilent 1100 series LC/MSD ion trap instrument on an analytical Phenomenex C18 Luna column (4.6 250 mm), at a flow rate of 1 ml/min. The detection wavelength was 254 nm and MS and MS/MS spectra were acquired in negative ion mode (range m/z 50–300). The product was then purified by reverse phase high performance liquid chromatography (RP-HPLC) on a Shimadzu LC-9A preparative HPLC equipped with a Phenomenex C18 Luna column (21.20 250 mm), at a flow rate of 15 ml/min. The purity of analog #10 used in all experiments was P95%. Extracts of analog #10 from the stability experiments were injected into an Agilent 1100 capillary chromatographic system with an Alltech C18 Hypercarb column (1 100 mm) coupled to an Agilent 1100 Series LC/MSD Trap mass spectrometer, equipped with an orthogonal geometry electrospray and an ion trap analyzer, at a flow rate of 0.03 ml/min. The solvent program used for both analytical and preparative HPLC was a gradient starting with 15% solvent A for 5 min, linearly increasing to 70% solvent B in 30 min and up to 100% B in 10 min. Solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile.
2.7. Competition binding of analog #10 on human granulocytes Granulocytes (7 106 per determination) were incubated in triplicate for 60 min at 25 °C in 100 ll of HBSS with 10 nM [3H]ABA (50 Ci/mmol, GE Healthcare), in the presence of increasing concentrations of analog #10 (0.01 nM to 100 lM), with or without excess (1 mM) unlabeled ABA. Cells were then centrifuged (30 s at 16,000 g), the supernatants were discarded, cell pellets were washed once in ice-cold HBSS and the radioactivity was determined on a Packard b-counter. The specific binding was calculated as the difference between total binding and binding in the presence of excess unlabeled ABA. 2.8. Competition binding of analog #10 on human LANCL2 Recombinant LANCL2–GST was purified and bound to a GSHcoupled magnetic resin as described before [5]. The protein (approximately 10 lg) was resuspended in 50 ll of ABA-binding buffer (25 mM Tris–HCl pH 8, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% Triton-X 100) and incubated, in triplicate, for 60 min at 25 °C with 100 nM [3H]-ABA (20 Ci/mmol, Biotrend Radiochemicals, Köln, Germany), in the presence of increasing concentrations of analog #10 (0.01 nM to 100 lM). Magnetic beads were then washed once with 1.5 ml of ice-cold binding buffer and separated from the supernatant with a magnet particle concentrator. LANCL2 resin was resuspended in 200 ll water and 4.0 ml Ultima-Gold (Perkin Elmer, Milano, Italy) and the radioactivity was determined after 18 h with a Packard b-counter. The specific [3H]-ABA binding was calculated as the difference between total binding and nonspecific binding, obtained with excess unlabeled ABA (2.5 mM). 2.9. Chemotaxis
2.3. MS and MS/MS spectrum of analog #10 m/z = 279.1 [MH]: rel. int. of fragment ions, ESI-MS [MH], m/z 260.9 (25%), m/z 234.9 (75%), m/z 216.9 (100%), m/z 198.8 (9%), m/z 190.9 (13%), m/z 180.8 (6%), m/z 168.8 (8%). 2.4. NMR spectra of analog #10 1
H NMR: 300 Hz, DMSO-d6, T = 25 °C; d 0.79 [s, 3H, 80 ]; 0.96 [s, 3H, 90 ];1.27 [s, 3H, 70 ]; 1.93 (d, 1H, 50 , J = 15.4 Hz]; 1.98 [d, 3H, 6, J = 1.2 Hz]; 2.74 [d, 1H, 50 , J = 15.5 Hz]; 3.22 [s, 1H, 30 ]; 5.66–5.68 [m, 1H, 2]; 6.22 [dd, 1H, 5, J = 15.8, 0.5 Hz]; 7.81 [dd, 1H, 4, J = 15.8, 0.7 Hz]. 13 C NMR: 300 Hz, DMSO-d6, T = 25 °C; d 19.65 [70 ]; 20.93 [6]; 24.92 [80 ]; 26.02 [90 ]; 41.55 [60 ]; 47.28 [50 ]; 62.11 [30 ]; 66.71 [20 ]; 76.15 [10 ]; 118.12 [2]; 126.9 [4]; 138.18 [5]; 149.31 [3]; 166.95 [1]; 206.00 [40 ].
ABA (1 lM) and analog #10 (0.1 nM to 5.0 lM), alone or in combination, were added to the bottom wells of chemotaxis chambers. Granulocyte suspensions (25 ll at 10 106/ml in HBSS, PBS, and 0.5% albumin, 39:16:1) were placed on top of the filters (3 lmdiameter), the plates were incubated for 90 min at 37 °C and transmigrated cells were quantified as described in [2]. 2.10. Phagocytosis Granulocytes (1 106/100 ll HBSS) were preincubated for 10 min at 25 °C with 20 lM ABA, without or with analog #10 (0.5 nM to 1 lM). Fluorescent latex beads (Sigma, Catalog No. L5655) were then added (2.5 105 beads/100 ll): after 0, 15 and 30 min, 100 ll of each suspension was centrifuged at 200g for 10 s. Cell pellets were washed with 1 ml of ice-cold HBSS and resuspended in 350 ll of ice-cold HBSS. The fluorescence of 100 ll aliquots was determined in triplicate with a plate reader (Fluostar Optima, BMG Labtechnologies, Offenburg, Germany).
2.5. Isolation of human granulocytes and monocytes 2.11. Determination of ROS production Granulocytes, monocytes and peripheral blood mononuclear cells (PBMC) were isolated from buffy coats [2,3], prepared from freshly drawn blood of healthy human volunteers.
Granulocytes were loaded with the ROS-sensitive fluorescent probe dichlorodihydrofluorescein diacetate (H2DCFDA) as described
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Fig. 1. Analog #10 inhibits the [cAMP]i rise induced by ABA and competes with ABA for binding to human granulocytes and to recombinant LANCL2. (A) Granulocytes were incubated for 1 min without (control) or with 1 lM ABA, without or with analog #10. The percentage of inhibition of the [cAMP]i rise was calculated as the ratio between the [cAMP]i increase over control values in cells incubated with both ABA and analog #10, and the [cAMP]i increase over control values in cells incubated with ABA alone. Incubation of granulocytes with analog #10 alone did not modify the [cAMP]i as compared to control values, at any one of the concentrations tested. (B) Granulocytes were incubated with 10 nM [3H]ABA, without or with excess unlabeled ABA, in the presence of analog #10. The specific binding was analyzed by nonlinear regression using the software GraphPad Prism v5. The calculated Ki was 0.46 nM (R2 = 0.99). Results are the mean ± SD of seven experiments. (C) Recombinant LANCL2–GST protein was incubated with 100 nM [3H]ABA, without or with excess unlabeled ABA, in the presence of analog #10. The specific binding was analyzed by nonlinear regression using the software GraphPad Prism v5. The calculated Ki was 6.21 nM (R2 = 0.99). Results are the mean ± SD of three experiments.
in [2] and 100 ll-aliquots of cells (1 106/ml in HBSS) were incubated in 96-well plates with 1 lM ABA, with or without analog #10 (0.01 nM to 1 lM). Fluorescence was monitored every 10 s for 10 min with a fluorescence plate reader.
the extraction yield, and samples were processed as described in [2] for the extraction of ABA. Extracts were analyzed by HPLC– ESI-MS and the concentration of analog #10 was calculated from the HPLC peak area.
2.12. Quantification of PGE2 and MCP-1
2.14. Cytotoxicity
Monocytes (2.5 107/dish in RPMI with 20% autologous serum) and granulocytes (1 107/500 ll in RPMI with 10% fetal calf serum, FCS) were cultured for 6 h at 37 °C with 100 nM ABA, with or without analog #10 (0.1–1.0 lM). The PGE2 and MCP-1 concentrations in the medium were determined by ELISA (Cayman Chemical Co. and GE Healthcare, respectively) and normalized to the cell protein content.
Human granulocytes or PBMC (4 105/well) were incubated in 96-well plates in a humidified atmosphere at 37 °C with 5% CO2 in RPMI containing 10% FCS, with or without analog #10. After 24 h, the number of live cells was estimated with the CellTiter Cell Proliferation Assay (Promega).
2.13. Stability of analog #10
Results from structure–function studies of ABA analogs in plants suggest that the structural requirements for the phytohormonal functions of ABA are the stereochemistries at the C-10 , the C-70 , the 2-Z-enoic acid side chain and the C-40 carbonyl group [7,8]. We previously reported that ABA biotinylated at the C-40 keto group binds to granulocyte membranes and is antagonized by excess unmodified ABA [2]. Thus, the C-40 carbonyl group does not
Analog #10 (10 lM) was incubated at 37 °C in deionized water, or in HBSS with Ca2+ and Mg2+, or in human plasma, or in HBSS containing 5 107 granulocytes/ml. Aliquots of the incubations were withdrawn at time zero and after 2 and 24 h. [3H]-ABA (5000 cpm, 50 fmol) was added as internal standard, to estimate
3. Results and discussion
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Fig. 2. Analog #10 inhibits ABA-induced chemotaxis, phagocytosis, ROS and PGE2 production in human granulocytes. (A) Granulocytes were allowed to migrate toward a solution containing 1 lM ABA, without or with analog #10. Results are expressed as Chemotaxis Index (CI) = no of cells migrated toward the chemoattractant/no of cells migrated toward medium. Analog #10 alone did not induce cell migration at any one of the concentrations tested. (B) Granulocytes were preincubated with ABA, or with ABA and analog #10 and then allowed to phagocytose fluorescent beads for the times indicated. The fluorescence increase over time zero values is shown (n = 3). (C) Granulocytes loaded with the ROS-sensitive fluorescent probe H2DCFDA were incubated for 10 min with 1 lM ABA, with or without analog #10. The fluorescence increase over time zero values is shown (n = 3). D. Granulocytes were incubated for 6 h without (control) or with ABA 1 lM, alone or together with analog #10. PGE2 release is shown relative to control (n = 3). ⁄⁄⁄p < 0.001, ⁄⁄0.001 < p < 0.01, ⁄0.01 < p < 0.5, ns p > 0.5 compared to ABA, by one way Anova with Dunnett’s Multiple Comparison post test (n = 3).
appear to be essential for ABA binding to granulocytes. On the basis of this result and of the studies in plants, four regions of the ABA molecule were modified for structure–activity analysis on human granulocytes: the C-1 carboxyl group (which was replaced with ester or amide moieties or with carbon chains of different length), the C-20 and C-30 configuration (the double bond was removed through epoxidation), the C-40 carbonyl group (which was reduced or substituted with amino groups), and the ring saturation (the cyclohexene was substituted with an aromatic ring). One of the 22 compounds synthesized, 20 a,30 a-dihydro-20 a, 30 a-epoxyabscisic acid (analog #10) (Supplementary data Fig. S1), had been already produced as an intermediate for the synthesis of other ABA analogs [6] and later shown to behave as a competitive inhibitor of the saturable uptake of (+)-[3H]ABA by barley cells, with similar efficacy as ABA [4]. We synthesized analog #10 in racemic form, starting from (±)ABA because previous results had shown that (+)-ABA and ()-ABA were similarly effective at stimulating granulocyte functions [2]. Also in plants, both ABA enantiomers display biological activity
[9,10]. Epoxidation of (±)-ABA with H2O2 in an alkaline environment afforded a racemic mixture of one diastereoisomer only, because addition of the hydrogen peroxide occurs at the less hindered a-face, opposite to the side chain [6]. As activation of adenylate cyclase is among the first molecular events triggered by ABA in human granulocytes [2], the 22 synthetic ABA analogs were tested for their ability to inhibit the ABA-triggered increase of the [cAMP]i in these cells. The [cAMP]i increased by 35 ± 5% (n = 20) in granulocytes incubated for 1 min at 25 °C with 1 lM ABA as compared to untreated cells. Analog #10, with an EC50 of 0.7 ± 0.2 nM, was the most effective among the compounds tested at inhibiting the [cAMP]i increase (Fig. 1A). For competition binding experiments, intact human granulocytes were incubated for 60 min at 25 °C with 10 nM [3H]-ABA and increasing concentrations of analog #10 (Fig. 1B). The calculated IC50 value was 0.87 nM (95% confidence intervals: from 0.48 to 1.58 nM). This value was used to calculate the Ki by applying the Cheng–Prusoff equation, Ki = IC50/1 + ([ABA]/KDABA), and considering KDABA = 11 nM [2]. The Ki of analog #10 was
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Fig. 3. Analog #10 abrogates MCP-1 and PGE2 production induced by ABA in human monocytes. Monocytes were incubated for 6 h without (control) or with 100 nM ABA, alone or with 1 lM analog #10. Release of PGE2 (white bars) and MCP-1 (black bars) is shown relative to control (n = 3). ⁄⁄⁄p < 0.001, ⁄⁄0.001 < p < 0.01, ⁄ 0.01 < p < 0.5, ns p > 0.5 compared to ABA, by one way Anova with Dunnett’s Multiple Comparison post test (n = 3).
0.46 nM (95% confidence intervals: from 0.25 to 0.83 nM), i.e. approximately 20 times lower than the KD of ABA for the same binding sites. LANCL2–GST was previously shown to bind [3H]-ABA with an apparent KD value of 2.4 lM and ABA was displaced by both (+)ABA and ()-ABA [5]. In competition binding experiments, analog 10 showed a higher affinity constant for LANCL2 compared to
ABA: the calculated IC50 value was 6.56 nM (95% confidence intervals: from 4.62 to 9.32) (Fig. 1C). This value was used to calculate the Ki by applying the Cheng–Prusoff equation, Ki = IC50/1 + ([ABA]/ KDABA), and considering KD ABA = 2400 nM [5]. The Ki of analog #10 was 6.21 nM (95% confidence intervals: from 4.37 to 8.82). Next, we investigated whether analog #10 antagonized the stimulatory effect of exogenous ABA on chemotaxis, phagocytosis, ROS and PGE2 production by human granulocytes. Analog #10 inhibited cell migration induced by 1 lM ABA in a concentrationdependent manner, with an approximate IC50 of 1 nM (Fig. 2A). Pre-incubation of granulocytes with increasing concentrations of analog #10 also inhibited phagocytosis of fluorescent beads induced by 20 lM ABA, with an approximate IC50 at 30 min of 0.5 nM (Fig. 2B). Analog #10 also inhibited ROS production and PGE2 release induced by 1 lM ABA, in a concentration-dependent manner, with an approximate IC50 of 0.5 and 1 nM, respectively (Fig. 2C and D). Altogether, these results indicate that analog #10 abrogates all pro-inflammatory effects induced by exogenous ABA on human granulocytes at concentrations 10–200 times lower than ABA. Finally, 1 lM analog #10 abrogated the stimulatory effect of 100 nM ABA on the release of both PGE2 and MCP-1 in monocytes (Fig. 3). The approximate IC50 value of analog #10 on PGE2 and MCP-1 release induced by 100 nM ABA was 1 nM. The stability of analog #10 in solution was assayed in deionized water, HBSS, human plasma and in HBSS containing human granulocytes at a cell density 10 times higher than the one present in human blood. No significant degradation of analog #10 was observed upon incubation for up to 24 h at 37 °C in any of the different media tested (Fig. 4A). In the presence of granulocytes, the concentration of analog #10 decreased slightly over time, although approximately 80% of the initial concentration was still present after 24 h incubation (Fig. 4A). Finally, no significant decrease of cell viability compared to untreated controls was observed upon incubation of granulocytes or of PBMC for 24 h in the presence of up to 1 mM analog #10 (Fig. 4B). This concentration is 6 logs higher than the highest IC50 value observed for analog #10 on the ABA-triggered pro-inflammatory activities of granulocytes and monocytes.
Fig. 4. Analog #10 is stable in solution and non toxic to human granulocytes and PBMC. (A) Analog #10 (10 lM) was incubated at 37 °C in deionized water (H2O), or in HBSS with Ca2+ and Mg2+ (HBSS), or in human plasma, or in HBSS containing 5 107 granulocytes/ml (granulocytes). After 2 and 24 h the concentration of analog #10 was determined by HPLC-MS. Results are expressed as percentage of analog #10 relative to time zero (n = 3). (B) Granulocytes (white bars) or PBMC (black bars) (4 105/well) were incubated without (control) or with analog #10 for 24 h. Cell viability was determined with a colorimetric assay. The percentage of live cells relative to control is shown (n = 4).
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In conclusion, here we show that analog #10 competes with ABA for binding to granulocyte membranes and to purified recombinant LANCL2 and inhibits the functional activation induced by exogenous ABA on human granulocytes and monocytes. Stability in solution and absence of cytotoxicity on granulocytes and on PBMC are important pre-requisites for testing analog #10 in animal models of inflammation, as a possible lead compound of a new family of anti-inflammatory drugs acting as ABA antagonists.
[2]
[3]
[4]
Acknowledgments This work was supported in part by the Italian Ministry of Education, University and Scientific Research, the University of Genova, the Liguria region, the Fondazione CARIGE, the Compagnia di S. Paolo and the European Union.
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