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PLoS One. 2010; 5(6): e10914.
Published online 2010 June 1. doi:  10.1371/journal.pone.0010914
PMCID: PMC2879362

Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion

Sudha Agarwal, Editor

Abstract

Group IIA secreted/synovial phospholipase A2 (GIIAPLA2) is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E2 (PGE2), the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA2, along with their chemical synthesis and results from PLA2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA2 affinities than did C1, and such predictions were confirmed by in vitro PLA2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1β-stimulated PGE2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints.

Introduction

Inflammation is a multi-faceted process involving numerous enzymes, such as phospholipases A2 (PLA2s) and cyclo-oxygenases (COXs) [1]. PLA2s catalyze the hydrolysis of cell-membrane glycerophospholipids at the sn-2 position leading to the generation of free fatty acids such as arachidonic acid. The later is subsequently metabolized into potent pro-inflammatory mediators such as eicosanoids (e.g. prostaglandin E2 [PGE2]) through a pathway involving COX-1 and COX-2 in part [2]. PGE2 is the main eicosanoid contributing to pain and inflammation in rheumatic diseases [3], [4]. Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the production of PGE2, which leads to a significant improvement in rheumatic symptoms. However, these drugs exhibit gastrointestinal toxicity mainly because of a marked decrease in COX-1 activity [4] and renal and blood pressure toxicities mainly because of a decrease in COX-2 activity. COX-1 is constitutively expressed in most tissues and appears to be responsible for maintaining normal physiological function. However, COX-2 is absent in most tissues under normal resting conditions but is induced in inflamed tissues and is responsible for increased PGE2 production. This activation has motivated the development of selective COX-2 inhibitors. However, these inhibitors also have severe side effects such as myocardial infarction [5], [6]. Overcoming this problem could involve the development of novel anti-inflammatory agents to efficiently inhibit the PLA2-dependent production of COX substrates without impairing the balance between COX-1 and COX-2.

PLA2s represent a growing family of enzymes of two main categories, intracellular and secreted. Among the 10 human secreted PLA2s (sPLA2s) known to date, the most studied is the non-pancreatic Group IIA, denoted GIIAPLA2, because of its involvement in the pathogenesis of many inflammatory diseases (for a review, see [7]). GIIAPLA2 was originally purified from the synovial fluid of patients with rheumatoid arthritis [8], [9], [10]. The number of rheumatoid arthritis-affected joints and the presence of destructive erosion correlate with the amount of GIIAPLA2 in the serum of patients [11]. Moreover, GIIAPLA2 induces an inflammatory response when injected in rabbit joints [12] and exacerbates rat adjuvant arthitis after intradermal injection [13].

The systemic implication of sPLA2s in inflammation has prompted a number of research groups to develop selective inhibitors of different types of these enzymes. Some potent candidates have been evaluated in phase II clinical trials. Surprisingly, no effect was observed when such inhibitors were used to treat patients with sepsis or rheumatoid arthritis [14], [15]. This failure could be due to the complexity of the inflammation process and the existence of compensatory pathways. However, these molecules have been tested only in high-level systemic inflammatory diseases, not in low-level inflammatory diseases such as atherosclerosis, diabetes, Alzheimer's, and osteoarthritis. Varespladib, a sPLA2 inhibitor, was recently found to reduce atherosclerosis in apolipoprotein-E-null mice [16]. Thus, the efficacy of sPLA2 inhibitors in these low-level inflammatory diseases should be re-examined.

We have developed various selective inhibitors of sPLA2s [17], [18], [19], [20], [21]. Previously, we reported on the computer-assisted design and synthesis of a series of novel oxadiazolone derivatives that were shown to exhibit potent inhibitory properties against GIIAPLA2 [20]. In this series, a Ca(II)-binding oxadiazolone ring was connected through a polymethylene chain of varying lengths to an indole ring, which has been shown to be involved in apolar and cation-π interactions with GIIAPLA2 residues. The optimal length of the linker was found to encompass 5 methylenes, and the corresponding compound, (3-{4-[5-(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one), is denoted C1 in the present study. In the current work, the indole moiety was replaced by other aromatic groups, which gave rise to compounds C2 to C8. Using molecular modeling, we computed and ranked energy balances for the binding of these inhibitors to GIIAPLA2. The inhibitory potencies of C2 to C8 against GIIAPLA2 was analyzed by enzymatic assay, and the anti-inflammatory activity of the most potent compound, C8, was evaluated in IL-1β-treated articular chondrocytes.

Results

Molecular modeling

We previously reported that one of the essential interactions between C1 and the target GIIAPLA2 is Ca(II) bidentate chelation by the oxadiazolone moiety in its anionic form [20]. Because C2-C8 are structurally similar to C1 (Fig. 1), docking was performed upon first anchoring the oxadiazolone ring in the same position as compound C1, followed by energy minimization and molecular dynamics. As was observed for compound C1, the lowest-energy frames of C2-C8/enzyme complexes are stabilized by π–π and cation-π interactions involving His6, Phe23, and Phe63 on the one hand and Arg7 and Arg33 of GIIAPLA2 on the other. Table 1 lists the energy values corresponding to the lowest-energy frames from molecular dynamics.

Figure 1
Synthesis scheme.
Table 1
Energy balances (ε = 4) from performing single-point Poisson-Boltzmann calculations of continuum solvation energies.

Chemistry

As outlined in Figure 1, 4-(5-bromopent-1-yloxy)benzyl cyanide 1 is prepared according to Dehaen and Hassner [22] by mono-substitution of 1,5-dibromopentane with 4-hydroxybenzyl cyanide in moderate yield. Compound 1 is then condensed in 25% to 50% yields, with 5-substituted indole derivatives or different aromatic alcohols, through their sodium salts prepared prior to use, to give 2a–g. The nitrile function of 2a–g is converted into amidoxime, by use of hydroxylamine released in situ from its HCl salt, to provide 3a–g in 35% to 80% yields. The action of phenyl chloroformate to the amidoximes 3a–g leads to the corresponding carbonate intermediates, which, when heated to reflux of toluene, cyclizes intra-molecularly to generate the substituted oxadiazolones C2-C8 in 34% to 50% yields.

In vitro inhibition of enzymatic activity of sPLA2s by C1-C8

The compounds C1-C8 were submitted to fluorimetric assay to determine their inhibitory potencies and selectivity towards human GIIAPLA2 (hGIIAPLA2) versus porcine group IB PLA2 (pGIBPLA2) (Table 2). GIBPLA2 is an enzyme of the same family as GIIAPLA2 (sPLA2) but is mainly involved in digestion of dietary phospholipids and is secreted by the pancreas [23]. Lipophilicity parameters, log P, of these products are calculated by use of Rekker's fragmental data [24] (Table 2). The molecules C1-C8 are specific inhibitors of hGIIAPLA2 because none inhibited pGIBPLA2 at the highest concentration tested (100 µM). Such selectivity implies that C1-C8 should not interfere with the digestion process.

Table 2
Inhibition of enzymatic activities of porcine pancreatic group IB (pGIB) and human group IIA (hGIIA) PLA2s by compounds C1 to C8 and their corresponding log P values.

The experimentally measured IC50s for hGIIAPLA2 (Table 2) are associated with the final energy balances, denoted δE2 in Table 1. The ranking of C1-C8 in terms of IC50 is the same as that of the δE2 magnitudes. In C2, the second phenyl ring is substituted with the ether O in the ortho position and in C6 in the para position. Both IC50 and δE2 values show C2 to have a significantly enhanced affinity for PLA2 as compared with C6, even though both are iso-lipophilic (Tables 1 and and2).2). In C2, the biphenyl group has favorable van der Waals interactions with both Phe23 and Val30 of the enzyme, but in C6, the interactions are limited to Phe23. Such interactions could be further optimized, as when the biphenyl ring was replaced by phenantrene in C8. The lowest-energy complex is now stabilized by an enhanced overlap of this ring with Phe63 (Fig. 2). However, the lipophilicity increases in parallel, which could possibly limit the bioavailability of C8. We found C8 indeed endowed with the most favorable δE2 value (Table 1), which was experimentally associated with the lowest IC50 value (0.62 µM vs. 5 µM for C1).

Figure 2
Representation of the most important interactions between C8 and the binding site of hGIIAPLA2 found from modeling.

At the other extreme, replacing the C1 indole ring by the smaller and less electron-rich phenyl ring, as in C7, resulted in a reduction of 10.3 kcal/mol in δE2 value. Thus, C7 can be predicted to have the least inhibitory potency in the series. This finding was confirmed by experimentation showing C7 to have the highest IC50 value (35 µM vs. 5 µM for C1).

Similar to C1, compounds C3-C5 have a bicyclic ring, whereas C3 possesses a benzo-1,3-thiazole instead of an indole ring. C4 and C5 have a chlorine and a methoxy substituent, respectively, in position 5 of the indole. In C3-C5, the aromatic rings interact simultaneously with His6, Arg7, and Val3, as was previously observed for C1 [20]. The difference in activity between C4 and C5 could be explained by additional electrostatic and/or van der Waals interactions contributed by methoxy substitution. C3 has anti-hGIIAPLA2 activity close to that of C1, along with substantially reduced lipophilicity (2.88 vs. 3.81 for C1).

Thus, in the C1-C8 series, C8 has the most favorable δE2 value and the lowest IC50 on human GIIAPLA2 activity, as evaluated by enzymatic assay. On the bases of the IC50 values we focused our cellular assays on the most potent compound C8, the sole compound with a sub-micromolar activity. We thus chose to evaluate the cytotoxicity and anti-inflammatory activity of C8 in primary cultured rabbit articular chondrocytes treated with the pro-inflammatory cytokine IL-1β, which is known to play a key role in rheumatic diseases such as osteoarthritis (for reviews see [25], [26]). Chondrocyte is the unique cell type in joints, and the cell model we chose is widely used to study the effect of inflammatory stress on joint cells.

Evaluation of the cytotoxicity of C8 on articular chondrocytes

We assessed the viability of the chondrocytes by MTT assay to evaluate the cytotoxic effects of C8 on these cells. Chondrocytes were treated for 20 h with 1 ng/mL IL-1β alone or 1 h after the addition of C8 at 0.31 to 9.92 µM, which corresponded to 0.5- to 5-fold the IC50 of C8 on human GIIAPLA2 activity (Table 2). Three different culture medium compositions were used: DMEM alone, or supplemented with 0.1% BSA or 2% FCS. IL-1β had no cytotoxic effects as compared with the untreated control condition for the three culture media tested (Fig. 3). In chondrocytes cultured in DMEM alone but with IL-1β, C8 had no cytotoxic effects at 0.31 to 2.48 µM (Fig. 3A). In chondrocytes cultured in DMEM with 0.1% BSA or 2% FCS and IL-1β, C8 had no cytotoxic effects at 0.31 to 9.92 µM (Fig. 3B and 3C). Thus, we evaluated the anti-inflammatory activity of C8 in culture conditions from 0.31 to 1.24 µM in DMEM alone and from 0.31 to 4.96 µM in DMEM supplemented with 0.1% BSA or 2% FCS.

Figure 3
Effect of IL-1β and C8 on viability of articular chondrocytes.

Effect of C8 on IL-1β-stimulated PGE2 secretion in articular chondrocytes

We tested the effect of C8 on the IL-1β-stimulated secretion of PGE2 in chondrocytes. PGE2 synthesis takes place mainly in response to cell activation by IL-1β, and its generation accounts for many of the actions induced by this cytokine [27]. In vitro, IL-1β induces the expression of COX-2 by chondrocytes, which results in increased PGE2 production [28]. PGE2 release thus represents a powerful IL-1β- and PLA2-dependent inflammatory marker in our cell model. Chondrocytes were treated for 20 h with IL-1β alone or 1 h after the addition of C8. As expected, IL-1β significantly stimulated PGE2 secretion by chondrocytes in the three different culture media: 23.3-, 18.3- and 2.8-fold induction as compared with untreated control conditions, in DMEM alone or supplemented with 0.1% BSA or 2% FCS, respectively (Fig. 4). In chondrocytes treated with IL-1β, C8 had a strong and statistically significant inhibitory effect on PGE2 secretion at all concentrations tested: from 0.31 to 1.24 µM in DMEM alone or from 0.31 to 4.96 µM in DMEM supplemented with 0.1% BSA or 2% FCS (Fig. 4). In DMEM alone, at concentrations of 0.31-, 0.62-, 0.94-, and 1.24-µM, C8 decreased the production of PGE2 induced by IL-1β by 59-, 58-, 74-, and 80-%, respectively (Fig. 4A). In DMEM supplemented with 0.1% BSA, at concentrations of 0.31-, 0.62-, 0.94-, 1.24-, 2.48-, and 4.96-µM, C8 decreased the production of PGE2 induced by IL-1β by 31-, 30-, 45-, 43-, 81-, and 92-%, respectively (Fig. 4B). In DMEM supplemented with 2% FCS, at concentrations of 0.31-, 0.62-, 0.94-, 1.24-, 2.48-, and 4.96-µM, C8 decreased the production of PGE2 induced by IL-1β by 26-, 48-, 49-, 54-, 68-, and 68-%, respectively (Fig. 4C). It is important to note that C8 down-regulated the IL-1β-stimulated secretion of PGE2 to the level of the control untreated condition at 4.96 µM in DMEM supplemented with 0.1% BSA and at 2.48 and 4.96 µM in DMEM supplemented with 2% FCS. The effect of C8 was then evaluated at the extreme concentrations (0.31- and 4.96-µM) in DMEM supplemented with 2% FCS and containing decreasing (1-, 0.5-, and 0.25-ng/mL) IL-1β concentrations (Table 3). The anti-IL-1β inhibitory effect of C8 at 0.31 µM increases when IL-1β concentration decreases. The inhibitory effect of C8 at 4.96 µM does not change when IL-1β concentration decreases. This is probably due to the fact that at 4.96 µM, the inhibitory effect of C8 on IL-1β-induced PGE2 production is maximal. A parallel cellular test was performed on the compound C1 whose IC50 is 5 µM (Table 2) and we observed that a 8 µM dose of C1, corresponding to 1.6-fold the IC50 of C1 on human GIIAPLA2 activity, does not decrease the stimulated PGE2 secretion by IL-1β at 1 ng/mL (data not shown). Thus, C8, but not C1, decreases the IL-1β-stimulated PGE2 secretion in a dose-dependent manner in the three culture medium compositions used.

Figure 4
Effect of IL-1β and C8 on PGE2 secretion by articular chondrocytes.
Table 3
Effect of C8 on PGE2 secretion by articular chondrocytes incubated with different IL-1β concentrations.

Effect of C8 on IL-1β-stimulated NO secretion in articular chondrocytes

We tested the effect of C8 on the IL-1β-stimulated secretion of NO in chondrocytes. NO is a mediator of immune and inflammatory responses. In vitro, IL-1β induces the expression of inducible NO synthase (iNOS) by chondrocytes, and consequently an increase in NO production [29]. NO secretion, evaluated by nitrite concentration in the cell culture medium, represents a reliable IL-1β-dependent and PLA2-independent inflammatory marker in our cell model. Chondrocytes were treated for 20 h with IL-1β alone or 1 h after the addition of C8. As expected, IL-1β significantly stimulated nitrite secretion by chondrocytes in the three different culture media: 8.1-, 2.6-, and 2.0-fold induction as compared with the control conditions, in DMEM medium alone or supplemented with 0.1% BSA or 2% FCS, respectively (Fig. 5). C8 did not significantly inhibit the IL-1β-stimulated nitrite secretion in chondrocytes cultured in DMEM medium alone or supplemented with 0.1% BSA (Fig. 5A, B). In DMEM supplemented with 2% FCS, C8 did not inhibit the IL-1β-stimulated nitrite secretion at 0.31-, 0.62-, and 0.94-µM and slightly decreased by 17-, 19-, 21-% the IL-1β-induced nitrite production at 1.24-, 2.48-, and 4.96-µM, respectively (Fig. 5C). Thus, C8 did not inhibit the IL-1β-stimulated NO secretion in DMEM alone or supplemented with 0.1% BSA and slightly inhibited IL-1β-stimulated NO secretion in DMEM supplemented with 2% FCS.

Figure 5
Effect of IL-1β and C8 on nitrite secretion by articular chondrocytes.

Effect of C8 on COX-1, COX-2, and iNOS protein levels in articular chondrocytes

We evaluated the effect of C8 on COX-1, COX-2 and iNOS protein levels in chondrocytes treated with IL-1β. Chondrocytes were treated for 20 h with IL-1β alone or 1 h after the addition of C8 (0.31-1.24 µM) in DMEM, and protein extracts were examined by western blot analysis. As expected, COX-1 protein was detectable but COX-2 and iNOS proteins were undetectable in untreated control conditions (Fig. 6A). Moreover, IL-1β treatment induced the expression of COX-2 and iNOS proteins without affecting the level of COX-1 protein (Fig. 6A). In the presence of IL-1β, C8 did not alter the COX-1, COX-2 and iNOS protein levels (Fig. 6A). Consequently, the protein ratio of COX-1 to COX-2 was not modified by C8 (Fig. 6B).

Figure 6
Effect of IL-1β and C8 on the COX-1, COX-2, and iNOS protein levels in articular chondrocytes.

Discussion

Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, and selective COX-2 inhibitors are currently used to reduce rheumatic symptoms. However, these drugs exhibit gastrointestinal, renal, blood pressure and cardiovascular toxicities. To overcome this problem, GIIAPLA2 inhibitors could be developed to inhibit the production of COX substrates without impairing the balance between COX-1 and COX-2. We designed and synthesized 7 new oxadiazolone derivatives (C2 to C8) derived from C1. Using molecular modeling, we computed and ranked energy balances for the binding of these inhibitors to GIIAPLA2. The energy balances (Table 1) taking into account solvation effects show a correlation between δE2, the overall energy balance for binding, and the experimentally measured IC50 for our novel compounds C1-C8. This finding should lend additional credence to our previous results [20], despite the approximations of the computational approach used in that study, which allows for only single-point computations of Poisson-Boltzmann solvation energies for the most stable minima of the molecular dynamics procedure. Our useful predictions made with the present simplified energy potential may be due to the very local changes we made in the C1-C8 series. These bear on the series' sole terminal aromatic group and target a limited number of amino acids, so that the accuracy of the energy potential may be sufficient. We plan to study such energy balances with the polarizable molecular mechanics procedure SIBFA [30], which, along with the Langlet-Claverie methodology for Continuum solvation [31], was recently used to investigate the binding of inhibitors to metalloenzymes [32]. This study should also allow for considering changes on other parts of the drugs as well.

One possible unfavorable feature of C8 is its enhanced lipophilicity as compared with the other compounds. Nevertheless, this feature did not prevent the pharmacological efficiency of C8 in chondrocytes. Reduction in Log P could be anticipated by replacing phenantrene with heterocyclic analogs and/or substitution with hydrophilic groups. Such reductions were seen on passing from compound C1 with an indole ring to C3 with a benzothiazole. Nevertheless, the high lipophilicity of C8 should be an interesting option for its prospective clinical development, considering the possibility of local administration (intra-articular infiltration).

The toxicity and anti-inflammatory activity of C8 were evaluated in rabbit articular chondrocytes in primary culture. The toxicity of C8 was assessed by MTT, which allows an evaluation of the cell number and/or metabolic activity in cells. C8 (from 0.31 to 9.92 µM) did not decrease cell viability in culture medium supplemented with 0.1% BSA or 2% FCS but did (at 4.96 and 9.92 µM) in culture medium alone. This observation is probably due to the cells being weakened in the absence of BSA or FCS. We also observed, as expected, an increase in cell number and/or metabolic activity in response to IL-1β. This effect increases in the presence of C8, at non toxic doses, whatever the culture conditions. Thus, depending on the culture conditions or C8 doses, C8 increases or decreases cell number and/or metabolic activity. Moreover, C8 from 0.31 µM inhibited IL-1β-induced secretion of PGE2 by chondrocytes, corresponding to half of the IC50 on human GIIAPLA2 activity evaluated in vitro by enzymatic assay. Therefore, C8 could be a potent anti-inflammatory drug in vivo. However, C8 did not inhibit IL-1β-induced NO secretion by chondrocytes cultured in DMEM alone or supplemented with 0.1% BSA and slightly inhibited IL-1β-stimulated NO secretion in DMEM supplemented with 2% FCS. These data suggest that the anti-inflammatory property of C8 in chondrocytes mainly depends on its capacity to inhibit PLA2 activity.

COX-1 is involved in normal physiological functions, whereas COX-2 is involved in the inflammatory response. Anti-inflammatory drugs such as NSAIDs and selective COX-2 inhibitors, used to treat rheumatic disease, have severe side effects owing to impairment in the balance between COX-1 and COX-2 [4], [5], [6]. Interestingly, the present work shows that the potent PLA2 inhibitor C8 decreases PGE2 production without impairing this balance. Consequently, C8 could be a useful candidate in developing new anti-inflammatory drugs lacking the side effects observed with NSAIDs and selective COX-2 inhibitors.

In summary, we report on the design, synthesis and testing of 7 C1 analogs that differ from C1 by indole substitution or by indole replacement by other aromatic rings, the largest being phenanthrene. Compounds C2-C8 show both inhibitory activity on secreted/synovial GIIAPLA2 and selectivity as compared with GIBPLA2, a pancreatic enzyme involved in the digestion of dietary phospholipids. The order of interaction energies predicted by molecular modeling of these compounds is associated with their experimental IC50 values with GIIAPLA2 used as a target. The most promising compound is C8 in terms of computed energy balance for binding GIIAPLA2 and experimental potency towards GIIAPLA2, namely one order of magnitude larger than that of C1. In addition, C8 is endowed with anti-inflammatory activity in articular chondrocytes by inhibiting IL-1β-stimulated PGE2 secretion in these cells. Furthermore, it does not modify the ratio between the COX-1 and COX-2 isoenzymes. C8 is therefore an attractive candidate for anti-inflammatory therapy in joints. Experiments in animal models of rheumatic diseases are in progress in our laboratory.

Materials and Methods

Ethics Statements

Experimental protocols using rabbits complied with French legislation on animal experimentation and were approved by INSERM (Intitut National de la Santé et de la Recherche Médicale)'s Committee for Animal Studies.

Molecular modeling

Molecular modeling is described in Supporting Information S1.

Synthesis of oxadiazolone derivatives

Synthesis of compounds C1-C8 is described in Supporting Information S1.

In vitro PLA2 assay

Fatty-acid free BSA and pancreatic PLA2 were from Sigma. hGIIAPLA2 was prepared as previously described [33]. The fluorescent substrate for PLA2 assay, 1-hexadecanoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) was from Molecular Probes (Eugene). PLA2 activity was evaluated as previously described [34] with β-py-C10-PG used as a substrate (2 µM final concentration). In a total volume of 1 mL, the standard reaction medium contained 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EGTA, 2 µM β-py-C10-PG, 0.1% fatty-acid free BSA and 6 ng/mL pancreatic PLA2 or 1 ng/mL hGIIAPLA2. The fluorescence (λex  = 342 nm and λem  = 398 nm) of the enzymatic reaction medium was recorded for 3 min with use of a spectrofluorimeter LS 50 (Perkin-Elmer) equipped with a Xenon lamp. The reaction was initiated by the addition of CaCl2 (10 mM, final concentration). The increase in fluorescence was continuously recorded for 1 min, and PLA2 activity was calculated as previously described [34]. When used, the inhibitor was added to the reaction medium after introduction of BSA. The activity is expressed in micromoles of fluorescent β-py-C10-PG hydrolyzed per min. The standard error of the mean of three independent experiments was less than 10%, which allows for the determination of the IC50 values (concentration of inhibitors producing 50% inhibition) of each compound.

Isolation and culture of chondrocytes from rabbit articular cartilage

Articular chondrocytes were isolated from 5-week-old Fauve de Bourgogne female rabbits (CPA, Orleans, France) and cultured at the first passage in conditions avoiding cell dedifferentiation as previously described [35]. Cells were cultured at 37°C in 12-well plates in Ham's F-12 medium containing 10% FCS, 20 IU/mL penicillin, and 20 µg/mL streptomycin (all from Invitrogen) until nearly confluent. Then medium was replaced with DMEM (Invitrogen) containing 20 IU/mL penicillin, and 20 µg/mL streptomycin and, if necessary, 0.1% fatty acid free BSA (Sigma) or 2% FCS. At this time the C8 compound dissolved in DMSO (Sigma) was added to the medium (the amount of DMSO was kept at 1‰ (v/v) in all the wells). 1 h after the addition of C8, IL-1β (PeproTech) was added to the medium. Consequently, chondrocytes were incubated for 20 h with IL-1β and for 21 h with C8.

Evaluation of cell viability

At 18 h after the addition of IL-1β, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma) was added to the cell culture medium at 0.5 mg/mL. Cells were incubated 2 more hours at 37°C. The medium was then removed, and DMSO was added to dissolve the formazan crystals. The absorbance of the resulting solution was spectrophotometrically measured at 570 and 690 nm (background). The value corresponding to absorbance570nm - absorbance690nm was directly proportional to the number and activity of the viable cells.

Determination of PGE2 and nitrite concentrations in culture medium

20 h after the addition of IL-1β to the chondrocytes, culture media were collected, and aliquots were stored at −80°C until PGE2 and nitrite quantification. PGE2 concentration in culture media was determined by use of an enzyme immunoassay (EIA) kit (PGE2 EIA Kit-monoclonal; Cayman Chemical). Nitrite concentration was determined by a spectrophotometry method based on the Griess reaction [36]. Briefly, 200 µL of culture medium or sodium nitrite (NaNO2, Merck) standard dilutions were mixed with 100 µL Griess reagent [0.5% (w/v) sulphanilic acid (Merck), 0.05% (w/v) N(1-naphtyl)ethylenediamine (Merck), 30% (v/v) acetic acid, 1.5 N HCl] and incubated for 10 min at 50°C. The absorbance was measured at 540 nm.

Preparation of whole-cell protein extracts, protein quantification and western blot analysis

Proteins were extracted from the cultured cells by addition of lysis buffer [10 mM Tris (pH 7.4), 0.5% (v/v) NP40, 150 mM NaCl, 1 mM PMSF, 0.1 mM Na3VO3, complete-EDTA-free protease inhibitor cocktail (Roche)]. Cell lysates were centrifuged for 15 min at 14000 rpm at 4°C and supernatants were collected. Protein concentrations were determined by the Bradford method [37] by use of the Protein Assay dye reagent (Bio-Rad). Protein extracts (20 µg) were size-separated by SDS-PAGE in a 10% (w/v) polyacrylamide gel and electroblotted to a nitrocellulose membrane. Equal protein loading and transfer was confirmed by staining the membrane with Ponceau Red [0.2% (w/v) in H2O:acetic acid 99[ratio]1]. The membrane was sequentially incubated with antibodies against COX-1 (1[ratio]200, Santa Cruz Biotechnology), COX-2 (1[ratio]500, Santa Cruz Biotechnology), iNOS (1[ratio]400, BD Biosciences) or α-tubulin (1[ratio]100, Santa Cruz Biotechnology) and then with peroxidase-conjugated donkey anti-goat IgG (1[ratio]20000) or donkey anti-rabbit IgG (1[ratio]200, both Santa Cruz Biotechnology). Immunocomplexes were detected by an enhanced chemiluminescence kit (Amersham Bioscience). The membrane was stripped by incubation in 0.2 M NaOH between successive immunodetections. Semi-quantitative scanning densitometry involved use of the ImageJ program (NIH, USA).

Statistical analysis

Results are expressed as means ± SEM for the number of experiments indicated. Statistical analysis involved use of the Kruskal-Wallis test, then the ANOVA Fisher's test. A P<0.05 was considered statistically significant.

Supporting Information

Supporting Information S1

Materials and Methods in chemistry and molecular modeling.

(0.12 MB DOC)

Acknowledgments

We thank the computer center CINES-Montpellier for computer time and technical support.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was supported in part by INSERM, University Paris Descartes, Arthritis Foundation Courtin and a program of Paris Centre University (Paris Descartes and Paris Diderot). The authors thank the computer center CINES-Montpellier for computer time and technical support. The authors also thank the Ligue Nationale Contre le Cancer for support. NT was supported by Assistance Publique-Hopitaux de Paris. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1. Murakami M, Kudo I. Recent advances in molecular biology and physiology of the prostaglandin E2-biosynthetic pathway. Prog Lipid Res. 2004;43:3–35. [PubMed]
2. Dennis EA. Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem. 1994;269:13057–13060. [PubMed]
3. McCoy JM, Wicks JR, Audoly LP. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J Clin Invest. 2002;110:651–658. [PMC free article] [PubMed]
4. Martel-Pelletier J, Pelletier JP, Fahmi H. Cyclooxygenase-2 and prostaglandins in articular tissues. Semin Arthritis Rheum. 2003;33:155–167. [PubMed]
5. Chen YF, Jobanputra P, Barton P, Bryan S, Fry-Smith A, et al. Cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs (etodolac, meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for osteoarthritis and rheumatoid arthritis: a systematic review and economic evaluation. Health Technol Assess. 2008;12:1–278, iii. [PubMed]
6. Solomon DH, Avorn J, Sturmer T, Glynn RJ, Mogun H, et al. Cardiovascular outcomes in new users of coxibs and nonsteroidal antiinflammatory drugs: high-risk subgroups and time course of risk. Arthritis Rheum. 2006;54:1378–1389. [PubMed]
7. Nevalainen TJ, Haapamaki MM, Gronroos JM. Roles of secretory phospholipases A(2) in inflammatory diseases and trauma. Biochim Biophys Acta. 2000;1488:83–90. [PubMed]
8. Hara S, Kudo I, Chang HW, Matsuta K, Miyamoto T, et al. Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis. J Biochem. 1989;105:395–399. [PubMed]
9. Kramer RM, Hession C, Johansen B, Hayes G, McGray P, et al. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem. 1989;264:5768–5775. [PubMed]
10. Seilhamer JJ, Plant S, Pruzanski W, Schilling J, Stefanski E, et al. Multiple forms of phospholipase A2 in arthritic synovial fluid. J Biochem. 1989;106:38–42. [PubMed]
11. Lin MK, Farewell V, Vadas P, Bookman AA, Keystone EC, et al. Secretory phospholipase A2 as an index of disease activity in rheumatoid arthritis. Prospective double blind study of 212 patients. J Rheumatol. 1996;23:1162–1166. [PubMed]
12. Bomalaski JS, Lawton P, Browning JL. Human extracellular recombinant phospholipase A2 induces an inflammatory response in rabbit joints. J Immunol. 1991;146:3904–3910. [PubMed]
13. Murakami M, Kudo I, Nakamura H, Yokoyama Y, Mori H, et al. Exacerbation of rat adjuvant arthritis by intradermal injection of purified mammalian 14-kDa group II phospholipase A2. FEBS Lett. 1990;268:113–116. [PubMed]
14. Bradley JD, Dmitrienko AA, Kivitz AJ, Gluck OS, Weaver AL, et al. A randomized, double-blinded, placebo-controlled clinical trial of LY333013, a selective inhibitor of group II secretory phospholipase A2, in the treatment of rheumatoid arthritis. J Rheumatol. 2005;32:417–423. [PubMed]
15. Zeiher BG, Steingrub J, Laterre PF, Dmitrienko A, Fukiishi Y, et al. LY315920NA/S-5920, a selective inhibitor of group IIA secretory phospholipase A2, fails to improve clinical outcome for patients with severe sepsis. Crit Care Med. 2005;33:1741–1748. [PubMed]
16. Fraser H, Hislop C, Christie RM, Rick HL, Reidy CA, et al. Varespladib (A-002), a Secretory Phospholipase A2 Inhibitor, Reduces Atherosclerosis and Aneurysm Formation in ApoE−/− Mice. J Cardiovasc Pharmacol 2009 [PubMed]
17. Assogba L, Ahamada-Himidi A, Habich NM, Aoun D, Boukli L, et al. Inhibition of secretory phospholipase A2. 1-design, synthesis and structure-activity relationship studies starting from 4-tetradecyloxybenzamidine to obtain specific inhibitors of group II sPLA2s. Eur J Med Chem. 2005;40:850–861. [PubMed]
18. Boukli L, Touaibia M, Meddad-Belhabich N, Djimde A, Park CH, et al. Design of new potent and selective secretory phospholipase A2 inhibitors. Part 5: synthesis and biological activity of 1-alkyl-4-[4,5-dihydro-1,2,4-[4H]-oxadiazol-5-one-3-ylmethylbenz-4′-yl(oyl)] piperazines. Bioorg Med Chem. 2008;16:1242–1253. [PubMed]
19. Dong CZ, Ahamada-Himidi A, Plocki S, Aoun D, Touaibia M, et al. Inhibition of secretory phospholipase A2. 2-Synthesis and structure-activity relationship studies of 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one (PMS1062) derivatives specific for group II enzyme. Bioorg Med Chem. 2005;13:1989–2007. [PubMed]
20. Plocki S, Aoun D, Ahamada-Himidi A, Tavarès-Camarinha F, Dong CZ, et al. Molecular modeling, design, and synthesis of less lipophilic derivatives of 3-(4-tetradecyloxybenzyl)-4H-1,2,4-oxadiazol-5-one (PMS1062) specific for group II enzyme. Eur J Org Chem 2005: 2005:2747–2757.
21. Touaibia M, Djimde A, Cao F, Boilard E, Bezzine S, et al. Inhibition of secreted phospholipase A2. 4-glycerol derivatives of 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one with broad activities. J Med Chem. 2007;50:1618–1626. [PubMed]
22. Dehaen W, Hassner A. Cycloadditions. 45. Annulation of heterocycles via intramolecular nitrile oxide-heterocycle cycloaddition reaction. J Org Chem. 1991;56:896–900.
23. Carey MC, Small DM, Bliss CM. Lipid digestion and absorption. Annu Rev Physiol. 1983;45:651–677. [PubMed]
24. Rekker RF, De Kort HM. The hydrophobic fragmental constant; an estimation to a 1000 data point set. Eur J Med Chem. 1979;14:479–488.
25. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002;39:237–246. [PubMed]
26. Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med. 2001;344:907–916. [PubMed]
27. Goetzl EJ, An S, Smith WL. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J. 1995;9:1051–1058. [PubMed]
28. Amin AR, Attur M, Patel RN, Thakker GD, Marshall PJ, et al. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide. J Clin Invest. 1997;99:1231–1237. [PMC free article] [PubMed]
29. Sakurai H, Kohsaka H, Liu MF, Higashiyama H, Hirata Y, et al. Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides. J Clin Invest. 1995;96:2357–2363. [PMC free article] [PubMed]
30. Gresh N, Cisneros GA, Darden TA, Piquemal JP. Anisotropic, Polarizable Molecular Mechanics Studies of Inter- and Intramolecular Interactions and Ligand-Macromolecule Complexes. A Bottom-Up Strategy. J Chem Theory Comput. 2007;3:1960–1986. [PMC free article] [PubMed]
31. Langlet J, Claverie P, Caillet J, Pullman A. Improvements of the continuum model. 1. Application to the calculation of the vaporization thermodynamic quantities of nonassociated liquids. J Phys Chem. 1988;92:1617–1631.
32. Roux C, Gresh N, Perera LE, Piquemal JP, Salmon L. Binding of 5-phospho-D-arabinonohydroxamate and 5-phospho-D-arabinonate inhibitors to zinc phosphomannose isomerase from Candida albicans studied by polarizable molecular mechanics and quantum mechanics. J Comput Chem. 2007;28:938–957. [PubMed]
33. Dong CZ, Romieu A, Mounier CM, Heymans F, Roques BP, et al. Total direct chemical synthesis and biological activities of human group IIA secretory phospholipase A2. Biochem J. 2002;365:505–511. [PubMed]
34. Radvanyi F, Jordan L, Russo-Marie F, Bon C. A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. Anal Biochem. 1989;177:103–109. [PubMed]
35. Francois M, Richette P, Tsagris L, Raymondjean M, Fulchignoni-Lataud MC, et al. Peroxisome proliferator-activated receptor-gamma down-regulates chondrocyte matrix metalloproteinase-1 via a novel composite element. J Biol Chem. 2004;279:28411–28418. [PubMed]
36. Evans CH, Watkins SC, Stefanovic-Racic M. Nitric oxide and cartilage metabolism. Methods Enzymol. 1996;269:75–88. [PubMed]
37. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. [PubMed]

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