Q C F H D a b c d e f a A R R A A K s C Q P M 1 c t f v a 2 T s ( ( ( ( ( 0 d Chemico-Biological Interactions 189 (2011) 9–16 Contents lists available at ScienceDirect Chemico-Biological Interactions journa l homepage: www.e lsev ier .com/ locate /chembio int uercetin as an inhibitor of snake venom secretory phospholipase A2 amila Aparecida Cotrima,∗, Simone Cristina Buzzo de Oliveiraa, Eduardo B.S. Diz Filhoa, abiana Vieira Fonsecaa, Lineu Baldissera Jr. b, Edson Antunesb, Rafael Matos Ximenesc, elena Serra Azul Monteiroc, Marcelo Montenegro Rabellod, Marcelo Zaldini Hernandesd, aniela de Oliveira Toyamae, Marcos Hikari Toyamaf Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brazil Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brazil Laboratório de Farmacologia de Venenos, Toxinas e Lectinas (LAFAVET), Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil Laboratório de Química Teórica Medicinal – LQTM, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Universidade Presbiteriana Mackenzie, CCBS, São Paulo, Brazil Campus Experimental do Litoral Paulista – UNESP, São Vicente, SP, Brazil r t i c l e i n f o rticle history: eceived 15 July 2010 eceived in revised form 27 October 2010 ccepted 29 October 2010 vailable online 4 November 2010 eywords: PLA rotalus durissus terrificus a b s t r a c t As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various phar- maceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity uercetin harmacological sites olecular docking by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other tructu s as s flavonoids with similar s potential use of flavonoid . Introduction Phospholipases A2 (PLA2, EC 3.1.1.4) are small proteins that atalyze the hydrolysis of glycerophospholipids at the sn-2 posi- ion in a Ca2+-dependent reaction, releasing lysophospholipids and atty acids [1–3]. These enzymes are the main component of snake enom and have been investigated not only because they have wide range of biological effects, but also due to their similar- ∗ Corresponding author at: Instituto de Biologia – UNICAMP, Rua Monteiro Lobato, 55 – Cidade Universitária Zeferino Vaz, Zip Code: 13083-862, Campinas, SP, Brazil. el.: +55 19 3521 6132. E-mail addresses: cami cotrim@yahoo.com.br (C.A. Cotrim), imonebuzzo@hotmail.com (S.C.B. de Oliveira), eduardodizfilho@gmail.com E.B.S. Diz Filho), fvmacieira@yahoo.com.br (F.V. Fonseca), libajunior@hotmail.com L. Baldissera Jr.), edson.antunes@uol.com.br (E. Antunes), rmximenes@hotmail.com R.M. Ximenes), hsazul@gmail.com (H.S.A. Monteiro), marcelorabello@globo.com M.M. Rabello), zaldini@ufpe.br (M.Z. Hernandes), gaveiraf@mackenzie.br D. de Oliveira Toyama), mhtjpn@yahoo.com (M.H. Toyama). 009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. oi:10.1016/j.cbi.2010.10.016 res could bind to sPLA2. Further research is warranted to investigate the PLA2 inhibitors. © 2010 Elsevier Ireland Ltd. All rights reserved. ity to mammalian phospholipases [4,5]. However, in contrast to their mammalian counterparts, several snake venom PLA2s are tox- ins that induce pharmacological effects [6] through arachidonic acid metabolism leading to the production of various lipid pro- inflammatory mediators such as prostaglandins, thromboxanes and leukotrienes [7]. Recent studies have shown that inhibition of cytosolic PLA2 (cPLA2) leads to a decrease in eicosanoid levels and, reduced inflammation [8]. Due to the role of PLA2s in the inflammatory process, there is pharmacological interest in PLA2 inhibitors, and among these, the flavonoids have been successfully studied. Flavonoids are widely produced in plants tissues making them suitable targets for phar- maceutical extraction and chemical synthesis [8,9]. The inhibitory effect of flavonoids on secretory PLA2 (sPLA2) was reported by Gil et al. [10], and Lindahl and Tagesson [11]. Their results showed that inhibition of sPLA2 from different sources following incu- bation with various flavonoids is dependent on the 5-hydroxyl group as well as the double bond and the double-bonded oxygen in the oxane ring, and that the hydroxyl groups at the 3′- and 4′- dx.doi.org/10.1016/j.cbi.2010.10.016 http://www.sciencedirect.com/science/journal/00092797 http://www.elsevier.com/locate/chembioint mailto:cami_cotrim@yahoo.com.br mailto:simonebuzzo@hotmail.com mailto:eduardodizfilho@gmail.com mailto:fvmacieira@yahoo.com.br mailto:libajunior@hotmail.com mailto:edson.antunes@uol.com.br mailto:rmximenes@hotmail.com mailto:hsazul@gmail.com mailto:marcelorabello@globo.com mailto:zaldini@ufpe.br mailto:gaveiraf@mackenzie.br mailto:mhtjpn@yahoo.com dx.doi.org/10.1016/j.cbi.2010.10.016 1 logica p t u o b m m t s s o a b b q I i D m d f q a 2 2 S s o A p C c e v 2 o d c d t c fl r S 2 m i w t 0 s a l t t 0 C.A. Cotrim et al. / Chemico-Bio osition are required for selective inhibition of PLA2 [11]. However, he exact mechanism by which flavonoids inhibit PLA2 remains nclear. Iglesias et al. [12] showed that morin modifies the sec- ndary structure of sPLA2 from Crotalus durissus cascavella venom, ut did not significantly affect its pharmacological activities. Although there are studies of flavonoids as PLA2 inhibitors, the ode of binding of flavonoids to PLA2 as well as their inhibitory echanism is still not clear. The aims of this article are to inves- igate the effect of quercetin, a widely spread flavonoid, on the tructure and function of a sPLA2 isolated from Crotalus duris- us terrificus, to increase our understanding of the mode action f polyphenolic compounds on the snake venoms, and to evalu- te therapeutic application against the symptoms caused by snake ites. In the last decades the action of quercetin on the PLA2 has een studied. In 1993 Lindahl and Tagesson [13] showed that uercetin is a potent inhibitor of PLA2 (group II) from Vipera russuli. n addition, Lättig et al. [8] showed through computational stud- es chemical interactions between humans’ sPLA2 and quercetin. ue to these characteristics, quercetin has been chosen as flavonoid odel. Moreover we also propose, through high-resolution three- imensional (3D) data (molecular docking), a structural model or understanding the molecular interactions between sPLA2 and uercetin, and how it can influence enzymatic and pharmacological ctivities of sPLA2. . Materials and methods .1. Venom, animals and reagents C. durissus terrificus venom was purchased from Bio-Agents erpentarium (Batatais, São Paulo, Brazil). Analytical HPLC- and equencing-grade solutes and solvents were purchased from vari- us suppliers (Bio Rad, Sigma Aldrich, Boehringer Mannheim, and pplied Biosystems). Female Swiss mice (18–20 g) used in the harmacological assays were obtained from the Multidisciplinary enter of Biological Investigations (CEMIB-UNICAMP) and male hicks were obtained from Itu farm in Campinas City. All animal xperiments were approved by the Ethics Committee of State Uni- ersity of Campinas (São Paulo, Brazil) under the number 1916-1. .2. Purification of sPLA2 Whole C. durissus terrificus venom was first fractioned as previ- usly described by Toyama et al. [14]. Dried venom (45 mg) was issolved in ammonium bicarbonate buffer (1.0 M; pH 8.0) and larified by centrifugation (4500 × g for 1 min). The sPLA2 from C. urissus terrificus was eluted using a non-linear gradient of ace- onitrile 66% in 0.1% of trifluoroacetic acid (TFA) by reverse-phase hromatography using a Supelco C5 column (0.10 cm × 25 cm) with ow rate of 1 mL/min with absorbance monitoring at 280 nm. The esulting PLA2 was termed sPLA2 and its purity was evaluated by DS-PAGE. .3. Incubation of sPLA2 with quercetin and purification of odified sPLA2 The incubation of sPLA2 with quercetin (mol:mol) was accord- ng to the procedure described by Zhao et al. [15]. Quercetin as dissolved in dimethyl sulfoxide (DMSO), and its concentra- ion never exceeded 1% during incubation. Quercetin (400 �L of a .1 mM solution) was added to 400 �L of a homogenized, purified PLA2 solution (1 mg/mL). The mixture was incubated for 90 min t room temperature, and 200-�L samples of this mixture were oaded onto a preparative reverse-phase column to separate the reated enzyme (sPLA2:Q) from quercetin. After column equilibra- ion with HPLC buffer A (aqueous 0.1% TFA), samples were eluted l Interactions 189 (2011) 9–16 using a discontinuous gradient of HPLC buffer B (66.6% of ace- tonitrile in 0.1% TFA) at a constant flow rate of 1.0 mL/min. The chromatographic run was monitored at 280 nm. 2.4. Electrophoresis Electrophoresis was carried out following the Laemmli method [16]. The degree of purity of fractions was assessed by discontinu- ous electrophoresis using a final acrylamide concentration of 12.5% in the resolving gels (1.0 M Tris–HCl, pH 8.8) and 5% in the stacking gel (0.5 M Tris–HCl, pH 6.8). Electrophoretic separation was carried out in a 250 Mighty Small (Hoefer Scientific Instruments) for SDS- PAGE. All samples and the molecular marker were treated with SDS and 1.0 M dithiothreitol (DTT), and the run was conducted at 60 mA for stacking gel and 90 mA for running gel. After electrophoresis, samples were stained with Coomassie brilliant blue R-250. 2.5. Circular dichroism spectroscopy sPLA2 and sPLA2:Q (sPLA2 + quercetin) were dissolved in 10 mM sodium phosphate buffer (pH 7.4) and final protein concentrations were adjusted to 8.7 mM. After centrifugation at 4000 × g for 5 min, samples were transferred to a 1-mm path length quartz cuvette. Circular dichroism spectra in the wavelength range 185–300 nm were acquired in-house with a J720 spectropolarimeter (Jasco Corp., Japan) using a bandwidth of 1 nm and a response time of 1 s. Data collection was performed at room temperature, with a scanning speed of 100 nm/min. Nine scans were obtained for each sample, and all spectra were corrected by subtracting buffer blanks. 2.6. Intrinsic fluorescence. The relative intrinsic fluorescence intensities of sPLA2 and sPLA2:Q were monitored with a Varian Cary Eclipse. The proteins were solubilized in water at room temperature. The measurements were performed in a 1.5-mL 1-cm path length quartz cuvette. Flu- orescence was measured between 300 and 450 nm after excitation at 280 nm. 2.7. Mass spectrometry The molecular mass of sPLA2 and sPLA2:Q were determined by matrix-assisted laser desorption ionization-time-of-flight (MALDI- TOF) mass spectrometry using a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems). One microliter of samples (sPLA2 and sPLA2:Q) in 0.1% TFA was mixed with 2 �L of the matrix �-cyano-4-hydroxycinnamic acid, 50% acetonitrile, and 0.1% TFA (v/v). The matrix was prepared with 30% acetonitrile and 0.1% TFA (v/v). Ion masses were determined with an acceleration voltage of 25 kV, the laser operated at 2890 �J/com2, a 300-ns delay, and the linear analysis mode. 2.8. Measurement of sPLA2 activity sPLA2 activity was measured following the protocol described by Lee et al. [17] and modified by Toyama et al. [14] in 96- well plates, using 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA, BIOMOL, USA) as substrate. Enzyme activity, expressed as the ini- tial velocity of the reaction (Vo), was calculated based on the increase in absorbance after 20 min. All assays were performed with absorbance at 425 nm using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). After the addition of native or treated sPLA2 (20 �g), the reaction mixture was incubated for up to 40 min at 37 ◦C and the absorbance read at 5-min intervals. logica 2 G f n a A f s n e p ( 2 t S t t w t h p e s 2 b r O C 1 t t a c p I n 1 t e 2 ( 2 T i m a o t ( 1 K 1 T t p sPLA2 was isolated from C. durissus terrificus venom through reverse-phase chromatography (Fig. 1a) and its enzymatic activity was evaluated using 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) as a substrate. SDS-PAGE revealed the presence of one protein band C.A. Cotrim et al. / Chemico-Bio .9. Antibacterial activity The antibacterial activity was assayed as described by Santi- adelha et al. [18] and the structural modification was done ollowing the method of Toyama et al. [19]. Clavibacter michiga- ensis michiganensis cells were harvested from fresh agar plates nd suspended in sterile distilled water (A600 nm = 3 × 108 CFU/mL). liquots of bacterial suspension were diluted to 103 colony- orming units/mL (CFU/mL) and incubated with sPLA2 or sPLA2:Q amples (75 �g/mL) for 60 min at 28 ◦C. Survival was assayed on utrient agar (Difco) plates (n = 5). For both antibacterial assays, lectron microscopy assessments of morphologic alterations were erformed in the presence of saline (negative control), quercetin 100 �M, quercetin control), sPLA2, and sPLA2:Q. .10. Paw edema assay Paw edema assays were performed with the aim of evaluate he inflammatory activity induced by sPLA2. For this assay female wiss mice were used, since they show a lower aggressive behavior han male mice. Paw edema was induced by a single subplan- ar injection of 25 �L sPLA2 or sPLA2:Q (25 �g/paw). Paw volume as measured immediately before the injection and at selected ime intervals thereafter (30, 60, 120, 180, and 360 min) using a ydroplethysmometer (model 7150, Ugo Basile, Italy). All sam- les were dissolved in sterile saline solution (0.9%). Results were xpressed as the increase in paw volume (�L) and calculated by ubtracting the basal volume from the volume following treatment. .11. Neurotoxic effect assay Male chicks (4–8 days old) were killed with ether, and the iventer cervicis muscle was removed [20] and mounted under a esting tension of 1 g in a 4 mL organ bath containing aerated (95% 2 + 5% CO2) Krebs solution (118.7 mM NaCl, 4.7 mM KCl, 1.88 mM aCl2, 1.17 mM KH2PO4, 1.17 mM MgSO4, 25.0 mM NaHCO2 and 1.65 mM glucose, pH 7.5) at 37 ◦C. A bipolar platinum ring elec- rode was placed around the tendon, which ran the length of he nerve trunk supplying the muscle. Indirect stimulation was pplied with a Grass S4 stimulator (0.1 Hz, 0.2 ms, 3–4 mV). Mus- le contractions and contractures were recorded by connecting the reparation to a force displacement transducer (Narco Biosystems nc.) coupled to a Gould RS 3400 recorder. Contractures to exoge- ous acetylcholine (ACh, 55 or 110 �M for 60 s) and KCl (5 mM for 20–130 s) were obtained in the absence of nerve stimulation prior o the addition of sPLA2 or sPLA2:Q (10 �g/mL) and at the end of the xperiment. The preparations were allowed to stabilize for at least 0 min before the addition of Ach, KCl, or a single concentration 10 �g/mL) of the compounds. .12. Platelet aggregation studies Platelet aggregation activities were assayed as described by oyama et al. [19]. Briefly, venous blood was collected with nformed consent from healthy volunteers who denied taking any edication in the previous 14 days. Collected blood was immedi- tely transferred into polypropylene tubes containing one-tenth f final volume of acid citrate dextrose (ACD-C; citric acid 3%, risodium citrate 4%, glucose 2%; 1:9 v/v). Platelet-rich plasma PRP) was obtained by centrifuging whole blood at 200 × g for 5 min. PRP was washed in a wash buffer solution (NaCl 140 mM, Cl 5 mM, sodium citrate 12 mM, glucose 10 mM and saccharose 2 mM; pH 6; 5:7 v/v) and centrifuged at 800 × g for 12 min at 20 ◦C. he platelet pellet was gently resuspended in Krebs–Ringer solu- ion and counts were performed on a Neubauer chamber. The final latelet suspension was adjusted to 1.2 × 108 platelets/mL. Platelet l Interactions 189 (2011) 9–16 11 aggregation was carried out using 400 �L of the washed platelet solution in a cuvette and incubated at 37 ◦C with constant stirring. The desired concentration of protein was added 3 min prior to the addition of a platelet aggregation inducer (thrombin). Aggregation was subsequently recorded for 5–10 min with an aggregometer (Chrono-log Lumi-Aggregometer model 560-Ca, Havertown, PA, USA). Aggregation experiments were performed with a concentra- tion of 15 �g/mL of sPLA2 and sPLA2:Q. 2.13. Myotoxic activity The liberation of creatine kinase (CK) from damaged muscle cells was followed by use of the CK-NAc kit (Laborlab) to mea- sure the enzyme activity in mice plasma. Five groups of animals (18–22 g) were injected in the right gastrocnemius muscle with 25 �L of 1.0 mg/mL of sPLA2, sPLA2:Q, or quercetin (n = 4) while the control group received an equal volume of 0.15 M NaCl. Blood was collected from the tail after 1 h into tubes containing heparin. The amount of CK was determined using 4 �L plasma, which was incubated for 3 min at 37 ◦C with 1.0 mL of the reagent according to the kit protocol. Activity was expressed in units per liter (U/L). 2.14. Molecular modeling (docking) The structural optimization of the quercetin ligand was initially achieved using the quantum chemical AM1 method [21] imple- mented in the BioMedCache program (BioMedCache, 1989) with default values for the convergence criteria. Docking calculations were performed with the GOLD 4.0 program [22] to obtain the in silico affinity of quercetin to the Crotoxin B target, a basic sPLA2 from C. durissus terrificus venom. This sPLA2 structure was taken from the RCSB Protein Data Bank [PDB] under the PDB code 2QOG. The structure of the B chain was chosen for calculations, maintain- ing the Ca2+ ion and the water molecule number 188, located 3.16 Å from Ca2+. Docking calculations were performed to consider the flexibility of the quercetin ligand in such a way that torsions were consid- ered active during the calculation. The active site was defined as all atoms within a 10-Å radius from His48, an important residue according to the literature [23,24]. 2.15. Statistical analyses Results were reported as means ± SEM of replicate experiments. The significance of differences between means was assessed by an analysis of variance, followed by a Dunnett’s test when sev- eral experimental groups were compared to the control group. The confidence limit for significance was 5%. 3. Results 3.1. Purification of sPLA2 and Incubation of sPLA2 with quercetin with a molecular mass of 14 kDa (Fig. 1c), corresponding to sPLA2. After incubation with quercetin, sPLA2:Q (sPLA2 + quercetin) eluted at 23.7 min whereas sPLA2 had a retention time of 24.3 min (Fig. 1b). This difference indicates an interaction between sPLA2 and quercetin, which changed the hydrophobicity of sPLA2. 12 C.A. Cotrim et al. / Chemico-Biologica Fig. 1. Purification and chemical modification of secretory phospholipase A2 (sPLA2). (a) Fractionation of whole venom was performed by reverse-phase HPLC ( o m t 3 b a fi H s m s s 3 t w l c t a q c t 3 m using C. michiganensis michiganensis (Gram-positive). As shown in Fig. 4b, sPLA2 has a higher inhibitory potential on bacterial growth than sPLA2:Q, since sPLA2 decreased CFU levels to 9.8% (90.2% inhi- bition) compared to 63.5% (only 36.5% inhibition) for sPLA2:Q. C5 column 0.10 cm × 25 cm) using a non-linear concentration gradient of buffer to btain a high purity protein. (b) Reverse phase HPLC profile of sPLA2 before and after odification with quercetin. (c) Tricine SDS-PAGE of sPLA2 from Crotalus durissus errificus. .2. Circular dichroism spectroscopy The effect of quercetin on the sPLA2 structure was evaluated y absorption spectra of sPLA2 and sPLA2:Q, circular dichroism, nd fluorescence spectroscopy. As shown in Fig. 2a, few modi- cations were observed in the 270–280 nm wavelength region. owever, some changes were observed on CD and fluorescence pectra after treatment with quercetin. CD spectra analysis showed odifications mainly in the region corresponding to the �-helices, uggesting that quercetin is able to induce changes in the secondary tructure of this enzyme (Fig. 2b). .3. Intrinsic fluorescence The presence of aromatic amino acids such as tryptophan and yrosine in the protein chain allows the use of fluorescence spectra, hich is sensitive for the investigation of protein conformation and igand binding. Fig. 3a shows an increase in the intensity of fluores- ence emission spectra after treatment with quercetin, suggesting hat this flavonoid is able to change the structure of the protein t the tertiary structure level, however, fluorescence spectrum of uercetin has shown that this compound has a peak of fluores- ence near the region of the tryptophan and possibly contributes o increase the fluorescence observed. .4. Mass spectrometry Mass spectrometry by MALDI-TOF indicated that the exact olecular mass of the native protein is 14,425.56 Da while that l Interactions 189 (2011) 9–16 of sPLA2:Q is 14,727.79 (Fig. 3b), an increase of 302.23 Da in sPLA2 treated with quercetin, suggesting that one molecule of quercetin is bound to the protein structure. 3.5. Measurement of sPLA2 activity and antibacterial activity The effect of quercetin on sPLA2 enzymatic activity was evalu- ated. Both enzymes, sPLA2 and sPLA2:Q exhibit allosteric behavior (Fig. 4a), and quercetin strongly inhibits sPLA2 activity. The max- imum velocity after 20 min for sPLA2 was 0.330 ± 0.04 vo/mol whereas for sPLA2:Q was 0.196 ± 0.02 vo/mol, showing a signif- icant decrease of 40%. To analyze possible correlations between the enzymatic activity of sPLA2 and its antibacterial activity, bacte- rial viability was tested by CFU counting. The assay was performed Fig. 2. UV/vis absorption and circular dichroism (CD) spectra. (a) Absorption spec- trum of sPLA2 and sPLA2 after treatment with quercetin (sPLA2:Q) at wavelength intervals 200–300 nm. The proteins were analyzed at 280 nm. (b) CD spectra of native sPLA2 and sPLA2:Q. Data over the range 185–280 nm are shown. The CD spectra are expressed in theta machine units in millidegrees. C.A. Cotrim et al. / Chemico-Biologica Fig. 3. Intrinsic fluorescence and mass spectrometry. (a) Intrinsic fluorescence of n m o c 3 a t t e d m 3 s f 1 n c o 3 s s i C 4. Discussion and conclusions In this study, sPLA2 from C. durissus terrificus was modified by quercetin, a flavonoid known for its anti-inflammatory activ- ative sPLA2 and sPLA2:Q was measured with excitation at 280 nm and emission onitoring between 300 and 450 nm. (b) MALDI-TOF mass spectrometry analysis f native sPLA2 and sPLA2:Q shows a difference between the molecular masses orresponding to one molecule of bound quercetin. .6. Paw edema assay Since flavonoids have shown a good capacity to inhibit sPLA2 nd consequently decrease its pro-inflammatory activity [13,25], he effect of quercetin on sPLA2 was evaluated. Following subplan- ar injections on Swiss mice, sPLA2 had a huge potential to induce dema after 60 min (Fig. 5a). Under the same experimental con- itions, sPLA2:Q did not show a decrease in edema effect, but the aximum edema was observed after 30 min. .7. Neurotoxic effect assay When the neurotoxic activity was evaluated, both sPLA2 and PLA2:Q induced neuromuscular blockage, but sPLA2 induced a aster effect at 80 min, whereas sPLA2:Q had a similar behavior at 00 min (Fig. 5b), suggesting that quercetin changed the velocity of ative sPLA2 binding to the neurotoxic site of chick biventer mus- le. However, this change is not able to abolish the neurotoxic effect f sPLA2. .8. Platelet aggregation studies In order to evaluate the anti-coagulant potential induced by PLA2 were performed platelet aggregation assays. The results have hown that sPLA2 from C. durissus terrificus has a moderate abil- ty to cause aggregation of washed platelets. While sPLA2 from . durissus cascavella induces about 85% aggregation at 3 �g/�L l Interactions 189 (2011) 9–16 13 [26], sPLA2 from C. durissus terrificus induced 70% aggregation at a concentration of 20 �g/�L. Treatment with quercetin decrease this effect, with 32% aggregation induced by sPLA2:Q at the same concentration (Fig. 5c). 3.9. Myotoxic activity The ability of sPLA2 to cause myonecrosis was also evaluated through measurement of released creatine kinase. Fig. 5d shows that sPLA2 induces an increase in plasma creatine kinase levels of 821.39 ± 107.8 U/L, indicating its ability to cause muscle dam- age. Treatment with quercetin significantly decreased the creatine kinase levels measured to 492.28 ± 71.5 U/L, a 40% decrease. 3.10. Molecular modeling (docking) In order to analyze possible intermolecular interactions between sPLA2 and quercetin, we performed in silico studies using molecular modeling (docking). The best docking solution for the quercetin ligand is shown in Fig. 6a. The GOLD score for this result was 43.94, showing good affinity for the target. The presence of several important intermolecular interactions such as (i) hydrogen bonds with residues Cys45 and His48 at 2.69 and 3.00 Å, respec- tively; (ii) hydrogen bond with water molecule 188 at 3.16 Å; (iii) one polar contact with Ca2+ ion at 3.19 Å; and (iv) hydrophobic interactions with residue Phe5 can account for the stability of the quercetin–sPLA2 complex (Fig. 6a and b). Fig. 4. Enzymatic and antibacterial assays. (a) Enzymatic activity was analyzed using 4N3OBA as substrate and monitored at wavelength 425 nm. sPLA2:Q shows a sig- nificant decrease compared to native sPLA2. (b) Effect of native sPLA2 and sPLA2:Q against Gram-positive bacteria. Quercetin decreases the ability of sPLA2 to inhibit Gram-positive bacteria. Error bars indicate SEM *P < 0.05 compared to saline control. 14 C.A. Cotrim et al. / Chemico-Biologica Fig. 5. Pharmacological assays. (a) Paw edema induced after the injection of sPLA2 and sPLA2:Q (25 �g/paw) into the right paw of Swiss mice. Measurements were done after 30, 60, 120, 180 and 240 min, with no differences observed after treat- ment with quercetin. (b) Neurotoxic effect of sPLA2 and sPLA2:Q on chick biventer cervicis muscle. Results were expressed as the percentage change in twitch tension. (c) Percent platelet aggregation caused by native sPLA2 and sPLA2:Q. The washed platelets assay was performed with venous blood collected from healthy volun- teers and the concentration of both proteins was 15 �g/mL (d) Myonecrosis was assayed based on the creatine kinase levels in Swiss mice. Twenty-five micrograms o m b i p a [ t c t q n t b e a results have showed that some pharmacological activities such as inflammatory and neurotoxicity were not inhibited after treatment with quercetin. This result corroborates the proposal by Ohno et al. [32], who suggested the existence of a distinct pharmacological site f native sPLA2, sPLA2:Q, and pure quercetin were injected into the gastrocnemius uscle. Results were expressed as units of enzymatic activity per liter (U/L). Error ars indicate the SEM. *P < 0.05 compared to sPLA2 activity. ties. Treatment with quercetin resulted in modification of the rotein secondary structure as observed in circular dichroism ssay (Fig. 2b). Similar results were observed by Iglesias et al. 12] with sPLA2 from C. durissus cascavella after modification with he flavonoid morin. Although a secondary structure modification ould be observed, the results do not allow concluding about ter- iary structure modification, once the fluorescence spectrum of uercetin has shown that this compound has a fluorescent peak ear the region of the tryptophan, suggesting that the increase in he total fluorescence of the protein might be caused due to the onding of quercetin. Treatment with quercetin led to a decrease of about 40% in sPLA2 nzymatic activity, similar to p-bromophenacyl bromide (p-BPB), PLA2 inhibitor. Docking studies suggest that quercetin binds in l Interactions 189 (2011) 9–16 the vicinity of the His48 residue, leading to inhibition of enzymatic activity. As shown by Verheij et al. [23], and Scott and Sigler [24], these amino acids are fundamental to enzymatic activity; His48 is involved in the first step of the enzymatic mechanism, and Asp49 is important for binding to Ca2+, a cofactor. It seems that quercetin bound to sPLA2 interferes with the binding between the protein and its substrate. Soares et al. [27] suggested that enzymatic activity is not required for antibacterial activity after chemical modification with BPB and EDTA in a C. durissus terrificus sPLA2 isoform, and the same was observed by Diz Filho et al. [28] for Crotalus durissus ruruima. However, our results show correlation between the enzy- matic and antibacterial activities against Gram-positive bacteria. Treatment of sPLA2 with quercetin decreased both these activities. According to Buckland and Wilton [29], mammalian PLA2 phos- pholipid hydrolysis is correlated with antibacterial activity because both are calcium dependent. We believe this isoform behaves as a mammalian PLA2 in this respect. Several studies have investigated the anti-inflammatory effects of flavonoids as well as their effect on both catalytic and phar- macological activities of PLA2 [8,11,12,30,31]. According to these models, quercetin molecules would interact with the enzymatic site thus affecting enzymatic and pharmacological activities. Our Fig. 6. Molecular docking. (a) Best docking solution for quercetin (stick model) and important interacting residues of the Crotoxin B target, besides Ca2+ ion and water oxygen. (b) Detailed view, with hydrogen bonds (distances in Å) shown as lines not involving the Ca2+ ion. Hydrogen atoms were omitted for clarity and the lines linked to the Ca2+ ion represent the coordination polyhedron. logica n A o t d w p t t a i b m i t m a o q i s m t t s s m f i n l o q q s f c c a a u C A m C ( P R [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ C.A. Cotrim et al. / Chemico-Bio ear the C-terminal region of PLA2 away from the catalytic site. fter modifications with 7-hydroxycoumarin, Toyama et al. [31] bserved that decreasing the enzymatic activity had not suppressed he pharmacological effect of PLA2, suggesting the presence of a istinct site. Although the inflammatory and neurotoxic effects ere unchanged after treatment with quercetin, the myotoxic and latelet aggregation effects were decreased by 40% and 55%, respec- ively, indicating dependency on the enzymatic activity. We believe hat perhaps both the myotoxic and the platelet aggregation sites re located in a second pharmacological site near the calcium bind- ng loop as proposed by Valentin and Lambeau [33]. Thus, after inding to the protein near that region, quercetin affects this site ore directly leading to considerable changes in these activities. Molecular docking has been used during the last decades due to ts importance in elucidating, through computational approaches, he best matches for protein–ligand interactions [34]. In this study, olecular docking was an important tool in evaluating the inter- ctions between sPLA2 and quercetin. Our result agrees with that bserved by mass spectrometry, showing that one molecule of uercetin is binding to the protein. Additionally, molecular dock- ng was useful in elucidating some important interactions between PLA2 and quercetin, which allowed better understanding of the olecular reasons for the effects of quercetin as well as other struc- urally similar flavonoids that could be studied in the future. The results shown here led us to conclude that quercetin binding o the protein decreased the catalytic activity of sPLA2 from C. duris- us terrificus. This sPLA2 probably has two distinct pharmacological ites: one responsible for effects such as platelet aggregation, yotoxicity, and antibacterial activities; and another responsible or inflammatory and neurotoxic effects. We believe the first one s located near the calcium-binding loop region and consequently ear the catalytic site where quercetin binds to the protein, thus eading to a loss of activities dependent on this site. The second ne, we believe is located near C-terminal region, away from where uercetin binds, and is not directly or indirectly affected by the uercetin binding to the catalytic site. Other flavonoids with very imilar chemical structures have a potential to interact with sPLA2 rom serpents. Further docking studies with new molecules of this lass could be useful in predicting how potent these compounds ould be as sPLA2 inhibitors. Although our study clarifies some spects of the chemical and pharmacological interactions between flavonoid and a sPLA2, further studies are necessary to better nderstand the action of flavonoids on sPLA2 activities. onflict of interest statement The authors have no conflict of interest to disclose. cknowledgements The authors are grateful to the Conselho Nacional de Desenvolvi- ento Científico e Tecnológico (CNPq) Proc. No: 133151/2009-3, oordenadoria de Aperfeiçoamento de Pessoal de Nível Superior CAPES), and Fundação de Amparo à Pesquisa do Estado de São aulo (FAPESP) for financial support. eferences [1] R. Kini, Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes, Toxicon 42 (8) (2003) 827–840. [2] T. Nevalainen, G. Graham, K. Scott, Antibacterial actions of secreted phospho- lipases A2. Review, Biochim. Biophys. Acta 1781 (1–2) (2008) 1–9. [3] J. Burke, E. Dennis, Phospholipase A2 biochemistry, Cardiovasc. Drugs Ther. 23 (1) (2009) 49–59. [4] J. Evangelista, A. Martins, N. Nascimento, C. Sousa, R. Alves, D. Toyama, M. Toyama, J. Evangelista, D. Menezes, M. Fonteles, M. Moraes, H. Monteiro, Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom, Toxicon 52 (7) (2008) 737–744. [ [ l Interactions 189 (2011) 9–16 15 [5] A. Soares, J. Giglio, Chemical modifications of phospholipases A2 from snake venoms: effects on catalytic and pharmacological properties, Toxicon 42 (8) (2003) 855–868. [6] R. Kini, Y. Chan, Accelerated evolution and molecular surface of venom phos- pholipase A2 enzymes, J. Mol. Evol. 48 (2) (1999) 125–132. [7] S. Chakraborti, Phospholipase A(2) isoforms: a perspective, Cell Signal. 15 (7) (2003) 637–665. [8] J. Lättig, M. Böhl, P. Fischer, S. Tischer, C. Tietböhl, M. Menschikowski, H. Gutzeit, P. Metz, M. Pisabarro, Mechanism of inhibition of human secretory phospholi- pase A2 by flavonoids: rationale for lead design, J. Comput. Aided Mol. Des. 21 (8) (2007) 473–483. [9] G. Di Carlo, N. Mascolo, A. Izzo, F. Capasso, Flavonoids: old and new aspects of a class of natural therapeutic drugs, Life Sci. 65 (4) (1999) 337–353. 10] B. Gil, M. Sanz, M. Terencio, R. Gunasegaran, M. Paya, M. Alcaraz, Morelloflavone, a novel biflavonoid inhibitor of human secretory phospholipase A(2) with anti- inflammatory activity, Biochem. Pharmacol. 53 (1997) 733. 11] M. Lindahl, C. Tagesson, Flavonoids as phospholipase A2 inhibitors: impor- tance of their structure for selective inhibition of group II phospholipase A2, Inflammation 21 (3) (1997) 347–356. 12] C. Iglesias, R. Aparicio, L. Rodrigues-Simioni, E. Camargo, E. Antunes, S. Marangoni, D. de Oliveira Toyama, L. Beriam, H. Monteiro, M. Toyama, Effects of morin on snake venom phospholipase A2 (PLA2), Toxicon 46 (7) (2005) 751–758. 13] M. Lindahl, C. Tagesson, Selective inhibition of group II phospholipase A2 by quercetin, Inflammation 17 (5) (1993) 573–582. 14] M. Toyama, D. de Oliveira, L. Beriam, J. Novello, L. Rodrigues-Simioni, S. Marangoni, Structural, enzymatic and biological properties of new PLA(2) isoform from Crotalus durissus terrificus venom, Toxicon 41 (8) (2003) 1033–1038. 15] H Zhao, L. Tang, X. Wang, Y. Zhou, Z. Lin, Structure of a snake venom phos- pholipase A2 modified by p-bromo-phenacyl-bromide, Toxicon 36 (6) (1998) 875–886. 16] U. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (5259) (1970) 680–685. 17] W. Lee, M. Toyama, A. Soares, J. Giglio, S. Marangoni, I. Polikarpov, Crys- tallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai, Acta Crystallogr. D: Biol. Crystallogr. 55 (Pt. 6) (1999) 1229–1230. 18] T. Santi-Gadelha, B. Rocha, C. Oliveira, K. Aragão, E. Marinho, C. Gadelha, M. Toyama, V. Pinto, C. Nagano, P. Delatorre, J. Martins, F. Galvani, A. Sampaio, H. Debray, B. Cavada, Purification of a PHA-like chitin-binding protein from Acacia farnesiana seeds: a time-dependent oligomerization protein, Appl. Biochem. Biotechnol. 150 (1) (2008) 97–111. 19] M. Toyama, D.O. Toyama, L. Passero, M. Laurenti, C. Corbett, T. Tomokane, F. Fonseca, E. Antunes, P. Joazeiro, L. Beriam, M. Martins, H. Monteiro, M. Fonte- les, Isolation of a new l-amino acid oxidase from Crotalus durissus cascavella venom, Toxicon 47 (1) (2006) 47–57. 20] B. Ginsborg, J. Warriner, The isolated chick biventer cervicis nerve-muscle preparation, Br. J. Pharmacol. Chemother. 15 (1960) 410–411. 21] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, Development and use of quantum mechanical molecular models. 76. AM1: a new general pur- pose quantum mechanical molecular model, J. Am. Chem. Soc. 107 (13) (1985) 8. 22] G. Jones, P. Willett, R. Glen, A. Leach, R. Taylor, Development and validation of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (3) (1997) 727– 748. 23] H. Verheij, J. Volwerk, E. Jansen, W. Puyk, B. Dijkstra, J. Drenth, G. de Haas, Methylation of histidine-48 in pancreatic phospholipase A2. Role of histi- dine and calcium ion in the catalytic mechanism, Biochemistry 19 (4) (1980) 743–750. 24] D. Scott, P. Sigler, Structure and catalytic mechanism of secretory phospholi- pases A2, Adv. Protein Chem. 45 (1994) 53–88. 25] B. Gil, M. Sanz, M. Terencio, M. Ferrándiz, G. Bustos, M. Payá, R. Gunasegaran, M. Alcaraz, Effects of flavonoids on Naja naja and human recombinant synovial phospholipases A2 and inflammatory responses in mice, Life Sci. 54 (20) (1994) PL333–338. 26] F. Fonseca, E. Antunes, R. Morganti, H. Monteiro, A. Martins, D. Toyama, S. Marangoni, M. Toyama, Characterization of a new platelet aggregating fac- tor from crotoxin Crotalus durissus cascavella venom, Protein J. 25 (3) (2006) 183–192. 27] A. Soares, A. Mancin, A. Cecchini, E. Arantes, S. França, J. Gutiérrez, J. Giglio, Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities, Int. J. Biochem. Cell Biol. 33 (9) (2001) 877– 888. 28] E. Diz Filho, S. Marangoni, D. Toyama, F. Fagundes, S. Oliveira, F. Fonseca, A. Calgarotto, P. Joazeiro, M. Toyama, Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima, Toxicon 53 (1) (2009) 104–114. 29] A. Buckland, D. Wilton, The antibacterial properties of secreted phospholipases A(2), Biochim. Biophys. Acta 1488 (1–2) (2000) 71–82. 30] H. Kim, H. Pham, V. Ziboh, Flavonoids differentially inhibit guinea pig epidermal cytosolic phospholipase A2, Prostaglandins Leukot. Essent. Fatty Acids 65 (5–6) (2001) 281–286. 31] D. Toyama, S. Marangoni, E. Diz-Filho, S. Oliveira, M. Toyama, Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edemato- 1 logica [ 6 C.A. Cotrim et al. / Chemico-Bio genic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom, Toxicon 53 (4) (2009) 417– 426. 32] M. Ohno, T. Chijiwa, N. Oda-Ueda, T. Ogawa, S. Hattori, Molecular evolution of myotoxic phospholipases A2 from snake venom, Toxicon 42 (8) (2003) 841–854. [ [ l Interactions 189 (2011) 9–16 33] E. Valentin, G. Lambeau, Increasing molecular diversity of secreted phospho- lipases A(2) and their receptors and binding proteins, Biochim. Biophys. Acta 1488 (1–2) (2000) 59–70. 34] I. Halperin, B. Ma, H. Wolfson, R. Nussinov, Principles of docking: an overview of search algorithms and a guide to scoring functions, Proteins 47 (4) (2002) 409–443. Quercetin as an inhibitor of snake venom secretory phospholipase A2 Introduction Materials and methods Venom, animals and reagents Purification of sPLA2 Incubation of sPLA2 with quercetin and purification of modified sPLA2 Electrophoresis Circular dichroism spectroscopy Intrinsic fluorescence. Mass spectrometry Measurement of sPLA2 activity Antibacterial activity Paw edema assay Neurotoxic effect assay Platelet aggregation studies Myotoxic activity Molecular modeling (docking) Statistical analyses Results Purification of sPLA2 and Incubation of sPLA2 with quercetin Circular dichroism spectroscopy Intrinsic fluorescence Mass spectrometry Measurement of sPLA2 activity and antibacterial activity Paw edema assay Neurotoxic effect assay Platelet aggregation studies Myotoxic activity Molecular modeling (docking) Discussion and conclusions Conflict of interest statement Acknowledgements References