Inhibition of Myotoxic Activity of Bothrops asper Myotoxin II by the Anti-trypanosomal Drug Suramin

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<ul><li><p>Inhibition of Myotoxic Activita</p><p>rus23</p><p>3Instituto Clodomiro Picado</p><p>San Jose, Costa Rica</p><p>4Center for Applied Toxinology</p><p>potrevs iv</p><p>tsibi</p><p>phenyl rings to the secondary amide backbone, the symmetrical suramin</p><p>imhergs</p><p>therapeutic agents developed and is used clinically species.15,16</p><p>structural motif or scaffold. Snake venom PLA2sexhibit a wide spectrum of activities including</p><p>PLA2s, phospholipases A2; suramin, 8,8 -[carbonylbis</p><p>doi:10.1016/j.jmb.2005.04.072[imino-3,1-phenylenecarbonylimino (4-methyl-3,1-phe-nylene) carbonylimino]] bis-1,3,5-naphtalenetrisulfonicin the treatment of African trypanosomiasis andonchocerciasis.14 The spectrum of suramin appli-cations now includes the clinical treatment ofangiogenesis, carcinomas of the kidney and pros-tate gland, breast cancer, inhibition of growthfactor/receptor interactions, inhibition of human</p><p>Phospholipases A2 (PLA2s: EC 3.1.1.4) are keyenzymes in the control, regulation, production andrelease of lipid mediator precursors that serve asmessengers in fundamental, highly regulated pro-cesses such as growth, adhesion, apoptosis,secretion, hemostasis and immune regulation. Thegroup II PLA2s encountered in viperid snakevenoms and mammalian fluids share significantsequence homology, and are based on a single</p><p>17Abbreviations used: Basp-II, Bothrops asper myotoxin II;</p><p>0Instituto Butantan, Sao PauloSP, Brazil</p><p>*Corresponding author</p><p>Introduction</p><p>Suramin (8,8 0-[carbonylbis [carbonylimino (4-methyl-3,1-pmino]] bis-1,3,5-naphtalenhexasodium salt), a highly chacompound, is one of the first0022-2836/$ - see front matter q 2005 E</p><p>acid hexasodium salt; CK, creatineethylene glycol; i-face, interfacial reextensor digitorum longus; PSS, phsolution; Hepes, 4-(2-hydroxyethyl)sulfonic acid; i.m., intramuscular.E-mail address of the correspond</p><p>arni@df.ibilce.unesp.brhydrophobic surfaces of the dimer and adopts a novel conformation thatlacks C2 symmetry in the dimeric crystal structure of the suraminBothropsasper myotoxin II complex. The simultaneous binding of suramin at thesurfaces of the two monomers partially restricts access to the nominalactive sites and significantly changes the overall charge of the interfacialrecognition face of the protein, resulting in the inhibition of myotoxicity.</p><p>q 2005 Elsevier Ltd. All rights reserved.</p><p>Keywords: suramin; heparin; myotoxicity; inhibition; crystal structure</p><p>ino-3,1-phenylene-enylene) carbonyli-trisulfonic acided polysulfonateduccessful synthetic</p><p>a-thrombin, protein-tyrosine phosphatases, G pro-teins, merozoite surface protein-1, and acidic andbasic fibroblast growth factors.514 Suramin pre-vents the development of muscle necrosis inducedby some snake venoms, since it inhibits themyotoxic and in vitro neuromuscular blockingactivities of Lys49 phospholipases A2 from BothropsFacultad de MicrobiologiaUniversidad de Costa Rica</p><p>molecule binds by an induced-fit mechanism complementing theMyotoxin II by the Anti-tryp</p><p>Mario T. Murakami1, Emerson Z. ArAna B. Martinez2, Sabrina Calil-EliaBruno Lomonte3, Jose M. Gutierrez</p><p>1Departament of PhysicsIBILCE/UNESP, Sao Jose do RioPreto, SP, Brazil</p><p>2Departamento de FarmacologiaBasica e Clinica, IBC/UFRJRio de Janeiro, RJ, Brazil</p><p>Suramin, a synthetictreatment of Africanfor the treatment of sand other polyanionA2 analogues both inas therapeutic agenconformational flexlsevier Ltd. All rights reserve</p><p>kinase; PEG, poly-cognition face; EDL,ysiological saline-1-piperazineethane-</p><p>ing author:y of Bothrops aspernosomal Drug Suramin</p><p>da2, Paulo A. Melo2</p><p>, Marcelo A. Tomaz2</p><p>and Raghuvir K. Arni1,4*</p><p>lysulfonated compound, developed initially for theypanosomiasis and onchocerciasis, is currently usederal medically relevant disorders. Suramin, heparin,nhibit the myotoxic activity of Lys49 phospholipaseitro and in vivo, and are thus of potential importancein the treatment of viperid snake bites. Due to itslity around the single bonds that link the central</p><p>J. Mol. Biol. (2005) 350, 416426myotoxic, neurotoxic, cardiotoxic, anticoagulant,platelet-aggregating, hypotensive and inflammatoryeffects.18 On the basis of sequence and structuralsimilarities, the snake venom group II PLA2s can besubdivided into (a) the catalytically active Asp49enzymes and (b) the basic, myotoxic Lys49 proteins,which possess no catalytic activity.17 Myotoxicity</p><p>d.</p></li><li><p>affects only muscle fibers without damaging othertissue structures such as connective tissue, nervesand vessels,19 and can lead to permanent tissue loss,disability, amputation and death.20</p><p>Suramin binds to a surprisingly wide variety ofproteins of different function, structure and size dueto its torsional flexibility and ability to stretch orcompress itself, which results in the two naphthylrings with the sulfonate groups being able tointeract simultaneously with a number of structu-rally distinct sites. Due to a lack of structuralinformation, the target sites, conformational flexi-bility and mode of binding of suramin to proteinsare still unclear at the atomic level.The crystal structure of the complex between</p><p>suramin and Bothrops asper myotoxin II representsthe first crystal structure of suramin bound to a</p><p>As illustrated in Figure 1(a), B. asper myotoxin IIcaused a time-dependent increase in the rate of</p><p>digitorum longus muscles. A more pronouncedeffect is observed after 90 minutes with 25 mg/ml ofB. asper myotoxin II alone. The presence of suramin</p><p>In vivo myotoxicity</p><p>Intramuscular (i.m.) injections of Basp-II aloneincreased plasma creatine kinase (CK) activity in adose and time-dependent fashion. Figure 1(b)indicates that an i.m. injection of Basp-II (2.0 mg/g) induced a significant increase in plasma CKactivity compared to physiological saline solution.CK levels increased from 65(G42) units/l (meanGSEM, nZ4) to 1368(G386) units/l (nZ4) after twohours. These values of plasma CK activity are inagreement with previously reported observations.21</p><p>Preincubation with suramin significantly (p!0.05)inhibits the increase in plasma CK activity inducedby Basp-II. Experiments performed following pro-tocol B (see Materials and Methods) indicate thatsuramin also significantly inhibits (p!0.05) the</p><p>Inhibition of B. asper Mytotoxin II by Suramin 417(10 mM) together with the toxin markedly inhibitsthe creatine kinase release induced by the B. aspermyotoxin II (Basp-II). After 90 minutes of exposureto the toxin, in the presence of suramin, the creatinekinase release was less than 15% of the valuemeasured in the absence of suramin.creatine kinase release from isolated extensorprotein. The structural details presented hereshould be of relevance in understanding the stericrequirements of suramin binding to proteins, andshould shed light on the mechanism of myotoxicitydisplayed by Lys49 phospholipases A2 and theirinhibition by polyanionic compounds.</p><p>Results</p><p>In vitro myotoxicityplasma CK activity increment induced by Basp-II.However, under these conditions, suramin was lesseffective in antagonizing myotoxicity than whenincubated with Basp-II prior to injection. Figure 2presents light micrographs of extensor digitorumlongus muscle exposed to either Basp-II (Figure 2(b)and (b 0)), Basp-II pre-incubated with suramin(1 mg/kg) (Figure 2(c) and (c 0)) or post-treatedwith suramin (1 mg/kg) 15 minutes after Basp-IIinjection. In all samples, normal muscle cells wereobserved in the central region of the muscle,whereas the peripheral fibers were in differentstages of necrosis, characterized by denselyclumped myofibrils and swollen cells. Pre-incu-bation or post-treatment with suramin resulted in areduction in the number of necrotic fibers(Figure 2(c) and (d)).</p><p>Crystal structure</p><p>Data collection and processing statistics arepresented in Table 1. Molecular replacement usinga single molecule of native Basp-II (PDB ID, 1CLP)as the search model resulted in clear rotation andtranslation solutions for the two molecules in theasymmetric unit, with a correlation coefficient of</p><p>Figure 1. In vitro and in vivoinhibition of myotoxicity of Basp-IIby suramin. (a) Experiments per-formed on isolated EDL mousemuscle exposed to Basp-II (25 mg/ml) alone or with 10 mM suramin.The toxinwas applied at time zero inthe absence (filled circles) or in thepresence of suramin (filled squares).(b)Theeffect of i.m. injectionofBasp-II alone (2.0 mg/g), pre-incubatedwith suramin (1.0 mg/kg), or post-treated with the same dose ofsuramin (1.0 mg/kg; intraperitonealroute), 15 minutes after the venom</p><p>i.m. injection. In (a) and (b), thedata represent the mean valuesGSEM (nZ4).</p></li><li><p>41875.8% and an R-factor of 30.5% after rigid-bodyrefinement. After four cycles of refinement andmodel building, the electron density peaks above2s in the Fourier difference maps were examined</p><p>Figure 2. Light micrographs of cross-sections of mouse ED(a) and (a 0) Panoramic and close-up views of the controlstructure, respectively. (b) and (b 0) Intense degeneration ofthe lesion induced by the Basp-II 24 hours after injectionmyofibrils (arrow) are indicated. (c) A section of muscle frsuramin (1 mg/kg). (d) Tissue from the group treated withThere are normal muscle cells in the central region (star) aSwollen cells (arrowhead) and clumped myofibrils (arrow)(a), (b) and (c) 200 mm; (a 0), (b 0) and (c 0) 50 mm.Inhibition of B. asper Mytotoxin II by Suraminand the additional electron density present in thevicinity of the calcium-binding loop was identifiedas belonging to the trisulphonate naphthalene ringof the suramin molecule. At all stages of the</p><p>L muscle 24 hours after the injection of PSS or Basp-II.muscle showing normal muscle cells (star) and musclethe peripheral muscle cells (asterisk), and the details of, respectively. Swollen cells (arrowhead) and clumpedom the group that received Basp-II pre-incubated withsuramin (1 mg/kg) 15 minutes after injection of Basp-II.nd an inflammatory reaction at the periphery (asterisk).are shown in detail in (c 0) and (d 0). Scale bars represent:</p></li><li><p>Table 1. Data collection, refinement statistics andhydrogen bond distances between Basp-II and suramin(atom numbering scheme based on the deposited atomiccoordinates 1Y4L)</p><p>Inhibition of B. asper Mytotoxin II by Suraminrefinement, the electron density maps indicated thepresence of a bound polyethylene glycol (PEG)molecule in both the monomers in the hydrophobicchannels that leads to the active sites. As in thecrystal structure of the highly homologous Bothrop-stoxin-I, a Lys49 phospholipase A2 (PLA2) from the</p><p>Basp-IICsuramin</p><p>A. Crystal preparationCryoprotectant solution Mother liquorC25% glycerolSoaking time 30 secondsB. Data collectionWavelength (A) 1.437Temperature (K) 100Detector MARCCDSynchrotron radiation source CPr beamline/LNLS-BrazilSpace group P212121Unit cell parametersa (A) 49.20b (A) 64.04c (A) 85.99Resolution (A) 30.01.70 (1.741.70)No. molecules in the asym-metric unit</p><p>2</p><p>Solvent content (%, v/v) 56VM (A</p><p>3 DaK1) 2.8No. reflections 253,143No. unique reflectionsa 15,224 (907)I/s(I) 26.0 (2.8)Multiplicity 6.5 (5.5)Completeness (%) 99.3 (97.4)Rmerge</p><p>b (%) 4.9 (50.7)C. Structure refinement statisticsRfactor (%) 20.6Rfree (%) 24.0rmsd from idealBond distances (A) 0.012Bond angles (deg.) 1.663Average B-factors (A2) 26.0Ramachandran plot analysisMost favoured regions (%) 91.5Additional allowed regions(%)</p><p>7.5</p><p>Generously allowed regions(%)</p><p>1.0</p><p>Hydrogen bondsProtein SuraminResidue Atom Atom Distance (A)Arg34 (A) N O29 2.97Arg34 (A) N3 O30 2.53Arg34 (A) NH2 O30 3.33Lys53 (A) Nz O28 3.00Lys53 (A) Nz O30 3.15Arg34 (B) NH1 O82 3.06Lys53 (B) Nz O80 2.64Lys53 (B) Nz O82 3.34Lys69 (B) Nz O54 2.75</p><p>Statistical values for the highest-resolution shells are given inparentheses.</p><p>a Multiplicities of the derivative data sets were calculated withthe Friedel-related reflections treated separately. Multiplicity ofthe native data set was calculated with the Friedel-pairs treatedas equivalent.</p><p>b RmergeZSjIhIK fIhgj=SfIhg, where Ih is the observedintensity of the ith measurement of reflection h and {I(h)} is themean intensity of reflection h calculated after scaling.venom of Bothrops jararacussu, the hydroxyl groupsof PEG form hydrogen bonds toHis48Nd1 (Figure 3)and the extended tails of the bound PEG moleculesoccupy the positions of the fatty acids (Figure 4(a))of the phospholipid analogue (PDB ID, 1POE).22</p><p>The refinement converged to a crystallographicresidual of 20.5% (RfreeZ24.0%) for all data between30.0 A and 1.70 A (Table 1). The final model consistsof the 242 amino acid residues, 170 solventmolecules, five isopropanol molecules, two frag-ments of PEG 3350 and a suramin molecule. Thecrystal structure is characterized by excellentstereochemistry, as indicated by an analysis of thedeviations from ideal values of the bond lengths,bond angles, planarity and non-bonded contacts(Table 1). The two molecules in the asymmetric unitare related by a 2-fold axis of rotation, and thehydrophobic surfaces surrounding the entrance tothe active sites form the dimer interface, resulting inthe burial of 3446 A2 of the surface area of eachmolecule (30.9%).The structure of group IIA PLA2s has been</p><p>reviewed extensively,17,23 and consists of threea-helices, a short b-wing and connecting loops(Figure 3). The principal, highly conserved struc-tural feature is a platform formed by the two longanti-parallel disulfide-linked (Cys29-Cys45 andCys51-Cys98) a-helices (helices 2 and 3, residues3754 and 90109) on which are located the aminoacid residues considered important for catalyticactivity (His48, Asp49, Tyr52, Tyr73 and Asp99).The interfacial recognition surface contains a widehydrophobic collar and provides access to thecatalytic site. In the case of catalytically activeAsp49 PLA2s, His48 and Asp99 together with astructurally conserved water molecule (hydrogenbonded to His48 Nd1) participate in the nucleophilicattack at the sn-2 position of the phospholipidsubstrate. The tetrahedral transition state inter-mediate is stabilized by a calcium ion and iscoordinated by Asp49 and the main-chain atomslocated in the calcium-binding loop. In the sub-group of Lys49 PLA2 analogues, substitution ofAsp49 by Lys results in the Nz atom of Lys49occupying the position of the calcium ion in Asp49PLA2s.</p><p>17,24 These Lys49 PLA2s are capable ofbinding fatty acids,25 stearic acid26 and PEG. Thecontroversy as to whether these enzymes possesscatalytic activity2729 has been clarified, since nosubstrate hydrolysis was detected with the wild-type recombinant protein.30</p><p>Suramin-binding site</p><p>NMR experiments indicate that the confor-mational flexibility at a3/a3 0 and a4/a4 0 and theC2 symmetry enable suramin to adopt multipleconformations wherein the two sulfonatednaphthyl rings are separated by distancesbetween 16 A and 30 A in the compact and fully</p><p>9</p><p>419extended conformations, respectively (Figure 5).In this structure, suramin adopts a novel confor-mation that lacks C2 symmetry, with an overall</p></li><li><p>Figure 3. Ribbon representationof the Basp-II dimer, suramin andPEG (atom colors) bound to His48.Figures 3 and 4 were generatedusing PYMOL (http://www.</p><p>420 Inhibition of B. asper Mytotoxin II by Sur...</p></li></ul>