Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor

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Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor
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  Acta Tropica 114 (2010) 97–102 Contents lists available at ScienceDirect ActaTropica  journal homepage: www.elsevier.com/locate/actatropica Crystal structure of   Schistosoma  purine nucleoside phosphorylase complexedwith a novel monocyclic inhibitor Humberto M. Pereira a , b , ∗ , Valério Berdini c , Mariana R. Ferri b , Anne Cleasby c , Richard C. Garratt a a Instituto de Física de São Carlos, Universidade de São Paulo, Brazil b Centro Universitário Central Paulista, UNICEP, Brazil c  Astex Therapeutics, Cambridge, UK  a r t i c l e i n f o  Article history: Received 12 February 2009Received in revised form18 December 2009Accepted 1 January 2010 Available online 1 February 2010 Keywords: SchistosomiasisPurine nucleoside phosphorylaseVirtual screeningCrystal structureInhibitors a b s t r a c t A novel inhibitor of   Schistosoma  PNP was identified using an “in silico” approach allied to enzyme inhi-bition assays. The compound has a monocyclic structure which has not been previously described forPNP inhibitors. The crystallographic structure of the complex was determined and used to elucidatethe binding mode within the active site. Furthermore, the predicted pose was very similar to thatdetermined crystallographically, validating the methodology. The compound Sm VS1, despite its lowmolecular weight, possesses an IC 50  of 1.3  M, surprisingly low when compared with purine analogues.ThisispresumablyduetotheformationofeighthydrogenbondswithkeyresiduesintheactivesiteE203,N245 and T244. The results of this study highlight the importance of the use of multiple conformationsfor the target during virtual screening. Indeed the Sm VS1 compound was only identified after flippingthe N245 side chain. It is expected that the structure will be of use in the development of new highlyactive non-purine based compounds against the  Schistosoma  enzyme. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Schistosomiasis is one of the world’s most neglected diseases(Hotez et al., 2006) and is the collective name for infection by one of five  Schistosoma  species (TDR, 2005), of which  Schistosoma man-soni ispredominantinAmericaandAfrica.In2005theWorldHealthOrganizationestimatedthat600millionpeopleworldwidewereatrisk of schistosomiasis, and close to 200 million people are actu-allyinfectedcontinuouslyorintermittently(TDR,2005).Morbidity due to  S .  mansoni  includes hepatosplenomegaly, liver fibrosis andascites, and as many as 130,000 individuals die per year from hae-matemesis due to related portal hypertension (Van Der Werf etal., 2003). The disease is associated with a chronic and debilitat-ing morbidity manifested by its consequences including cognitiveimpairment, lassitude, and growth stunting (Abdulla et al., 2007).In the absence of a vaccine, efficient vector control and water san-itation, the treatment and control of schistosomiasis rely heavilyon a single drug, praziquantel (PZQ). This drug is active against allschistosome species, and over 25 years of use has established thedrug as safe and effective (Caffrey, 2007). The main problem with PZQisthatitispracticallyinactiveagainstimmatureschistosomes, ∗ Correspondingauthorat:IFSC-USP,AvTrabalhadorSaocarlense400,Centro,CP369,13560-970SãoCarlos-SP,Brazil.Tel.:+551533739868;fax:+551633739881. E-mail address:  hmuniz.pereira@gmail.com (H.M. Pereira). itsfullactivitybeingdisplayedonly6–8weeksafterinfection(TDR,2005).Furthermore,treatmentfailureisoftenthefirstindicativeof theendoftheeffectivelifeofadrug(Haganetal.,2004).Inthecase ofpraziquantelseveralreportsoftreatmentfailureorthedevelop-mentofresistantstrainsofschistosomeshavebeenreported(Silvaet al., 2005; Wolfe, 2003; Lawn et al., 2003; Danso-Appiah and DeVlas, 2002; Caffrey, 2007; Fallon and Doenhoff, 1994; Ismail et al.,1994;Doenhoffetal.,2002).Clearlyitwouldbestrategicallyunwiseto depend on a single agent for sustainable control of schistoso-miasis. Ideally, a range of treatment options should be available,preferably including the use of different drug classes (Hagan et al.,2004). The World Health Organization in their Special ProgrammeFor Research and Training in Tropical Diseases reports that severalmillion PZQ doses are administered every year and the number isgoing to rise in the immediate future, increasing the risk of thedevelopment of resistance. It should therefore be apparent that itis now time to start looking for new alternatives for the treatmentof schistosomiasis (TDR, 2005).It has been demonstrated that  S .  mansoni  is unable to syn-thesize purine nucleotides  de novo , and that Schistosomes havemultiple mechanisms for incorporating preformed purine basesandnucleotidesintothepurinenucleotidepool(Doveyetal.,1984;Senft and Crabtree, 1977; Senft et al., 1972, 1973). Together withnew data from the  Schistosoma  genome projects for two species(  japonicum  and  mansoni ) (Hu et al., 2003; Verjovski-Almeida etal., 2003) this suggests that the purine salvage pathway may bea potential target for novel anti-Schistosomal agents. 0001-706X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2010.01.010  98  H.M. Pereira et al. / Acta Tropica 114 (2010) 97–102 Purinenucleosidephosphorylase(EC2.4.2.1)isacentralenzymeof this pathway and catalyzes the cleavage of the glycosidic bondin purine ribo and deoxyribonucleosides, in the presence of inor-ganic orthophosphate (Pi) as a second substrate, generating thecorresponding purine base and ribose(deoxyribose) 1-phosphate.In the last two decades there has been considerable interest innew approaches to drug discovery that offer improvements to theprocess of identifying new therapeutic agents. Technologies suchas high-throughput screening (HTS) and combinatorial chemistryallow for the testing of large libraries of compounds against a pro-tein target (Hartshorn et al., 2005). Furthermore, recent advances in drug discovery have enabled a dramatic increase in the numberof synthetic molecules and natural products that are available fortesting  in vitro  in biochemical and cellular assays (Mcinnes, 2007).Virtualscreening(VS),thatistheuseofcomputationaltoolstoiden-tify a reduced number of molecules with increased potential forbioactivity to be evaluated experimentally, has emerged both as acomplementaryandalternativemethodtoHTS(Alvarez,2004).The developmentofvirtualscreeningmethodsextendsthepossibilitiestostudiesofmoleculesthatdonotnecessarilyexistphysicallyinaninvestigator’scollectionbutwhichcanbereadilyobtainedthroughpurchase or synthesis. The other obvious advantage is that, as aresult of computational prediction of binding affinity, only a rel-atively small subset of compounds requires testing and thereforeactivities can be quantified in low or medium throughput assayformat (Mcinnes, 2007).Among the latest developments in drug discovery is a conceptcalled fragment based design, or fragment-based screening (FBS).IncontrasttoconventionalHTS,wherefullybuilt,“drugsize”chem-ical compounds are screened for activity, FBS identifies very smallchemical sub-structures or fragments (usually 100–250Da) thatmay only exhibit weak binding affinity ( Jahnke and Daniel, 2006),but which may be more readily identified in a physical chemical(crystallographic) screen rather than a biological one.Here we describe the virtual screening of a small library of lowmolecular weight compounds using the three-dimensional struc-ture of   S .  mansoni  PNP. Despite the relatively small size of boththe library and its component compounds, three completely novelcompounds with inhibitor activity were readily identified. One of these compounds has a monocyclic structure of a type never pre-viously described as a PNP inhibitor, and its structure in complexwith SmPNP is reported here. 2. Materials and methods  2.1. SmPNP virtual screening  Using the SmPNP structure in the form of its complex withNDSB-195 and acetate (PDB code 1TCV) (Pereira et al., 2005) a vir- tual screen was performed in order to identify compounds thatmay be potential binders. For this purpose the coordinates corre-spondingtotheNSDB-195andacetateligandswereremovedfromthe structure file and the key residues in the SmPNP active sitewereidentifiedandassignedforuseinthevirtualscreening.Theseincludedallresidueswhichinteractdirectlywithoneoftheligands:G34,S35,H66,R86,H844,Y90,A118,A119,Y202,E203,M221,S222,T244, N245, S247 and H259. The residue E203 was used in its pro-tonatedstateandrotamerswereconsideredforN245inwhichtheN  1  and O  2  atoms were exchanged by flipping   2 . The maximummolecularweightofthecompoundsusedinthesearchwas280Da,which are part of a proprietary ASTEX database including 30,000compounds. The program GOLD ( Jones et al., 1997) was used for virtual screening employing the GOLD virtual screening parame-ters and goldscore as a scoring function. The best scored solutionswereinspectedmanuallyand22compoundsthatinteractedfavor-ably with key residues in the active site were selected for activityassays.  2.2. SmPNP activity assays Activity assays for the compounds identified during VSemployed the coupled xanthine oxidase method of Kalckar(Kalckar, 1947). This method consists of the measurement of the hypoxanthinereleasedduringphosphorolysisofinosineordeoxyi-nosineindirectlybymonitoringtheuricacidproducedbyxanthineoxidase on its oxidation. The reaction mixture (final volume 1mL)contained 50mM potassium phosphate buffer pH 7.0, 100  L of DMSO (in which the potential inhibitors identified during virtualscreening were dissolved at a concentration of 100  M), 100  Minosineand0.02unitsofxanthineoxidase.Thereactionwasstartedby addition of 550ng of SmPNP to the reaction mixture and theOD 293  was monitored for 2min.The active compounds in the assay were subjected to IC 50 determination. In this assay an automated method using theBIOMEK3000 workstation was employed. The method used wasbased on that of  Erion et al. (1997), which itself is derived from the method of Kalckar but employs 2-(4-iodophenyl)-3-(4-nitro-phenyl)-5-phenyltetrazoliumchloride(INT).TheadditionofINTtothe coupled assay resulted in the formation of the highly coloredformazan product which absorbs within the range 490–500nm(Erion et al., 1997). For the IC 50  determinations, the assay mix-ture(200  L)contained1mMINT,20mUxanthineoxidase,10  Minosine, 100mM Tris–HCl buffer pH 7.0 and 50mM potassiumphosphatepH7.0anddifferentconcentrationsoftheVScompound.For Sm VS1 the concentrations used were 25.00, 12.50, 6.25, 3.12,1.60, 0.80 and 0.40  M. The reaction was started with the addi-tion of SmPNP ( ∼ 500ng) and monitored in the microplate readerat 500nm for up to 2min. For calculation of the IC 50  value, a non-linearfour-parameterfitwasusedaccordingtothesigmoidal I  maxmodel using the Sigma Plot Software.  2.3. Crystallization and soaking of SmPNP  The purification and crystallization protocols used were aspreviously described (Pereira et al., 2003, 2005). The soaking parameters for SmPNP crystals consisted of 20% PEG 1500, 10%DMSO (in which the compound was dissolved at a concentrationof 5mM), 15mM sodium acetate buffer pH 4.9 or 5.0 (at the pH of crystalgrowth)and20%glycerol.Thissolutionisalsoacryoprotec-tantallowingfortherapidfreezingofthecrystalsdirectlyfromthesoakingsolutions.Thecrystalscouldbemaintainedinthissolutionfor 96h before freezing.  2.4. X-ray data collection X-ray diffraction data were collected at 100K to a resolutionof 1.9Å from an SmPNP crystal after soaking with the compoundSm VS1. Measurements were made on Station PX14.2 of the SRS(Daresbury-UK)using  =0.980Å,over1 ◦ incrementsin ϕ foratotalrotationof65 ◦ .Thedatawereindexedandintegratedusingthepro-gram MOSFLM (Leslie, 1999) and scaled using the program SCALA from the CCP4 suite (1994).  2.5. Structure solution and refinement  Crystals of the Sm VS1 complex were isomorphous with otherPNPcomplexesandthereforethestructurecouldbesolvedbyrigidbodyrefinementusingSmPNPincomplexwithacetate(1TD1)asaninitial model using the Refmac program (Murshudov et al., 1997).Full refinement was carried out using both Refmac (Murshudovet al., 1997) and Phenix (Adams et al., 2002) and the model  H.M. Pereira et al. / Acta Tropica 114 (2010) 97–102 99 Fig. 1.  Structures of the active compounds identified during virtual screening. (A) Sm VS1, (B) Sm VS2 and (C) Sm VS3. building performed with COOT (Emsley and Cowtan, 2004), using   a -weighted2Fo-FcandFo-Fcelectrondensitymaps.Atotalof763water molecules were included using both COOT and Phenix andthe Sm VS1 and a sulphate ion were added using the Find Ligandroutine of COOT.In all cases, the behaviour of   R  and  R Free  was used as theprincipalcriteriaforvalidatingtherefinementprotocolanditscon-vergence. The stereochemical quality of the model was evaluatedwith Procheck (Laskowski et al., 1993). The coordinates and struc- turefactorshavebeendepositedwiththePDBunderthefollowingcode: 3E0Q. 3. Results and discussion  3.1. Virtual screening  As described previously (Pereira et al., 2007), a database of  30,000 compounds with molecular weight below 280Da, wasused in the virtual screening (VS). Compounds with the highestscores (about 100) were visually inspected and 22 compoundswere selected on the basis of their drug-like properties and poten-tial favourable interactions with the key residues of the activesite. We only describe here the compounds that demonstratedinhibitory activity against SmPNP. The compound Sm VS1 (6-amino-5-bromo-1,2,3,4-tetrahydropyrimidine-2,4-dione) shownin Fig. 1, has a monocyclic structure and its preferred pose forms six hydrogen bonds with residues of the active site,all within the base binding pocket. Compounds Sm VS2 (N-[4-(2,4-dimethylphenyl)-1,3-thiazol-2-yl]guanidine) and Sm VS3(N-[4-(4-methylphenyl)-1,3-thiazol-2-yl]guanidine) have similarstructures, differing only by an additional methyl substitution inSm VS2 (Fig. 1). The predicted binding mode is also very similar in both cases. However, in Sm VS2 the dimethylphenyl group pos-sesses a different orientation in the ribose binding site due to thepresence of the second methyl group in comparison with Sm VS3.The guanidine group of both compounds interacts directly withE203 and N245 of the base binding site. In the case of Sm VS2,two hydrogen bonds are formed with both E203 and N245, andthe dimethylphenyl group is accommodated within the ribosebinding site, the most hydrophobic part of the PNP active site.Compound Sm VS3, despite its similarity with Sm VS2, makesonly one hydrogen bond with E203 whilst its methylphenyl groupalso occupies the ribose binding site, making hydrophobic con-tacts.  3.2. Activity assays and IC  50  determination Twenty-two of the selected compounds were used in a singlepoint inhibition assay. Two of the compounds, being insoluble inDMSO, were not tested. The assay served to discriminate false hitsfromtrueligandsandasafilterforchoosingcompoundsforsubse-quent soaking experiments and IC 50  determinations. Three of the20remainingcompoundsshowedinhibitoractivityagainstSmPNPand one of them, Sm VS1, inhibited completely the enzyme at aconcentrationof100  M.Sm VS2andSm VS3,whicharequitesim-ilar (differing only in the presence of a methyl group as describedabove), inhibited 79.5% and 70.6% respectively. These three com-pounds were selected for IC 50  determination. Compound Sm VS1possessesanIC 50 of1.30 ± 0.07  M(aresultwhichissurprisinglowforamonocycliccompound)andisabletoformH-bondswithbothbase and ribose binding site. In comparison, the  K  M  value for thesubstrate inosine is about 7  M (Pereira et al., 2003). Sm VS2 and Sm VS3, when submitted to the IC 50  determination proved to bepromiscuous inhibitors due to protein precipitation and we havesofarbeenunabletoobtaincrystalstructuresforthesecomplexes.It should be pointed out that purine analogues have been usedpreviously in the treatment of Schistomiasis, including tubercidinwhichataconcentrationof100  Minducestheseparationofmaleand female adult worms (Dovey et al., 1985; El Kouni, 1991; ElKouni et al., 1987). Furthermore immucillins have shown greatpromise for the treatment of parasitic diseases such as malaria,having K  i  valueswithinthenanomolartopicomolarrange(Evansetal., 2003; Schramm, 2002; Fedorov et al., 2001; Clinch et al., 2009).However,theenzymefrom Plasmodium belongstothehighmolec-ular weight class of PNPs which are significantly different to thatfromthehumanhost,facilitatingthedevelopmentofparasitespe-cificinhibitors.Thereis,therefore,stillaneedforfindingalternativelead compounds particularly for parasites such as Schistosomeswhich,liketherehosts,possesslowmolecularweightPNPs.Sm VS1therefore appeared to merit further attention.Monocyclic compounds have yet to be described as efficientPNPsinhibitors,andtheidentificationofSm VS1opensuppossibil-itiesforthedesignofmorepowerfulcompoundsbasedinthisnovelnon-purine sub-structure that could be useful in the treatment of schistosomiasis.  3.3. Structure description The SmPNP structure in complex with Sm VS1 is similar to oth-ers previously described (Pereira et al., 2005). The RMSd for all C  s between the Sm VS1 complex structure and that with acetate(1TD1) was only 0.18Å. Some residues could not be located in theelectron density and therefore were removed from the final struc-ture(subunitAresidues1–4,63–65;subunitBresidues1–3,62–65,253–267;subunitCresidues1–2).WhencomparedwithSmPNPincomplex with hypoxanthine (PDB code 3FNQ) the RMSd is 0.52Åand there are no remarkable differences between the structuresfrom the point of view of the polypeptide chain. The similar bind-ing mode suggests that Sm VS1 may act as a competitive inhibitorwith respect to natural substrates.After rigid body refinement it was possible to identify elec-tron density corresponding to the Sm VS1 molecule in all threeactive sites and the structure was placed into this density usingthe program Coot. The Fo-Fc electron density map showing boththe Sm VS1 molecule and the base binding site of SmPNP can beseen in Fig. 2.The final structure has 6315 protein atoms, 584 watermolecules, 1 molecule of Sm VS1 and 1 molecule of DMSO persubunit. The final  R  and  R Free  values are 18.1% and 24.2% respec-tively, and 91.1% of the residues are in the most favoured regionsof the Ramachandran plot. The full data collection and processingstatistics are given in Table 1.  100  H.M. Pereira et al. / Acta Tropica 114 (2010) 97–102 Fig. 2.  Fo-Fc electron density omit map (contoured at 3   ) for the Sm VS1 bound tosubunit C of SmPNP. Hydrogen bonds between the ligand and active site residuesandwatermoleculesareshown.IntotalSm VS1makeseightdirectcontactswithinthe active site explaining its affinity for SmPNP.  3.4. The active site description The active site of the binary complex contains one moleculeof Sm VS1 and one water molecule. Despite its low molecularweight ( ∼ 206Da) difference density for the Sm VS1 compoundwas clearly visible indicating the ligand form a large number of H-bond interactions with active site residues, all situated withinthe base binding site. In total, Sm VS1 makes eight H-bonds withkey residues (four with E203, three with N245 and one with T244)as well as two H-bonds with water molecules (Fig. 2). When com- pared with the unliganded structure, the base binding site showsslight differences mainly involving N245, whose C   and C   atomsare shifted by 0.6Å and 0.8Å respectively. This is the result of   Table 1 Full data collection and refinement statistics.Full data collection and refinement statisticsSpace group  P  2 1 2 1 2 1 Cell dimensions (Å)  a =49.85;  b =120.012;  c  =131.40Detector ADSC Quantum 4X-ray source SRS PX14.2Wavelength (Å) 0.980Resolution range (Å) 44.46–1.9 (2.0–1.9)Redundancy 2.6 (2.3)Rmeas (%) 8.8 (56.3)Rsym (%) 4.9 (34.1)Completeness (%) 94.8 (92.9)Total reflections 146321Unique reflections 56943 I  /   I   9.7 (2.0)Refinement parameters R  (%) 18.1 R Free (%) 24.2Ramachandran plotMost favoured region (%) 91.1No. of residues in disallowed regions 3Overall B-factor (Å 2 ) 26.0Ligand B-factor (Å 2 ) 18.34No. of protein atoms 6315No. of water molecules 763No. of ligand atoms 42r.m.s. bond lengths (Å) 0.006r.m.s. bond angles (Å) 0.780 Fig. 3.  Superposition of the base binding sites of both liganded (white) and unli-ganded structures (green). The binding of Sm VS1 caused small rearrangements tothe base binding site, principally to N245, which included the flipping of its sidechain enabling the formation of three hydrogen bonds with Sm VS1. (For interpre-tation of the references to color in this figure legend, the reader is referred to theweb version of the article.) an altered orientation (Fig. 3), which includes flipping of the side chain, a movement which allows N245 to interact tightly withSm VS1.When all three subunits have been superposed it is possible toperformacomparisonofthebindingmodeofSm VS1intheactivesites.InsubunitsAandC,Sm VS1bindssimilarly.However,insub-unit B the ligand binds more deeply within the active site beingshiftedinthedirectionofN245,andforthisreasonthesidechainsof T244andN245moveslightlyincomparisonwithsubunitA(0.42Åand0.54Å).Nevertheless,thesameH-bondingnetworkisretainedin all cases and we have no specific explanation for the differencesseen in subunit B. This is consistent with our (and others) obser-vation of a high degree of flexibility and conformational variabilitywithin the PNP active site.Whenthevirtualscreeningandcrystallographicposesarecom-pared,itcanbeseenthattheGOLDprogramhasfaithfullypredictedthe binding mode of Sm VS1 (Fig. 4). The RMSd for all heavy atoms between the pose and crystallographic structure is 1.06Å and theposecorrectlypredictssixoutoftheeightexperimentallyobservedH-bonds to active site residues.In fact two virtual screens were performed, the first one withthe srcinal structure derived from the acetate complex and thesecondwiththesidechainofN245flipped.Onlyinthesecondcaseis Sm VS1 a high scoring hit presenting the expected pose in thebase binding site. This emphasizes the importance of target flex-ibility at all levels (side chains, main chains, domain movement,etc.) which is clearly a critical limitation for the success of virtualscreening.Onepossiblesolutionistheuseofmultiplestructuresinthe virtual screening process. This was possible in the present caseduetotheknownflexibilityoftheactivesite.Thismaybelesseasyin other cases where little may be known about target flexibility.However it emphasizes the need for the use of multiple structureswherever possible. Indeed, one of the practical advantages of theincreased rate at which protein structures can now be obtained(andthepotentialofhighthroughputmethods)isthepossibilitytobetter sample energetically accessible conformations via multiplestructuredetermination.Indeed,theuseofmultiplestructureshas  H.M. Pereira et al. / Acta Tropica 114 (2010) 97–102 101 Fig.4.  Comparisonofthevirtualscreeningpose(green)andthecrystallographicallydetermined structure (white). The program GOLD correctly predicted the bindingmode of Sm VS1 in the active site (RMSd between the two positions was 1.08Å).This difference is probably due to the small rearrangements observed in the activesite of the crystal structure which seem to be necessary in order to accommodatethe ligand. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of the article.) Fig. 5.  Comparison of the mode of binding of hypoxanthine (green) and Sm VS1(white).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderis referred to the web version of the article.) beenusedinthedesignoffocusedfragmentlibraries(Hartshornetal., 2005, Howard et al., 2006). ComparisonwithSmPNPincomplexwithhypoxanthine(Fig.5)reveals that the latter forms only four hydrogen bonds with activesite residues (one with E203, two with N245 and one with A118)plus three with two water molecules. No significant differencesare observed in the active site residues of the two complexes, andSm VS1 occupies essentially the same volume of space and is co-planar with the hypoxanthine molecule (Fig. 5).The orientation of Sm VS1 in the active site of SmPNP furthershowsthebromineatomtobepointingtowardstheribosebindingsite. It is well known that bromine can be readily substituted byother groups during synthetic chemistry and this could be poten-tiallyexploitedinordertobuildanewgenerationofSm VS1-basedcompounds by “growing” into the ribose binding pocket. It is to beexpected that such larger compounds will have improved affinity.However it is already encouraging that a rapid virtual screeningcoupled to a simple enzyme assay was able to detect a novel non-nucleotide based inhibitor of    M affinity which has the potentialto be developed into a lead compound.  Acknowledgments We acknowledge the grants from FAPESP-Cepid (98/14138-2)and FAPESP (Grants HMP, 99/09304-3 and 06/60280-3). 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