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Chapter 7| Activity Based Serine Hydrolase Profiling Reveals Broad

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Chapter 7| Activity Based Serine Hydrolase Profiling Reveals Broad Synthetic studies on kinase inihbitors and cyclic peptides : strategies towards new antibiotics Tuin, A.W. Citation Tuin, A. W. (2008, December 16). Synthetic studies on kinase inihbitors and cyclic peptides : strategies towards new antibiotics. Retrieved from https://hdl.handle.net/1887/13365 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral thesis in the License: Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/13365 Note: To cite this publication please use the final published version (if applicable). Chapter7| ActivityBased Serine Hydrolase Profiling Reveals Broad Reactivity of OAlkyl Methylphosphonofluoridates INTRODUCTION Activitybased profiling of enzymes has emerged as a powerful and broadly applicable techniquetoreportonenzymeactivitiesincomplexbiologicalsamples,andtorevealthe inhibitorypotentialandselectivityofnewlydesignedinhibitorsaimedat(asubsetof)the enzymes.1Thegenerallayoutofanactivitybasedprobeisasfollows.Arecognitionelement directstheprobetotheselectedenzyme,orenzymefamily,andspecificitycanbegeared suchthateitherasingleenzymeorabroadfamilyofenzymesistargeted.Thesearethen covalentlyandirreversiblyboundthroughreactionofnucleophilicfunctionalgroupsresiding within the active site with an electrophilic trap that matches the reactivity of the nucleophile. 127 Chapter7 In cases where an amino acid side chain is involved in catalysis, active site labelling is relatively easy and a variety of electrophilic traps aimed at serine hydrolases,2 cysteine hydrolases3 and threonine hydrolases4 have been described. Some enzyme families (metalloproteases,5 phosphatases6) however employ water as the nucleophile and here covalent modification is aimed for by installation of a photoreactive group within the activitybasedprobe.Thethirdgeneralelementthatdistinguishesanactivitybasedprobeis anidentificationand/orenrichmentelement,normallybiotinorafluorophore.Hereactivity basedprobescomeintwoflavours.Inthefirstthebiotinorfluorophoreisattacheddirectly totheprobe,2whereasinthesecond,forreasonsofcellpermeabilityorenzymerecognition, a2steplabelingapproachisappliedwhereafunctionalisedfluorophoreorbiotinismadeto react with the covalently and irreversibly modified enzymes by means of a bioorthogonal ligationreaction.7 Thegeneraloutlineofactivitybasedprobesasdescribedabovedictatesthattheseare mostsuitedforthestudyofenzymesthatemployanucleophilicaminoacidsidechainwithin their active sites. This holds true for the serine dependent enzymes, a family that encompasses such diverse activities as esterases, serine proteases and ester synthetases.8 Indeed,serinehydrolasesareidealtargetsforactivitybasedprofilingandoneoftheseminal studies defining the field of activitybased enzyme profiling is the development and application, by Cravatt and coworkers, of the biotinylated organophosphorus compound, FPbiotin(1,Figure1).2,9Ourinterestinserinehydrolaseenzymefamilyliesintheirreactivity towards small molecule Oalkyl methylphosphonofluoridates such as the illreputed nerve agentsarin2.10 Figure1;StructureofCravatt’scarbonmodifiedfluorophosphonate1,sarin2 andoxygenmodified methylfluorophoshonate3 128 ActivityBasedSerineHydrolaseProfiling Thetoxicityof2andrelatedcompoundstothemammaliannervoussystemisascribedto theirhighreactivitytowardsacetylcholinesterase(AChE).11However,recentreportsreveala broaderreactivityofthistypeofcompoundstowardsotherserinedependentenzymes.12We heredescribeourresultsintheactivitybasedprofilingmediatedestablishmentofarangeof sarinreactive serinedependent enzymes in liver and long tissue. FPbiotin 1wouldbe suitableforthispurposebutwefeelthatthedesignof3isbettersuitedasamimickofsarin 2. Both 2and3 contain a methyl substituent directly attached to the phosphorous atom whichhasbeenprovenbeneficialinbindingtoAChE.13 Werevealthebroadreactivityof3toarangeofserinedependentenzymesandshow that labelling of eight specific activities can be abolished by the addition of 2 in a competition experiment. Finally, we demonstrate that the enzymatic activity of butyrylcholinesterase(BuChE),14whichiscurrentlyunderinvestigationinseverallaboratories asabioscavengeragainstorganophosphorusbasednervepoisoning,15canberestoredinan experimentinwhichtheenzymeisfirstactivitytaggedwith3,thenliberatedbytreatment with an oxime16 or fluoride containing reactivationcocktail.17 Together with biotin streptavidinpulldown,thistechnologycouldimprovecurrentisolation/purificationmethods ofBuChE.18 RESULTSANDDISCUSSION The synthesis of fluorophosphonate 3 is depicted in Scheme 1 and commences with a standardthreestepconversionof11bromoundecan1ol4intoNboc11aminoundecan1 ol5.Treatmentofthelattercompoundwithdipnitrophenylmethylphosphonate19andDBU gave methylphosphonate 6 in good overall yield. At this stage the pnitrophenol in 6 was substitutedbyfluorineusingtetrabutylammoniumtriphenyldifluorosilicate(TBAT)asthedry fluorinesource,givingfluorophosphonate7in70%yield.EnsuingacidicNdeprotectionand treatment with biotinOSu20 provided the target compound 3 in a near quantitative yield overthelasttwosteps.Inthisfashionweroutinelypreparedactivitybasedprobe3ona20 mgscale. 129 Chapter7 Scheme1;Reagentsandconditions.(i)a:2eq.NaN3,DMF80°C,16h.; b: PMe3,THF/H2O;c:Boc2O,THF,0°C, 73%overthreesteps.(ii)1.5eq.dipnitrophenylmethylphosphonate,1.0eq.DBU,DCM,1h.65%.(iii)3.7eq. TBAT,THF,90%.(iv)50%TFA/DCM,30min.(v)0.9eq.biotinOSu,3.0eq.DiPEA,DMF,99%. Forthelabellingandcompetitionstudiesweselectedthreerelevantbiologicalsamples, namely, rhesus monkey liver homogenate, human A549 lung cell lysates and intact A549 cells(Figure2ACrespectively).Thesesampleswereincubatedwithprobe3at10M,either intheabsenceorinthepresence(120M)ofsarin2.Afterstreptavidinpulldownofthe thus biotinylated proteins, these were resolved by SDSPAGE, the protein bands were excised from the gel and trypsin digested after which the resulting oligopeptides were identified by MALDITOF mass spectral analysis. The images of the gel slabs of the three tissue labelling experiments are depicted in Figure 2 and represent in each case labelling without(left)andinthepresenceof(right)sarin2. Boththemonkeyandhumantissuesgaveadistinctlabellingpattern(lanes1,3and5)for probe 3 and, whereas lysate labelling (lane 3) gives more bands than that of the correspondinglivingcells(lane5),thebandsvisualizedinlane5arealsopresentinlane3. Thecombinedlabelledproteinsconstitute(seeTable1)awidearrayofdifferentenzymes, butthecommondenominatoris,asexpected,thattheyallcontainaserineresidueintheir activesite.Infact,thelabellingprofileandtherangeofidentifiedproteinscompareswellto theliteraturereports2,9ontheactivitybasedprofilingexperimentsusingFPbiotin1andits derivativesandweconcludethatmethylfluorophosphonate3iswellsuitedforitsintended purpose,thatistheestablishmentofthereactivityprofileofthenerveagentsarin2.The three samples reveal some differences in the serinedependent enzymes of which the 130 ActivityBasedSerineHydrolaseProfiling activitybasedlabellingwith3isabolished(seelanes2,4and6)uponadditionofsarin2.In themonkeyliverhomogenate,fiveenzymesareinhibitedbysarininacompetitivemanner, fourofwhich(indicatedbyarrowsinFigure2)couldbeidentifiedbyexcisionofthebands followedbyMALDITOFmassspectralanalysis.Thefouridentifiedenzymesareacylpeptide hydrolase, liver carboxyl esterase, esterase D (or Sformyl glutathione hydrolase) and PAF acetylhydrolase (see lane 2). From the experiments involving the A549, lysate six sarin reactiveproteinsarecompetedinthisfashion.Fiveoftheseenzymescouldbeidentifiedand are carboxylesterase, PAF acylhydrolase, fatty acid synthase, dipeptidyl peptidase 9 and prolyloligopeptidase(seelane4).TheexperimentsusingA549tissuerevealfourenzymesas sarin targets (lane 6) that could be identified. In the intact A549 lung cell, the fatty acid synthaseandthedipeptidylpeptidase9arenotfound,buttheenzymelongchaincoenzyme Athioesteraseis.Intotalwehaveidentifiedeightenzymesthataresensitivetowardssarin, allbelongingtotheserinehydrolasesuperfamily.Likely,moreorganophosphorusreactive proteins will be identified in this fashion when one starts from a different tissue. Of all identified proteins, literature data exists that denote them to be reactive towards organophosphorouscompoundsingeneral,whichcorroboratesthevalidityofouractivity basedserinehydrolaseprofilingapproach.21Thus,althoughweareawareofthehighdoses ofsarin2usedinourexperimentswefeelitissafetoconcludefromourexperimentsthat thereactivityofthisnerveagentismuchbroader. 131 Chapter7 Proteinscovalently Liver Intact A549 LysedA549cells boundtosarinbiotin homogenate cells Fattyacidsynthase(1) + +,# + CarbamoylphosphatesynthetaseI + R R Pyruvatecarboxylase + + + Acylpeptidehydrolase(2) +,# R R Dipeptidylpeptidase9(3) R +,# + Prolyloligopeptidase(4)* + +,# +,# Carboxylesterase(5) +,# +,# +,# Serinehydroxylmethyltransferase + R R LongchainacylcoenzymeAthioesterase(6) + R +,# KIAA13263=arylacetamidedeactylaselike1 R + R Aldehydedehydrogenase R + R PAFacetylhydrolase1b(7)** +,# +,# +,# Actin + + R Alcohol dehydrogenase 1B/ fumarylacetoacetate + R R hydrolase ProstaglandinFsynthase R + + Aldoketoreductasefamily1,memberC1isoform2 + R R EsteraseD/Sformylglutathionehydrolase(8) +,# R R LysophospholipaseIandII R R + GlutathioneSRTransferase + + R Table1;Proteinsfromrhesusmonkeyliver,humanA549cellsorlysedhumanA549cellsthatwerebiotinylated uponexposureto10Mofprobe3for1h.Proteinswereidentifiedbymassspectrometryfrombandsexcised fromonedimensionalSDSPAGEgels.Proteinsthatarespecifictargetsforsarinarebold,andthenumbers(in
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