Formulation | DMSO |
MolecularWeight | 846 |
Storage | -20°C |
Purity | HPLCandTLCanalysis |
Compound | FFPR-CK.1HCl |
Assay | |
ShelfLife(properlystored) | 12months |
ChemicalFormula | C42H42O9N6Cl2 |
ThestructureofbiotinylatedFPRchloromethylketones(BFPRCK)(TOP)andbiotinylatedEGRchloromethylketone(BEGRCK)(BOTTOM)areshown.TheSPACERrepresentacarbon-spacerusedtooptimizethereactivityofthebiotin-groupaftertheprobehasbeenreactedwiththeactivesiteofaserineprotease.
Overview:
Tri-peptidechloromethylketoneshavebeenutilizedextensivelytoirreversIBLyinhibitvariousserineproteases(1-5).AmongthemostcommonchloromethylketonesareFPRCK(Phe-Pro-Arg-chloromethylketone;commonlyreferredtoasPPACK),whichisarapidthrombininhibitorandEGRCK(Glu-Gly-Arg-chloromethylketone;commonlyreferredtoasGGACK),whichisarapidfactorXainhibitor(1).BothFPRCKandEGRCKareusedextensivelyduringproteinisolationprocedurestoinhibitserineproteaseactivityandpreventfurtherconversionofzymogenstoactiveenzymes.Recently,themodificationofthesetri-peptidechloromethylketoneswithreportinggroups,suchasfluorescentprobes(6-8,14),rADIoactivelabels(9)orthioreactive-labels(10),hasprovidedauniqueapproachtothestudyofvariousserineproteases.Theseprobesareusefulbecausetheyallowameansofreportingmolecularchangesinanenzyme,andnotitszymogen,whilealsoinhibitingtheenzymaticactivity.
TheuseofbiotinasareportinggrouphasbeenusedextensivelywithantibodiesinELISAbasedassaysandinwesternblotting.Thebiotin,inconjunctionwithavidin,createsahighlysensitivemethodfordetectingantibodies,andtherefore,antigens.Bymodifyingthetripeptide-chloromethylketoneswithabiotingroup,thesensitivityoftheavidin/biotinsystemcanbeextendedtostudyserineproteaseswithouttheneedforspecificantibodiestotheactiveenzymes.
Biotinylatedtripeptidechloromethylketonescanbeusedinavarietyofways(11-13).First,thecompoundscanbereactedwithunwantedserineproteasesinasampleorpreparation,andcanthenberemovedalongwiththeproteaseusingavidin-Sepharose(11).Second,thebiotinylated-serineproteasecanbevisualizedonablotwithouttheuseofspecificantibodies(11).Third,thebiotinylatedserineproteasecanbequantitatedinanactive-sitespecificimmunoassay(12,13,15),suchasthetPA-CASSIA(seeAssayKits).ThespacerutilizedonthesecompoundshasbeenoptimizedtoallowgoodreactivityofthebiotinylatedFPRCKandthebiotinylatedEGRCKintheabovementionedprocedures.
Inadditiontobiotinylatedchloromethylketones,fluoresceinlabelledcompoundsarealsoavailable.ThefluoresceinlabelledcompoundsareusefulinbothWesternblotandfluorescentimagingapplications.
BiotinylatedandfluoresceinlabelledFPRCKandEGRCKarepreparedbythemethodofWilliamsetal.(11).
Properties:
SpecialProperties | Tri-peptidechloromethylketones(CMK)areverypotentandirreversibleinhibitorsofserineproteases.BFPRCKisespeciallyusefulforinhibitionofthrombinandtPA,whileBEGRCKisusefulforinhibitionoffactorXa.ThebiotinmoietyprovidestheABIlitytousethepeptide-CMKsasspecificprobesfordetectionand/orcaptureofserineproteasesviatheavidin/biotininteraction. |
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SpecialNotes | FPRCKandEGRCKaresuppliedlyophilized,andshouldbestoredat4oC.BiotinylatedCMKsaresuppliedin10mMHClandshouldbestoredfrozedat-20oCorcolder.FluorosceinCMKsaresuppliedinDMSO,andshouldalsobestoredat-20oCorcolder. |
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NaturalProteinStopsDeadlyHumanBrainCancerInMice
ScientistsfromJohnsHopkinsandfromtheUniversityofMilanhaveeffectivelyproventhattheycaninhibitlethalhumanbraincancersinmiceusingaproteinthatselectivelyinducespositivechangesintheactivityofcellsthatbehavelikecancerstemcells.ThereportispublishedinNature.
Themostcommontypeofbraincancer-glioblastoma-ismarkedbythepresenceofthesestem-cell-likebraincells,which,insteadoftriggeringthereplacementofdamagedcells,formcancertissue.Stemcells,unlikeallothercellsinthebody,arecapableofformingalmostanykindofcellwhentheright"signals"triggertheirdevelopment.
Fortheirtreatmentexperiment,theresearchersreliedonaclassofproteins,bonemorphogenicproteins,thatcauseneuralstem-cell-likeclusterstolosetheirstemcellproperties,whichinturnstopstheirABIlitytodivide.
Firsttheypretreatedhumanglioblastomacellswithbonemorphogenicprotein4(BMP4),theninjectedthesetreatedcellsintomousebrains.Inmiceinjectedwithcellsthatwerenotpretreated,large,invasivecancersgrew.InthemicewithBMP4-treatedcells,nocancersgrewatall.Threetofourmonthsafterinjection,allmicethatgotuntreatedcellsdied,andnearlyallmicewithBMP4-treatedcellswerealive.
Next,thescientistsdeliveredslow-releaseBMP4-containing"beads"directlyintomousebrainswithimplantedglioblastomacells.Micethatgotemptybeadsdevelopedlargemalignanttumorsanddied.MicewithBMP4beadssurvivedmuchlonger,and80percentsurvivedfourmonthsaftercancercellimplants.
"OurideaistotreatpatientswithBMP4orsomethinglikeitrightaftersurgerytoremoveglioblastomainhopesofpreventingtheregrowthofthecancerandimprovingsurvivaltime,"saysAlessandroOlivi,M.D.,directoroftheDivisionofNeurosurgicalOncologyatHopkinsandacontributortothestudy.
OlivisaysclinicalstudiesusingBMP4couldbeginwithinayearand,ifsuccessful,drugtherapiescouldbeavailabletothepublicwithinthreetofouryears.
"ThiswasproofoftheideathatBMPscouldstopglioblastomabydepletingthestem-cell-likepopulationthatfeedsit,"saysHenryBrem,M.D.,chairmanoftheDepartmentofNeurosurgeryatHopkinsandacollaboratorinthestudy."Thisopensexcitingdoorstofutureresearchintotreatmentsandtherapiesforsuchadevastatingdisease."