Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation 英文参考文献.docVIP

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Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation 英文参考文献.doc

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Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation 英文参考文献

ScreeningforNovelLRRK2InhibitorsUsingaHigh- ThroughputTR-FRETCellularAssayforLRRK2Ser935 Phosphorylation SpencerB.Hermanson1,CobyB.Carlson1,StevenM.Riddle1,JingZhao2,KurtW.Vogel1 ,R. JeremyNichols2,KunBi1* 1PrimaryandStemCellSystems,LifeTechnologiesCorporation,Madison,Wisconsin,UnitedStatesofAmerica,2TheParkinson’sInstitute,Sunnyvale,California,United StatesofAmerica Abstract Background:Mutationsintheleucine-richrepeatkinase-2(LRRK2)havebeenlinkedtoParkinson’sdisease.Recentstudies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicatingthatthesesitesareprimetargetsforcellularreadoutsofLRRK2inhibition. Methodology/Principal Findings: Using Time-Resolved Fo¨rster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbiumlabeledanti-phospho-Ser935antibodythatgeneratesaTR-FRETsignalbetweenterbiumandGFP.LRRK2wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reducedfortheR1441Cmutantandlittlecouldbedetectedforthekinase-deadmutantD1994A.TheTR-FRETcellularassay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2canreducethephosphorylationlevelatSer935.Todemonstratetheutilityofthisassayforscreening,weprofileda smalllibraryof1120compounds.ThreeknownLRRK2inhibitorswereidentifiedand16hitswerefollowedupintheTR-FRET andacytotoxicityassay.Interestingly,outofthetop16hits,fiveareknowninhibitorsofIkBphosphorylation,twoCHK1and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysisfortheireffectsonthephosphorylationofSer910,Ser935,Ser955andSer973. Conclusions/Signifi