Alzheimer Drug Mechanisms

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Alzheimer Drug Mechanisms

Alzheimer Drug MechanismsNov 18, 2010Pharmaceutical Challenges to Alzheimer’s DiseaseFIP Pharmaceutical Sciences 2010Department of Neuropathology and NeuroscienceGraduate School of Pharmaceutical SciencesThe University of TokyoTaisuke Tomita


millionsIncreasing number ofAlzheimer disease patients115.465.735.6


Pathology of Alzheimer’s disease patients brainSilver staining Electron microscopy ImmunostainingNeurofibrillary tangles: Paired helical filaments consisted ofhyperphosphorylated tauSenile plaques: amyloid deposits consisted of Ab peptides


AmyloidHypothesisADLuminalSenile plaquesAb oligomerPHFSynapticdysfunctionNeuronal lossAbCytosolAPPb-secretaseg-secretase


g-Secretase mediated intramembrane cleavageAbAlzheimer’sdiseasePen-2Aph-1APPPSSignalingCancerNctNICDNotchTakasugi et al.,Nature (2003)


1 st generation g-secretase inhibitor (GSI)OOHNOHOHNONHONH 2FFOOHInhibitionNONof HONotchsignalingL-685,458DAPTFOHNONHONHOOHNONHONFDBZJBC(2003)LY450139Semagacestat


Failure of Phase III trial of semagacestat• Announced on Aug 17, 2010• More than 2,600 patients with mild-to-moderate Alzheimer'sdisease have been enrolled.• Preliminary findings show that not only did semagacestat failto slow disease progression, but that it was actuallyassociated with “worsening of clinical measures of cognitionand the ability to perform activities of daily living”.Furthermore, the incidence of skin cancer was significantlyhigher in the treatment group than the placebo group.Specific inhibition of Ab is mandatory


Notch-sparing GSINH 2OCF 3F N OSNOO NClCF 3OHOCF 3O SN ONH SOSNClClNHFFHNNHONHNNBMS-708163 GSI-953 ELN475516PF-3084014Notch-sparing GSIs preferentially inhibit the Abgeneration compared to the Notch signalingBMS-708163 and GSI-953 enter the clinical phase


g-secretase modulator (GSM)OOHOOHNFCF 3NOCF 3OHNSNNFClFFNFR-Flurbiprofen(Tarenflurbil)GSM-1Page et al., JBC 2008JanssenWO2009/052126Compound 4Kounnas et al., Neuron 2010Selective decrease in toxic Ab42 levels without affectingNotch signaling; “modulation” of proteolytic activityDevelopment of Tarenflurbil was discontinued (2008)


Mechanism-based drug developmentProtein expression/Enzymatic analysisGeneRational design of the compoundsbased on the structure-function relationshipsStructural analysisDrug developmentChemistry


For the rational design of the compounds…HIV protease + inhibitor


Approaching the structure and function ofg-secretase• Compounds used as “bioprobe” to identify thefunctionally-important domain in the g-secretase.– Chemical biology approach using photoaffinity probe– Cysteine chemistry-based structural analysis


Protease inhibitors targetthe functional domain in the enzymeTransition state analoguetargets catalytic siteH2OH2OH2ONon-Transition state analoguetargets catalyticallyimportantsiteH2OAllosteric inhibitortargets structurallyimportantsiteIdentification of the binding site revealsthe “functional domain” in the proteaseH2O


Identification of the molecular target of compoundsParentcompoundPhotoaffinityprobePhotoactivatable groupe.g., BenzophenoneBiotinUVAvidinpull downEnzyme SourceTarget identification


Benzophenone and Biotin moieties for PALFunctionalprotein/domaining-secretaseLigandBenzophenoneBiotinKan et al, Chem Comm 2003


DAP-BpB directly bound to PS1 CTFDAP Bp B


PS1 is major target for GSIsHelical peptide-type GSI→PS1 NTF/CTFTSA-type GSI→PS1 NTF/CTFPen-2Aph-1DAPT↓PS1CTFMorohashi et al.,JBC 2006PresenilinNctCompound E↓PS1NTFFuwa et al., ACSChme Biol 2007


BMS-299897 is a sulfonamide-typeNotch-sparing GSIFOHFOOBarten et al., 2005NSOFClBMS-299897IC 50 for Ab = 7.1 nMIC 50 for NICD = 105.9 nM


Design and synthesis of AS-BpBBenzophenoneBiotinHF14057(Parent compound)AS-BpBBMS-299897-likearylsulfonamide


AS-BpB directly targets PS1 NTF


HF14057HF14057AS-BpB binding was completely abolished byother Notch-sparing inhibitorsNS2034Competitor - 1 10 (mM)FCF 3ClOSOFHNCF 3 SCF 3HNOSOHOSPS1NTFOOClNS2046NS2034WO 2009/131906NS2046BegacestatCompetitor - 1 10 (mM)PS1NTF


HF14057Helical peptide-type GSI failed to abolishedthe labeling of PS1 NTF by AS-BpBAc–Val–Gly–Aib–Val–Val–Ile–Aib–O-benzyl-Thr–Val–Aib–Val–Val–Aib–NH 2D-13D-13Competitor - 0.1 1 10 (mM)PS1NTFPAL and competition assay provide functional information ofthe compounds and the target molecules


Approaching the structure and function ofg-secretase• Compounds used as “bioprobe” to identify thefunctionally-important domain in the g-secretase.– Chemical biology approach using photoaffinity probe‣We can identify the functional role of the subunit.‣We can analyze the mode of actions of novel compounds.– Cysteine chemistry-based structural analysis


Substituted Cysteine Accessibility Method◆Cysteine substituted mutants are biologically functional◆MTSEA-biotin・thiol reactive・hydrophilic・membrane impermeable・no chargeCysSolubilizationPull down byStreptoavidinBeadsCys+ +++-Cys---CysCysCysCysCys


TMD8SCAM analysis of TMD8, 9 and C terminusMTSEA-biotinTMD9-C terminusATIVILAFGCCLY FLTFL VLL L LGA I FFKK A L AP LPIIDF M QQ Q P FHFY V LL A YDTIASITMTSEA-biotin


WTWAL ILAVPICSVYLVALVDGXGD motifDGRLGIGFLKVSAFKAATGV L VY SSAGDWNTTIIVLAFGCCLY FLTFL VLL L LGA I FFKK A L AP LPIPAL motifIResults of SCAMDF M QQ Q P FHFY V LL A YDTIASIT• TMD8 is buried within thelipid bilayer.• Several residues at TMD9and C terminus arehydrophilic.• Consecutive residuesaround PAL motif are wateraccessible.


Mapping of TMD arrangement by crosslinkingTMD6250WTWAL ILVAI SVYLVALVCPDDGRGIGFLKVSAFKAATGV L VY SL~5.2ÅKKSAGDWNLAPTMD9TTDIF450 MAQ Q PC Y V LAV FLD~16.9ÅITL I ALGCY F 446L TFL VL L LL 443GA I F~24.7ÅF435LPINo cross-linkSITAFH QFIYCross-linkerLength (Å)5.2 24.7NTFCTF


L-685,458, a transition-state analogue,targets to catalytic aspartatesMTSEA-biotinWTWAL ILAVRSAKATAGV L VI SIFY SVYD D FL GL-685,458VALLVGCLSubsites that are involved in the recognition of thePKsubstratesshould be occupied by KL-685,458VG


Labeling competition analysis by L-685,458TMD6KWTWAL ILAVPICSVYLVALVDDGRLGIGFLKVSAFKAATGV L VY SKKSFAAGDWNTTIIVLLAFGIAPCCLY FLTFL VLL L LGA I FDF M QQ Q P FHFY V LL A YDTIALPISITTMD9GXGD motifPAL motif


TMD1Mechanistic implications to theintramembrane proteolysis by g-secretaseTMD6TMD7YD/GxGD motifA STMD8TGADSW 404ATMD9T459 462K 406464FGPQ Q394Q 454 455F 456 FYADAY246450 451461IL467250 I387FY 446D L443D385257G G383 L L 432 F440260 A GP TALI435 LKPLMIVHSI437PAL motifMembraneCytosolExtracellular / LuminalLateral gateSato et al., J Neurosci (2006)Sato et al., J Neurosci (2008)Takagi et al., J Neurosci (2010)


Approaching the structure and function ofg-secretase• Compounds used as “bioprobe” to identify thefunctionally-important domain in the g-secretase.– Chemical biology approach using photoaffinity probe‣We can identify the functional role of the subunit.‣We can analyze the mode of actions of novel compounds.– Cysteine chemistry-based structural analysis‣We can reveal the structure of the catalytic subunit.‣We can annotate the binding site of the compounds.‣We can speculate the dynamic structural changes.


Further direction of the development forthe g-secretase-based therapeuticsStructural biologyComputer scienceMolecular biologyCell biologyBiochemistryOrganic chemistryChemical biologyStructural biochemistry


Collaborators• Takeda Pharmaceutical CompanyNobuhiro Suzuki, Asano Asami-Odaka, Hiroaki Fukumoto, Masaomi Miyamoto• School of Pharmaceutical Sciences, University of ShizuokaToshiyuki Kan• Laboratory of Synthetic Organic and Medicinal Chemistry, Faculty of PharmaceuticalSciences, Teikyo UniversityHideaki Natsugari• Laboratory of Biostructural Chemistry, Graduate School of Life Sciences, TohokuUniversityHaruhiko Fuwa, Makoto Sasaki• Department of Synthetic Natural Products Chemistry, Graduate School ofPharmaceutical Sciences, The University of TokyoSatoshi Yokoshima, Tohru Fukuyama• VIB, KU LeuvenBart De Strooper• The University of ChicagoGopal Thinakaran• Division of Cellular Therapy, Advanced Clinical Research Center, Institute of MedicalSciences, The University of TokyoToshio Kitamura• Memorial Sloan-Kettering Cancer CenterYueming Li


2010 members of Tomita groupSupport: KAKENHI, NIBIO, MHLW, MEXT, JST (Target Proteins Research Program, CREST)SfN 101116

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