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Evotec Munich

Building innovativedrug discovery alliancesEvotec MunichQuantitative Proteomics to Support theDiscovery & Development of Targeted DrugsEvotec AG, Evotec Munich, June 2013


About Evotec MunichA leader in chemical proteomics and phosphoproteomicsEvotec MunichEvotec’s Center of Excellence for proteomics and oncologyEmerged from Kinaxo Biotechnologies, a Max Planck spin-off foundedby the renowned cancer researcher Prof. Axel UllrichCombines highest service quality standards with powerful technologicalinnovation developed by leading proteomics scientists such as Dr.Henrik Daub, Evotec Munich’s SVP Technology & ScienceCollaborates with leading academic research laboratories including theMatthias Mann lab at the Max Planck InstituteProf. Dr. Axel Ullrich, Max-Planck DirectorHas worked with numerous global pharma and biotechnology companies such asPAGE 1


Evotec MunichTechnology OfferingGlobal Proteome Profiling- Deep Proteome- PhosphoScout ®- AcetylomicsChemical Proteomics- Cellular Target Profiling ®- KinAffinity ®Targeted ProteomicsSRM Assay DevelopmentBiomarker Discoveryunbiased discovery ofpredictive biomarkersPAGE 2


Evotec Munich Proteomics TechnologiesDedicated to support the development of targeted drugsTarget deconvolutionCellular mode-of-actionanalysis to support LODrug repositioningTargetdiscoveryHTscreens /ScaffoldselectionOn/offtargetprofilingMode ofActionanalysisResponsePredictionBiomarkerAssaysTarget ID &validationScreeningH2L LO Preclinic PhI/IICellular Target Profiling tosupport H2LProteomic signatures forbiomarker developmentCellular Target Profiling ® Subproteomic Cellular Target Profiling ®Omics Technologies (Phosphoproteomics, Acetylomics,Proteomics)PAGE3


Evotec Munich TechnologiesOverview of general workflowsSampleProteins or PeptidesSeparationIonizationMass SpectrometerData AnalysisData InterpretationIsotopic Labeling(SILAC, DiMe, mTRAQ, iTRAQ, TMT)Pre-fractionation / Enrichment(Affinity beads, SCX-IMAC/TiO 2 , antibodies,SAX, high pH RP)Liquid Chromatography (LC)(EASY nLC1000, 20-50 cm, 75 µm ID, 1.9 µm beads,column ovens)Mass Spectrometry (MS)(LTQ-Orbitrap Velos, Q-Exactive, TSQ Vantage)Identification/Quantification(MaxQuant)Bioinformatics/Statistics(Spotfire, in-house programs, e.g. SubExtractor)PAGE4


Cellular Target ProfilingSummaryIdentification of small molecule targets in any cell type or tissue of choiceDetermination of a compound‘s proteome-wide binding affinities to discriminatebetween high, medium and low affinity cellular targetsProfiling of a compound against native, endogenously expressed, posttranslationallymodified full length proteins in the presence of cellular co-factorsand native complex partnersExtensive, non-target class restricted track record in target deconvolution andprofiling of various small molecule compounds (e.g. kinase inhibitors, antibiotics,epigenetic drugs, HDM2 inhibitors & small molecules targeting metabolic enzymes,ligases, reductases, transferases, heat shock proteins)PAGE 6


KinAffinity ®Chemical proteomics for kinome analysisTest compoundK D [µM]0,010,1110Target Kinase 1Target Kinase 2Target Kinase 3Target Kinase 4Target Kinase 5Target Kinase 6Target Kinase 7(1) Cell or tissuesample(2) Mixture of beads ofbroadly selective kinaseinhibitors for enrichment ofkinases(3) Competition of targetkinases by test compound(4) LC-MS/MSanalysis(5) List of targetkinases rankedaccording to theirK d valuesLysate of the cell line or tissue of interest is prepared (1). A matrix comprising ofimmobilized broad-spectrum kinase inhibitors is used to enrich the expressed kinomeof the cell or tissue (2). Competition assays with the free test compound (3) andquantitative mass spectrometry analysis (4) reveal the compound’s affinity profile,ranking all protein kinase targets according to their K d values to the free compound (5).Sepharose matrixImmobilised kinase inhibitorTest cpdTarget kinaseTarget kinase bound to test cpdPAGE 7


PhosphoproteomicsMode of action analysis of kinase inhibitorsPhosphoScout ® is a quantitative mass spectrometry basedplatform to monitor dynamic phosphorylation events in vivo on aglobal scale• Allows identification and quantification of phosphorylation sites in livingcells, animal models and patient samplesIdentification of regulatedphosphorylation networks• Currently 10,000 to 15,000 phosphorylation sites can be measured in asingle experiment using Evotec Munich’s PhosphoScout technology• Enables comprehensive investigation of (tumor) signaling pathways andtheir response to drugs• Applications include identification of pharmacodynamic read-outs and(comparative) mode of action analysis of kinase-selective drugsPAGE 8


AcetylomicsMode of action analysis of HDAC and sirtuin inhibitorsIMSIMS/MSAcEnzymaticcleavageAcAcAcm/zm/zG-V-L-P-A-W-RAc PAcMaxQuant Software(Dept. M. Mann, MPI)ProteinsPeptidesLC-MSData AnalysisIsotopiclabelingEnrichmentQuantitative & globallysine acetylation analysisQuantitative and global measurement of 1000+ site-specific lysineacetylations, can be combined with global proteome quantificationHighly optimized workflow with respect to sensitivity and selectivityAcetylomics is an ideal tool for cellular mode of action analyses that gofar beyond the field of chromatin biologyIdentification of regulated acetylation networksPAGE 9


Deep Proteome AnalysisIndustry-leading capabilities in protein abundance profilingQuantitative mass spectrometry-based approach to accurately measure protein abundance levels in vivo ona global scale• Allows large-scale protein identification and quantification in any biological material• 7,000+ distinct proteins can be detected from cell or tissue samples with MS sample pre-fractionation• ~5,000 distinct proteins can be detected from cell or tissue samples in single-shot experiments• Applications include target identification and biomarker discovery in animal or patient samples (tissues and bodyfluids)Deep Proteome ProfilingPPeptidefractionationLC-MS/MSIheavylightheavylightMSm/zData Analysis7000+proteinsPIheavylightheavylightMSm/z~5000proteinsIsotope Labelingor Label-freeExtraction ofProteinsEnzymaticCleavageLC-MS/MSData AnalysisSingle-shot Proteome ProfilingPAGE 10


Biomarker DiscoveryEvotec’s three-tier strategy to support oncology biomarker discoveryPAGE 11


ResponderNon-resp.ResponderNon-resp.Proteome and phosphoproteomebiomarker discoveryComplementary approachesTest sample set of drugresponders and nonresponders(cultured cells, xenografts,patient samples)QuantitativeproteomeprofilingProtein expressionPhosphositesGlobal analysis of cellularprotein levelsTarget class independenceOnly µg protein amounts requiredfor MS analysisEasier transition from discovery tovalidation/clinical analysisQuantitativephosphoproteomeprofilingGlobal analysis of cellularkinase activityDirect linkage with kinase functionOther PTMs possible, such aslysine acetylation for HDACsComprehensive proteome and/or phosphoproteome profiling across responder and non-responder samplesPAGE 12


Key AdvantagesMultivariate protein-basedstratification markersProteomics-based biomarker discoveryBiomarker discovery on functional protein level (instead of mRNA analysis) toensure higher predictivityUnique capabilities in proteome-wide analysis (6000+ proteins) to identifymore predictive biomarkers than by targeted or lower coverage analysisIdentication of multivariate markers with higher predictive power thanunivariate markerClinical response prediction for Flt3inhibitor AC220 in 12 AML patientsPhosphosignature of 5 sites with 92%prediction accuracyValidation with independent AMLpatient samples revealed 7/8 predictionaccuracyPAGE 13


Building innovativedrug discovery alliancesThank you for your attentionYour contact:Dr. Klaus GodlVice President, Proteomics & Chemical Biology+49 89 4524465-12klaus.godl@evotec.com

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