Building innovative drug discovery alliances. Evotec Munich. Quantitative Proteomics to Support the Discovery & Development of Targeted Drugs

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1 Building innovative drug discovery alliances Evotec Munich Quantitative Proteomics to Support the Discovery & Development of Targeted Drugs Evotec AG, Evotec Munich, June 2013

2 About Evotec Munich A leader in chemical proteomics and phosphoproteomics Evotec Munich Evotec s Center of Excellence for proteomics and oncology Emerged from Kinaxo Biotechnologies, a Max Planck spin-off founded by the renowned cancer researcher Prof. Axel Ullrich Combines highest service quality standards with powerful technological innovation developed by leading proteomics scientists such as Dr. Henrik Daub, Evotec Munich s SVP Technology & Science Collaborates with leading academic research laboratories including the Matthias Mann lab at the Max Planck Institute Prof. Dr. Axel Ullrich, Max-Planck Director Has worked with numerous global pharma and biotechnology companies such as PAGE 1

3 Evotec Munich Technology Offering Global Proteome Profiling - Deep Proteome - PhosphoScout - Acetylomics Chemical Proteomics - Cellular Target Profiling - KinAffinity Targeted Proteomics SRM Assay Development Biomarker Discovery unbiased discovery of predictive biomarkers PAGE 2

4 Evotec Munich Proteomics Technologies Dedicated to support the development of targeted drugs Target deconvolution Cellular mode-of-action analysis to support LO Drug repositioning Target discovery HT screens / Scaffold selection On/off target profiling Mode of Action analysis Response Prediction Biomarker Assays Target ID & validation Screening H2L LO Preclinic PhI/II Cellular Target Profiling to support H2L Proteomic signatures for biomarker development Cellular Target Profiling Subproteomic Cellular Target Profiling Omics Technologies (Phosphoproteomics, Acetylomics, Proteomics) PAGE 3

5 Evotec Munich Technologies Overview of general workflows Sample Proteins or Peptides Separation Ionization Mass Spectrometer Data Analysis Data Interpretation Isotopic 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, 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) PAGE 4

6 Cellular Target Profiling Compound Affinity Chromatography combined with quantitative mass spectrometry Test compound K D [µm] Target Protein 1 0,01 Target Protein 2 0,1 1 Target Protein 3 10 (1) Cell or tissue sample (2) Enrichment of target proteins by compound affinity chromatography (3) Competition of target proteins by test compound (4) LC-MS/MS analysis (5) List of target proteins ranked according to their K d values Lysate of the cell line or tissue of interest is prepared (1). A linker derivative of the test compound is immobilized on Sepharose beads and used to enrich compound target proteins (2). Competition assays with the free test compound (3) and quantitative mass spectrometric analysis (4) reveal the compound s affinity profile, ranking all protein targets according to their K d values for free compound (5). Sepharose resin Linker derivative of test cpd Test cpd Target proteins PAGE 5

7 Cellular Target Profiling Summary Identification of small molecule targets in any cell type or tissue of choice Determination of a compound s proteome-wide binding affinities to discriminate between high, medium and low affinity cellular targets Profiling of a compound against native, endogenously expressed, posttranslationally modified full length proteins in the presence of cellular co-factors and native complex partners Extensive, non-target class restricted track record in target deconvolution and profiling 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

8 KinAffinity Chemical proteomics for kinome analysis Test compound K D [µm] 0,01 0, Target Kinase 1 Target Kinase 2 Target Kinase 3 Target Kinase 4 Target Kinase 5 Target Kinase 6 Target Kinase 7 (1) Cell or tissue sample (2) Mixture of beads of broadly selective kinase inhibitors for enrichment of kinases (3) Competition of target kinases by test compound (4) LC-MS/MS analysis (5) List of target kinases ranked according to their K d values Lysate of the cell line or tissue of interest is prepared (1). A matrix comprising of immobilized broad-spectrum kinase inhibitors is used to enrich the expressed kinome of the cell or tissue (2). Competition assays with the free test compound (3) and quantitative 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 matrix Immobilised kinase inhibitor Test cpd Target kinase Target kinase bound to test cpd PAGE 7

9 Phosphoproteomics Mode of action analysis of kinase inhibitors PhosphoScout is a quantitative mass spectrometry based platform to monitor dynamic phosphorylation events in vivo on a global scale Allows identification and quantification of phosphorylation sites in living cells, animal models and patient samples Identification of regulated phosphorylation networks Currently 10,000 to 15,000 phosphorylation sites can be measured in a single experiment using Evotec Munich s PhosphoScout technology Enables comprehensive investigation of (tumor) signaling pathways and their response to drugs Applications include identification of pharmacodynamic read-outs and (comparative) mode of action analysis of kinase-selective drugs PAGE 8

10 Acetylomics Mode of action analysis of HDAC and sirtuin inhibitors I MS I MS/MS Ac Enzymatic cleavage Ac Ac Ac m/z m/z G-V-L-P-A-W-R Ac P Ac MaxQuant Software (Dept. M. Mann, MPI) Proteins Peptides LC-MS Data Analysis Isotopic labeling Enrichment Quantitative & global lysine acetylation analysis Quantitative and global measurement of site-specific lysine acetylations, can be combined with global proteome quantification Highly optimized workflow with respect to sensitivity and selectivity Acetylomics is an ideal tool for cellular mode of action analyses that go far beyond the field of chromatin biology Identification of regulated acetylation networks PAGE 9

11 Deep Proteome Analysis Industry-leading capabilities in protein abundance profiling Quantitative mass spectrometry-based approach to accurately measure protein abundance levels in vivo on a 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 body fluids) Deep Proteome Profiling P Peptide fractionation LC-MS/MS I heavy light heavy light MS m/z Data Analysis proteins P I heavy light heavy light MS m/z ~5000 proteins Isotope Labeling or Label-free Extraction of Proteins Enzymatic Cleavage LC-MS/MS Data Analysis Single-shot Proteome Profiling PAGE 10

12 Biomarker Discovery Evotec s three-tier strategy to support oncology biomarker discovery PAGE 11

13 Non-resp. Responder Non-resp. Responder Proteome and phosphoproteome biomarker discovery Complementary approaches Test sample set of drug responders and nonresponders (cultured cells, xenografts, patient samples) Quantitative proteome profiling Protein expression Phosphosites Global analysis of cellular protein levels Target class independence Only µg protein amounts required for MS analysis Easier transition from discovery to validation/clinical analysis Quantitative phosphoproteome profiling Global analysis of cellular kinase activity Direct linkage with kinase function Other PTMs possible, such as lysine acetylation for HDACs Comprehensive proteome and/or phosphoproteome profiling across responder and non-responder samples PAGE 12

14 Key Advantages Multivariate protein-based stratification markers Proteomics-based biomarker discovery Biomarker discovery on functional protein level (instead of mrna analysis) to ensure higher predictivity Unique capabilities in proteome-wide analysis (6000+ proteins) to identify more predictive biomarkers than by targeted or lower coverage analysis Identication of multivariate markers with higher predictive power than univariate marker Clinical response prediction for Flt3 inhibitor AC220 in 12 AML patients Phosphosignature of 5 sites with 92% prediction accuracy Validation with independent AML patient samples revealed 7/8 prediction accuracy PAGE 13

15 Building innovative drug discovery alliances Thank you for your attention Your contact: Dr. Klaus Godl Vice President, Proteomics & Chemical Biology

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