Category: Drug Target Strategies
The discovery of innovative, safe and efficacious drugs is hampered by a multitude of challenges. For example inefficient target engagement as well as off-target effects are frequent causes for late stage failure of candidate drug molecules. This raises the critical need for strategies to identify the mechanisms of action of bioactive molecules. To address some of these fundamental challenges in drug discovery, we have developed the Thermal Proteome Profiling (TPP), which enables the hypothesis-free identification of drug targets and off-targets in live cells or cell extracts. TPP is based on the Cellular Thermal Shift Assay (CETSA) introduced by the group of Paer Nordlund in 2013 (Molina et al. Science 2013) enabling to monitor drug-protein interactions in living cell. We demonstrated that the combination of CETSA with quantitative mass spectrometry and a dedicated bioinformatic analysis platform enables the determination of thermal stability data on a proteome-wide scale (Savitski et al. Science 2013). We refined TPP by combining temperature dependent and concentration dependent experiments into one experimental scheme (2D-TPP) to allow the comprehensive determination of drug target affinities across the proteome (Becher et al. Nat Chem Biol. 2016). Using the histone deacetylase inhibitor panobinostat as a model system we compared how experimental parameters in the 2D-TPP setup influence the ability to identify its targets and off-targets and to distinguish changes in thermal stability caused by direct target binding from indirect effects. Experiments performed in live HepG2 cells were compared to three different cell extract protocols. We found that membrane proteins were underrepresented if cell extracts were generated without addition of detergents. Better coverage of membrane proteins was achieved when the detergent was either added prior to or after the heat treatment. The latter can be applied to ‘crude’ cell extracts where cells were opened by shear force but not cleared for insoluble particles. Whilst heating of detergent treated cell extracts led to altered melting points of target proteins and incomplete coverage of targets, detergent solubilization of heat-treated extracts enabled similar coverage as live cell treatment and did not substantially alter thermal stability of target proteins. This new protocol for 2D-TPP with cell extracts will enable the detection of the drug effects on their targets/off- target extracted from primary material allowing to measure personalized target engagement.
Christina Rau– Group leader, Cellzome GmbH, a GSK Company, Heidelberg, Baden-Wurttemberg, Germany
Cellzome GmbH, a GSK Company
Heidelberg, Baden-Wurttemberg, Germany
Christina Rau received her PhD at the Freiburg University investigating the biochemical pathway of toluene degradation by anaerobic bacteria. In 2001 she joined the Cellzome AG, at that time a privately-owned drug discovery company that was acquired by GlaxoSmithKline in 2012.
After initial work on the yeast interactome, that led to a landmark study in Nature, Christina focused on establishing chemoproteomics assays, mainly in the frame of large collaborations with pharma industry.
In 2008 Christina moved to the Cellzome Array Platform Team, which she supervised from 2012 on. In this role, she established chemoproteomics screening assays based on the kinobeads technology and was responsible for running several screening campaigns as well as supporting lead optimization projects.
Since 2015 Christina leads the Chemoproteomics group at Cellzome, which is mainly focused on performing proteomics assays to assess affinity and selectivity as well as target engagement of small molecules.