Potent, selective and cell-permeable inhibitors that target key regulators of cellular signalling ("chemical probes") are valued reagents in both fundamental and applied biological research, and they are essential tools for target validation and provide starting points for translational research projects. We established family wide screening platforms for comprehensive inhibitor profiling for human protein kinases, acetyl-lysine recognition domains of the bromodomain family as well as lysine demethylases.
Protein Kinases: A particular research focus of the groups is on structural mechanisms of protein kinase regulation (3, 4), family wide structural analysis (5) and development of specific inhibitors (6). The structural biology team of the group has solved so far more than 60 novel kinase crystal structures and developed expression systems for close to 200 human kinases. These data offers a valuable resource for family wide structural comparisons and assay development as well as for structure based inhibitor design. Recent efforts have been focussed on the development of target specific inhibitors that can be used as tools for the functional annotation of kinases (7). The development of selective inhibitors is a challenging task considering the large size and the largely conserved catalytic domains of the protein family. However, detailed comparison of crystal structures of this protein family identified unique structural features within and outside the kinase active site that can be explored for the development of inhibitors with high target selectivity.
Epigenetic effector domains: Epigenetic mechanisms are defined as heritable and acquired changes in gene function that occur without alteration of the DNA sequence. These processes have been associated with a large diversity of cellular processes and dysfunction of epigenetic control of gene expression leads to development of disease (8). A key mechanism of gene expression control is regulated by histone post translational modifications that are controlled by families of “Readers, Writers and Eraser” proteins. We are particularly interested in developing inhibitors that inhibit protein interactions mediated by “reader domains” that recognize lysine acetylation marks (Bromodomains). Recently we discovered that bromodomains can be selectively targeted by small molecules. We comprehensively characterized the structure and substrate requirements of the human bromodomain family (9) developed assay systems that allowed family wide profiling of inhibitor selectivity. Using these tools we were able to develop highly specific, potent and cell active inhibitors for a number of bromodomains (2).
Chemical Libraries: We developed several chemical libraries including a kinase focussed library (~ 5000 compounds), the GSK kinase tool kit (PKIS) as well as bromodomain targeted libraries. These libraries have already been comprehensively screened in vitro and are now available for phenotypic screens. Several public domain libraries are also available.
Drug Target Discovery and Target Deconvolution
(Chemical Proteomics & Thermal Stability Profiling)
In order to understand the cellular targets of small molecules and drugs we use a combination of compound affinity chromatography coupled to protein mass spectrometry called "chemical proteomics" or "chemoproteomics". This technology allows us to identify the proteins which bind to compounds in cell or tissue lysates which, for example, can help to uncover the mechanism of action of compounds that have emerged from phenotypic screens or drugs that exhibit so-called "polypharmacology". The power of this approach is that in contrast to classical biochemical in vitro screening assays here the compound is exposed to an entire and competitive cellular proteome (~6,000 natural full-length proteins with all posttranslational modifications) which provides a much more phyisologically relevant context for evaluating the cellular effects of compound.
Recently, we have established a new methodology termed "Thermal Stability Profiling" which enables the profiling of small molecules and metabolites in intact living cells. Here we take advantage of the ligand-induced thermal stabilisation of proteins (see "Thermal shift assays" below) to unravel the molecular targets of drugs and drug candidates.
High Throughput Structure Determination
The chemical biology group has access to high throughput protein production and crystallization facility located at the SGC. High resolution crystal structures of targets proteins are essential for rational design of selective inhibitors. The long history of the team in protein crystallization enables rapid determination of protein ligand complexes as well as fragment screening by protein crystallography.
Amplified Luminescent Proximity Homogeneous Assays (ALPHA) have been developed in particular for screening of protein interaction inhibitors for instance for bromodomain and other epigenetic reader domains. The assay measures an energy transfer from one bead to the other of targets immobilized on beads, ultimately producing a luminescent/fluorescent signal. The PPI inhibitor will conceivably disrupt complex formation in concentration dependent manner resulting in loss of the fluorescent signal (10).
Differential Scanning Fluorimetry (Thermal Shift Assays)
The assay principle is based on the stabilization of the native protein structure upon the ligand binding. The advantage of this assay is that it can be used for any protein that is stable in solution with minimal optimization. Comparison with enzymatic assays as well as with direct ligand binding assays revealed usually good correlation between the measured DTm shift and directly determined binding constants or IC50 values (11).
Biolayer Interferometry (BLI)
BLI is a label-free direct detection method for study protein-protein and protein-ligand interaction similar to the more widely used surface plasmon resonance method (SPR). OctetRed384 is 16-channel medium to high throughput machine operating with 384-well sample plates. For specific immobilization of proteins we developed a large number of expression systems that allow for in vivo biotinylation in bacteria. The method can be used for fragment screening as well as for determination of binding constants and binding kinetic parameters.
Isothermal Titration Calorimetry (ITC)
ITC is a versatile method for the determination of ligand binding constants (KB’s) in solution by measuring the binding heats that are released (enthalpic) or consumed (entropic). ITC is a very direct method which can be applied to a large diversity of ligand- receptor systems. The generated data reveal also the thermodynamic driving forces that give rise to ligand binding. It is therefore a very informative technology for structure based design of inhibitor molecules. An example of a set of ITC experiments is shown on the left. The figures shows the raw titration heats (upper panel) as well as the normalized binding heats (binding isotherm) for each injection on the lower panel as well as a non-linear least squares fit (solid line). Shown are three titration experiments (Bromodomain of BRD4): A) Bank titration (ligand into buffer, B) Inactive (-)-JQ1 stereo isomer. As expected no significant heat effects have been observed C) Active (+)-JQ1 showing exothermic strong binding (2).