Chemical Biology

To identify, explore, and validate new targets, the Huber Laboratory integrates a wide range of discovery approaches - including small-molecule and phenotypic screening, biochemical and structural biology, protein–protein interaction and chemical proteomics, medicinal chemistry, and genetic perturbation methods such as RNAi and CRISPR-based editing.

By combining these complementary techniques, we aim to generate high-quality chemical probes that illuminate fundamental biology and provide starting points for drug discovery.

For an overview of our collaborative efforts with our academic and industry partners please also see a recent article from WIRED.

Research

Chemical biology bridges chemistry and biology to create “chemical probes” that explore how proteins work and assess their potential as drug targets.

 Technologies

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.

 ALPHA Screen

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 (K_B’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).