CeNS Colloquium

Dr. Dennis Breitsprecher
NanoTemper Technologies GmbH

MicroScale Thermophoresis (MST) is an exceptionally powerful technique to quantify biomolecular interactions. It is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell and conformation. Thus, this technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen. Recent progress and developments in MST technology and new MST applications beyond standard biomolecular interaction studies will be presented, spanning a range from straight-forward determination of picomolar dissociation constants (Kds) to the analysis of thermodynamic parameters of interactions as well as monitoring unfolding mechanisms of proteins. In addition, MST was successfully used in an in-house fragment screen at NanoTemper Technologies, using the newly developed NT.Automated instrument for high-throughput applications. In close collaboration with Sanofi R&D, a library containing 193 pre-selected fragments was screened for their interaction with the kinase MEK1. More than 70 binders with dissociation constants ranging from the low µM to low mM range were identified. Importantly, 7 out of 8 previously determined hits were among the top-fifteen fragments from the MST-ranking and yielded x-ray co-crystal structures with MEK1. Thus, high-throughput binding analysis by MST is a valuable tool that compliments qualitative screening methods and circumvents false-positive results which are commonly picked up by other biophysical screening methods. In addition to advantages such as little sample consumption (4 µl of sample per data point, only a few picogram of target protein are required for the entire screening), easy handling, rapid assay optimization and straight-forward data analysis, MST moreover provides information about different binding sites, protein stability and aggregation. Thus, high-throughput MST analysis is a perfect assed in both, modern drug discovery processes as well as basic research.