Discussion

Biopyhsical approaches are an integral part of the drug discovery process to asses binding constants of molecular interactions, which are in turn required for efficient lead optimization. Here we show that MicroScale Thermophoresis (MST) is a powerful technique to rapidly determine binding constants in fragment screening approaches. MST detects the directed movement of fluorescent molecules in microscopic temperature gradients. Small changes in the hydration shell of these molecules, caused for instance by binding of small molecules, results in changes in their thermophoretic movement which can be used to derive binding constants. MST is a highly sensitive method that can detect fluorophores at low picomolar concentrations and requires only a few µl of sample material.
Using the new NT.Automated instrument, we screened a library containing 193 pre-selected fragments for their interaction with the drug target mitogen-activated protein kinase 1 (MEK1). We identified >70 binders with dissociation constants (Kds) ranging from the low µM to low mM range. 16 fragments displayed Kds below 100 µM. Importantly, 7 out of 8 previously determined hits were among the top-fifteen fragments from our MST-ranking and yielded x-ray co-crystal structures with MEK1. Moreover, the MST ranking showed a very strong correlation with a qualitative DSF screening, while a SPR screening failed to identify positive hits and showed no correlation with the DSF ranking. Instead, SPR picked up several false-positives, which were easily identified by MST due to their protein aggregation/denaturating effects. 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 asset in modern drug discovery processes.