An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

Abstract

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

“…The thermal unfolding of 5 g/L PPI13 in different formulations was studied with nanoDSF ® . 21,22 The samples were filled in standard glass capillaries, the capillaries were sealed and placed in a Prometheus ® NT.48 (NanoTemper Technologies, Munich, Germany). The device was used to linearly change the sample temperature from 25 C to 100 C with a ramp of 0.1 C/min.…”

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

“…In order to ensure that the variation in uorescence was associated with binding, we performed a standard denaturation test (SD test) as described previously. 23 For the SD test of full-length AQP2 with LIP5, four samples were prepared in the same manner as for the MST experiment above, corresponding to high, medium and low concentration of LIP5 (2450 nM, 322 nM and 28 nM) as well as labelled full-length AQP2 alone. For the AQP0 peptide, three samples were prepared corresponding to high and low concentrations of AQP0 peptide (5.375 nM and 0.67 nM) and labelled CaM alone.…”

Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective

“…26–28 The use of MST is increasingly being reported in fragment-based drug discovery, 23,24,30–33 including kinases such as p38α 30 and MEK1, 31 and the bromodomain BRD9. 32 In both the MEK1 study by Sanofi in collaboration with NanoTemper and the BRD9 study by Boehringer Ingelheim, MST was used to confirm the binding of fragments identified by SPR and DSF, as well as to identify additional hits not detected by the other techniques.…”

“…25 This explains why MST is gaining popularity as a technique for fragment screening, because it allows the study of weak-affinity fragments titrated up to 10 mM. 31,32 In addition, MST is described as offering a wide detection range, from low-millimolar to picomolar affinities, when using NanoTemper’s dedicated Monolith NT.115 Pico instrument. 28 In general, dissociation constants (K d ) within twofold of the protein concentration can be accurately quantified.…”

“…Usefully, MST can often also identify false positives and the mechanism by which they interfere, such as photobleaching, photoenhancement, and autofluorescence (apparent in the magnitude and shape of the MST trace), and aggregation (apparent as irregular bumpy traces). 31,42 The MST assay optimization procedure is usually rapid and the analysis of binding interactions and determination of binding affinities (K d or EC 50 ) simple. We have established a stepwise workflow to developing MST assays and screening hit compounds ( Fig.…”