Objective

Fragment-based (FBDD) has become widely used as an alternative to traditional high throughput screening (HTS).  As fragment hits have relatively weak affinity, they are often identified using biophysical techniques such as NMR, SPR, DSF, ITC, or X-ray crystallography. A new technique, Microscale Thermophoresis (MST), is well suited to characterising the binding of fragments - or even ions - to biomolecules due to its large detection range from pM to mM affinities. MST measures the movement of fluorescently-labelled molecules in temperature gradients created by laser within microliter-volume glass capillaries. The thermophoretic movement of a molecule is determined by its size, charge, and hydration shell. Ligand binding affects these properties, resulting in changes in the thermophoretic characteristics of the molecule. These changes can be used to derive dissociation constants (K_d) within minutes.  This method offers a number of benefits for FBDD , notably its fast, efficient and precise ability to characterise fragments with a low number of false positives and false negatives, whilst using very small amounts of protein.

Here, we report an affinity-based FBDD approach using MST to screen a library of 320 fragments against histone-lysine methyltransferase G9a (also known as EHMT2). We identified 17 fragments hits by single-shot screening at 1 mM concentration (5.3% hit rate) using MST; whereas screening the same library with Differential Scanning Fluorimetry (DSF) and AlphaScreen, afforded only one hit with each of the techniques. We used NMR Saturation-Transfer Difference (STD) to confirm hits and X-ray crystallography to obtain structural information on the positioning of the fragment hits for hit-to-lead development. This highlights the advantages of MST for working with ternary systems, which can be difficult using some other biophysical techniques such as ITC and DSF.