Objective

Domainex performs fully-integrated structure-based drug design programs. We show how we use structural information to find small-molecule ligands by both virtual screening (LeadBuilder) and fragment screening (FragmentBuilder). These technologies will be illustrated with a case study on the lysine methyltransferase enzyme, G9 (also known as EHMT2).

Firstly, a proprietary crystal structure of G9a bound to peptide was used to set up LeadBuilder, our virtual screening platform to identify small molecule starting points for drug discovery programs. LeadBuilder has two key elements: the first is our in-house ‘NICE’ curated database of commercially-available lead-like compounds; the second is a proprietary two-stage virtual screening protocol based on searches against multiple Target Site Pharmacophore Models (TSPM), followed by docking into the protein target site.  Typically 500-1500 virtual hits are then acquired and tested in an appropriate biochemical assay.  This approach has a much better success rate than HTS, and delivers highly-developable hit compounds that enable an accelerated hit to lead process.

 We also report a fragment-based drug design (FBDD) approach using Microscale Thermophoresis (MST) - an affinity-based technique - to screen a library of low molecule weight compounds (‘fragments’). FBDD has become widely used as an alternative to traditional high throughput screening (HTS).  Since fragment hits have relatively weak affinity, they are often identified using biophysical techniques such as NMR, SPR, DSF, ITC, or X-ray crystallography. 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 (Kd) 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.

 Out of a library of 320 fragments, we identified 17 fragments hits against G9a 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.