Fragment Screening

At the heart of the platform is screening of fragments by MicroScale Thermophoresis (MST, Nanotemper Technologies GmbH). This capillary-based, homogenous technology is able to quantify even weak binding events in a solution-based manner. The Domainex fragment collection can be screened in a matter of a few days to determine initial Kd values of fragment hits. Domainex has alternative screening methods available if required, such as HTRF, DSF and direct-binding mass spectrometry.

MST Advantages Over Alternative Methods

  • Minimal assay development
  • Little protein required
  • Solution-based, so no immobilization
  • Measure up to quaternary biological systems
  • Capture orthosteric and allosteric binders
  • Sensitive across the nM-mM range
  • A high throughput technique
  • Eliminates false positives early

Case Study: Lysine methyltransferase, G9a

G9a is a lysine methyltransferase (KMT) involved in epigenetic gene regulation by covalent modification of histones. It catalyses the transfer of methyl groups from S-adenosyl methionine (SAM) to lysine residues on histone proteins (Fig 1).

Figure 1: G9a catalyses the transfer of methyl groups to histones

Literature1-4 supports the role of G9a in mechanisms of carcinogenesis, making it an attractive oncology target. Part of the fragment library was screened at 1 mM against a G9a-SAM complex using MST. Fragments were declared as hits if a significant shift in the response compared to the reference was observed (Fig 2).

Figure 2: Hits identified from the MST screen

The thermophoresis traces allow easy identification of false-positive fragments such as aggregators and compounds effecting the fluorescence signal (Figure 3 and 4, respectively).

Figure 3: MST raw data showing aggregation
Figure 4: MST raw data showing auto-fluorescence (Green: DMSO, Blue: test compound)

A 5.3% hit rate was obtained. Screening the same fragments using Differential Scanning Fluorimetry (DSF) or the activity based AlphaScreen both showed only a 0.3% hit rate.

Hits were taken into secondary screening to determine their binding affinities (Kd) to the G9a-SAM complex using MST (examples shown in Table 1).

Table 1: Screening Summary

Hit Validation

Orthogonal confirmation of hit binding to G9a was demonstrated by Saturation Transfer Difference (STD) NMR spectroscopy (Fig. 5). Three G9a-fragment structures were solved in-house in the presence of co-factor SAM with a resolution of 1.5- 2.0 Å (Fig 6) which revealed different fragment binding modes.

Figure 5: STD-NMR confirmation spectra for five fragments. Spectrum a (black) shows the NMR-STD confirmation for each fragment, as well as the SAM co-binder. Spectrum b (red) shows the reference spectrum of each fragment and the SAM co-binder (8.16 ppm) in PBS, pH 8.5 (10 % D2O). Spectrum c (grey) is the false positive control. No signal indicates there was no aggregation of the fragment, and no direct excitation of the fragment or SAM with the on-resonance pulse.
Figure 6: In-house G9a-Fragment structures (Orange –Fragment, Yellow –SAM)

References

  1. Copeland et al., Nature Reviews, 2012, (8), 724-732;
  2. Hamamoto et al., Nature Cell Biology, 2004, (6), 731-740;
  3. Hamamoto et al., Cancer Sci., 2006, (97), 113-118;
  4. Liu et al. J. Natl. Cancer. Inst., 2013, doi: 10.1093/jnci/djt30