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). Literature supports the role of G9a in mechanisms of carcinogenesis, making it an attractive oncology target.1-4 Domainex has solved the key technical drug discovery challenges associated with KMTs, including generating a number of proprietary crystal structures, assays and a novel screening library of small molecule inhibitors. A fragment-based hit-finding approach using Microscale Thermophoresis (MST) for the ternary G9a-SAM-fragment system was employed. Hits were confirmed using protein X-ray crystallography and subsequently optimized.
Figure 1: G9a catalyses the transfer of methyl groups to histones
Domainex's fragment library contains >1,000 fragments and has been carefully designed to maximise the chances of finding suitable chemical starting points. The library gives good coverage of ‘ideal fragment space’ by optimising a number of parameters, is ‘Rule of 3’ compliant, has good chemical diversity and is SP3 rich.
Additional filters were applied to remove:
- Compounds containing atoms other than H, C, N, O, S, F, Cl
- Reactive functional groups
All compounds in the library show > 1 mM aqueous solubility in 1% DMSO
Screening by MicroScale Thermophoresis (MST)
MST is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target. These changes can be used to derive dissociation constants (Kd) within minutes.
G9a Fragment Screen
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
Orthogonal 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. This resulted in several options for FBDD to provide alternative inhibitor chemotypes.
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)
Three series were progressed and after one round of fragment elaboration (~50 compounds) a 10-fold increase in affinity was achieved. Figure 7 shows the efficient process used by the medicinal and computational chemists.
Figure 7: Rapid elaboration of the fragment hits
- MST was used to successfully screen a sub-set of our fragment library against the KMT G9a with a hit rate of 5.3%, identifying fragments with high ligand efficiencies.
- MST allows easy false-positive identification by highlighting compounds that induce protein aggregation or cause fluorescent effects.
- Low and high affinity binders could be identified using the same technique.
- Three fragment hits were successfully crystallised bound to G9a, which enabled a SBDD program for this target.
- In one round of fragment elaboration a 10-fold increase in affinity was achieved.
 Copeland et al., Nature Reviews, 2012, (8), 724-732;
 Hamamoto et al., Nature Cell Biology, 2004, (6), 731-740;
 Hamamoto et al., Cancer Sci., 2006, (97), 113-118;
 Liu et al. J. Natl. Cancer. Inst., 2013, doi: 10.1093/jnci/djt30