BioFocus has compared and evaluated a number of assay technologies for epigenetic targets. The gold standard remains the radiometric assay format, which allows use of highly relevant substrates. In the specific case of HMTs, BioFocus has used nucleosome preparations, recombinant histone preparations, and other protein substrates or peptides in the radiometric assay format.
For certain members of this target family no peptide substrate has yet been identified, and some protein targets only show enzymatic activity when nucleosome preparations are used as substrate.
Besides the feasibility of assay development, the pharmacological relevance of the screening assays supports the use of endogenous protein substrates. However, there are a number of target family members where homogeneous assay technologies have led to highly comparable screening results. As these technologies are often more cost- and time-efficient they are preferred for primary high-throughput screening (HTS).
In addition to radiometric assays as an HTS method for epigenetic drug discovery for HMTs, histone demethylases, and bromodomains, BioFocus has focused on Almac’s FLEXYTE® fluorescence lifetime technology (FLT) and Caliper’s mobility shift assays. The details of these assay technologies are described in Figure 3 and in Wigle et al.2
TR-FRET, AlphaScreen®, fluorescence polarization (FP), and ELISA-based methods are also available on the market with various advantages and disadvantages concerning reproducibility, specificity, throughput, false-positive/false-negative issues, label limitations, and flexibility of substrate use.
BioFocus has designed and executed case studies for the HMT G9a, a target relevant to oncology and HIV. Further case studies focus on the histone demethylase LSD1, a target expected to be relevant to oncology and bromodomain BRD4, where a number of lead compounds have been published and could be used as templates for the initiation of further ideas (not shown here).
The G9a case study started with the use of computational chemistry tools to select a small number of compounds from the BioFocus compound library, which consists of 900,000 small molecules and about 100,000 natural product samples.
For G9a, extensive information concerning protein structures and tool compound availability allowed us to select compounds using ligand-based as well as docking technologies, covering both the substrate-binding and the SAM-binding sites. Based on this approach, a total of 2,112 test compounds were selected for physical screening.
This small selection was screened three times using i) a FlashPlate® radiometric assay with recombinant full-length histone H3, ii) a FLT assay (as described in Figure 3), and iii) a mobility shift assay (as described in Wigle et al2). Both FLT and mobility shift assays applied a similar peptide substrate. The sensitivity of all three assays was confirmed using S-adenosylhomocysteine (SAH) and sinefungin as reference inhibitors, yielding highly similar IC50 values.
While no common compounds were identified in all three primary screens, we could conclude that the FLT and mobility shift assays primarily generated substrate competitive inhibitors, while the preference for the hit compounds from the radiometric assay was toward the SAM-binding site.
The SAM competitive inhibitors, which were identified by the radiometric and FLT assay, showed a broad range of selectivities against a small panel of HMTs. The most potent hits in the Caliper mobility shift assay all showed substrate competitive behavior, in accordance with the virtual screening source annotation and the mapping to the substrate-binding site.
Our studies have allowed us to evaluate the advantages and limitations of these different technologies in the context of HTS and mode-of-action studies. More details are available in Ahrens et al.3