Development of high-throughput assays to identify allosteric inhibitors of kinase activation cascades
The creation of an assay for compounds, which allosterically inhibit the activation of ERK2 by MEK1 kinase.
Since this assay shall indirectly measure kinase activity, common assays that detect the direct kinase activity could not be used. Because this drug discovery project was aimed to create a new allosteric inhibitor, a traditional ATP competition assay could also not be used.
We created a cascade assay in which ERK2 kinase was converted from its non-active to the active conformation by its physiological upstream activator, MEK1. The activation enabled ERK2 to phosphorylate its substrate, which could be quantified via our high-throughput 33PanQinase assay technology based on the transfer of radioactive 33P from ATP to the substrate.
A high-throughput capable method with the potential to identify allosteric inhibitors of protein kinase activation cascades
Development of a novel methyltransferase activity assay format with high-throughput capacity
To enable the pioneering of methyltransferase drug discovery, high-throughput capable assays for the detection of methyltransferase activity were dearly needed by the epigenetic drug discovery community in the early 2010‘s. At this time, tool compounds which are crucial for successful assay development were not available.
We have produced de-novo methyltransferase enzymes and applied a modified miniaturized radioisotope-based filter-binding assay, HotSpot, originally developed for kinases. The HotSpot assay format detects total methylation of a substrate on both lysine and arginine residues. Substrates ranging from peptides to nucleosomes can be used without the need for modification; therefore, this platform is suitable for ultra-high-throughput screening (uHTS) and selectivity profiling against a large methyltransferase panel. We have determined the linearity and kinetic of the assay and investigated the substrate specificity of 22 methyltransferases. To demonstrate the capability of the HotSpot platform, we screened a small library of 1,280 compounds against selected methyltransferases with their specific substrate and tritiated S-adenosyl-l-methionine (SAM) as the methyl donor.
Assay Development for Histone Methyltransferases