Objective

Kinetic characterisation of compounds is critical to determine how a compounds mechanism of action (MoA) effects potency in cellular assays and DMPK profiles. Detailed mechanistic characterisation of compounds tends to be performed on a limited number of compounds during lead optimisation. However, there is substantial interest in techniques that will enable more-detailed characterisation of compounds earlier in the drug discovery process. In this work, we have used the PhosphoSens® platform from AssayQuant Technologies to quickly develop kinetic assays for a range of kinases, enabling us to determine compound MoA for a range of targets during both the lead identification and lead optimisation stages of drug discovery projects.The AssayQuant technology uses chelation-enhanced fluorescence; specifically a SOX chromophore in peptide or protein substrates, to create real-time sensors of phosphorylation. The result is a simple yet powerful method to measure the activity of protein kinases using a homogeneous and continuous format, where the level of fluorescence is directly proportional to the amount of phosphorylated product. This format allows multiple parameters to be varied in a matrixed manner to facilitate detailed characterisation with minimal effort and thereby greatly improving confidence with which new compounds are selected, optimised and brought forward to the clinic.