Recent reviews of successful Phase II and III molecules highlighted that an integrated understanding of the fundamental pharmacokinetic/pharmacodynamic principles of exposure at the site of action, target binding and expression of functional pharmacological activity were key. Implementing these principles at the earliest possible stage of drug discovery would be the ideal scenario; however, there are a large number of targets and target classes, e.g. epigenetic readers, where cellular penetration or target engagement is often difficult to determine. Epigenetic reader proteins, such as Chromodomain and Tudor domain containing proteins, often fall into this category. These proteins are involved in detecting specific post-translational modifications to histones and then subsequently enabling modification of local chromatin structure and recruiting other proteins to initiate gene transcription. Historically, designing assays for these targets is often limited to recombinant protein-based assays, which can be problematic depending on the target, and phenotypic assays if available.

Bromodomain containing proteins are a class of epigenetic readers that selectively bind acetylated lysines found in the amino-terminal tails of histone proteins. Bromodomains are small protein interaction modules made up of ~110 amino acids, consisting of four alpha-helix bundles that form a central hydrophobic pocket that recognises and binds acetylated lysines. Recently, the therapeutic relevance of disrupting the interaction between bromodomains and histones in a number of diseases has been demonstrated by the discovery of potent BET (bromodomain and extra-terminal) family inhibitors.

InCELL Hunter™ is a novel, powerful cellular ligand binding assay platform to determine compound binding in a cellular environment. InCELL Hunter™ platform is a method for determining the interaction of a test molecule with an intracellular protein target in intact, recombinant, mammalian cells. This technology offers the potential to compliment protein-based assays as well as deliver assays for targets with poor tractability.

Here we evaluate the InCELL Hunter™ technology and describe the use of these assays to characterise the cellular permeability and target engagement for a number of bromodomain interacting-inhibitors.