Objective

The emergence of proteolysis targeting chimeras (PROTACs) as well as small molecules that inherently modulate target stability, such as selective estrogen receptor degraders (SERDs), has established target degradation as a key mechanism of action (MoA) for consideration in drug discovery campaigns. Target degradation often has additional benefits over target inhibition and can in some cases enable targets that were previously considered intractable. Western blotting is commonly used to monitor protein levels in cells but it is low throughput and only semi-quantitative, making it challenging to deliver accurate IC50s at multiple timepoints and impossible to profile large HTS outputs for the degradation MoA. High-throughput plate-based cellular assays for identification of protein degraders are therefore required. Here we present a case study where there is a phenotypic disconnect between genetic knockdown and chemical inhibition of the target. In such instances, compounds that degrade the target can be used as validation tools to address the disconnect between the genetics and the pharmacology and, if they recapitulate the genetic knockdown phenotype, could provide an efficacious mechanism of action, where other small molecules have failed. We show that antibody-based methods for quantification of target protein levels in cells, such as immunofluorescence imaging and AlphaLISA (Perkin Elmer), can suffer from a phenomenon known as ‘epiturbance’. This is when small molecule binders of a target disturb antibody binding to the target, thus appearing as if they degrade the target, even if they don’t. We therefore present the deployment HiBiT tagging (Promega) to enable development of antibody-independent high-throughput target degradation assays.