Identify Effective Therapeutics with Simple, Quantitative Cell-Based Assays to Measure Receptor Internalization

Both multi- and single-pass membrane receptors constitutively exhibit complicated and dynamic membrane receptor trafficking as well as internalization induced by ligand or compound binding. Measuring how therapeutic molecule binding to membrane receptors affects receptor internalization can provide insight into drug tolerance, unwanted side effects, diseases, and ultimately help with identifying safer drugs. Additionally, antibody-based therapeutics that target specific receptors such as the immuno-oncology checkpoint receptors can induce high levels of internalization and serve as candidates for conjugation to cytotoxic compounds. These therapeutic Antibody-drug conjugates (ADCs) take advantage of both antibody specificity for tumor antigens and antibody induction of receptor internalization to deliver cytotoxic drugs to cancer cells effectively. We present here a collection of cell-based assays that use the industry-validated Enzyme Fragment Complementation (EFC) technology to monitor receptor internalization activated by small molecules or biologics. These assays are easy-to-use and offer a quantitative means of measuring receptor internalization that is amenable to high-throughput screening. PathHunter^® Internalization Assays provide an effective alternative to more cumbersome, less quantitative techniques that rely on imaging, antibodies, or dyes and labels to determine internalization. Assays for both single-pass membrane receptors – such as receptor tyrosine kinases (RTK) and checkpoint receptors – and multi-pass membrane receptors, such as G-protein coupled receptors (GPCRs), have been developed using this generic platform. An additional assay format specific for GPCRs measures activated GPCR internalization using β-arrestin recruitment. Results from such membrane receptor internalization assays can provide important safety and efficacy information during drug discovery and development.