Evaluation of peptidomimetic inhibitors

Abstract Scientific research in drug discovery relies on robust, versatile, and well-established methodologies. One effective strategy for targeting disease-relevant proteins, such as E3 ligases, involves rational drug design using degron peptides as starting points for compound development. Here, we present a comprehensive set of complementary assays for the evaluation of degron-based binders, enabling their integration into an efficient drug discovery pipeline. We introduce screening strategies such as Differential Scanning Fluorimetry (DSF) and Fluorescence Polarization (FP), alongside both solution-based and immobilization-based biophysical techniques, including Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) for reliable affinity determination. To assess intracellular interactions with full-length target proteins, we also employ Nano Bioluminescence Resonance Energy Transfer (NanoBRET). Together, these methods establish a robust framework for the discovery and characterization of degron-based peptidomimetic compounds.