High-Throughput Screen for Candidate PROTAC Identification

The identification of potent and selective proteolysis targeting chimeras (PROTACs) presents unique screening challenges compared to traditional small-molecule inhibitor discovery, due to linker-dependent structure-activity relationships, ternary complex cooperativity, cellular permeability constraints, and the need to connect target engagement with productive ubiquitination and degradation. This chapter provides a methodological guide to the high-throughput screening (HTS) strategies for candidate PROTAC identification. We outline the workflow from the construction of focused chemical libraries to the implementation of orthogonal screening cascades. Key biochemical and biophysical approaches, including time-resolved fluorescence energy transfer (TR-FRET), AlphaLISA, fluorescence polarization (FP), surface plasmon resonance (SPR), and affinity selection-mass spectrometry (ASMS), are discussed for their utility in the rapid triage of PROTAC hits. We further discuss the role of cell-based assays, including NanoBRET, HiBiT, Cytoblot/in-cell immunodetection, high-throughput flow cytometry, and high-content imaging, which enable assessment of intracellular target engagement, degradation potency, selectivity, and mechanism of action. Emerging phenotypic, chemoproteomic, and computational strategies are also considered as complementary approaches for expanding the degradable target space and prioritizing high-quality candidates. Together, these screening modalities provide an integrated framework for accelerating the discovery and validation of PROTAC candidates.