Proximity Biosensor Assay for PROTAC Ternary Complex Analysis

Ternary complexes, consisting of two proteins connected by small molecules like PROTACs or molecular glues pose new challenges for the analysis of molecular interactions, because they depend not only on binary affinities, but are orchestrated by cooperativity and avidity effects. Here, we introduce a proximity binding assay for the simultaneous measurement of binary and ternary interaction kinetics on a biosensor surface. Target proteins and ubiquitin E3 ligase substrate receptors are tethered to mobile swivel arms of a Y-shaped DNA scaffold, which presents them in close proximity to PROTAC analytes flown across the sensor. PROTAC-induced ternary complex formation is measured by fluorescence energy transfer (FRET), while binary interactions are detected by fluorescence quenching. The assay is applied to cereblon (CRBN) and von Hippel-Lindau (VHL) as E3 ligase substrate receptors, a range of compounds including AT1, MZ1, dBETs, and ARV-825 as PROTACs, and the two bromodomains of Brd2, Brd3, Brd4, and BrdT proteins as targets. Automated workflows enable the measurement of 384 real-time sensorgrams in a single run using picomo-lar sample quantities. Ternary and binary binding kinetics and proximity-mediated binding enhancements are analyzed. Ternary complex stability is shown to arise from a dynamic interplay of associations and dissociations, suggesting that proximity assays can be utilized to identify weak interactions. The insights into proximity-mediated binding kinetics can enable the development of PROTACs and molecular glues with improved properties for targeted protein degradation.