Abstract
Heterobifunctional small-molecule degraders known as Proteolysis Targeting Chimeras (PROTACs) serve as a chemical bridge bringing into direct association a target protein with an active E3 ligase complex, called the ternary complex, to facilitate targeted protein degradation. This ternary complex formation is the first key mechanistic step in a cascade of events that results in ubiquitination and subsequent degradation of the target protein via the ubiquitin-proteasome pathway. The ternary complex, however, is a nonnative cellular complex; therefore, PROTAC compound design has many challenges to overcome to ensure successful formation, including achieving structural and electrostatic favorability among target and ligase. Due to these challenges, finding successful PROTACs typically requires testing of extensive libraries of heterobifunctional compounds with varying linkers and E3 handles. As PROTAC ternary complex formation is also critically dependent on cellular context, live cell assays and technologies for rapid and robust screening are highly enabling for triaging of early stage compounds. Here, we present cellular assays utilizing NanoBRET technology for the study of ternary complexes, showing examples with two most popular PROTAC E3 ligase components, VHL (von Hippel-Lindau disease tumor suppressor) and CRBN (Cereblon). These assays can be run in either endpoint or real-time kinetic formats, are compatible with high-throughput workflows, and provide insight into how altering the PROTAC chemical composition affects the formation and stability of the ternary complex in live cells.
Published Version
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