Abstract
Introduction Proteolysis Targeting Chimaeras (PROTACs) offer a powerful strategy to degrade cancer-associated proteins. PROTACs recruit an E3 ubiquitin ligase, such as von Hippel-Lindau (VHL), to a protein of interest (POI) to form a ternary complex. Measuring the formation of this ternary species is important for understanding PROTAC mode of action. Proximity AlphaLISA assays can be used for this purpose but have, to date, focused on using purified recombinant protein in vitro . Here, we report the development and optimisation of an AlphaLISA assay for monitoring ternary complex formation using full-length POI within cell lysates. Material and methods AlphaLISA assays were used to profile PROTAC-induced complex formation of FLAG-tagged full-length POI in cell lysate to purified biotinylated VCB. Signal was measured from close proximity of FLAG-tagged acceptor beads to streptavidin donor beads and benchmarked against a well-characterised PROTAC, MZ1, purified His-tagged Brd4 bromodomain 2 (BD2) and biotinylated VCB. A FLAG-tagged full-length POI containing an insert of Brd4 BD2 (POI-fusion) was utilised, such that MZ1 could be employed as a tool compound for assay development. Results and discussions Signal intensity was dependent on total lysate protein and VCB concentration, allowing for optimisation of component concentrations for maximal signal. A mild, detergent-free hypotonic cell lysis buffer gave enhanced signal over harsher buffers containing detergents. Additionally, the cell lysate cytosolic fraction provided superior signal compared to nuclear fractions and whole cell lysates, further highlighting the reliance of transient ternary complex formation on assay conditions. By utilising these optimal assay conditions determined with the POI-fusion, it is now possible to detect ternary complex formation between POI-targeting PROTACs and full-length POI. Conclusion To our knowledge, this optimised cell lysate AlphaLISA assay provides the first report and proof of concept for the interrogation of PROTAC-induced complex formation of full-length proteins with E3 ligases, which will be a valuable tool to the burgeoning PROTAC field. Consideration of assay parameters that permit optimal conditions for ternary complex formation in a POI-dependent manner allows the application of the AlphaLISA method as a screening platform. This application could profile PROTAC efficiency for a wide range of full-length, cell-derived proteins that are more relevant to the endogenous context encountered by the PROTAC intracellularly.
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