Oct 25, 2018

SPR-measured kinetics of PROTAC ternary complexes influence target degradation rate

BioRxiv : the Preprint Server for Biology
Michael J RoyAlessio Ciulli


Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, via a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no method to monitor ternary complex kinetics has been reported, and it remains to be addressed how this might impact on the kinetics of protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that form either cooperative or non-cooperative complexes. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular d...Continue Reading

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Mentioned in this Paper

Protein Degradation, Regulatory
Complex (molecular entity)
BRD4 protein, human
Chimera Organism
Protein Degradation, Metabolic
Protac snake venom protein C activator
Binding (Molecular Function)

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