SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate.

Michael J Roy,Scott J Hughes,Michael Galant,Claire Whitworth,Alessio Ciulli,Sandra Winkler,William Farnaby,Klaus Rumpel

doi:10.1021/acschembio.9b00092

Michael J Roy, Scott J Hughes + Show 6 more

Open Access

https://doi.org/10.1021/acschembio.9b00092

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Abstract

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted 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 in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. 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 degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

Highlights

Proteolysis-targeting chimeras (PROTACs) are bivalent molecules consisting of ligands for each of a target protein and an E3 ligase, joined via a linker.[1,2] PROTAC behavior can be modeled by three-body binding equilibria.[3]
Cooperativity assays based on competitive binding have been used to determine binding and cooperativity of PROTACs.[11]
Promising cell-based assays have been reported that enable real-time kinetic monitoring of PROTAC ternary complexes in cells, including the kinetic EGFP separation of phase-based protein interaction reporter (SPPIER)[18] and kinetic bioluminescence resonance energy transfer (BRET)[19] approaches

Summary

Introduction

Proteolysis-targeting chimeras (PROTACs) are bivalent molecules consisting of ligands for each of a target protein and an E3 ligase, joined via a linker.[1,2] PROTAC behavior can be modeled by three-body binding equilibria.[3]. Using a Biacore T200 SPR instrument and streptavidinimmobilized biotin−VHL, we measured the kinetics and affinity of VHL binding for a concentration series of either PROTAC alone (to form a binary complex with VHL, KDbinary) or PROTAC pre-incubated with near-saturating concentrations of target protein (to form a ternary complex with VHL, KDternary; Figure 1).

Results

Conclusion