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

Michael Roy, Sandra Winkler, Scott J Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio Ciulli (Lead / Corresponding author)

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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 Brd4 BD2 or Brd2 BD2 and VHL. Equivalent complexes with Brd3 BD2 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.

Original languageEnglish
Pages (from-to)361-368
Number of pages8
JournalACS Chemical Biology
Volume14
Issue number3
Early online date5 Feb 2019
DOIs
Publication statusPublished - 15 Mar 2019

Fingerprint

Proteolysis
Enzyme kinetics
Degradation
Ubiquitin-Protein Ligases
Assays
Proteins
Kinetics
Benchmarking
Ligases
Kinetic parameters
Half-Life
Tuning
Amino Acids
Molecules

Keywords

  • PROTACs
  • ternary complexes
  • surface plasmon resonance
  • kinetics
  • protein-protein interactions

Cite this

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title = "SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate",
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 Brd4 BD2 or Brd2 BD2 and VHL. Equivalent complexes with Brd3 BD2 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.",
keywords = "PROTACs, ternary complexes, surface plasmon resonance, kinetics, protein-protein interactions",
author = "Michael Roy and Sandra Winkler and Hughes, {Scott J} and Claire Whitworth and Michael Galant and William Farnaby and Klaus Rumpel and Alessio Ciulli",
note = "This project has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007−2013) as a Starting Grant to A.C. (grant agreement no. ERC-2012-StG-311460 DrugE3CRLs), and by Boehringer Ingelheim. Biophysics and drug discovery activities at Dundee were supported by Wellcome Trust strategic awards (100476/Z/12/Z and 094090/Z/10/Z, respectively) to the Division of Biological Chemistry and Drug Discovery.",
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SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. / Roy, Michael; Winkler, Sandra; Hughes, Scott J; Whitworth, Claire; Galant, Michael; Farnaby, William; Rumpel, Klaus; Ciulli, Alessio (Lead / Corresponding author).

In: ACS Chemical Biology, Vol. 14, No. 3, 15.03.2019, p. 361-368.

Research output: Contribution to journalArticle

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AU - Hughes, Scott J

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AU - Farnaby, William

AU - Rumpel, Klaus

AU - Ciulli, Alessio

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