Oral Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Measuring the kinetics of PROTAC ternary complexes. (#18)

Michael J Roy 1 , Sandra Winkler 2 , Scott J Hughes 1 , Claire Whitworth 1 , Michael Galant 2 , William Farnaby 1 , Klaus Rumpel 2 , Alessio Ciulli 1
  1. Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom
  2. Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

Proteolysis-targeting chimeras (PROTACs), bifunctional degrader molecules, represent an exciting modality for the development of novel chemical probes and therapeutics that induce protein degradation.1 PROTACs function by simultaneously binding to a target protein and an E3 ligase, forming an intermediate species consisting of a target:PROTAC:ligase ternary complex, leading ultimately to proximity-dependent ubiquitylation and degradation of the target protein via the Ubiquitin Proteasome System (UPS).2-4

Despite the essential role of this ternary intermediate, there is still an incomplete understanding of how the stability and dynamics of the PROTAC ternary complex may impact on degradation efficiency or be used as an optimisation parameter in PROTAC design.

Towards addressing this, we have recently developed an SPR-based assay to measure for the first time the kinetics of target:PROTAC:ligase ternary complex formation and dissociation. We have benchmarked the assay using PROTACs that bind to the bromodomain (BDs) of BET proteins and the E3 ligase VHL. Our data reveals marked differences in rates of ternary complex dissociation (off-rates) for different PROTAC/BD pairs. Further, we observe an apparent correlation between measured differences in ternary complex stability and relative initial intracellular rates of target degradation in response to PROTAC treatment. These studies establish a novel assay to inform future PROTAC development.

  1. Fisher, S. L.; Phillips, A. J., Targeted protein degradation and the enzymology of degraders. Curr Opin Chem Biol 2018, 44, 47-55.
  2. Douglass, E. F., Jr.; Miller, C. J.; Sparer, G.; Shapiro, H.; Spiegel, D. A., A comprehensive mathematical model for three-body binding equilibria. J Am Chem Soc 2013, 135 (16), 6092-9.
  3. Hughes, S. J.; Ciulli, A., Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem 2017, 61 (5), 505-516.
  4. Gadd, M. S.; Testa, A.; Lucas, X.; Chan, K. H.; Chen, W.; Lamont, D. J.; Zengerle, M.; Ciulli, A., Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 2017, 13 (5), 514-521.