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Characterizing PROTAC ternary complex formation with mass photometry 

The PROTAC technology 

 

PROTACs (PROteolysis-TArgeting Chimeras) are emerging therapeutic modalities that degrade unwanted or harmful proteins in cells. A PROTAC molecule consists of three elements: a moiety that binds to the target protein of interest, a moiety that binds to a E3 ligase (an enzyme responsible for targeted protein degradation), and a linker connecting these moieties. By drawing a protein of interest into a ternary complex with the E3 ligase, PROTACs induce the target protein degradation through the cellular proteasome machinery.

Protac cellular sketch print_edited.jpg

How to measure PROTAC ternary complex formation? 

Illustration of a protein complex.

When applied to PROTAC assays, mass photometry – a label-free technology – offers insights into the dynamics of ternary complex formation. With the ability to measure the mass of single biomolecules in solution, it enables the characterization of PROTAC-driven interactions with the target protein and the E3 ligase. This means that you can use mass photometry to assess a range of behaviors that are important for the PROTAC mechanistic function: ternary complex formation, cooperativity, stoichiometry and ‘hook effect’. 

Biophysical characterization of the PROTAC ‘hook effect’

 

Mathematical models predict a bell-shaped dependency on PROTAC concentration. At high concentrations, ineffective binary complexes are observed, which compete with effective ternary complexes. Competition leads to a decline in the formation of ternary complexes, ultimately impacting the potency of the PROTACs. This is known as the ‘hook effect’. Mass photometry can be used to quantify relative concentrations of intermediate species and assess their cooperativity effects. More specifically, you can determine the concentration range where maximal complex formation occurs and identify PROTAC compounds with significant positive cooperativity in the ternary complex, which do not present an observable ‘hook effect’.  

To learn how mass photometry can help characterize protein interactions and complexes 

Mass photometry for the study of PROTAC ternary complexes

Image of application note - Formation of PROTAC ternary complexes measured with mass photometry

Learn how mass photometry can be used to inform on aspects of PROTAC ternary complex formation in solution, without requiring protein labelling or immobilization. These aspects involve a range of behaviors important for PROTAC mechanistic function: ternary complex formation, cooperativity, stoichiometry, and hook effect. Discover how you can apply mass photometry to visualize the possible intermediate interactions, due to cooperative effects, in a three-part PROTAC system. The system presented in the application note includes the target protein BRD4 (implicated in the development of several cancers), the VHL/ElonginC/ElonginB ligase and the PROTAC compound MZ1.

Additional resources​

BLOG

Exploring the structural dynamics of shelterin with mass photometry

Learn how mass photometry can be used to study the compositional heterogeneity, stoichiometry, and stability of a multimeric complex. Find out how it complements other bioanalytical techniques, such as native mass spectrometry and negative-stain EM. 

Image depicting the webinar title : The evolution of carbon fixation: Studying Form I Rubisco using ancestral sequence reconstruction and mass photometry
Image depicting John Zinder, superimposed with the title of the webinar "Exploring the structural dynamics of shelterin with mass photometry"

WEBINAR​

The evolution of carbon fixation: Studying Form I Rubisco using ancestral sequence reconstruction and mass photometry

Discover how mass photometry can be used in combination with ancestral sequence reconstruction to determine when an ancestral enzyme gained its structural complexity and how it started to interact with a novel subunit.

To discuss how mass photometry can be applied to the characterization of PROTAC complex formation

More Application Notes

Browse through our catalogue of application notes highlighting some recent case studies featuring mass photometry.

Investigating lentivirus titer with macro mass photometry

 

Mass photometry supports membrane protein purification protocols 
 

Characterizing protein oligomerization with automated mass photometry

Quantifying heterogeneous AAV capsid loading using mass photometry
 

Download the application note

Download application note
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