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.
How to measure PROTAC ternary complex formation?
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’.
Mass photometry for the study of PROTAC ternary complexes
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.
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