Monitoring PROTAC interactions in biochemical assays using Lumit immunoassays.

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Discovering novel Proteolysis Targeting Chimeras (PROTACs) requires efficient assays to assess PROTAC-protein interactions and ternary complex formation.
• Existing methods may not be suitable for high-throughput screening, hindering the pace of new PROTAC molecule development.

Purpose of the Study:

• To optimize and implement Luminex immunoassays for measuring PROTAC binding and ternary complex formation in a biochemical format.
• To demonstrate the utility of Luminex assays in ranking small molecule and PROTAC affinities for specific protein targets.
• To validate Luminex assays for assessing PROTAC-mediated ternary complex formation involving target proteins and E3 ligases.

Main Methods:

• Optimization and execution of Luminex immunoassays in a biochemical setting.
• Utilizing Luminex assays to determine the binding affinities of small molecules and PROTACs to Bromodomain 4 (BRD4) isoforms (BD1, BD2).
• Measuring PROTAC-induced ternary complex formation between BRD4(BD1, BD2) and the VHL E3 ligase using Luminex technology.

Main Results:

• The Luminex immunoassays were successfully optimized for measuring PROTAC binding and ternary complex formation.
• The assays effectively ranked the binding affinities of various small molecules and PROTACs against BRD4(BD1, BD2).
• Biochemical assay results demonstrated strong correlation with data obtained from live and lytic cell-based assays.

Conclusions:

• Luminex immunoassays provide a robust and high-throughput compatible biochemical screening methodology for evaluating novel PROTAC molecules.
• These assays can accurately assess PROTAC-protein interactions and ternary complex formation, crucial for PROTAC discovery.
• The correlation with cell-based assays validates Luminex immunoassays as a reliable tool in the early stages of small molecule drug discovery.