Single-Molecule-Based, Label-Free Monitoring of Molecular Glue Efficacies for Promoting Protein-Protein Interactions...

Area of Science:

• Biophysics
• Nanotechnology
• Drug Discovery

Background:

• Modulating protein-protein interactions (PPIs) is crucial for drug discovery, particularly for targeting previously undruggable proteins.
• Molecular glues are bifunctional molecules that promote PPIs, offering a promising therapeutic strategy.
• Conventional methods for discovering molecular glues are limited, hindering efficient drug development.

Purpose of the Study:

• To introduce a novel YaxAB nanopore system for probing molecular glue efficacy in inducing PPIs.
• To demonstrate single-molecule, label-free monitoring of protein complex formation using the YaxAB nanopore.
• To enable ultrasensitive screening and structure-activity relationship (SAR) analysis of molecular glues.

Main Methods:

• Utilized a funnel-shaped YaxAB nanopore with a wide entrance and adjustable pore size.
• Employed electro-osmotic flow (EOF) for simultaneous detection and quantification of multiprotein states.
• Applied single-molecule-based, label-free detection to monitor protein complex formation.

Main Results:

• Successfully monitored protein complex formation between mTOR and FKBP proteins induced by the molecular glue rapamycin.
• Achieved simultaneous detection and single-molecule quantification of single, binary, and ternary protein complexes.
• Demonstrated sensitive discrimination between rapamycin-induced complexes and those induced by its analogues, based on subtle mass differences.

Conclusions:

• The YaxAB nanopore system provides a proof-of-concept for single-molecule, label-free, and ultrasensitive screening of molecular glues.
• This approach facilitates efficient discovery and rational design of molecular glues and other bifunctional drugs.
• The method holds potential for low-cost, highly efficient drug development targeting complex diseases.