Allosteric nanobodies to study the interactions between SOS1 and RAS.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions, but tools for their characterization are limited.
• Understanding and modulating PPIs is key for deciphering cell metabolism and developing therapeutics.

Purpose of the Study:

• To develop novel, broadly applicable methods for discovering nanobodies that modulate PPIs.
• To identify nanobodies targeting the SOS1•RAS complex for potential therapeutic applications.

Main Methods:

• Development of Cross-link PPIs and immunize llamas (ChILL) for nanobody discovery.
• Implementation of Display and co-selection (DisCO) strategies for nanobody selection.
• In vitro and in cellulo validation of nanobody function on the SOS1•RAS complex and RAS signaling.

Main Results:

• ChILL and DisCO successfully identified diverse nanobodies with inhibitory or stabilizing effects on the SOS1•RAS complex.
• Identified nanobodies modulated RAS nucleotide exchange and downstream signaling.
• A connective nanobody with a long CDR3 occupied a therapeutic lead compound binding pocket.

Conclusions:

• ChILL and DisCO are powerful, versatile tools for discovering functional nanobodies against PPIs.
• The identified nanobodies offer new avenues for modulating RAS signaling and developing targeted therapies.
• The unique CDR3 of a discovered nanobody presents a novel pharmacophore for drug discovery.