Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Targeting intracellular protein interactions is crucial for drug development.
• Variable domains of the immunoglobulin heavy chain (VH domains) are promising miniproteins but face challenges with intracellular stability and expression.

Purpose of the Study:

• To engineer a stable, disulfide-free VH domain suitable for intracellular applications.
• To develop a high-diversity phage display library using this engineered VH domain scaffold.
• To identify high-affinity VH domains targeting eukaryotic initiation factor 4E (eIF4E), a protein frequently overexpressed in cancer.

Main Methods:

• Construction of a high-diversity phage display library based on an autonomous, disulfide-free VH domain.
• Affinity maturation techniques to select VH domains with high binding affinity for eIF4E.
• Structural analysis to determine the binding site and pose of VH domains on eIF4E.
• Assessment of the effects of intracellular VH domain overexpression on cancer cell proliferation and malignancy-related protein expression.

Main Results:

• An autonomous, disulfide-free VH domain was successfully developed for intracellular applications.
• Picomolar affinity VH domains targeting eIF4E were identified using the engineered library.
• These VH domains bind to eIF4E at the eIF4G binding site through a unique structural interaction.
• Intracellular overexpression of these VH domains significantly reduced cancer cell proliferation and malignancy-related protein expression.

Conclusions:

• Engineered VH domains are suitable for intracellular applications, overcoming previous stability limitations.
• The developed VH domains targeting eIF4E demonstrate therapeutic potential for cancer treatment.
• This approach facilitates the discovery of intracellularly applicable VH domains for various biological targets.