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Protease-Containing Nanobodies for Detecting and Manipulating Intracellular Antigens Using Antiviral Drugs
Quan Le1,2, John T Ngo1,2
1Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
ACS Chemical Biology
|May 12, 2025
Summary
Researchers developed a novel protease-based method to control protein-protein interactions (PPIs) using small molecules. This strategy utilizes self-cleaving nanobodies engineered with hepatitis C virus NS3 protease, enabling drug-dependent modulation of biological systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Controlling protein-protein interactions (PPIs) is crucial for understanding and engineering biological processes.
- Existing tools for modulating PPIs often lack precise temporal and dose-dependent control.
- Nanobodies offer a versatile scaffold for protein engineering, but their stability and function can be challenging to regulate.
Purpose of the Study:
- To develop a novel chemogenetic system for inducible control of PPIs using small molecules.
- To engineer self-cleaving nanobodies whose antigen-binding activity can be modulated by protease inhibitors.
- To demonstrate the application of this system for controlling intracellular signaling pathways.
Main Methods:
- Engineered nanobody scaffolds by inserting the hepatitis C virus (HCV) NS3 cis-protease.
- Developed chimeric nanobodies that undergo self-cleavage in the presence of NS3 protease activity.
- Utilized NS3 protease inhibitors to control the antigen-binding ability of the engineered nanobodies in a dose-dependent manner.
- Validated the system using nanobodies targeting fluorescent proteins (mCherry, eGFP), a peptide tag (ALFA), and a G-protein coupled receptor (β2-adrenergic receptor).
Main Results:
- Demonstrated that antigen-binding of engineered nanobodies can be precisely controlled by NS3 inhibitors.
- Successfully designed and validated drug-controllable nanobodies against various targets, including mCherry, eGFP, and the ALFA tag.
- Showed that an NS3-containing nanobody targeting the β2-adrenergic receptor could modulate endogenous G-protein-mediated signaling.
- Established a generalizable chemogenetic platform for controlling intracellular PPIs.
Conclusions:
- Introduced a novel protease-based strategy for creating drug-controllable nanobodies.
- This system allows for precise, small-molecule-mediated regulation of PPIs and cellular signaling.
- The developed chemogenetic components utilize clinically approved antiviral drugs, offering a promising tool for biological research and therapeutic applications.
