Development of a Split Esterase for Protein-Protein Interaction-Dependent Small-Molecule Activation

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Split reporters (fluorescent proteins, luciferases) are vital for measuring biological interactions.
• Biosensors are key for engineered gene circuits in synthetic biology.
• A general method for generating small molecules based on biological signals is currently lacking.

Purpose of the Study:

• To develop a proximity-dependent split esterase for interaction-dependent small molecule unmasking.
• To demonstrate the versatility of ester-protected small molecules for generating various probes.
• To explore applications in protein-protein interaction studies, drug development, and cell-based therapies.

Main Methods:

• Developed a proximity-dependent split esterase system.
• Utilized ester-protected small molecules for probe generation (fluorescent, chemiluminescent, pharmacological).
• Integrated the split esterase with ester-masked probes for interaction detection and cell killing.

Main Results:

• Demonstrated fluorescent, chemiluminescent, and pharmacological probe generation via ester unmasking.
• Successfully measured protein-protein interactions and inhibitor engagement using the split esterase system.
• Showcased selective small-molecule-dependent cell killing and intercellular killing via bystander effect.
• Confirmed compatibility with other split reporter systems for multiplexed detection.

Conclusions:

• The split esterase system is a versatile addition to the split protein toolbox.
• This system offers significant potential for synthetic biology applications, including targeted therapies.
• Enables precise control over small molecule generation in response to biological signals.