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Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

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Proligands with protease-regulated binding activity identified from cell-displayed prodomain libraries.

Jerry M Thomas¹, Patrick S Daugherty

¹Department of Chemical Engineering, University of California, Santa Barbara, 93106, USA.

Protein Science : a Publication of the Protein Society

|August 5, 2009

Summary

Researchers developed a method to discover protease-activated binding ligands (proligands) using engineered E. coli. This technique enhances ligand specificity for targeted therapies and diagnostics by requiring protease activation for binding.

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Area of Science:

Biotechnology
Molecular Biology
Drug Discovery

Background:

Developing targeted therapeutic and diagnostic agents requires ligands with high specificity.
Protease-activated ligands offer a mechanism for conditional binding, enhancing targeting precision.
Existing methods for discovering such ligands are limited.

Purpose of the Study:

To develop a general method for discovering protease-activated binding ligands (proligands).
To engineer combinatorial prodomain libraries displayed on E. coli for ligand discovery.
To identify proligands with protease-mediated switching activity for improved targeting specificity.

Main Methods:

Constructed combinatorial peptide libraries of candidate prodomains fused to a matrix metalloprotease-2 substrate linker and a vascular endothelial growth factor-binding peptide.
Employed a two-stage flow cytometry screening procedure to isolate proligands requiring protease treatment for binding.
Utilized two unique library design strategies and three sorting cycles to identify effective prodomains.

Main Results:

Successfully identified prodomains exhibiting protease-mediated switching activity.
The most effective proligand demonstrated a 100-fold enhancement in apparent binding affinity upon protease exposure.
Validated the efficacy of the developed screening method for proligand discovery.

Conclusions:

The developed method provides a generalizable approach for discovering protease-activated binding ligands.
This technique holds significant potential for creating novel therapeutic and diagnostic ligands with enhanced systemic targeting specificity.
The proligand discovery platform can be applied to various protease targets and binding domains.