Agonist Discovery for Membrane Proteins on Live Cells by Using DNA-encoded Libraries

Area of Science:

• Chemical Biology
• Molecular Biology
• Drug Discovery

Background:

• Identifying biologically active ligands for membrane proteins is crucial in chemical biology.
• Existing methods for discovering small molecule agonists often face challenges in directly targeting membrane proteins on live cells.

Purpose of the Study:

• To develop and demonstrate a novel approach for directly identifying small molecule agonists against membrane proteins using DNA-encoded libraries (DELs) on live cells.
• To validate the methodology using three key membrane proteins: epidermal growth factor receptor (EGFR), thrombopoietin receptor (TPOR), and insulin receptor (INSR).

Main Methods:

• Utilizing DNA-encoded libraries (DELs) for selection on live cells, connecting extracellular ligand binding with intracellular biochemical transformation.
• Employing large-scale DEL screening (up to 1.033 billion compounds) against selected membrane protein targets.
• Characterizing identified agonists for binding affinity, cellular activity, and downstream signaling pathway activation.

Main Results:

• Successfully discovered novel agonists with subnanomolar affinity and low micromolar cellular activities for EGFR, TPOR, and INSR.
• Identified insulin receptor (INSR) agonists that activate the receptor possibly via an allosteric site, showing synergistic effects with insulin and activating downstream pathways.
• Demonstrated that discovered INSR agonists do not activate the highly homologous insulin-like growth factor 1 receptor (IGF-1R), mitigating potential tumor progression risks.

Conclusions:

• Developed a "functional" DNA-encoded library selection approach for cell surface targets, enabling direct agonist identification.
• The presented methodology offers a widely applicable strategy for the discovery of agonists against various membrane proteins.
• This work advances the field of chemical biology by providing a powerful tool for functional screening and drug discovery targeting membrane proteins.