Activity-Based DNA-Encoded Library Screening for Selective Inhibitors of Eukaryotic Translation.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Most human proteins lack druggable binding pockets, limiting small molecule probe development to ~2% of the proteome.
• Traditional high-throughput screening assays are often not feasible for many protein targets.

Purpose of the Study:

• To develop a universal screening assay for discovering small molecule probes targeting proteins with challenging or unknown functions.
• To circumvent limitations of canonical druggability and assay development.

Main Methods:

• Developed an in vitro transcription-translation (IVTT) activity assay by fusing reporter genes (e.g., GFP) to target sequences.
• Screened a 5,348-member DNA-encoded library (DEL) for translation inhibitors using microfluidic picoliter-scale droplets.
• Validated hits using a PCSK9-GFP reporter, including cellular assays and preliminary selectivity profiling.

Main Results:

• Identified multiple inhibitors of PCSK9-GFP IVTT, with the lead compound reducing PCSK9 levels in HepG2 cells.
• Demonstrated a plug-and-play approach by screening the DEL against diverse targets like RPL27, KRASG12D, MST1, and USO1.
• Achieved IC50 values between 1-20 μM for validated hits.

Conclusions:

• The microfluidic IVTT DEL screening platform offers a scalable solution for probe discovery against a wider range of protein targets.
• This approach significantly expands the potential for identifying chemical probes for the human proteome and beyond.
• Selective translation modulation provides a viable strategy for overcoming traditional drug discovery constraints.