Activity-Based\nDNA-Encoded Library Screening for Selective\nInhibitors of Eukaryotic Translation

Small molecule probes\nexist for only ∼2% of human\nproteins\nbecause most lack functional binding pockets or cannot be assayed\nfor high-throughput screening. Selective translation modulation circumvents\ncanonical druggability and assay development constraints by using\nin vitro transcription–translation (IVTT) as a universal biochemical\nscreening assay. We developed an IVTT activity assay by fusing a GFP\nreporter to various target gene sequences and screened the target\nsequences for inhibitors in microfluidic picoliter-scale droplets\nusing a 5,348-member translation inhibitor DNA-encoded library (DEL).\nScreening a proof-of-concept PCSK9-GFP reporter yielded many hits;\n6/7 hits inhibited PCSK9-GFP IVTT (IC<sub>50</sub> 1–20 μM),\nand the lead hit reduced PCSK9 levels in HepG2 cells. Preliminary\nselectivity was informed by counterscreening the DEL against a frameshift\nmutant PCSK9-GFP reporter. A plug-and-play approach to assay development\nand screening was demonstrated by scouting the DEL for activity using\nreporter genes of targets with difficult-to-assay or even unknown\nfunction (RPL27, KRAS<sup>G12D</sup>, MST1, USO1). This microfluidic\nIVTT DEL screening platform could scale probe discovery to the human\nproteome and perhaps more broadly across the tree of life.