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

Overview

Journal ACS Cent Sci

Specialty Chemistry

Date 2024 Oct 28

PMID 39463829

Authors

Huda Barhoosh

Anjali Dixit

Wesley G Cochrane

Valerie Cavett

Robin N Prince

Brooke O Blair

Fred R Ward

Kim F McClure

Phillip A Patten

Margot G Paulick

Brian M Paegel

Affiliations

Soon will be listed here.

Abstract

Small molecule probes exist for only ∼2% of human proteins because most lack functional binding pockets or cannot be assayed for high-throughput screening. Selective translation modulation circumvents canonical druggability and assay development constraints by using in vitro transcription-translation (IVTT) as a universal biochemical screening assay. We developed an IVTT activity assay by fusing a GFP reporter to various target gene sequences and screened the target sequences for inhibitors in microfluidic picoliter-scale droplets using a 5,348-member translation inhibitor DNA-encoded library (DEL). Screening a proof-of-concept PCSK9-GFP reporter yielded many hits; 6/7 hits inhibited PCSK9-GFP IVTT (IC 1-20 μM), and the lead hit reduced PCSK9 levels in HepG2 cells. Preliminary selectivity was informed by counterscreening the DEL against a frameshift mutant PCSK9-GFP reporter. A plug-and-play approach to assay development and screening was demonstrated by scouting the DEL for activity using reporter genes of targets with difficult-to-assay or even unknown function (RPL27, KRAS, MST1, USO1). This microfluidic IVTT DEL screening platform could scale probe discovery to the human proteome and perhaps more broadly across the tree of life.

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