A High-Throughput Assay to Identify Specific Nascent Chain Inhibitors

"Undruggable" proteins without surface-accessible binding sites pose significant challenges to target-based drug discovery. Innovative approaches are needed to tackle these proteins. One promising strategy is targeting them in their nascent chain form at the ribosome, where they have a different conformation than in the folded form. A systematic approach to screen for such compounds has however been lacking. Here, we present a high-throughput assay to identify small molecules that specifically inhibit a protein of interest in its nascent-chain form. The assay employs a human in vitro transcription/translation system and monitors expression of the protein in real time via fluorescence detection. Specific inhibitors for the nascent chain of interest can then be identified by comparison with a counter screen. The assay was optimized to maximal sensitivity and reaction costs of ~$0.01 per well, enabling large-scale screens. We validated performance with the reference compound PF846 on the nascent chain of the protein PCSK9. Feasibility for high-throughput screening was demonstrated using a library of 1,760 compounds against the oncogenic KRAS variant A146T and the protein ApoC3, with mean Z' scores of 0.69 and 0.88, respectively. Sixteen global translation inhibitors were identified in each campaign, while no compounds met the criteria for POI-selective inhibition. The assay thus provides a robust platform for larger-scale screening campaigns.