On-DNA Binder Confirmation: Increasing Confidence in DEL Hits.

DNA-encoded libraries (DELs) are used throughout small-molecule drug discovery to identify new lead compounds for protein targets. DEL hits are traditionally evaluated via off-DNA resynthesis and subsequent biological testing. This approach can be time- and resource-intensive, limiting the number of putative hits selected for follow-up. Additionally, these hits often fail to confirm off-DNA, leading to lost time and effort. Here, we introduce GSK's on-DNA hit resynthesis and binder confirmation workflow, which increases throughput and emulates the original library synthesis, thereby enabling identification of side product binders and increasing confidence in DEL hits. We utilized thermal shift, microscale thermophoresis, activity, and compound-immobilized SPR assays to develop a robust platform for the derisking of DEL hits. This is followed by a mass-spectrometry-based assay to identify specific binders in mixtures of on-DNA compounds. We share GSK's application of on-DNA binder confirmation to both evaluate and expand hits from DEL screens.