Development of a Selection Method for Discovering Irreversible (Covalent) Binders from a DNA-Encoded Library
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Updated: Feb 3, 2026

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay
Published on: February 28, 2025
Zhengrong Zhu1, LaShadric C Grady1, Yun Ding1
11 GlaxoSmithKline, Cambridge, Massachusetts, USA.
Abstract:
DNA-encoded libraries (DELs) have been broadly applied to identify chemical probes for target validation and lead discovery. To date, the main application of the DEL platform has been the identification of reversible ligands using multiple rounds of affinity selection. Irreversible (covalent) inhibition offers a unique mechanism of action for drug discovery research. In this study, we report a developing method of identifying irreversible (covalent) ligands from DELs. The new method was validated by using 3C protease (3CP) and on-DNA irreversible tool compounds (rupintrivir derivatives) spiked into a library at the same concentration as individual members of that library. After affinity selections against 3CP, the irreversible tool compounds were specifically enriched compared with the library members. In addition, we compared two immobilization methods and concluded that microscale columns packed with the appropriate affinity resin gave higher tool compound recovery than magnetic beads.
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