Development of a Selection Method for Discovering Irreversible (Covalent) Binders from a DNA-Encoded Library

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.

Exploring the Lower Limit of Individual DNA-Encoded Library Molecules in Selection

“…The binding process is determined by the reversible interaction of the small molecules with targets. In some studies, the DEL molecules were with covalent warheads, 22 cross-linking ligands, 23 or target proteins conjugated with complementary DNA tags 24 ; the selection strategies in these cases were very different, and our conclusion does not apply.…”

Recent Advances on the Selection Methods of DNA‐Encoded Libraries

“…Indeed, DELs have been used to identify covalent inhibitors for c-Jun N-terminal kinase (JNK-1) [46] and 3C protease (3CP). [47] In addition, the Winssinger group used PNA (peptide nucleic acid)-encoded libraries and identified novel covalent inhibitors for bromodomains, [48] MEK2, and ERBB2, [49] although DNA microarray was used for hit identification. By using an in-solution selection method named IDUP (Figure 3b), [50] Liu and co-workers discovered that ethacrynic acid, a known ligand for glutathione S-transferase, is a covalent inhibitor for MAP2 K6 kinase.…”

“…[51] To discover covalent inhibitors, electrophilic "warheads", such as the α,β-unsaturated carbonyl motif, are often incorporated in the library to react with the nucleophilic side chains of lysines and cysteines, and the selections are performed under stringent conditions to remove non-covalent binders, such as SDS washes [49] or heating. [47]…”

Encoded Library Technologies as Integrated Lead Finding Platforms for Drug Discovery

“…Some of the abovementioned limitations may be overcome when using covalently binding ligands. In this case, enrichment is not primarily governed by reversible binding constants, making the identification of less frequently occurring binding events possible [97,98]. Both platforms require the target to be well behaved under screening conditions.…”