From Photo-crosslinking to Covalent: A Cascade DNA-Encoded Library Strategy for Pin1 Inhibitor Discovery

Peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (Pin1) plays vital biological roles in vivo and has been studied as a therapeutic target for a variety of human diseases. Covalent inhibitors represent the mainstream strategy in current Pin1 drug discovery, however, existing covalent molecules still rely on known affinity scaffolds or direct screening. Herein, we developed an innovative cascade DNA-encoded library (DEL) strategy that integrates photo-crosslinking with covalent targeting, enabling the de novo design of novel Pin1 covalent inhibitors. Photo-crosslinking DEL (PDEL) selection was first used to identify low-affinity binding fragments. These fragments then served as the beginning for covalent focused DELs (Co-FDEL) construction. Finally, covalent DEL selection led to the identification of two hits, LWH-1 and LWH-2, which covalently modify Pin1 at Cys113, with IC50 values of 447.4 nM and 273.7 nM. This strategy facilitates the rational evolution of low-affinity fragments to potent covalent inhibitors, offering an accelerated pathway for targeting challenging proteins.