Deep seeking covalent DNA encoded library for novel JAK3 inhibitor discovery

To better understand how pre-installed covalent warheads affect the ligand discovery in DNA encoded library (DEL), we have designed three individual covalent DELs incorporating 7, 32 and 64 cysteine-targeting covalent warheads respectively, and screened these DELs against JAK3 purified protein. The experiments resulted 6 novel series of covalent inhibitors with drug-like properties, where the most potent compounds achieved picomolor IC₅₀ and good selectivity against a mini panel of kinases. The mass spec study confirmed their covalent MOAs by targeting JAK3 Cys909. More importantly, we confirmed the synergistic effect of the binding moiety and warhead by comparing the activities with their close analogs, suggesting that these compounds may not able to be designed by installation of covalent warheads to reversible binders. Further analysis revealed that 7 warheads were sufficient for identifying JAK3 covalent ligands. This work deepens our understanding of the design and screening of covalent DEL, and also demonstrate the power of DEL in the identification of diverse inhibitors.