Tao Chen, Longying Cai, Xiaofei Dong, Lifang Zhang, Xuemin Cheng, Jingsong Qu, Guanyu Yang, Sen Gao, Linfu Luo, Huiyong Ma, Shuai Xia, Guansai Liu, Jin Li, Jianyou Shi, Dengfeng Dou

Abstract

To better understand how pre-installed covalent warheads affect the ligand discovery in DNA-encoded libraries (DELs), three individual covalent DELs incorporating 7, 32, and 64 cysteine-targeting covalent warheads, respectively, were designed and screened against JAK3 purified protein. The experiments resulted in 6 novel series of covalent inhibitors with drug-like properties, with the most potent compounds achieving picomolar IC50 and good selectivity against a mini panel of kinases. Mass spectrometry studies confirmed their covalent mechanisms of action (MOAs) by targeting JAK3 Cys909. Importantly, the synergistic effect of the binding moiety and warhead was confirmed by comparing the activities with their close analogs, suggesting that these compounds may not be designed by simply installing covalent warheads onto 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 DELs and demonstrates the power of DEL in the identification of diverse covalent inhibitors.

Summary

This study explores the impact of covalent warheads in DNA-encoded libraries (DELs) on ligand discovery by designing and screening three covalent DELs with varying numbers of cysteine-targeting warheads against JAK3. The results identified six novel series of covalent inhibitors with drug-like properties, achieving picomolar IC50 values and good selectivity. Mass spectrometry confirmed the covalent binding to JAK3 Cys909. The study highlights the necessity of covalent warheads for effective screening and demonstrates that a library with 7 warheads was sufficient for hit identification. This work provides insights into optimizing covalent DEL design and screening strategies for discovering potent and selective covalent inhibitors.

Highlights

- Covalent DNA-encoded libraries (CoDELs) with 7, 32, and 64 cysteine-targeting warheads were designed and screened against JAK3.

- Six novel series of covalent inhibitors with picomolar IC50 values and good selectivity were discovered.

- Mass spectrometry confirmed covalent binding to JAK3 Cys909.

- The study demonstrated the necessity of covalent warheads for effective screening and identified 7 warheads as sufficient for hit identification.

- The findings provide a robust framework for optimizing covalent DEL design and screening strategies.

Conclusion

This research demonstrates the power of DNA-encoded library (DEL) technology in discovering novel covalent inhibitors for JAK3. By incorporating covalent warheads into DELs, we identified six series of potent and selective covalent inhibitors with drug-like properties. The study highlights the importance of covalent warheads in achieving effective screening and confirms that a library with 7 distinct warheads is sufficient for identifying JAK3 covalent ligands. The findings provide valuable insights into optimizing covalent DEL design and screening strategies, paving the way for the discovery of novel covalent inhibitors for other challenging targets.