Warhead Strategy forTargeted Protein S‑Nitrosation

Protein post-translational modifications (PTMs) are critical factors in disease progression. While progress has been made in regulating disease-associated PTMs such as phosphorylation and acetylation, the approach to achieving targeted S-nitrosation for disease treatment, particularly in vivo, remains largely uncharted. This challenge arises because S-nitrosation is primarily mediated by nitric oxide (NO), a gaseous free radical that complicates efforts to confine the effects of NO to a specific protein. Herein, we designed and synthesized α-(NONOate-O²-yl) methyl acrylamides as in situ NO-releasing warheads linked to a Bruton’s tyrosine kinase (BTK)-specific skeleton, resulting in targeted S-nitrosation agents (TSNO1–6). These TSNO compounds covalently bind to BTK, releasing NO in situ, which then reacts with BTK to achieve targeted S-nitrosation. Notably, cellular and tissue proteomic studies confirmed that TSNO1 effectively accomplished targeted S-nitrosation of BTK at Cys527, demonstrating promising in vitro and in vivo antitumor activity. Mechanistic studies indicated that S-nitrosation at Cys527 enhanced phosphorylation inhibition at Tyr551 compared to ibrutinib, impeding BTK activation and providing an additional therapeutic benefit beyond covalent inhibition. In addition, we generally extended this strategy to FGFR4 and HER2. Collectively, utilizing the warhead strategy enables us to implement targeted modulation of S-nitrosation.