Covalent Affibody‐Molecular Glue Drug Conjugate Nanoagent for Proximity‐Enabled Reactive Therapeutics

Abstract Sulfur‐fluoride exchange (SuFEx) reaction is an emerging class of click chemistry reaction. Owing to its efficient reactivity under physiological conditions, SuFEx reaction is used to construct covalent protein drugs. Herein, a covalent affibody‐molecular glue drug conjugate nanoagent is reported, which can irreversibly bind with its target protein through proximity‐enabled SuFEx reaction. As a proof of concept, a latent bioreactive unnatural amino acid fluorosulfate‐L‐tyrosine (FSY) is first introduced at site 36 of the affibody with cysteine mutation (Z HER2:342 ‐Cys) to produce Z HER2:342 ‐36 FSY ‐Cys. Subsequently, Z HER2:342 ‐36 FSY ‐Cys is coupled with a molecular glue drug (CR8) to yield an amphiphilic conjugate of Z HER2:342 ‐36 FSY ‐CR8, which can self‐assemble into affibody–drug conjugate nanoagent (Z HER2:342 ‐36 FSY ‐CR8 ADCN) in PBS. When Z HER2:342 ‐36 FSY ‐CR8 ADCN specific binds to human epidermal growth factor receptor 2 (HER2) on cancer cells, the FSY36 of Z HER2:342 approaches to the His490 of HER2 and ultimately reacts with each other to form a covalent bond via SuFEx reaction. Such a covalent binding mode endows Z HER2:342 ‐36 FSY ‐CR8 ADCN with permanent binding ability to effectively increase the concentration of drugs in tumor. Eventually, the covalent Z HER2:342 ‐36 FSY ‐CR8 ADCN exhibits an outstanding tumor inhibition ratio of 90.03 ± 4.29% in HER2‐positive ovary tumor models, strikingly higher than that of the noncovalent one (64.25 ± 7.71%).