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

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%).