A pH-Sensitive NIR Fluorescent Probe as a Protagonist in the Discovery of New LC3 Ligands Facilitating the Construction of ATTECs.

Autophagy-tethering compounds (ATTECs) as a new targeted protein degradation technology could directly bind targets to LC3 (a key autophagosome-associated protein) and subsequently result in the degradation of targets via the autophagolysosomal pathway. Herein, we developed a new LC3 ligand screening strategy using an NIR fluorescent probe with both pH-sensitive and LC3-targeted features. The presence of both the probe and a potential LC3 ligand leads to competitive binding to LC3 in cells and hinders the probe from entering and being activated in acidic lysosomes via the autophagy pathway. Notably, LD5, an in-house compound of our lab, was screened out as a potential LC3 ligand by the strategy, and its capacity of binding to LC3 was further verified by SPR technology. By using LD5 as the LC3 binding moiety, two ATTECs were synthesized, which exhibited significant activities in degrading PCSK9 and lipid droplets, respectively, and further validated the feasibility of our LC3 ligand screening strategy.