Orthogonal Light- and Enzyme-Triggered and-Gate Prodrugs for Senolytic Therapy.

Senescence-associated β-galactosidase (SA-β-gal) is widely used to target senescent cells, but its activity in non-senescent tissues can limit selectivity. Here, we developed a light-enzyme AND-gate strategy in which an o-nitrobenzyl photocage blocks β-galactoside cleavage until 390 nm irradiation. The strategy was evaluated using a fluorescent probe (LS-C), a doxorubicin prodrug (LS-D), and an ARV-771-based PROTAC (LS-A). LS-C exhibited dual-trigger-dependent fluorescence activation, while LS-D preferentially induced apoptosis and LS-A promoted BRD4 degradation in senescent cells following light activation. Across multiple senescence models, LS-D and LS-A showed higher senolytic indices than their corresponding parent compounds. In senescent lung cancer patient-derived organoids, LS-D combined with light increased organoid death, whereas either treatment alone had minimal effects. These results provide proof-of-concept evidence for sequential light-enzyme AND-gating as a strategy for controlling payload activation in senescent cells under the tested in vitro and ex vivo conditions.