IF a peracetylated β-D-galactopyranoside–doxorubicin prodrug (herein GalDox), comprising an acetyl-masked galactose trigger covalently linked to doxorubicin via a self-immolative p-hydroxybenzyl carbamate (PHB-carbamate) spacer, is administered systemically (intraperitoneal, 5–10 mg/kg, bi-weekly × 4 weeks) to naturally aged C57BL/6J mice (22–24 months, both sexes),

THEN selective depletion (≥50% reduction) of SA-β-gal-positive senescent cells across multiple tissues (liver, lung, white adipose, skeletal muscle), accompanied by ≥40% reduction in circulating SASP factors (IL-6, MMP3, GDF15), restoration of grip strength and rotarod performance toward young-reference baselines, and <20% cytotoxicity to passage-matched non-senescent primary fibroblasts in parallel in vitro selectivity assays will be observed,

BECAUSE the following causal chain operates:

1. Lysosomal SA-β-gal as the activation gate: Senescent cells overexpress GLB1-encoded lysosomal β-galactosidase at pH ~4.5, the biochemical basis of the SA-β-gal assay universally used to mark senescence. GLB1 knockdown abolishes the senolytic selectivity of β-galactosidase-activated prodrugs, confirming it is the functional activating enzyme (Knockdown of GLB1 diminishes selective toxicity of β-gal-activated prodrug SSK1)[https://doi.org/10.1038/s41422-020-0314-9].

2. Peracetylation enables cell permeability and lysosomal routing: Acetylation of the galactose hydroxyl groups renders the prodrug lipophilic and cell-permeable; cytosolic esterases deacetylate the sugar, restoring its hydrophilicity and trapping the prodrug within the endolysosomal compartment, where GLB1 is concentrated. This is the exact mechanism validated for Nav-Gal (Nav-Gal is cell-permeable due to acetylation and pharmacologically inactive in non-senescent cells)[https://doi.org/10.1111/acel.13142].

3. Self-immolative PHB-carbamate linker releases free doxorubicin intralysosomally: Upon GLB1-mediated glycosidic bond hydrolysis, the exposed phenol of the PHB spacer undergoes spontaneous 1,6-elimination, releasing unmodified doxorubicin. This linker chemistry is well-precedented in β-galactosidase-cleavable prodrug systems for GDEPT and has been adapted here for the senolytic context (Galactose-triggered self-immolative linker design is the basis of GMD prodrug cleavage)[https://doi.org/10.1111/acel.13133].

4. [SPECULATIVE] Elevated lysosomal membrane permeabilization (LMP) in senescent cells facilitates nuclear doxorubicin translocation: Senescent cells accumulate lipofuscin—an oxidatively cross-linked protein–lipid aggregate—inside lysosomes, imposing chronic lysosomal stress and partial LMP. This LMP vulnerability (documented in the GalNP system, where endolysosomal uptake is the first step toward intracellular drug release) (GalNPs are internalized by endocytosis and trafficked to lysosomes in senescent cells)[https://doi.org/10.15252/emmm.201809355] creates a senescence-specific "second gate" enablin...

SENS category: RepleniSENS

Key references: • doi.org/10.1038/s41422-020-0314-9]. • doi.org/10.1111/acel.13142]. • doi.org/10.1111/acel.13133]. • doi.org/10.15252/emmm.201809355] • doi.org/10.1007/s10522-020-09893-9].