A galactose-caged BCL-xL PROTAC activated exclusively by senescent-cell-high lysosomal β-galactosidase, with an AI-op...
IF GalPROTAC-753b — a bifunctional degrader comprising: (i) an ABT-263-derived BCL-xL warhead, (ii) a VHL-recruiting ligand with an AI-optimized binding interface (designed via AlphaFold2/ProteinMPNN pipeline), and (iii) a β-D-galactose cap conjugated via a self-immolative linker to the VHL-recruiting arm, administered intraperitoneally at 5 mg/kg twice weekly for 8 weeks — is administered to 24-month-old male C57BL/6J mice,
THEN hepatic senescent cell burden will be reduced by ≥40% relative to vehicle, representing a ≥2-fold improvement in the senescent-to-non-senescent hepatocyte killing selectivity ratio compared to unmodified 753b PROTAC, measured by: (a) SA-β-gal+ hepatocyte frequency (X-gal histochemistry and C12FDG flow cytometry), (b) hepatic Cdkn2a (p16^INK4a^) and Cdkn1a (p21^CIP1^) mRNA by RT-qPCR, (c) circulating platelet counts (to assess thrombocytopenia-free window), and (d) SASP marker reduction (IL-6, IL-1α, MCP-1) in liver homogenate and plasma,
BECAUSE the following causal chain operates:
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Lysosomal SA-β-gal (GLB1) activity is markedly elevated in senescent hepatocytes relative to proliferating or quiescent non-senescent hepatocytes, enabling intracellular cleavage of the galactose cap on GalPROTAC-753b and liberation of the active VHL-recruiting domain exclusively within senescent cells — a mechanism directly validated for galactose-masked cytotoxic payloads in the GMD prodrug class, where senolytic activity was shown to depend on lysosomal GLB1 activity in a cell-autonomous manner (GLB1-dependent senolytic prodrug activation)[https://doi.org/10.1111/acel.13133].
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In non-senescent hepatocytes and platelets — which possess baseline-low lysosomal GLB1 activity — the galactose cap remains intact, sterically occluding the VHL ligand from binding Cullin2-VHL E3 ubiquitin ligase, preventing ternary complex (BCL-xL / GalPROTAC-753b / VHL) formation and thus rendering the compound pharmacologically inert in these populations [SPECULATIVE — cap occlusion steric model not yet experimentally validated for PROTAC galactose-caged architectures; inferred by analogy to GMD occlusion mechanism (https://doi.org/10.1111/acel.13133)].
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Following GLB1-mediated galactose cleavage, the self-immolative linker undergoes 1,6-elimination, releasing fully active 753b PROTAC intralysosomally / cytosolically; the VHL-binding arm engages the Cullin2-RING E3 complex, the BCL-xL warhead simultaneously binds BCL-xL's BH3-binding groove, and the resulting ternary complex catalyzes K48-linked polyubiquitination of BCL-xL — a mechanism confirmed for the unmodified VHL-recruiting BCL-xL PROTAC series (PROTACs degrade SCAP components via the ubiquitin-proteasome system)[https://pubmed.ncbi.nlm.nih.gov/41558613/].
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Senescent hepatocytes depend on BCL-xL upregulation as a primary senescent cell anti-apoptotic pathway (SCAP) component to evade apoptosis; catalytic BCL-xL degradation (rather than competitive inhibition) removes this survival signal a...
SENS category: GlycoSENS
Key references: • doi.org/10.1111/acel.13133]. • doi.org/10.1111/acel.13133 • doi.org/10.1038/s41467-020-18039-x]. • doi.org/10.1016/j.ebiom.2017.04.013]. • doi.org/10.1038/s41467-020-18039-x]