A uPAR-targeted BCL-xL PROTAC fusion construct exploits senescence-specific uPAR surface induction to deliver catalyt...

IF a bispecific fusion construct — comprising a high-affinity uPAR Domain 1-binding peptide (KD <50 nM) covalently linked via a cleavable linker to a BCL-xL-recruiting PROTAC warhead (VHL or CRBN E3 ligase recruiter fused to a navitoclax-derived BCL-xL warhead) — is administered at 10–250 nM for 48 hours in vitro, or 10 mg/kg IV twice weekly for 4 weeks in vivo to 20-month-old male C57BL/6J mice,

THEN selective ubiquitin-proteasomal degradation of BCL-xL protein (≥70% reduction by quantitative Western blot normalized to GAPDH) and Annexin V-positive apoptosis will be observed specifically in therapy-induced senescent IMR90 ER:RAS fibroblasts but not in proliferating IMR90 controls (<15% BCL-xL degradation), and in vivo treatment will reduce p16Ink4a mRNA burden by ≥50% in at least two of three aged tissues (liver, lung, epididymal white adipose tissue) with platelet counts remaining within ≥80% of vehicle baseline — outcomes not achievable by untargeted ABT-263 or Nav-Gal at equivalent doses —

BECAUSE of the following mechanistic chain:

1. uPAR (PLAUR) is broadly and selectively upregulated on the surface of senescent cells across diverse tissue types, including therapy-induced, oncogene-induced, and naturally aged senescent cells, but is expressed at low or undetectable levels on quiescent, proliferating, or post-mitotic cells including platelets. This surface differential creates a targetable molecular address that concentrates the construct on senescent cells while sparing non-senescent bystanders. (uPAR is broadly induced during senescence and uPAR-targeted CAR T cells are effective senolytics)[https://doi.org/10.1038/s41586-020-2403-9]

2. The uPAR-binding peptide moiety engages uPAR Domain 1 on the senescent cell surface, triggering receptor-mediated endocytosis or direct membrane-proximal delivery of the attached BCL-xL PROTAC warhead into the intracellular compartment of senescent cells, achieving locally concentrated BCL-xL degrader payload without systemic BCL-xL inhibition at apoptosis-competent concentrations in non-senescent tissues. [SPECULATIVE: endocytic trafficking of uPAR ligand-drug conjugates sufficient for intracellular PROTAC delivery has not been directly demonstrated for this configuration; supported by analogy to antibody-drug conjugate and receptor-mediated PROTAC delivery precedent.]

3. Once internalized in senescent cells, the PROTAC warhead recruits an E3 ubiquitin ligase (VHL or CRBN) to BCL-xL, forming a ternary complex that drives polyubiquitination and proteasomal degradation of BCL-xL — a catalytic mechanism that produces sub-stoichiometric, deep, and sustained BCL-xL depletion that is fundamentally superior to the competitive occupancy mechanism of ABT-263/navitoclax, which requires continuous high-drug-concentration binding to BCL-xL and accounts for the platelet toxicity of those agents. (PROTAC-mediated BCL-xL degradation reduces navitoclax platelet toxicity while improving senolytic activity)...

SENS category: LysoSENS

Key references: • doi.org/10.1038/s41586-020-2403-9] • doi.org/10.1038/s41467-020-15838-0] • doi.org/10.1038/s41467-020-15838-0]. • doi.org/10.1038/s41586-020-2403-9], • doi.org/10.1038/s41467-020-15838-0],