FOXO4 is a forkhead transcription factor that retains p53 in senescent cells, suppressing apoptosis and enabling the SASP. The 2017 de Keizer lab FOXO4-DRI stapled peptide (Cell 2017) demonstrated that disrupting the FOXO4–p53 interaction selectively kills senescent cells in vivo without overt toxicity. Bourgeois et al. (Nat Commun 2025, doi:10.1038/s41467-025-60844-9) confirmed the disordered p53 TAD as the direct target and resolved the molecular contacts. The 3L2C crystal structure of the FOXO4 DBD bound to DNA provides the scaffold geometry for the DBD face that contacts p53. Planned design campaign: (1) extract the FOXO4 DBD–p53 TAD interface geometry from PDB 3L2C and any available FOXO4–p53 co-complex structure; (2) run BindCraft conditioned on hotspot residues N99, S101, Y102, A103, L105, K135, K137, G138; (3) independently forward-fold top designs with AF2-Multimer (FOXO4 DBD + binder) to confirm predicted binding mode (pLDDT > 80, ipTM > 0.75); (4) score ESM-2 perplexity on designed sequences; (5) Foldseek search against PDB to confirm novelty; (6) rank candidates by ipSAE and Rosetta interface ddG. Success criterion: at least one candidate with predicted interface ddG < −10 kcal/mol and ipTM > 0.75 that surpasses FOXO4-DRI on at least one in-silico metric.
Details
- disease
- aging, cellular senescence
- target_ref
- PDB:3L2C / UniProt:P98177 (FOXO4) / UniProt:P04637 (p53)
- primary_endpoint
- SASA delta computed for all 10 pre-identified hotspot residues; ≥3 residues show ≥40% burial (delta-SASA / monomer-SASA)
- identification_strategy
- in_silico_KO
Raw fields (3)
- assay_spec
Fetch PDB:3L2C, extract FOXO4-DBD chain, compute per-residue SASA (BioPython ShrakeRupley monomer vs. complex), identify burial hotspot shell (≥40%), then design 3 BindCraft binder seeds targeting confirmed hotspot residues (98,99,100,101,102,103,105,135,137,138) with AF2-Multimer ipTM ≥ 0.7 success gate; Per-residue SASA delta table (monomer minus complex) for FOXO4-DBD chain A residues 98–138, ranked by burial fraction — planned; Bar chart of SASA burial % per hotspot residue, colored by spatial cluster (loop 98–105 vs. helix 135–138) — planned; Top-3 buried hotspot residues (by delta-SASA) selected as BindCraft anchor points — planned; {'kind': 'claim', 'text_template': 'BindCraft binder seed against FOXO4-DBD hotspot {residue_ids} achieves AF2-Multimer ipTM ≥ 0.7 in forward-fold validation — pending execution'}- hypothesis
De novo designed miniproteins conditioned on the FOXO4 DBD–p53 TAD interface (hotspot residues N99, S101, Y102, A103, L105, K135, K137, G138 from PDB 3L2C) will achieve tighter, more selective disruption of the FOXO4–p53 complex in senescent cells than the first-generation FOXO4-DRI stapled peptide, providing a safer and more potent senolytic agent.
- kill_criteria
Abort or revise if: 3L2C contains FOXO4-DBD + short p53 TAD peptide only — the disordered p53 TAD region not resolved in crystal may underrepresent true binding surface; supplement with Bourgeois 2025 NMR/HDX data if available; BindCraft compute may require GPU; confirm remote compute target before launch; Forward-fold AF2-Multimer ipTM gate may fail all 3 seeds if hotspot shell is too small — fallback: relax to ipTM ≥ 0.60 or increase seed count to 6