FOXO4-DRI (Baar et al. 2017, Cell) established that disrupting the FOXO4–p53 interaction in senescent cells triggers p53-dependent apoptosis selectively in those cells. The CR3 (C-terminal transactivation) domain of FOXO4 binds the N-terminal transactivation domain (TAD1/TAD2) of p53; this interface is the design target. This plan proceeds in four stages: (1) PDB structure curation of FOXO4-CR3 and p53-TAD — including NMR/crystal contacts (planned: PDB 2LZB for FOXO4 CR3, 2MEI for p53-TAD context) and hotspot profiling of the ~8 key contact residues; (2) BindCraft de novo binder campaign conditioned on FOXO4-CR3 hotspot surface (success criterion: ≥10 designs with AF2-Multimer ipTM > 0.70 and pAE_interaction < 10 Å); (3) AF2-Multimer independent forward-fold validation of all BindCraft outputs (success criterion: AF2 pLDDT > 80 on binder chain, AF2-Multimer ipTM > 0.65 independent of design model); (4) ESM-2 perplexity filter and Foldseek novelty screen against PDB to confirm no close structural prior art. Reference benchmark: FOXO4-DRI (Baar 2017) — any candidate must outperform DRI on at least one in-silico metric (predicted interface buried surface area, ipTM, or ESM likelihood). Open questions flagged for Claire: immunogenicity of helical peptide scaffold in aged mice; for Jerome: CNS penetration if systemic senolytic clearance is desired.
Details
- disease
- aging, cellular senescence
- target_ref
- UniProt:P98177 (FOXO4 CR3 domain) × UniProt:P04637 (p53 TAD1/TAD2)
- timeline_weeks
- 8
- primary_endpoint
- ≥1 BindCraft sequence with ESM-2 perplexity ≤ 8.0 (log-likelihood threshold consistent with natural protein-like sequences)
- identification_strategy
- in_silico_KO
Raw fields (3)
- assay_spec
Interface hotspot extraction from CD38–substrate complex (PDB 2I65) followed by BindCraft binder design targeting the NAD-binding pocket, with ESM-2 perplexity filter on top-10 sequences; Ranked table of ≤10 BindCraft binder sequences with ESM-2 perplexity score, predicted length, and hotspot coverage — status: planned; {'kind': 'claim', 'text_template': 'BindCraft candidate CD38-BC-{N} achieves ESM-2 perplexity ≤ 8.0 and covers ≥4 of 6 defined hotspot residues — status: planned'}; FASTA of top-3 sequences passing perplexity filter, ready for AF2-Multimer ipTM evaluation next tick — status: planned- hypothesis
De novo helical peptides or miniproteins targeting the FOXO4 CR3 domain at its p53-TAD binding interface will disrupt FOXO4–p53 nuclear retention in senescent cells with higher potency and selectivity than the reference FOXO4-DRI peptide, enabling senolytic clearance without off-target p53 perturbation in non-senescent tissues.
- kill_criteria
Abort or revise if: PDB 2I65 contains cADPR product, not NAD+ — hotspot geometry may differ from catalytic-state substrate complex; verify with 4TMQ (NAD+ bound) as fallback; BindCraft without GPU will be slow — confirm compute availability before launch; ESM-2 perplexity threshold of 8.0 is heuristic; if all candidates fail, lower to ≤ 10.0 and flag for manual review