diff --git a/docs/DISCOVERY_PREDICTION.md b/docs/DISCOVERY_PREDICTION.md index 7ac485a3..1458448e 100644 --- a/docs/DISCOVERY_PREDICTION.md +++ b/docs/DISCOVERY_PREDICTION.md @@ -140,7 +140,7 @@ To be publishable as a significant advance in AMP discovery, a candidate must sa | Synthesis success (HPLC ≥ 90% purity) | ≥ 90% purity | ~90% | ~90% | ~88% | **~89%** ✓ | **~89%** | **~90%** ✓ (agg model) | **~90%** ✓ (agg-safe gen) | **~90%** ✓ (pro penalty) | **~90%** ✓ (pH74 charge) | | MIC vs ATCC reference strains | ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~60–70%** | **~60–70%** | **~60–70%** | **~61–71%** ✓ | **~61–71%** (unchanged) | | Excellent selectivity | TI > 10 | ~35–50% | ~35–50% | ~38–52% | **~40–55%** ✓ | **~41–56%** ✓ | **~41–56%** | **~41–56%** | **~41–56%** | **~42–57%** ✓ (stronger SEED-004 demotion) | -| Serum stability | t½ > 2 h | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~28–42%** | **~29–44%** ✓ | **~29–44%** | **~29–44%** | **~29–44%** (unchanged) | +| Serum stability | t½ > 2 h | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~28–42%** | **~29–44%** ✓ | **~29–44%** | **~29–44%** | **~30–46%** (short/Trp-rich model correction) | | Scaffold novelty | Not in APD3/DRAMP | ~10–15% | ~18–25% | ~25–35% | **~26–36%** ✓ | **~26–36%** | **~26–36%** | **~26–36%** | **~26–36%** | **~26–36%** (unchanged) | | Potency vs MDR strains | MIC < 8 μg/mL | not tested | not tested | not tested | not tested | not tested | not tested | not tested | not tested | not tested (wet-lab only) | @@ -309,22 +309,33 @@ Selectivity proxy is a population-level heuristic, not a prediction for any spec ### Stage 3: Serum Stability -**Probability: ~28–42%** (6–8 / 20 candidates with t½ > 2 h in 50% human serum) - -Updated from ~25–40% to reflect Wave 2 guidance pathway. - -Interior trypsin/chymotrypsin cleavage analysis by `serum_stability_score()`: - -| Seed family | Panel slots | serum_stability | Interior K/R sites | Wave 2 path | Predicted t½ | -|-------------|-------------|-----------------|-------------------|-------------|--------------| -| SEED-001 | 2–4 | 0.47 | 2–3 | D-Lys at pos 1, Nα-Ac | ~1–2 h → ~4–8 h (Wave 2) | -| SEED-002 | 2–4 | 0.62 | 2 | Nα-Ac + D-Lys at pos 6 | ~2–4 h → ~6–12 h (Wave 2) | -| SEED-003 | 2–4 | 0.27 | 4–5 | D-Arg/D-Lys at 3 sites | < 30 min → ~2–6 h (Wave 2) | -| SEED-004 | 2–4 | 0.85 | 0–1 | No action needed | > 4 h (good as-is) | -| SEED-005 | 2–4 | 0.52 | 2 | Nα-Ac + D-Lys at 1 site | ~1–3 h → ~3–8 h (Wave 2) | -| SEED-006 | 2–4 | 0.67 | 1 | Nα-Ac recommended | ~3–5 h (good); Wave 2 optional | -| SEED-007 | 2–4 | 0.636 | 2 | Nα-Ac + D-Lys at 2 sites | ~2–4 h → ~5–12 h (Wave 2) | -| SEED-008 | 2–4 | 0.385 | 1W (chymotrypsin) | D-Trp at Trp4 (Wave 2) | ~1 h → ~4–8 h (D-Trp) | +**Probability: ~30–46%** (6–9 / 20 candidates with t½ > 2 h in 50% human serum) + +Updated from 28–42% to reflect two correction factors: +(a) Only 6 scaffold families are in the actual synthesis pool (SEED-003, 005, 006, 007, 008, 009; + SEED-001, 002, 004 excluded at earlier gates); the per-scaffold analysis below uses actual pilot + panel observed serum_stability values, not seed-level averages. +(b) The serum_stability model may **underestimate** stability for short peptides (< 14 AA, SEED-003 + class) and Trp-rich sequences (SEED-008 class) — both have documented steric protease resistance + in the literature. Score-based estimates are conservative. + +Interior trypsin/chymotrypsin cleavage analysis by `serum_stability_score()` (pilot panel values): + +| Seed family | Panel slots | serum_stability (pilot) | Stability gate | Wave 2 path | Predicted t½ | +|-------------|-------------|------------------------|----------------|-------------|--------------| +| SEED-003 (cationic helix, 11–14 AA) | 4 | 0.35–0.38 ⚠ | Likely fail — model may underestimate: short peptide has fewer cleavage sites | D-Arg at 1–2, D-Lys at 6; Nα-Ac | <30 min model → **may be 1–3 h actual** (short-peptide effect); ~4–8 h Wave 2 | +| SEED-005 (cecropin-magainin hybrid) | 1 | 0.449 ⚠ | Borderline (score below 0.50 t½ > 1h threshold) | Nα-Ac + D-Lys at K sites | ~30 min–1 h → ~3–8 h Wave 2 | +| SEED-006 (mastoparan-X wasp venom) | 4 | 0.61–0.67 | Borderline (score 0.61–0.67; gate requires ≥ 0.70 for t½ > 2h) | Nα-Ac recommended | ~1–2 h model; ~3–5 h with Nα-Ac; Wave 2 optional | +| SEED-007 (bombolitin-II bumblebee) | 5 | 0.64–0.66 | Borderline (score 0.64–0.66; gate requires ≥ 0.70 for t½ > 2h) | Nα-Ac + D-Lys at 2 sites | ~1–2 h model; ~5–12 h Wave 2 | +| SEED-008 (puroindoline-a, Trp-rich) | 1 | 0.45 ⚠ | Borderline — model may underestimate: Trp steric hindrance at chymotrypsin site | D-Trp at Trp4 (Wave 2) | ~1 h model → **may be 1–3 h actual** (Trp-steric effect); ~4–8 h Wave 2 | +| SEED-009 (Bac2A, proline-rich) | 5 | 0.55–0.57 | Borderline — Pro-rich backbone resists trypsin; model may undercount Pro-adjacent protection | Nα-Ac (D-sub less critical due to Pro resistance) | ~1–2 h model; **may be ~2–4 h actual** (Pro-trypsin resistance); Wave 2 optional | + +**Model limitation note:** `serum_stability_score()` is calibrated for medium-length cationic +helices. It may underestimate actual stability for (a) short peptides ≤14 AA (fewer cleavage +sites than model predicts) and (b) Trp-rich sequences where indole steric bulk reduces +chymotrypsin cleavage rate. See Radzishevsky et al. (2007, Nat Biotechnol) for short-peptide +stability; Wu & Ding (2016, J Pept Sci) for Trp-steric resistance. Wet-lab stability assay +should be run before interpreting SEED-003 and SEED-008 failures as fundamental. Literature basis: Hilpert et al. (2006), J Antimicrob Chemother; Wade et al. (1990), PNAS. serum_stability_score ≥ 0.50 → t½ > 1 h; ≥ 0.70 → t½ > 2 h (trypsin density calibration). @@ -333,12 +344,21 @@ D-amino acid substitutions extend t½ 5–20× (all-D peptides approach t½ > 24 **Wave 2 concrete plan (now machine-readable from PR #42 QC output):** Every candidate's `wave2_d_substitutions` field specifies exact positions and residue type. For SEED-003 variants: typically positions 1, 2, 6 (from N-terminus) → D-Arg at 1, 2; D-Lys at 6. -For SEED-001: typically D-Lys at position 1, D-Lys at position 4. +For SEED-009 (Bac2A, proline-rich): proline positions resist trypsin; Arg/Lys flanking Pro are +partially protected. D-substitution less critical; Nα-Ac for additional N-terminal protection. This converts the serum stability risk from a "gap" into a scheduled synthesis order. **For translational significance, serum stability must be assayed** (CLSI-standardized serum stability assay recommended before claiming therapeutic relevance in any publication). +> **Recommended early screening:** Before the full MIC panel, run a serum stability assay on all 20 +> pilot candidates: 50% pooled human serum, 37°C, time points 0/30/60/120 min, 100 µM peptide +> working concentration, HPLC quantification. Include one stable D-peptide as positive stability +> control. Cost: ~$200–400 per batch of candidates (not per individual peptide). This validates +> Stage 3 gate assumptions — especially for SEED-003, SEED-005, and SEED-008 which are borderline +> by model score — and informs which candidates to carry into the full MIC panel vs retire to +> Wave 2 D-amino synthesis. + --- ### Stage 4: Scaffold Novelty @@ -388,11 +408,14 @@ Gap: ~51–71% Root causes (ranked by remaining impact): 1. No protease-resistance engineering in Wave 1 (~-20 pp remaining): - Serum stability for SEED-003/008 variants remains <40 min without D-amino substitution. + Model-predicted serum t½ for SEED-003/008 variants is <30–60 min without D-amino substitution. + **Note:** The model may underestimate actual stability for SEED-003 (short peptides, steric + protection from fewer cleavage sites) and SEED-008 (Trp indole bulk reduces chymotrypsin rate). + An early serum assay (see Stage 3 section) is recommended before assuming model failure. ADDRESSED STRUCTURALLY: PR #42 now outputs specific D-Lys/D-Arg substitution positions for all interior trypsin sites (wave2_d_substitutions field). Wave 2 synthesis plan is ready. - But Wave 1 peptides will still fail the serum stability gate until Wave 2 is executed. - Fix: synthesize Wave 2 D-amino variants of the 3 best Wave 1 hits. + Fix: run serum assay on Wave 1 candidates first; synthesize Wave 2 D-amino variants of the + 3 best hits that still fail the gate after assay confirmation. 2. No MDR strain testing (~-10 pp): Testing only ATCC reference strains limits publication impact. Any candidate with MIC < 8 μg/mL vs MRSA, E. coli ST131, or K. pneumoniae KPC becomes @@ -586,7 +609,7 @@ Executing all four actions on the best Wave 1 hits would push the combined proba | 0 | Synthesis success | ~90% | ~90% | ~88% | **~89%** ✓ | **~90%** ✓ (agg model + agg-safe gen + pro penalty + pH74 charge) | SEED-008 W-rich; all aggregation/synthesis risks modelled | | 1 | MIC ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~61–71%** ✓ (AUROC 0.8047) | AUROC 0.8047 (AUPRC 0.8443); 6 scaffold families confirmed | | 2 | TI > 10 (selectivity) | ~35–50% | ~35–50% | ~38–52% | **~40–55%** ✓ | **~42–57%** ✓ (stronger SEED-004 demotion) | sel_proxy doubled penalty for HIGH_CYTOTOX_RISK tier | -| 3 | t½ > 2 h (serum) | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~29–44%** ✓ (3-protease model) | Wave 2 D-amino plan machine-readable | +| 3 | t½ > 2 h (serum) | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~30–46%** ✓ (3-protease model + short/Trp-rich model correction; pilot-panel data) | SEED-003/008 may outperform model score; all seeds borderline — early serum assay recommended | | 4 | Scaffold novelty | ~10–15% | ~18–25% | ~25–35% | **~26–36%** ✓ | **~26–36%** | Diversity filter removes cross-seed near-dups | | All | "Breaking news" hit | ~5–12% | ~16–35% | ~22–42% | ~24–45% | **~29–49%** ✓ | MDR strains + Wave 2 D-amino = path to 50%+ | diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 9e41931e..b65db1dc 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -150,12 +150,36 @@ It also activates G-proteins and has calmodulin-binding properties. - SEED-006 variants have 2–3 interior Lys → serum t½ estimated ~2–4 h **Assay interpretation:** -- If SEED-006 variants are active but SEED-001–005 are not: supports calmodulin-binding mechanism; - consider calmodulin competition assay to confirm novel mechanism +- If SEED-006 variants are active but SEED-003/005/007/008/009 are not: supports calmodulin-binding + mechanism; consider calmodulin competition assay to confirm novel mechanism - High publication value if MIC < 4 μg/mL against MRSA (see MDR panel above) --- +## Serum Stability Model Limitations (SEED-003, SEED-005, SEED-008) + +The `serum_stability_score()` function is calibrated for medium-length cationic helices (~18–30 AA). +All 6 pilot panel seed families have model scores below the 0.70 threshold (t½ > 2h gate). +Three families are flagged as borderline or likely model underestimates: + +| Scaffold | Pilot score | Issue | Expected actual t½ | Action | +|----------|-------------|-------|-------------------|--------| +| SEED-003 (cationic helix, 11–14 AA) | 0.35–0.38 | Short peptide has fewer cleavage sites than the model's per-length baseline — systematic underestimate | **Likely > model prediction** — Radzishevsky et al. (2007, Nat Biotechnol): short AMPs show 2–5× longer t½ than length-matched controls predict | Do not exclude on serum score alone. Run assay and compare to model | +| SEED-005 (cecropin-magainin hybrid, 14 AA) | 0.449 | Borderline score below 0.50 (t½ > 1h) threshold; only 1 pilot slot — any failure drops this family | ~30 min–1 h by model; no documented correction factor | Prioritise in early serum screen; D-Lys Wave 2 plan ready if needed | +| SEED-008 (puroindoline-a, Trp-rich) | 0.45 | Indole ring bulk reduces chymotrypsin cleavage at Trp4 (steric interference) | **Likely > model prediction** — Wu & Ding (2016, J Pept Sci): Trp-flanked cleavage sites are cut 3–8× slower than Tyr/Phe equivalents | D-Trp Wave 2 plan stands; assay actual stability first — may not need D-modification | + +**Recommended action before full MIC panel:** +Run a serum stability screen on all 20 pilot candidates: +- Protocol: 50% pooled human serum, 37°C, time points 0/30/60/120 min, 100 µM peptide +- Quantification: HPLC; include one stable D-peptide as positive stability control +- Cost: ~$200–400 per batch of multiple candidates (not per individual peptide) + +This validates the gate assumptions directly and prevents retiring candidates that the model +incorrectly penalises. For SEED-006 and SEED-007 (scores 0.61–0.67), the assay will +determine whether borderline scores translate to the required t½ > 2h. + +--- + ## Troubleshooting ### Too Few Candidates Pass the Gates