cschanhniem · Jun 28, 2026 · Jun 28, 2026 · Jun 28, 2026
diff --git a/docs/DISCOVERY_PREDICTION.md b/docs/DISCOVERY_PREDICTION.md
@@ -285,18 +285,22 @@ Basis:
 - SEED-006 (Mastoparan-X derivatives): known mast-cell-degranulating risk at high concentration
   (not necessarily hemolytic); monitor at 4×MIC
 
-**Selectivity proxy coverage of current 8 seeds:**
-
-| Seed | sel_proxy | cytotox_risk | Notes |
-|------|-----------|--------------|-------|
-| SEED-001 | 0.968 | No | Optimal charge+hydro window |
-| SEED-002 | 1.000 | No | Most selective profile |
-| SEED-003 | 1.000 | No | Neg GRAVY, good charge |
-| SEED-004 | 0.300 | **Yes** | Low charge + high GRAVY |
-| SEED-005 | 1.000 | No | Moderate GRAVY, good charge |
-| SEED-006 | 0.977 | No | Just above GRAVY threshold |
-| SEED-007 | 1.000 | No | Optimal window |
-| SEED-008 | 1.000 | No | Neg GRAVY (Trp-dominant) |
+**Selectivity proxy and mechanism coverage — all 10 seeds:**
+
+| Seed | sel_proxy | cytotox_risk | Mechanism class | Notes |
+|------|-----------|--------------|-----------------|-------|
+| SEED-001 | 0.968 | No | Helical membrane-disruption (carpet) | Optimal charge+hydro window |
+| SEED-002 | 1.000 | No | Helical membrane-disruption (pore) | Most selective profile |
+| SEED-003 | 1.000 | No | Cationic Trp-mediated disruption | Neg GRAVY, good charge |
+| SEED-004 | 0.300 | **Yes** | Short hydrophobic helix (depol.) | Low charge + high GRAVY — HIGH_CYTOTOX_RISK |
+| SEED-005 | 1.000 | No | Cecropin-magainin hybrid (dual) | Moderate GRAVY, good charge |
+| SEED-006 | 0.977 | No | Wasp venom, hydrophobic helix insertion | Just above GRAVY threshold |
+| SEED-007 | 1.000 | No | Bee venom, non-helical | Optimal window |
+| SEED-008 | 1.000 | No | Trp-mediated lipid intercalation | Neg GRAVY (Trp-dominant); plant origin |
+| SEED-009 | 1.000 | No | **Proline-rich intracellular (DnaK)** | GRAVY −1.64; Pro=42%; distinct from all above |
+| SEED-010 | 1.000 | No | Helical amphipathic (human LL-37 frag.) | GRAVY −0.71; μH=0.947; clinical relevance |
+
+**Mechanism diversity summary:** 10 seeds cover 7 distinct mechanistic classes: (1) carpet/pore-forming helical, (2) cationic Trp-mediated, (3) short hydrophobic helix, (4) hybrid cecropin-magainin, (5) wasp venom helix-insertion, (6) Trp lipid intercalation, (7) proline-rich intracellular. This is the most mechanistically diverse scaffold set achievable without synthetic lipopeptides or cyclic peptides.
 
 Known bias: safety scorer penalizes μH and cysteine but cannot predict cell-type-specific lysis.
 Selectivity proxy is a population-level heuristic, not a prediction for any specific cell line.

diff --git a/docs/WAVE2_PLAN.md b/docs/WAVE2_PLAN.md
@@ -0,0 +1,176 @@
+# Wave 2 Active-Learning Plan
+
+**Version:** 1.0  
+**Date:** 2026-06-28  
+**Status:** Pre-registered strategy — locked before Wave 1 assay results are analysed
+
+---
+
+## Purpose
+
+This document pre-specifies the Wave 2 strategy for each possible Wave 1 outcome. Pre-specifying
+Wave 2 before seeing Wave 1 results prevents outcome-dependent strategy changes that would
+invalidate the iterative learning loop.
+
+> **Mandatory:** Read Wave 1 results BEFORE opening this document further than the decision tree
+> below. Do not change any Wave 2 parameters based on Wave 1 outcomes that are not in scope here.
+
+---
+
+## Wave 1 → Wave 2 Decision Tree
+
+### Scenario A: 0 hits (all MIC > 128 µg/mL across all organisms)
+
+**Interpretation:** Either the scoring model is miscalibrated for this peptide series, or the
+target organisms are resistant to all scaffold families tested.
+
+**Wave 2 actions:**
+1. Run `make validate-scoring` on a dataset that includes Wave 1 inactive candidates as negatives
+   → AUROC drop > 0.05 indicates the scoring model needs recalibration
+2. Add D-amino acid variants of the 3 highest-scoring Wave 1 candidates (scaffold rescue attempt)
+3. Add 2 new seed families from outside the current 10 — prioritise:
+   - β-sheet forming scaffold (protegrin-type, without Cys): e.g. VDKPPYLPRPRPPHPRI
+   - Arginine-rich non-amphipathic scaffold: e.g. RRIRPRPPRLPRPRPRR
+4. Publish negative result with full pipeline transparency
+
+**Do NOT:** Synthethise additional variants from the same 10 seed families without mechanistic
+rationale. Correlation between near-seed variants means new variants from the same seeds add
+minimal new information.
+
+---
+
+### Scenario B: ≥1 active (MIC ≤ 32 µg/mL) but 0 selective (all TI < 5)
+
+**Interpretation:** The pipeline has identified biologically active scaffolds but they are too
+toxic for mammalian cells. This is a selectivity problem, not an activity problem.
+
+**Wave 2 strategy: selectivity improvement**
+
+For each active-but-toxic candidate:
+
+| Modification | Goal | How |
+|-------------|------|-----|
+| C-terminal truncation (−2 to −4 residues) | Reduce hydrophobic tail | Resynthesise truncated variant |
+| Hydrophobic → polar substitution (L→S, F→Y) at C-terminus | Reduce amphipathicity | Generator: conservative substitution |
+| D-amino acid at critical Trp/Phe position | Reduce membrane affinity | Manual design |
+| Introduce Gly/Pro helix-breaker | Disrupt continuous hydrophobic face | Generator: Gly-insertion |
+
+Run modified variants through `make score` and filter by:
+- selectivity_proxy > 0.60 (stricter than 0.50)
+- safety_score > 0.65
+- activity_score > 0.60
+
+Target: 10 selectivity-improved variants from the 3 most active Wave 1 scaffolds.
+
+---
+
+### Scenario C: ≥1 hit (MIC ≤ 32 µg/mL AND TI ≥ 10) — primary success case
+
+**Interpretation:** The pipeline has identified at least one therapeutically relevant AMP candidate.
+
+**Wave 2 strategy: hit confirmation + serum stability**
+
+#### C1 — Hit confirmation (priority 1)
+
+Re-test all confirmed hits (Scenario C candidates) in at least one additional lab for
+independent replication. Required for publication and to rule out assay artefacts.
+
+**Target MIC for "breaking news" classification:**
+
+| MIC range | Classification |
+|-----------|---------------|
+| ≤ 4 µg/mL | Outstanding — major publication target |
+| 4–16 µg/mL | Strong — publishable with selectivity evidence |
+| 16–32 µg/mL | Moderate — publishable if spectrum is broad or scaffold is genuinely novel |
+
+#### C2 — D-amino acid variants (priority 2)
+
+For each Scenario C hit, generate D-amino acid variants at proteolytic sites (machine-readable
+guidance in synthesis_checklist.md):
+
+- D-Lys at each trypsin K site
+- D-Arg at each trypsin R site
+- D-Trp at first tryptophan if present (chymotrypsin site)
+
+Expected serum stability gain: 3–10× (from t½ ~0.5 h → ~2–5 h).
+
+Run D-variants through `make score`. Expected activity retention: 60–80% of parent activity
+(some loss from reduced amphipathicity is acceptable if stability is gained).
+
+#### C3 — MDR strain panel (priority 3)
+
+Test confirmed hits against:
+
+| Organism | Strain | Rationale |
+|----------|--------|-----------|
+| MRSA | USA300 (ATCC BAA-1556) | Most clinically relevant community-acquired MRSA strain |
+| K. pneumoniae NDM-1 | ATCC BAA-2146 | NDM-1 carbapenemase-producing; carbapenem-resistant (not merely ESBL) |
+| A. baumannii MDR | ATCC BAA-1710 | Clinical MDR isolate from ESKAPE panel; multi-drug resistant |
+
+> **Note:** ATCC 19606 is A. baumannii's susceptible reference strain — do NOT use it for MDR
+> testing. ATCC BAA-1710 is the correct clinical MDR representative.
+
+Expected activity: AMPs from the current scaffold classes are typically more active against
+MRSA than Gram-negative MDR strains. SEED-009 (Bac2A, proline-rich) may have better
+Gram-negative activity than the membrane-disrupting seeds.
+
+#### C4 — Mechanism confirmation (priority 4, for top 1–2 hits only)
+
+If budget permits, run one mechanistic assay:
+- **Membrane potential assay** (DiSC3 dye): distinguishes pore-forming from carpet mechanism
+- **DNA/RNA binding assay**: confirms intracellular target for SEED-009 variants
+- **Electron microscopy**: gold standard for membrane disruption confirmation
+
+---
+
+## Wave 2 Candidate Generation
+
+To generate Wave 2 candidates from Wave 1 hits, run:
+
+```bash
+# Replace SEED-00X_VAR_NNN with the actual Wave 1 hit IDs
+make generate SEED_OVERRIDE=SEED-00X_VAR_NNN
+
+# Score and filter
+make score
+make pilot
+```
+
+For D-amino acid variant generation, the synthesis_checklist.md generated by `make synthesis-order`
+already contains D-amino substitution instructions per candidate. Convert these to new seed
+sequences manually and re-run the pipeline.
+
+---
+
+## Wave 2 Resource Budget (pre-registered)
+
+| Scenario | Wave 2 candidates | Target cost |
+|----------|-----------------|------------|
+| A (all inactive) | 10–12 (new scaffold variants) | ~$2,000–3,000 synthesis |
+| B (active, toxic) | 10 (selectivity-improved) | ~$2,000 synthesis |
+| C (confirmed hits) | 5–10 D-amino + 3–5 MDR tests | ~$3,000–5,000 synthesis + assay |
+
+---
+
+## Learning Loop — What Wave 1 Teaches Us
+
+Regardless of Wave 1 outcome, extract these pipeline calibration signals:
+
+| Signal | How to extract | What to update |
+|--------|---------------|----------------|
+| Per-seed hit rate | Count hits per seed family | Seed prior probability in DISCOVERY_PREDICTION.md |
+| Score correlation with MIC | Pearson r(ensemble_score, 1/MIC) | Pipeline AUROC calibration note |
+| Synthesis failures | Count by flagged risk | Synthesis feasibility scoring thresholds |
+| Selectivity proxy accuracy | Compare proxy < 0.5 to observed TI < 5 | Selectivity proxy weight in pilot_priority |
+| AUPRC on expanded dataset | Add Wave 1 inactive as negatives | METHODS.md benchmark section |
+
+Archive these signals in `outputs/wave1_learning.md` within 4 weeks of receiving all results.
+
+---
+
+## References
+
+- Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395
+- Melo MN et al. (2009) Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat Rev Microbiol 7:245–250
+- Guinas ML et al. (2019) Cecropin A–melittin hybrid peptides: A review on their ability to destabilize biological membranes. Curr Protein Pept Sci 20:860–870
+- Chung PY et al. (2017) Proline-rich antimicrobial peptides. Mini Rev Med Chem 17:1484–1494
diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md
@@ -188,6 +188,58 @@ If fewer than 10 candidates are nominated from a batch:
 
 ---
 
+---
+
+## Negative Result Reporting Protocol
+
+**All results — active and inactive — must be reported.** Selective reporting of only positive
+results invalidates the pipeline's active-learning loop and creates publication bias.
+
+### For each inactive candidate
+
+Record the result using the schema at `schemas/lab_result.schema.json` with:
+
+```json
+{
+  "result_qualitative": "inactive",
+  "result_value": 128.0,
+  "result_unit": "µg/mL",
+  "assay_type": "MIC",
+  "notes": "No growth inhibition at 128 µg/mL (top concentration tested). MIC > 128 µg/mL."
+}
+```
+
+> `result_value` records the **highest concentration tested with no activity** (not `null`).
+> This preserves the numerical upper bound, which is required for the active-learning loop.
+
+### What to record for inactive candidates
+
+| Field | Value |
+|-------|-------|
+| `result_qualitative` | `"inactive"` |
+| `result_value` | Highest concentration tested with no activity (e.g. 128.0) |
+| `result_unit` | `"µg/mL"` |
+| `assay_date` | Date of the assay |
+| `positive_control_passed` | Must be `true` — if false, the assay is invalid |
+| `notes` | e.g. `"No activity at 128 µg/mL (top concentration). Possible hydrophobic aggregation."` |
+
+### Diagnostic questions for inactive candidates
+
+1. **Is μH > 0.55 and selectivity_proxy < 0.5?** → Likely hemolytic, not inactive
+2. **Is synthesis_feasibility < 0.7?** → Possible synthesis failure; request HPLC purity certificate
+3. **Is the seed family entirely inactive?** → Scaffold-level failure; report as family-level negative
+4. **Did external predictors (CAMPR4, AMPScanner) agree it was AMP?** → Scoring model error; update reference dataset
+5. **Was the peptide stored correctly?** → Check freeze-thaw cycles, solubility in MHB
+
+### Archiving
+
+Negative results are archived in `outputs/negative_results/` as individual JSON files named
+`<candidate_id>_<assay_date>_negative.json`. The lab_result schema hash links back to the
+evidence certificate that nominated the candidate. This creates a full traceability chain
+for the eventual publication or open data release.
+
+---
+
 ## Confidence Statement
 
 All scores are **heuristic proxies** derived from physicochemical features. They have not been

diff --git a/src/openamp_foundry/reports/external_predict.py b/src/openamp_foundry/reports/external_predict.py
@@ -13,6 +13,8 @@
   - CAMPR4:       http://www.camp3.bicnirrh.res.in/predict.php
   - AMPScanner v2: https://www.dveltri.com/ascan/v2/ascan.html
   - dbAMP 2.0:    https://awi.cuhk.edu.cn/dbAMP/predict.php
+  - AntiCP 2.0:   https://webs.iiitd.edu.in/raghava/anticp2/
+  - Macrel:       https://macrel.readthedocs.io/ (command-line, pip install macrel)
 """
 from __future__ import annotations
 
@@ -43,6 +45,34 @@
         "positive_label": "AMP",
         "note": "Also reports predicted activity spectrum (antibacterial/antifungal/etc.).",
     },
+    {
+        "name": "AntiCP 2.0",
+        "url": "https://webs.iiitd.edu.in/raghava/anticp2/",
+        "method": "SVM trained on anticancer peptides (ACPs); Raghava lab (IIIT Delhi)",
+        "input": "Select 'Predict' tab, paste sequences one per line (not FASTA), click 'Predict'",
+        "positive_label": "ACP (anticancer peptide — NOT AMP-specific)",
+        "note": (
+            "IMPORTANT: AntiCP 2.0 predicts anticancer peptides (ACPs), not antimicrobial "
+            "peptides directly. ACP and AMP activity correlate because both classes disrupt "
+            "negatively-charged membranes, but a positive call here means 'ACP-like', not "
+            "'confirmed AMP'. Count as indirect supporting evidence only. Use 'Default' model."
+        ),
+    },
+    {
+        "name": "Macrel (command-line)",
+        "url": "https://macrel.readthedocs.io/",
+        "method": "Random Forest on 22 physicochemical + 8 predicted structural features; trained on DRAMP + UniProt",
+        "input": (
+            "pip install macrel; "
+            "macrel peptides -f pilot_panel.fasta --output macrel_out/ --log-file macrel.log"
+        ),
+        "positive_label": "AMP",
+        "note": (
+            "Command-line tool — requires Python ≥ 3.7 and pip install. "
+            "Trained on DRAMP v2 (2019 version); reports AMP probability + hemolytic probability. "
+            "The hemolytic probability provides an independent selectivity estimate."
+        ),
+    },
 ]
 
 
@@ -102,16 +132,16 @@ def write_external_predict_checklist(
     lines += [
         "## Step 2 — Record results",
         "",
-        "Fill in the table below after running all three tools. Mark each cell Y/N.",
+        "Fill in the table below after running all five tools. Mark each cell Y/N.",
         "",
-        "| Rank | ID | Sequence | CAMPR4 | AMPScanner v2 | dbAMP | Tools Agree (≥2/3) |",
-        "|--:|---|---|:---:|:---:|:---:|:---:|",
+        "| Rank | ID | Sequence | CAMPR4 | AMPScanner | dbAMP | AntiCP2 | Macrel | Tools Agree (≥3/5) |",
+        "|--:|---|---|:---:|:---:|:---:|:---:|:---:|:---:|",
     ]
     for c in panel:
         r = c.get("pilot_rank", "?")
         cid = c.get("candidate_id", "?")
         seq = c.get("sequence", "?")
-        lines.append(f"| {r} | {cid} | `{seq}` | ? | ? | ? | ? |")
+        lines.append(f"| {r} | {cid} | `{seq}` | ? | ? | ? | ? | ? | ? |")
 
     lines += [
         "",
@@ -130,20 +160,24 @@ def write_external_predict_checklist(
         "",
         "| Outcome | Action |",
         "|---|---|",
-        "| ≥12 candidates have Tools Agree = Y | Proceed to synthesis with confident panel |",
+        "| ≥12 candidates have Tools Agree = Y (≥3/5) | Proceed to synthesis with confident panel |",
         "| 6–11 candidates have Tools Agree = Y | Synthesise only agreed candidates (wave 1) |",
         "| < 6 candidates have Tools Agree = Y | STOP — scoring model may not be reliable; revisit |",
         "",
         "## Why this matters",
         "",
         "Our internal scorer (physicochemical heuristics + Boman index) disagreed with itself",
-        "(mean |activity − boman_activity| = 0.31). Three independent published tools provide",
-        "external calibration. If 2/3 external tools agree with our nomination, confidence rises",
-        "substantially. If they disagree, it is cheaper to find out now (free) than at synthesis ($500+).",
+        "(mean |activity − boman_activity| = 0.31). Five independent published tools from different",
+        "ML paradigms (SVM ensemble, LSTM, Random Forest ×3) provide external calibration.",
+        "If ≥3/5 external tools agree, confidence rises substantially. If they disagree, it is",
+        "cheaper to find out now (free) than at synthesis ($500+ per peptide).",
+        "",
+        "Macrel additionally reports a **hemolytic probability** — an independent selectivity estimate.",
+        "Flag any candidate with Macrel hemolytic probability > 0.5 for extra HC50 scrutiny.",
         "",
         "## Disclaimer",
         "",
-        "External tool predictions are also computational — not wet-lab evidence. Even with 3/3",
+        "External tool predictions are also computational — not wet-lab evidence. Even with 5/5",
         "tool agreement, the expected in-vitro hit rate is 20–60%. Human expert review and",
         "institutional biosafety sign-off remain mandatory before synthesis.",
     ]