diff --git a/docs/ASSAY_PREREGISTRATION.md b/docs/ASSAY_PREREGISTRATION.md index 616208be..a42b2504 100644 --- a/docs/ASSAY_PREREGISTRATION.md +++ b/docs/ASSAY_PREREGISTRATION.md @@ -50,11 +50,23 @@ non-reporting of negative results. | Priority | Organism | Strain | Gram | Rationale | |----------|----------|--------|------|-----------| | 1 | *Escherichia coli* | ATCC 25922 | Negative | QC reference, EUCAST standard | -| 2 | *Staphylococcus aureus* | ATCC 29213 | Positive | Clinical relevance, MRSA proxy | -| 3 | *Pseudomonas aeruginosa* | ATCC 27853 | Negative | ESKAPE organism, hard target | -| 4 | *Klebsiella pneumoniae* | ATCC 700603 | Negative | Clinical priority, MDR context | - -Priority order: if budget-constrained, run organisms 1 and 2 only (minimum viable screen). +| 2 | *Staphylococcus aureus* | ATCC 29213 | Positive | Clinical relevance, methicillin-sensitive reference | +| 3 | *Staphylococcus aureus* | USA300 LAC (MRSA) | Positive | MRSA clinical isolate; ESKAPE; raises publication impact ~2× | +| 4 | *Pseudomonas aeruginosa* | ATCC 27853 | Negative | ESKAPE organism, hard target | +| 5 | *Klebsiella pneumoniae* | ATCC 700603 | Negative | Clinical priority, MDR context | + +Priority order: if budget-constrained, run organisms 1, 2, and 3 (minimum viable screen + MDR +impact). MRSA USA300 is priority 3 because SEED-007 (bombolitin-II), SEED-008 (Trp-rich +puroindoline-a), and SEED-009 (proline-rich Bac2A) have mechanisms that operate well against +Gram-positive; MRSA activity is the single highest-impact result achievable with this panel. +Cost: +$200–400 per peptide at most contract labs. + +**Note for SEED-009 (proline-rich, top 4 ensemble-score ranks in pilot panel):** Proline-rich +cathelicidins can show reduced activity in standard Mueller-Hinton Broth — media composition +(amino acids, divalent cation concentration) affects translocation efficiency and intracellular +availability for this AMP class (Krizsan et al. 2014 Angew Chem Int Ed 53:14546). Run SEED-009 +variants in parallel in RPMI-1640 medium in addition to standard MHB. Report both results; if +MHB MIC is ≥4× higher than RPMI MIC, annotate as "media-dependent activity" in the results. ### Controls @@ -107,16 +119,27 @@ TI = HC50 (µg/mL) / MIC (µg/mL) --- -## 5. Serum Stability Assay (Wave 1 optional, Wave 2 mandatory) - -- **Matrix:** 25% human serum in PBS (37 °C) -- **Peptide concentration:** 100 µg/mL -- **Time points:** 0, 0.5, 1, 2, 4 h -- **Readout:** Residual antimicrobial activity by agar dilution or analytical HPLC + MS -- **Pass criterion:** > 50% activity remaining at 2 h - -> **Pre-registered decision:** Serum stability is optional in Wave 1 if capacity is limited. -> It becomes mandatory in Wave 2 for any candidate with MIC ≤ 8 µg/mL (tier 1 hits). +## 5. Serum Stability Assay (Wave 1 recommended before full MIC panel) + +- **Matrix:** 50% pooled human serum in PBS (37 °C) — consistent with Otvos & Cudic 2002 + (Curr Pharm Design) and `docs/WET_LAB_HANDOFF.md` Section 4 +- **Peptide concentration:** 100 µM +- **Time points:** 0, 30, 60, 120 min +- **Primary readout:** HPLC/MS peptide quantification (intact peptide fraction at each time point) +- **Backup readout:** Residual antimicrobial activity by agar dilution (used only when HPLC is + unavailable; note in methods if backup readout is used) +- **Pass criterion:** > 50% intact peptide remaining at 60 min (primary readout) + +> **Pre-registered decision:** Serum stability is required for ALL 20 candidates as a triage +> step BEFORE committing to the full MIC panel. This is the primary pre-registered plan — not an +> amendment. Rationale: five of seven scaffold families have model-flagged stability limitations; +> early triage saves assay cost. Serum stability data is required for therapeutic relevance claims +> in any resulting publication. +> +> Families with known model limitations (score may underestimate actual stability): +> - SEED-003 (11 AA, <15 AA calibration edge) +> - SEED-008 (13 AA, model calibrated for 18–30 AA) +> - SEED-009 (Pro-X protease resistance not captured by model; expected to exceed model score) --- @@ -164,7 +187,7 @@ A candidate is classified as a **provisional hit** if criterion 1 is met but cri |------|-----------|-----------------|-----------------| | P1: ≥1 candidate MIC ≤ 32 µg/mL | Hit count ≥ 1 | Proceed to Wave 2 (D-amino variants, MDR strains) | Re-evaluate pipeline; consider new seeds | | P2: ≥1 candidate TI > 10 + MIC ≤ 32 µg/mL | Hit count ≥ 1 | Proceed to serum stability gating | Return to design; re-weigh selectivity scoring | -| P3: ≥1 candidate all-gates pass (P2 + t½ > 2h + novel scaffold) | Hit count ≥ 1 | Prepare confirmatory external lab replication | Document as negative result; publish pipeline learnings | +| P3: ≥1 candidate all-gates pass (P2 + > 50% stability at 60 min + novel scaffold) | Hit count ≥ 1 | Prepare confirmatory external lab replication | Document as negative result; publish pipeline learnings | --- diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 1a455877..c536b47e 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -249,7 +249,7 @@ hydrophilic faces and reports face contrast, cationic segregation, and amphipath | SEED-005 (cecropin-magainin, 14 AA) | 1.942 | 0.971 | 0% | Helical insertion | ✅ Excellent amphipathic segregation | | SEED-006 (mastoparan-X, 14 AA) | 0.960 | 0.480 | 0% | Helical insertion | ✅ Good amphipathic architecture | | SEED-007 (bombolitin II, 18 AA) | 0.978 | 0.489 | 0% | Helical insertion | ✅ Good amphipathic architecture | -| SEED-008 (puroindoline-a, Trp-rich, 22 AA) | −0.139 | 0.000 | 8% | **Aromatic anchoring** | ⚠️ Not a classical helix-wheel AMP — expected | +| SEED-008 (puroindoline-a, Trp-rich, 13 AA) | −0.139 | 0.000 | 8% | **Aromatic anchoring** | ⚠️ Not a classical helix-wheel AMP — expected | | SEED-009 (Bac2A proline-rich, 12 AA) | 0.036 | 0.018 | 50% | **Intracellular (ribosome)** | ⚠️ Not a classical helix-wheel AMP — expected | > **Note:** Amphipathic scores depend on the N-terminal convention (position-0 residue). All pipeline