From 47c6440c745c0e526d9561ee46d8400d18f1cde1 Mon Sep 17 00:00:00 2001 From: j Date: Sun, 28 Jun 2026 23:04:51 +0700 Subject: [PATCH 1/2] docs: add MRSA USA300 to preregistration, align serum stability protocol MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit ASSAY_PREREGISTRATION.md: - Add S. aureus USA300 LAC (MRSA) as priority 3 organism — required for MDR impact claims; SEED-007, SEED-008, SEED-009 mechanisms are Gram-positive- relevant; MRSA activity doubles publication impact potential (~2× as per WET_LAB_PROBABILITY.md Section 4) - Add SEED-009 media note: run in RPMI-1640 parallel to MHB for proline-rich variants (Gagnon 2016; protein-rich MHB interferes with Pro-rich uptake) - Change serum stability from "Wave 1 optional" → "Wave 1 recommended before MIC panel" — early attrition on unstable candidates saves assay cost - Change 25% human serum → 50% pooled human serum (consistent with WET_LAB_HANDOFF.md Section 4 and Otvos & Cudic 2002 Bac2A reference) - Change time points 0/0.5/1/2/4h → 0/30/60/120 min (consistent with WET_LAB_HANDOFF.md; saves one time point, matches standard cathelicidin protocol) - Note SEED-003, SEED-008, SEED-009 model calibration limitations in section --- docs/ASSAY_PREREGISTRATION.md | 50 ++++++++++++++++++++++++----------- 1 file changed, 35 insertions(+), 15 deletions(-) diff --git a/docs/ASSAY_PREREGISTRATION.md b/docs/ASSAY_PREREGISTRATION.md index 616208be..0d643186 100644 --- a/docs/ASSAY_PREREGISTRATION.md +++ b/docs/ASSAY_PREREGISTRATION.md @@ -50,11 +50,22 @@ 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, ranks 1–2, 6–7):** Proline-rich cathelicidins show reduced +activity in Mueller-Hinton Broth (protein-rich media interferes with intracellular uptake). +Run SEED-009 variants in parallel in RPMI-1640 medium (or 2× diluted MHB) in addition to +standard MHB. Report both; if MHB MIC is high but RPMI MIC is low, note the media effect +explicitly in the results (Gagnon et al. 2016 Cell 167:471; Krizsan et al. 2014 Angew Chem). ### Controls @@ -107,16 +118,25 @@ 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 +- **Readout:** Residual antimicrobial activity (agar dilution) or HPLC/MS peptide quantification +- **Pass criterion:** > 50% intact peptide (or activity) remaining at 60 min + +> **Pre-registered decision (updated):** Serum stability is recommended for ALL 20 candidates +> as a triage step BEFORE the full MIC panel, not as an optional add-on. Rationale: five of +> seven scaffold families have model-flagged stability limitations; early attrition here saves +> synthesis/assay cost for low-stability candidates. Serum stability data is also required +> for any publication reporting therapeutic relevance. +> +> 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) --- From 9f0e008dcfe824209f5b4fc4c420a7490023732c Mon Sep 17 00:00:00 2001 From: j Date: Sun, 28 Jun 2026 23:10:31 +0700 Subject: [PATCH 2/2] fix: address code review findings on preregistration and handoff docs MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit ASSAY_PREREGISTRATION.md: - [HIGH] Gate P3 criterion aligned with Section 5: 't½ > 2h' → '> 50% stability at 60 min' (both now match the pre-registered pass criterion) - [MEDIUM] Remove 'Pre-registered decision (updated)' wording; use 'Pre-registered decision' only — the document is not yet locked so this is the original plan, not an amendment - [MEDIUM] Fix SEED-009 media note: replace imprecise 'protein-rich media interferes' with accurate 'media composition (amino acids, divalent cations) affects translocation efficiency' per Krizsan et al. 2014 Angew Chem Int Ed 53:14546 - [MEDIUM] Remove Gagnon 2016 Cell citation from target organisms note (uncertain journal/volume; Krizsan 2014 is sufficient and on solid ground) - [LOW] Fix serum stability dual readout: primary = HPLC/MS, backup = bioactivity (eliminates post-hoc readout flexibility that would undermine pre-registration) - [LOW] Remove vague "ranks 1–2, 6–7" parenthetical from SEED-009 note (not defined in document scope) WET_LAB_HANDOFF.md: - [HIGH] Fix SEED-008 helix-wheel table length: 22 AA → 13 AA (all 4 SEED-008 variants in pilot panel are 13 AA: FPVTWRFWRWWKG etc.; 22 AA was incorrect) --- docs/ASSAY_PREREGISTRATION.md | 31 +++++++++++++++++-------------- docs/WET_LAB_HANDOFF.md | 2 +- 2 files changed, 18 insertions(+), 15 deletions(-) diff --git a/docs/ASSAY_PREREGISTRATION.md b/docs/ASSAY_PREREGISTRATION.md index 0d643186..a42b2504 100644 --- a/docs/ASSAY_PREREGISTRATION.md +++ b/docs/ASSAY_PREREGISTRATION.md @@ -61,11 +61,12 @@ puroindoline-a), and SEED-009 (proline-rich Bac2A) have mechanisms that operate 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, ranks 1–2, 6–7):** Proline-rich cathelicidins show reduced -activity in Mueller-Hinton Broth (protein-rich media interferes with intracellular uptake). -Run SEED-009 variants in parallel in RPMI-1640 medium (or 2× diluted MHB) in addition to -standard MHB. Report both; if MHB MIC is high but RPMI MIC is low, note the media effect -explicitly in the results (Gagnon et al. 2016 Cell 167:471; Krizsan et al. 2014 Angew Chem). +**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 @@ -124,14 +125,16 @@ TI = HC50 (µg/mL) / MIC (µg/mL) (Curr Pharm Design) and `docs/WET_LAB_HANDOFF.md` Section 4 - **Peptide concentration:** 100 µM - **Time points:** 0, 30, 60, 120 min -- **Readout:** Residual antimicrobial activity (agar dilution) or HPLC/MS peptide quantification -- **Pass criterion:** > 50% intact peptide (or activity) remaining at 60 min - -> **Pre-registered decision (updated):** Serum stability is recommended for ALL 20 candidates -> as a triage step BEFORE the full MIC panel, not as an optional add-on. Rationale: five of -> seven scaffold families have model-flagged stability limitations; early attrition here saves -> synthesis/assay cost for low-stability candidates. Serum stability data is also required -> for any publication reporting therapeutic relevance. +- **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) @@ -184,7 +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