From bc368c944cda9a9c6109a64b8754b78dc2987ca3 Mon Sep 17 00:00:00 2001 From: j Date: Mon, 29 Jun 2026 01:46:36 +0700 Subject: [PATCH] fix(docs): correct SEED-009 Met omission + add PR #105/#106 tracking MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit WET_LAB_HANDOFF.md: SEED-009 synthesis guidance said "No Met, no Cys" for all variants, but SEED-009_VAR_033 (RRLPRPGYMPRP) has Met at position 9. Corrected to: - VAR_033: Met×1 at position 9 — Nle substitution + HPLC purity + −80°C inert-atmosphere storage (same as SEED-007 protocol); both MET×1 and PROLINE_RICH_INTRACELLULAR flags fire simultaneously - VAR_027/017/039: no Met, no Cys → standard Fmoc SPPS ROADMAP.md + DISCOVERY_PREDICTION.md: tracking notes for PR #105 (PYROGLUTAMATE_RISK flag) and PR #106 (this correction) --- docs/DISCOVERY_PREDICTION.md | 11 +++++++++++ docs/ROADMAP.md | 2 ++ docs/WET_LAB_HANDOFF.md | 11 ++++++++--- 3 files changed, 21 insertions(+), 3 deletions(-) diff --git a/docs/DISCOVERY_PREDICTION.md b/docs/DISCOVERY_PREDICTION.md index 72b88be1..89b8db43 100644 --- a/docs/DISCOVERY_PREDICTION.md +++ b/docs/DISCOVERY_PREDICTION.md @@ -803,6 +803,17 @@ No biological activity has been demonstrated. The lab is the judge.* --- +**Tracking note (post-PR #106):** WET_LAB_HANDOFF.md: factual correction — SEED-009 synthesis +guidance previously stated "No Met, no Cys → standard Fmoc SPPS" for all 4 variants; corrected +to note that VAR_033 (RRLPRPGYMPRP) has Met at position 9 (same Nle substitution + HPLC purity +requirement as SEED-007 variants). Other 3 SEED-009 variants (VAR_027/017/039) have no Met. + +**Tracking note (post-PR #105):** presynth_check.py: PYROGLUTAMATE_RISK flag added for N-terminal +Q (t½ hours–days at pH 7.4, 5–50× MIC loss); E1 excluded (acid-catalysed, negligible at +physiological pH); Nα-acetylation or Q1→K1/R1 substitution surfaced as remediation. None of the +20 pilot candidates are affected (no Q-terminal sequences in the pilot panel), but the check +protects future seed generation batches. + **Tracking note (post-PR #103):** ASSAY_PREREGISTRATION.md: serum stability model limitations block extended to cover all 5 affected families — SEED-003 (pre-existing, 11 AA length edge), SEED-007 (added: Met/Nle substitution + 11 AA length edge), SEED-008 (pre-existing, Trp steric, diff --git a/docs/ROADMAP.md b/docs/ROADMAP.md index 7e787a37..8e1facdf 100644 --- a/docs/ROADMAP.md +++ b/docs/ROADMAP.md @@ -89,6 +89,8 @@ Implemented during the pre-wet-lab improvement loop (PRs #31–#54): - WET_LAB_HANDOFF.md completed with dedicated special notes for all 7 seed families: SEED-007 (bombolitin-II, 4 pilot slots, all NOVEL, best serum stability, Met oxidation guidance, breakthrough-candidate framing), SEED-005 (cecropin-magainin hybrid, safety/serum risk note), SEED-008 (puroindoline-a, Trp aggregation handling, aromatic-mechanism CD interpretation), SEED-009 (Bac2A proline-rich, Pro coupling guidance, RPMI-parallel assay note restated as standalone); Serum Stability section header updated to "All 7 Seeds" with SEED-006 and SEED-007 table rows added; DISCOVERY_PREDICTION.md tracking updated (PR #100) - presynth_check.py Met oxidation flag strengthened: Nle (norleucine) substitution explicitly recommended, inert-atmosphere storage, mandatory HPLC purity at receipt — actionable guidance now surfaces at QC report level for all SEED-007 pilot variants (PR #102) - ASSAY_PREREGISTRATION.md serum stability model limitations block extended to cover all 5 affected families: SEED-003 (pre-existing, 11 AA length edge), SEED-007 (added: Met/Nle substitution + 11 AA length edge), SEED-008 (pre-existing, Trp steric, 13 AA length edge), SEED-009 (pre-existing, Pro-bond protease resistance), SEED-005 (added: safety gate, hemolysis at MIC/3 mandatory, exclude if HC₅₀ < 10× MIC); all limitations pre-registered before synthesis order (PR #103) +- presynth_check.py: PYROGLUTAMATE_RISK flag added for N-terminal Q (cyclises to pGlu at pH 7.4, t½ hours–days, 5–50× MIC loss); E1 intentionally excluded (acid-catalysed only, negligible at physiological pH); Nα-acetylation (zero extra cost) or Q1→K1/R1 substitution surfaced as remediation; 7 tests including Q+QG double-flag interaction guard (PR #105) +- WET_LAB_HANDOFF.md: SEED-009 synthesis guidance corrected — VAR_033 (RRLPRPGYMPRP) contains Met at position 9 (Nle substitution + HPLC purity required); the "No Met, no Cys" statement now scoped to VAR_027/017/039 only (PR #106) ## v1.0 — Validated dry-lab-to-wet-lab loop diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 6954d09f..9ad2b1ad 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -406,9 +406,14 @@ Pro ring conformational constraint (Vanhoof *et al.* 1995, *FASEB J* 9:736–744 Pro-rich AMPs show > 2 h stability in 50% human serum (Otvos & Cudic 2002, *Curr Pharm Design*). **Synthesis guidance:** -- No Met, no Cys → standard Fmoc SPPS. -- Pro-rich sequences (50% Pro in some variants): use extended coupling times or double-coupling - at Pro positions; pseudoproline (Ψ-Pro) dipeptides at Pro-Ser/Thr junctions if yield is low. +- No Cys in any variant → no disulfide risk. +- **VAR_033 (RRLPRPGYMPRP) contains Met at position 9** — same oxidation risk as SEED-007: + request Nle (norleucine) substitution for oxidation-resistant synthesis; mandatory HPLC purity + at receipt; store at −80°C under inert atmosphere. The presynth QC report for VAR_033 will + carry both `MET×1` and `PROLINE_RICH_INTRACELLULAR` flags simultaneously. +- VAR_027, VAR_017, VAR_039: No Met, no Cys → standard Fmoc SPPS. +- Pro-rich sequences (33–42% Pro): use extended coupling times or double-coupling at Pro + positions; pseudoproline (Ψ-Pro) dipeptides at Pro-Ser/Thr junctions if yield is low. - C-terminal amidation optional (Pro-rich AMPs rely less on C-terminal charge). **Assay interpretation:**