From b699e065537884fb35aa0a03757826be4c7e5453 Mon Sep 17 00:00:00 2001 From: j Date: Mon, 29 Jun 2026 21:01:01 +0700 Subject: [PATCH 1/2] =?UTF-8?q?feat(panel):=20integrate=20wave=200.5b=20to?= =?UTF-8?q?p=20picks=20=E2=80=94=20SEED-020=20external=20predictor=20confi?= =?UTF-8?q?rmation?= MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit External predictor screening (CAMPR4, AMPScanner v2, Macrel, AMPActiPred, HemoFinder, AntiCP2) of 23 wave 0.5b candidates confirmed SEED-020_VAR_004 (RLRIRVLKRLLK) and SEED-020_VAR_002 (KVRIRVLKRLLK) as the best safety-activity leads in the batch. Broad novelty check confirms both are NOVEL (< 50% similarity to the 72-AMP curated reference database; max similarity 41.7% vs REF-RLK-001 and 37.5% vs REF-LL37-003). Key external predictor metrics: - SEED-020_VAR_004: AMPScanner 0.9928, HemoFinder LOW, Non-AntiCP 0.29 (cleanest in batch) - SEED-020_VAR_002: AMPScanner 0.9960 (highest in batch), HemoFinder LOW, Non-AntiCP 0.32 Both outperform SEED-019_VAR_004 on the AntiCP off-target safety criterion while maintaining equivalent activity. Both are NOVEL — confirmed activity is publishable. Changes: - docs/WET_LAB_HANDOFF.md: new SEED-019/020 section — scaffold description, synthesis guidance (Nα-Ac REQUIRED for 5 trypsin sites, C-amide recommended, SHORT_PEPTIDE serum note, Macrel hemolysis false-positive warning), assay priority note (SAR triad comparison with SEED-019_VAR_004 within same assay plate) - docs/EXPERT_REVIEW_PACK.md: §5 family table extended with SEED-019/020 row (P(≥1 active) ~78–87%, best ext-predictor confirmed); wave 0.5b addendum block added below table summarising the confirmation rationale - docs/ROADMAP.md: PR tracking entries for PR #104 (SEED-006 Met fix) and this PR wave1_final_panel.csv updated locally (outputs/ is gitignored): SEED-020_VAR_004 and SEED-020_VAR_002 added as BALANCED_LEAD (WAVE0_5B). Tests: 1372 passed. --- docs/EXPERT_REVIEW_PACK.md | 9 ++++++ docs/ROADMAP.md | 2 ++ docs/WET_LAB_HANDOFF.md | 60 ++++++++++++++++++++++++++++++++++++++ 3 files changed, 71 insertions(+) diff --git a/docs/EXPERT_REVIEW_PACK.md b/docs/EXPERT_REVIEW_PACK.md index c382854a..25999c72 100644 --- a/docs/EXPERT_REVIEW_PACK.md +++ b/docs/EXPERT_REVIEW_PACK.md @@ -174,6 +174,15 @@ not a claim that they are inactive. | SEED-007 (bombolitin) | 4 | Short amphipathic helix | Genuine (✓ NOVEL) | Low (11 AA; good) | ~70–82% | Novel short helix | | SEED-008 (Trp-rich) | 4 | Indole ring stacking; not helical | Genuine (✓ NOVEL) | Moderate (13 AA model edge) | ~70–82% | Novel mechanism; high disagreement | | SEED-009 (Pro-rich) | 4 | Ribosome exit + DnaK | Genuine (✓ NOVEL) | **Best** (Pro-X bonds resist protease) | ~60–75% | Novel; run RPMI parallel | +| SEED-019/020 (Arg-Ile/Val/Leu) | 3 | Cationic amphipathic helix | Genuine (✓ NOVEL all 3) | Short (12 AA model edge) | **~78–87%** | Best ext. predictor confirmed leads | + +> **Wave 0.5b addendum (2026-06-29):** External predictor screening (CAMPR4 × 4 votes, +> AMPScanner v2, Macrel, AMPActiPred, HemoFinder, AntiCP2) of 23 new SEED-020/021/023/024 +> candidates confirmed SEED-020_VAR_004 (RLRIRVLKRLLK) and SEED-020_VAR_002 (KVRIRVLKRLLK) +> as the best safety-activity tradeoff in the batch: HemoFinder LOW, AMPScanner > 0.99, +> Non-AntiCP. Both confirmed NOVEL by broad novelty check (< 50% similarity to 72-AMP database). +> Both added to `outputs/wave1_final_panel.csv` as BALANCED_LEAD candidates (source: WAVE0_5B). +> Full synthesis notes: `docs/WET_LAB_HANDOFF.md §SEED-019/SEED-020`. **SEED-007 reviewer note:** Bombolitin-II (*Megabombus pennsylvanicus* venom) is the **largest selected family** (26 variants, 4 pilot slots). All 4 pilot candidates are NOVEL diff --git a/docs/ROADMAP.md b/docs/ROADMAP.md index 5c7d6a47..682b7409 100644 --- a/docs/ROADMAP.md +++ b/docs/ROADMAP.md @@ -95,6 +95,8 @@ Implemented during the pre-wet-lab improvement loop (PRs #31–#54): - EXPERT_REVIEW_PACK.md: Pipeline Limitations table updated — PYROGLUTAMATE_RISK row added (N-terminal Q, physiological pH, none of 20 pilot candidates affected); SEED-009_VAR_033 Met row added (PR #108) - test: close remaining 1% coverage gap — 6 modules to 100% branch coverage; 1321 tests total; only 6 structural CLI guard lines remain uncovered; all source modules at 100% branch coverage (PR #109) - feat(bench+qc): expanded benchmark (95 AMPs + 96 decoys, n=191, AUROC=0.7832, CI₉₅: 0.72–0.84); 52 new public-domain AMPs across 12 classes (defensins, proline-rich, lantibiotics); DKP_RISK flag for N-terminal X-Pro diketopiperazine cyclization — all 4 SEED-008 pilots (F-Pro) flagged with dynamic mass (244 Da), MS receipt check, Nα-Ac REQUIRED; WET_LAB_HANDOFF.md SEED-008 synthesis guidance updated; ASSAY_PREREGISTRATION.md SEED-008 DKP note added; 1337 tests (PR #110) +- fix(docs+qc): correct SEED-006 Met synthesis error — ALL 4 SEED-006 pilots contain Met at position 9 (inherited from mastoparan-X); Nle/M9Nle REQUIRED in synthesis order; ASSAY_PREREGISTRATION.md SEED-006 Met pre-registered; EXPERT_REVIEW_PACK.md: SHORT_PEPTIDE count corrected (6→10 candidates), DKP_RISK + Met rows added, AUROC stale values corrected; regression guard `test_seed006_all_pilots_have_met_at_position9` with positional seq[8]=='M' assertion in TestOxidationRisk (PR #104) +- feat(panel): wave 0.5b external-predictor confirmation — SEED-020_VAR_004 (RLRIRVLKRLLK, AMPScanner 0.9928, HemoFinder LOW, Non-AntiCP 0.29) and SEED-020_VAR_002 (KVRIRVLKRLLK, AMPScanner 0.9960, HemoFinder LOW, Non-AntiCP 0.32) confirmed NOVEL by broad novelty check (72-AMP DB, <50% similarity); both added to wave1_final_panel.csv as BALANCED_LEAD (WAVE0_5B source); SEED-019/020 synthesis notes added to WET_LAB_HANDOFF.md; EXPERT_REVIEW_PACK.md §5 updated with SEED-019/020 family row and wave 0.5b addendum ## v0.5.7 — Novelty Audit v2: Proper Prior-Art Scan ✓ (2026-06-29) diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 30fdf59b..77074cdd 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -441,6 +441,66 @@ Pro-rich AMPs show > 2 h stability in 50% human serum (Otvos & Cudic 2002, *Curr --- +## SEED-019/SEED-020 (Arg-Ile/Val/Leu Alternating Scaffold) Special Notes + +SEED-019 and SEED-020 form a tight SAR cluster of short cationic amphipathic helices with the +motif **R-X-R-I-R-V-L-K-R-L-L-K** (X = V, L, K). All candidates in this cluster are 12 AA. +SEED-020 variants are design-inspired mutations of SEED-019_VAR_004 (RVRIRLVKRLLK) confirmed +by external predictor screening (wave 0.5b; 2026-06-29). + +**Best candidates added to final panel (wave 0.5b, external predictor confirmed):** + +| Candidate | Sequence | AMPScanner | HemoFinder | AntiCP | Broad novelty | +|-----------|----------|:----------:|:----------:|:------:|:-------------:| +| SEED-020_VAR_004 | RLRIRVLKRLLK | 0.9928 (AMP) | LOW | Non-AntiCP 0.29 | NOVEL (41.7% max) | +| SEED-020_VAR_002 | KVRIRVLKRLLK | 0.9960 (AMP) | LOW | Non-AntiCP 0.32 | NOVEL (37.5% max) | +| SEED-019_VAR_004 | RVRIRLVKRLLK | panel anchor | LOW | Non-AntiCP | NOVEL (38.1% max) | + +SEED-020_VAR_004 is the **best safety-activity tradeoff in the entire wave 0.5b batch** by +combined external predictor consensus. SEED-020_VAR_002 has the highest AMPScanner score (0.9960) +of any candidate in the batch. + +**Novelty:** All 3 confirmed NOVEL by broad novelty check (72-AMP curated reference database; +< 50% similarity to any known published AMP). Closest match is REF-RLK-001 (cationic helix +class) or REF-LL37-003 (LL-37 fragment) — sequence identity is low enough that confirmed +activity would be publishable as a novel amphipathic helix subclass. + +**Mechanism:** Cationic amphipathic helix — alternating hydrophobic (Leu/Val/Ile) and cationic +(Arg/Lys) faces interact with anionic bacterial membranes, causing rapid membrane depolarization. +Expected to be Gram-negative and Gram-positive active (broad-spectrum). The high Arg density +(≥4 Arg in 12 AA) creates a charge density of +4 to +5 at pH 7.4, which is above the minimum +required for E. coli outer membrane permeation. + +**Hemolysis warning:** Macrel hemolysis predictor scores 0.93 for all SEED-020 variants. However, +**Macrel hemolysis is known to flag all cationic helices as hemolytic regardless of actual activity** +(systematic bias documented in the pipeline; Melittin false-negative caveat in `EXPERT_REVIEW_PACK.md`). +HemoFinder (a separate predictor) classifies all SEED-019/020 variants as LOW hemolysis risk. +**Run empirical HC50 assay — do not exclude on Macrel score alone.** + +**Synthesis guidance:** +- No Met, no Cys → standard Fmoc SPPS, no oxidation risk, no disulfide risk. +- **N-terminal acetylation (Nα-Ac) REQUIRED** on all variants: 5 interior Lys/Arg trypsin sites; + acetylation blocks aminopeptidase exopeptidase entry (specify "N-terminal acetylation (Ac-)" + in synthesis order — zero extra cost). +- **C-terminal amidation (CONH₂) RECOMMENDED**: adds +0.5 net charge, improves serum exopeptidase + resistance, standard for short cationic helices (specify "C-terminal amide" in synthesis order). +- SHORT_PEPTIDE (12 AA): serum stability model calibrated for 18–30 AA — model score likely + **underestimates** actual t½; measure empirically (50% human serum, 37°C, 0/30/60/120 min HPLC). +- 5 trypsin sites at R/K positions → WAVE2_D_AMINO substitution plan generated; if serum t½ < 30 min, + proceed directly to D-Arg substitution at positions R1, R3, R5 in Wave 2. +- Storage: lyophilized at −20°C (no oxidation-sensitive residues; standard cationic peptide storage). + +**Assay priority:** +- Screen SEED-020_VAR_004 and SEED-020_VAR_002 in the same plates as SEED-019_VAR_004 for + direct SAR comparison. +- If both SEED-020 variants and SEED-019_VAR_004 are active, this SAR triad enables immediate + QSAR analysis of R→K and L→R substitution effects — a publishable structure-activity insight + even from a single wet-lab run. +- MRSA USA300 is a priority target for this scaffold class (Arg-rich AMPs disrupt Gram-positive + membranes; no intracellular target required unlike SEED-009). + +--- + ## Serum Stability Model Limitations (All 7 Seeds) The `serum_stability_score()` function is calibrated for medium-length cationic helices (~18–30 AA). From 5d35cce87854b1c61eaef87121955c809eba6a0e Mon Sep 17 00:00:00 2001 From: j Date: Mon, 29 Jun 2026 21:13:05 +0700 Subject: [PATCH 2/2] fix(review): address all HIGH/MEDIUM issues from code review of wave05b PR MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit HIGH — SEED-019_VAR_004 classified NOVEL in new docs but is CLOSE_RELATIVE with POSSIBLE_PATENT_RISK in official novelty audit v2 (66.7% to DRAMP05502). Added explicit IP NOTE block in WET_LAB_HANDOFF.md; added patent risk flag to EXPERT_REVIEW_PACK.md §5 row and wave 0.5b addendum; publishability claim scoped to SEED-020 pair only. HIGH — Arg count/charge density errors: "≥4 Arg" doesn't apply to KVRIRVLKRLLK (3 Arg + 3 Lys = 6 cationic); net charge "+4 to +5" incorrect — should be "+5 to +6". Fixed to "3–4 Arg plus 2–3 Lys, 6 total cationic, net charge ~+5 to +6." HIGH — Nα-Ac rationale conflated exopeptidase + endopeptidase protection. Added explicit clarification: Nα-Ac blocks aminopeptidase (exopeptidase) from the free α-amine; trypsin endoproteolysis at 5 K/R sites requires D-amino Wave 2. MEDIUM — Motif descriptor removed (had positional errors: K at pos1 for VAR_002, V/L order mismatch for SEED-019); replaced with prose description of the pattern. MEDIUM — Macrel bias characterization changed from "all cationic helices" to "non-discriminating in this dataset — 60/60 candidates flagged regardless of class" (matches documented evidence in METRICS_CURRENT.md). MEDIUM — P(≥1 active) range adjusted from 78–87% to 75–85% in EXPERT_REVIEW_PACK §5; "best ext. predictor confirmed leads" clarification added to reviewer focus. LOW — Melittin cross-reference removed; now cites docs/METRICS_CURRENT.md. LOW — SEED-022 documented in addendum (excluded at pre-screen, no viable candidates after safety scoring). Tests: 1372 passed. --- docs/EXPERT_REVIEW_PACK.md | 12 +++++--- docs/WET_LAB_HANDOFF.md | 58 ++++++++++++++++++++++---------------- 2 files changed, 42 insertions(+), 28 deletions(-) diff --git a/docs/EXPERT_REVIEW_PACK.md b/docs/EXPERT_REVIEW_PACK.md index 25999c72..bbde245f 100644 --- a/docs/EXPERT_REVIEW_PACK.md +++ b/docs/EXPERT_REVIEW_PACK.md @@ -174,15 +174,19 @@ not a claim that they are inactive. | SEED-007 (bombolitin) | 4 | Short amphipathic helix | Genuine (✓ NOVEL) | Low (11 AA; good) | ~70–82% | Novel short helix | | SEED-008 (Trp-rich) | 4 | Indole ring stacking; not helical | Genuine (✓ NOVEL) | Moderate (13 AA model edge) | ~70–82% | Novel mechanism; high disagreement | | SEED-009 (Pro-rich) | 4 | Ribosome exit + DnaK | Genuine (✓ NOVEL) | **Best** (Pro-X bonds resist protease) | ~60–75% | Novel; run RPMI parallel | -| SEED-019/020 (Arg-Ile/Val/Leu) | 3 | Cationic amphipathic helix | Genuine (✓ NOVEL all 3) | Short (12 AA model edge) | **~78–87%** | Best ext. predictor confirmed leads | +| SEED-019/020 (Arg-Ile/Val/Leu) | 3 | Cationic amphipathic helix | ✓ NOVEL (SEED-020 pair); CLOSE_RELATIVE + ⚠ patent risk (SEED-019_VAR_004) | Short (12 AA model edge) | **~75–85%** (SEED-020 pair); patent clearance needed for SEED-019_VAR_004 | Best ext.-predictor confirmed leads; IP check before SEED-019 synthesis | > **Wave 0.5b addendum (2026-06-29):** External predictor screening (CAMPR4 × 4 votes, > AMPScanner v2, Macrel, AMPActiPred, HemoFinder, AntiCP2) of 23 new SEED-020/021/023/024 > candidates confirmed SEED-020_VAR_004 (RLRIRVLKRLLK) and SEED-020_VAR_002 (KVRIRVLKRLLK) > as the best safety-activity tradeoff in the batch: HemoFinder LOW, AMPScanner > 0.99, -> Non-AntiCP. Both confirmed NOVEL by broad novelty check (< 50% similarity to 72-AMP database). -> Both added to `outputs/wave1_final_panel.csv` as BALANCED_LEAD candidates (source: WAVE0_5B). -> Full synthesis notes: `docs/WET_LAB_HANDOFF.md §SEED-019/SEED-020`. +> Non-AntiCP. Both confirmed NOVEL by broad novelty check (72-AMP curated DB; < 50% max +> similarity; no patent DB matches). Both added to `outputs/wave1_final_panel.csv` as +> BALANCED_LEAD candidates (source: WAVE0_5B). SEED-019_VAR_004 (already in panel) carries +> a POSSIBLE_PATENT_RISK flag (66.7% to DRAMP05502 in full 27,234-sequence audit) — require +> IP clearance before synthesis. SEED-022 was excluded at pre-screen (no viable candidates +> after safety scoring; poor AntiCP / HemoFinder profile across all variants). Full synthesis +> notes: `docs/WET_LAB_HANDOFF.md §SEED-019/SEED-020`. **SEED-007 reviewer note:** Bombolitin-II (*Megabombus pennsylvanicus* venom) is the **largest selected family** (26 variants, 4 pilot slots). All 4 pilot candidates are NOVEL diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 77074cdd..9c52a381 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -443,45 +443,55 @@ Pro-rich AMPs show > 2 h stability in 50% human serum (Otvos & Cudic 2002, *Curr ## SEED-019/SEED-020 (Arg-Ile/Val/Leu Alternating Scaffold) Special Notes -SEED-019 and SEED-020 form a tight SAR cluster of short cationic amphipathic helices with the -motif **R-X-R-I-R-V-L-K-R-L-L-K** (X = V, L, K). All candidates in this cluster are 12 AA. -SEED-020 variants are design-inspired mutations of SEED-019_VAR_004 (RVRIRLVKRLLK) confirmed -by external predictor screening (wave 0.5b; 2026-06-29). +SEED-019 and SEED-020 form a tight SAR cluster of short cationic amphipathic helices sharing +the alternating Arg-Ile/Val/Leu pattern. All candidates are 12 AA with 5–6 combined Lys/Arg +residues. SEED-020 variants are design-inspired mutations of SEED-019_VAR_004 (RVRIRLVKRLLK) +confirmed by external predictor screening (wave 0.5b; 2026-06-29). **Best candidates added to final panel (wave 0.5b, external predictor confirmed):** -| Candidate | Sequence | AMPScanner | HemoFinder | AntiCP | Broad novelty | -|-----------|----------|:----------:|:----------:|:------:|:-------------:| -| SEED-020_VAR_004 | RLRIRVLKRLLK | 0.9928 (AMP) | LOW | Non-AntiCP 0.29 | NOVEL (41.7% max) | -| SEED-020_VAR_002 | KVRIRVLKRLLK | 0.9960 (AMP) | LOW | Non-AntiCP 0.32 | NOVEL (37.5% max) | -| SEED-019_VAR_004 | RVRIRLVKRLLK | panel anchor | LOW | Non-AntiCP | NOVEL (38.1% max) | +| Candidate | Sequence | AMPScanner | HemoFinder | AntiCP | Broad novelty (72-AMP DB) | Full-DB novelty | +|-----------|----------|:----------:|:----------:|:------:|:-------------:|:---:| +| SEED-020_VAR_004 | RLRIRVLKRLLK | 0.9928 (AMP) | LOW | Non-AntiCP 0.29 | NOVEL (41.7% max) | NOVEL | +| SEED-020_VAR_002 | KVRIRVLKRLLK | 0.9960 (AMP) | LOW | Non-AntiCP 0.32 | NOVEL (37.5% max) | NOVEL | +| SEED-019_VAR_004 | RVRIRLVKRLLK | panel anchor | LOW | Non-AntiCP | 38.1% max vs non-patent AMPs | **CLOSE_RELATIVE ⚠ patent risk** | + +> **IP NOTE — SEED-019_VAR_004:** Full 27,234-sequence novelty audit v2 (APD6, DRAMP general, +> DRAMP patent, UniProt) classifies SEED-019_VAR_004 as **CLOSE_RELATIVE (66.7% identity to +> DRAMP patent sequence DRAMP05502)**. **IP clearance is required before ordering synthesis +> or publishing results for SEED-019_VAR_004.** The SEED-020 pair (VAR_004 and VAR_002) were +> not flagged in the patent database and carry no identified IP risk. SEED-020_VAR_004 is the **best safety-activity tradeoff in the entire wave 0.5b batch** by combined external predictor consensus. SEED-020_VAR_002 has the highest AMPScanner score (0.9960) of any candidate in the batch. -**Novelty:** All 3 confirmed NOVEL by broad novelty check (72-AMP curated reference database; -< 50% similarity to any known published AMP). Closest match is REF-RLK-001 (cationic helix -class) or REF-LL37-003 (LL-37 fragment) — sequence identity is low enough that confirmed -activity would be publishable as a novel amphipathic helix subclass. +**Novelty:** SEED-020_VAR_004 and SEED-020_VAR_002 confirmed NOVEL by broad novelty check +(72-AMP curated reference database; < 50% similarity). Closest matches are in the REF-RLK-001 +(cationic helix) and REF-LL37-003 (LL-37 fragment) classes — confirmed activity in either +would be publishable. SEED-019_VAR_004 carries a patent proximity flag (see IP NOTE above); +publishability claims for SEED-019_VAR_004 require IP clearance. **Mechanism:** Cationic amphipathic helix — alternating hydrophobic (Leu/Val/Ile) and cationic (Arg/Lys) faces interact with anionic bacterial membranes, causing rapid membrane depolarization. -Expected to be Gram-negative and Gram-positive active (broad-spectrum). The high Arg density -(≥4 Arg in 12 AA) creates a charge density of +4 to +5 at pH 7.4, which is above the minimum -required for E. coli outer membrane permeation. - -**Hemolysis warning:** Macrel hemolysis predictor scores 0.93 for all SEED-020 variants. However, -**Macrel hemolysis is known to flag all cationic helices as hemolytic regardless of actual activity** -(systematic bias documented in the pipeline; Melittin false-negative caveat in `EXPERT_REVIEW_PACK.md`). -HemoFinder (a separate predictor) classifies all SEED-019/020 variants as LOW hemolysis risk. +Expected to be Gram-negative and Gram-positive active (broad-spectrum). Each sequence carries +3–4 Arg plus 2–3 Lys, for a combined Lys/Arg density of 6 in 12 AA; net charge at pH 7.4 +is approximately **+5 to +6** depending on terminal modifications — well above the +3 threshold +for E. coli outer membrane permeation. + +**Hemolysis warning:** Macrel hemolysis predictor scores 0.93 for all SEED-020 variants. +However, **Macrel hemolysis is non-discriminating in this dataset — 60/60 wave 0.5 candidates +were flagged regardless of sequence class** (documented in evidence JSONs; see +`docs/METRICS_CURRENT.md`). HemoFinder (a separate predictor) classifies all SEED-019/020 +variants as LOW hemolysis risk. **Run empirical HC50 assay — do not exclude on Macrel score alone.** **Synthesis guidance:** - No Met, no Cys → standard Fmoc SPPS, no oxidation risk, no disulfide risk. -- **N-terminal acetylation (Nα-Ac) REQUIRED** on all variants: 5 interior Lys/Arg trypsin sites; - acetylation blocks aminopeptidase exopeptidase entry (specify "N-terminal acetylation (Ac-)" - in synthesis order — zero extra cost). +- **N-terminal acetylation (Nα-Ac) REQUIRED**: blocks N-terminal aminopeptidase (exopeptidase) + from degrading the free α-amine terminus (specify "N-terminal acetylation (Ac-)" in synthesis + order — zero extra cost). Note: Nα-Ac does NOT protect against endopeptidase (trypsin) cleavage + at the 5 interior K/R sites; endoproteolytic protection requires D-amino substitution (Wave 2). - **C-terminal amidation (CONH₂) RECOMMENDED**: adds +0.5 net charge, improves serum exopeptidase resistance, standard for short cationic helices (specify "C-terminal amide" in synthesis order). - SHORT_PEPTIDE (12 AA): serum stability model calibrated for 18–30 AA — model score likely