STRC h01 Phase 6c Full 6-Target Panel 2026-04-24
Extends STRC h01 Phase 6c COX Selectivity Panel 2026-04-24 from 2/7 → 6/6 dockable targets after literature curation of bound-ligand PDB IDs (none guessed, all verified bound HETATM resnames). Same 10-ligand probe panel, same ensemble pipeline. 60 docks delivered in 4 min 42 s.
Receptor curation — Stage A revision
| Target | New PDB | Bound ligand | Class | Source |
|---|---|---|---|---|
| TRPM4 | 8RD9 | A1H0C | anthranilic-class pore blocker | Ekundayo 2025 Nat Commun 16:833 |
| Cx50 (via Cx36 paralog) | 8QOJ | YMZ (mefloquine) | quinoline antimalarial / pan-connexin blocker | Ding 2024 Cell Discov 10:68 |
| KCNQ4 | 7BYM | FBX (retigabine) | KCNQ-family activator | Li T 2021 Mol Cell 81:25 |
| TMEM16A | 7ZK3 | JRF (1PBC) | aromatic pore blocker | Lam, Rutz & Dutzler 2022 Nat Commun 13:2798 |
| COX-1 | 1Q4G | BFL (bromoflufenamic) | fenamate | (existing) |
| COX-2 | 5IKR | ID8 | fenamic-class | (existing) |
Old IDs replaced: TRPM4 7MF0 (404), Cx50 7JN0 (apo), KCNQ4 7BYL (was KCNQ1), TMEM16A 5OYB (apo). BK channel skipped — no NSAID/blocker-bound PDB exists in RCSB; only NS1619 activator-bound is available, which is not a fenamate-class proxy. Cross-paralog warning logged for Cx36→Cx50.
Boxes derived from cif HETATM centroid (TRPM4 obabel pdb conversion drops HETATMs, falls back to cif parser); 22 Å cube (channel pockets are wider than h01 18 Å).
Stage B — full 6-target dock results
Per-ligand selectivity matrix (Kd in µM)
| Ligand | h01 mut Kd (5e) | COX-1 | COX-2 | TMEM16A | TRPM4 | Cx50 (via Cx36) | KCNQ4 |
|---|---|---|---|---|---|---|---|
| niflumic-acid | 41.1 | 0.42 | 0.27 | 1.67 | 44.6 | 218.7 | 410.7 |
| flufenamic-acid | 32.7 | 0.51 | 0.37 | 0.77 | 24.1 | 56.1 | 278.5 |
| diflunisal | 104.2 | 0.72 | 1.04 | 3.09 | 43.2 | 238.4 | 675.9 |
| meclofenamic-acid | 94.0 | 0.91 | 3.81 | 5.45 | 75.9 | 270.6 | 882.6 |
| tafamidis | (untested) | 1.52 | 1.54 | 4.72 | 55.1 | 127.2 | 461.5 |
| benzox-3CN-COOH (4h #1) | (untested) | 1.04 | 1.74 | 2.88 | 84.1 | 167.2 | 441.6 |
| benzox-3OMe-COOH (4h #2) | (untested) | 1.00 | 0.99 | 2.60 | 82.3 | 166.1 | 559.4 |
| iododiflunisal | (untested) | 9.42 | 2.95 | 4.28 | 51.1 | 247.9 | 614.9 |
| aspirin (neg ctrl) | — | 40.3 | 22.1 | 36.1 | 666.8 | 1066.4 | 2736.6 |
| DMSO (proxy neg) | — | 17600 | 14107 | 27396 | 69947 | 29360 | 63420 |
Bold = sub-5 µM Kd → off-target liability at therapeutic 10 µM dose.
Pipeline calibration (vs published IC₅₀)
| Target | Parent | Predicted Kd | Published IC₅₀ | Within 1 OOM? |
|---|---|---|---|---|
| COX-2 | niflumic | 0.27 | 0.1–0.3 | ✅ |
| COX-2 | flufenamic | 0.37 | 0.5–3 | ✅ |
| TRPM4 | flufenamic | 24.1 | 2.8 | ⚠ 9× weaker (anthranilic-class A1H0C site is more permissive than flufenamic site) |
| TMEM16A | flufenamic | 0.77 | 12 | ⚠ 16× tighter (1PBC pore-blocker site is the JRF aromatic anchor, fenamates fit) |
| Cx50 (Cx36 proxy) | flufenamic | 56.1 | 3 (Cx50, not Cx36) | ⚠ 19× weaker (cross-paralog underestimate, expected) |
| KCNQ4 | flufenamic | 278.5 | 56 (KCNQ-family) | ⚠ 5× weaker (retigabine site is activator-targeting, fenamates likely block elsewhere) |
Two calibration take-aways:
- COX scoring is robust (within 2× of published) — interpretable Kd-level differences.
- Channel scoring shows site-specific deviations of 5-19× — interpret rank order, not absolute Kd. Use relative selectivity (ligand_X / ligand_Y at same target) as the primary metric, not absolute Kd.
Real findings
Finding 1 — TMEM16A is a SECOND off-target wall, not just COX
Phase 6c COX-only proof note flagged the COX selectivity wall. Full panel reveals TMEM16A is just as bad:
- Tafamidis TMEM16A Kd 4.7 µM, COX-2 Kd 1.5 µM (3× ratio, both sub-10)
- Benzoxazole-3CN TMEM16A 2.9 µM, COX-2 1.7 µM
- Benzoxazole-3OMe TMEM16A 2.6 µM, COX-2 0.99 µM
Both COX (Arg120 carboxylate salt bridge) and TMEM16A (pore-blocker JRF aromatic anchor) accept the bicyclic-COOH pharmacophore at sub-5-µM. The tafamidis-playbook Tier-1/Tier-2 set is a double-off-target liability, not single.
For Misha’s pediatric target this matters because TMEM16A is highly expressed in stria vascularis (key cochlear epithelium for endocochlear potential maintenance) — TMEM16A inhibition at 1-5 µM would risk ototoxicity beyond just COX-driven blood-flow effects.
Finding 2 — KCNQ4 cleanly de-selected by all bioisosteres
| Compound | KCNQ4 Kd | Therapeutic 10 µM bound fraction |
|---|---|---|
| tafamidis | 461 µM | 2% |
| benzoxazole-3CN-COOH | 442 µM | 2% |
| benzoxazole-3OMe-COOH | 559 µM | 2% |
| niflumic | 411 µM | 2% |
| flufenamic | 278 µM | 4% |
The retigabine (FBX) pocket on KCNQ4 (at S5/S6 interface) is dimensionally and chemically incompatible with the carboxylate-anchored fenamate/benzoxazole pharmacophore. KCNQ4 = DFNA2 paralog — this is the most safety-critical channel for a hearing-loss pediatric drug, and Phase 6c says we’re clean on it.
Finding 3 — TRPM4 mildly de-selected by bicyclic bioisosteres
| Compound | TRPM4 Kd | Improvement vs flufenamic (24 µM) |
|---|---|---|
| flufenamic | 24.1 | 1× (baseline) |
| diflunisal | 43.2 | 1.8× |
| niflumic | 44.6 | 1.8× |
| iododiflunisal | 51.1 | 2.1× |
| tafamidis | 55.1 | 2.3× |
| meclofenamic | 75.9 | 3.1× |
| benzoxazole-3OMe | 82.3 | 3.4× |
| benzoxazole-3CN | 84.1 | 3.5× |
Modest 3-4× selectivity gain from anthranilic→benzoxazole pivot. At 10 µM therapeutic, tafamidis TRPM4 bound-fraction = 15% — borderline, would still register on patch-clamp as ~20% inhibition. Better than parents but not transformative.
Finding 4 — Cx50 (via Cx36 proxy): bicyclic bioisosteres mildly preferred
Cross-paralog caveat applies (the 8QOJ Cx36-mefloquine pocket is a different ligand class than fenamic-class Cx50 binding). Pattern: flufenamic 56 µM (best, but Cx36 not Cx50), tafamidis/benzoxazoles 127-167 µM, diflunisal/iododiflunisal/meclofenamic 238-271 µM. Bicyclic bioisosteres are 2-4× tighter than non-fenamic NSAIDs but 2-3× weaker than flufenamic. Insufficient resolution for actionable selectivity claim — Phase 8 wet-lab dye-transfer assay is the authoritative gate.
Selectivity heatmap — what dies, what survives
For each compound at therapeutic 10 µM dose, classify off-target as:
- KILL if bound-fraction ≥ 50% (sub-10 µM Kd) → would register on activity assay
- WARN if bound-fraction 20-50% (10-40 µM Kd) → may register on sensitive assay
- CLEAN if bound-fraction < 20% (Kd > 40 µM) → safe at therapeutic
| Compound | COX-1 | COX-2 | TMEM16A | TRPM4 | Cx50 | KCNQ4 | Verdict |
|---|---|---|---|---|---|---|---|
| tafamidis | KILL | KILL | KILL | WARN | CLEAN | CLEAN | 3 kills — not viable as-is |
| benzox-3CN-COOH | KILL | KILL | KILL | CLEAN | CLEAN | CLEAN | 3 kills — not viable |
| benzox-3OMe-COOH | KILL | KILL | KILL | CLEAN | CLEAN | CLEAN | 3 kills — not viable |
| diflunisal | KILL | KILL | KILL | WARN | CLEAN | CLEAN | 3 kills |
| iododiflunisal | KILL | KILL | KILL | WARN | CLEAN | CLEAN | 3 kills |
| meclofenamic | KILL | KILL | KILL | WARN | CLEAN | CLEAN | 3 kills |
| niflumic | KILL | KILL | KILL | WARN | CLEAN | CLEAN | 3 kills |
| flufenamic | KILL | KILL | KILL | WARN | KILL | CLEAN | 4 kills |
Every Phase 4h Tier-1/Tier-2 candidate has ≥ 3 sub-10 µM off-targets. None are wet-lab viable as currently designed.
Implication for v4 in-flight (Stage 1 700/1572 as of writeup)
v4 fragment-grow library uses the same locked cores (3-amino-benzofuran-2-COOH + benzoxazole + benzothiazole) and same -COOH / -CONHOH / -CONHSO₂Me / tetrazole acid bioisosteres as the Phase 4h playbook. Top-N v4 GREENs/YELLOWs will inherit the COX + TMEM16A liabilities unless they break the carboxylate engagement entirely.
Decision for v4 post-verdict:
- If v4 returns GREEN/YELLOW → cross-dock top-30 against this Phase 6c 6-target panel (5 min wall) BEFORE any Phase 8 ordering. Real shortlist = compounds that pass both K1141 affinity AND COX/TMEM16A selectivity. Likely 0/30 will pass currently — drives next-step toward v4b carboxylate-deselect sub-strategy.
- If v4 returns RED → COX/TMEM16A liability is moot, focus on Phase 3c v5 RFdiffusion de novo pocket design.
v4b proposed sub-strategy (queue, do not launch yet):
- Lock cores 3-amino-benzofuran-2-COOH + benzoxazole-5-COOH (v3b/v4 winners)
- Replace -COOH with non-anionic K1141-engaging bioisosteres: amidoxime, N-hydroxyacetamide, 1,2,4-oxadiazol-5-ol, tetrazole (already partial), N-acylcyanamide
- Each candidate must (a) keep K1141 ε-NH₃⁺ H-bond geometry, (b) drop the COX Arg120 salt bridge, (c) drop the TMEM16A pore-blocker aromatic-anchor charge interaction
- ~500-1000 ligand library, focused enumeration, ~30-45 min wall
Action items
- Cross-dock v4 top-30 against Phase 6c panel as soon as v4 lands (~50 min from this writeup)
- Phase 4h playbook revision (already started in-place); add TMEM16A as second deselect target
- Phase 8 SOP: promote both COX and TMEM16A activity assays to G2.5 kill gate (alongside MST), not deferred to G3
- Decide on v4b carboxylate-deselect launch after v4 verdict (likely yes)
- Replace BK channel skip with patch-clamp Phase 8 G3 gate (no NSAID-bound PDB exists; cannot dock-screen)
- Cx50 native PDB needed (Cx36 paralog uncertainty) — wait for new connexin literature or run Cx50 AF3 + dock against pore-lining residues
Ranking delta
Tier A held. mech 3 / deliv 4 / misha_fit 4 all held.
- Phase 4h “tafamidis-class bioisostere kills off-targets” thesis falsified for two off-targets (COX + TMEM16A), not just COX. KCNQ4 (DFNA2 paralog, most safety-critical) cleanly de-selected — partial validation.
- Pipeline now has 6-target self-consistent off-target Kd matrix usable for medchem triage.
- Material constraint: no current Tier-1/Tier-2 Phase 4h compound passes the 3-of-3 (h01 Kd + COX + TMEM16A) selectivity gate. Any wet-lab order of these compounds is a calibration purchase, not a lead-development purchase.
next_stepunchanged: v4 still primary; v4 verdict triggers either cross-dock-then-Phase-8 (if YELLOW with selective compounds) or v4b carboxylate-deselect launch (if v4 chemistry inherits same off-target pattern).lit_audit: fixedheld; this is calibrated dock against verified bound-ligand PDBs.
Connections
[part-of]h01 hub[supersedes]STRC h01 Phase 6c COX Selectivity Panel 2026-04-24 — extends from 2/7 → 6/6 targets[see-also]STRC h01 Phase 4h Tafamidis Playbook Library 2026-04-23 — design thesis falsified on COX + TMEM16A; KCNQ4 validated[see-also]STRC h01 Fenamic Scaffold Tox Audit 2026-04-23 — IC₅₀ priors mostly reproduced (5-19× site-specific deviation)[see-also]STRC h01 Phase 5e Mutant Re-Dock Delivery 2026-04-24 — h01 mutant Kd reference[see-also]STRC h01 Phase 8 Wet-Lab Triage SOP — TMEM16A joins COX as G2.5 kill gate- Misha Compound-Het Therapy Stack Model
- STRC Hypothesis Ranking