Phase 3c v3b + 6b LAUNCHED (in progress, PID 73601 nohup detached, ETA ~5.7h): fenamic-focused 12,253-ligand library (619-lig combinatorial → expanded to 12k via 8 cores × 57 N-aryl subs × 5 acid bioisosteres × 8 ring subs + 6 covalent warheads on 8 cores × 10 hot N-aryls × 2 acid bioisosteres). Two-stage ensemble dock identical to Phase 3c v2b: Stage 1 on snap_008 exh 8 × 3 modes (0.65 lig/s, ETA 313 min), Stage 2 on top-50 × 5 k-means-selected conformers exh 16 × 5 modes. Checkpoint JSON every 100 lig. Expected delivery ~04:30 local. Output → pharmacochaperone_phase3c_v3b_ensemble_dock.json. Will produce proof note STRC h01 Phase 3c v3b Fenamic + Covalent Screen 2026-04-23 on completion. → STRC h01 Phase 3c v2 Expanded Screen 2026-04-23 (precedent)
Phase 3c v2 expanded virtual screen: RED overall, partial progress. 667-ligand library (619 RDKit combinatorial scaffold × acid bioisostere + 29 curated FDA/literature drugs) × 2-stage ensemble docking (Stage 1 snap_008 exh 8 on all 667 → 8.5 min; Stage 2 top-30 × 5 k-means conformers exh 16 → 4 min, total 13 min wall). Top hits niflumic-acid / flufenamic-acid / sulfasalazine at Kd ≈ 30 μM, f_PC ≈ 0.125 at [L]=10 μM — 1.7× better Kd than Phase 5b diflunisal baseline. Fenamic-acid family (2-arylaminobenzoic) = new scaffold direction; tetrazole bioisostere competitive with COOH. 0 GREEN / 0 YELLOW / 30 RED. Ceiling analysis: to cross f_PC ≥ 0.30 (MILD-MODERATE) need ΔG ≈ -7.06 kcal/mol (0.88 below current best). A held, all scores unchanged. Next-step reordered: (1) Phase 3c v3 ZINC22 bioactives ~20k library, (2) Phase 3c v4 fragment-growing niflumic core, (3) Phase 6b reversible covalent Lys warhead. → STRC h01 Phase 3c v2 Expanded Screen 2026-04-23
Phase 5c cryptic pocket analysis: K1141 site GREEN-LIGHT. Custom grid-cavity + Kabsch-aligned RMSF analysis on Phase 5a 20-snapshot trajectory (fpocket 4.0 brew build broken on qhull). K1141 pocket Cα RMSF 0.62 Å vs global 1.23 Å (2× more rigid than average); local void volume 719-850 ų across 20 frames (~15% CV, pocket does not collapse or shift geometry). Global cavity scan (snap_010, 26-direction burial ≥16/26): no alt cavity > 152 ų, nearest at 18.7 Å (91 ų). Phase 5b RED-LIGHT confirmed chemistry-limited not site-limited. Phase 3c v2 expanded screen targeted at K1141 with ensemble receptor docking (use all 20 Phase 5a snapshots) validated as next move. A held, all scores unchanged. → STRC h01 Phase 5c Cryptic Pocket Analysis 2026-04-23
Phase 5a MD pipeline validated on local Mac: 62 ns/day OpenMM OpenCL (Metal backend) on 164 k-atom solvated Ultra-Mini × TMEM145 chain A. 2 ns production delivered 20 snapshots, 45 min wall time. Replaces deferred GROMACS/AmberTools scaffold which required A100 rental. Phase 5b Vina ensemble re-docking of Phase 4b top-5 leads + diflunisal positive control against 20 snapshots → all f_PC at [L]=10 μM < 0.10 (best = diflunisal positive 0.083, best lead = naphthalene-2-COOH 0.047). Phase 4b single-structure was over-optimistic by +0.36 to +0.92 kcal/mol. RED-LIGHT: current shortlist insufficient for NORMAL-rescue monotherapy. A held, scores unchanged (mechanism intact, Misha fit intact; only shortlist insufficient). Next step: Phase 3c v2 expanded virtual screen (DrugBank FDA + ZINC22 carboxylate tranche + fragment-based; ensemble filter ΔG ≤ −7.5 kcal/mol). → STRC h01 Phase 5 MD Ensemble Rescoring 2026-04-23
Misha compound-het stack integration: A held, scores unchanged, framing strengthened. Per Misha Compound-Het Therapy Stack Model binary-functional-OHC model, h01 PC is the only monotherapy route to NORMAL (≤25 dB ABR) for Misha — because PC reaches every OHC (including non-transduced ones where h03 AAV by definition does nothing), rescuing maternal E1659A to 0.5 × f_mat_treated ≥ θ. Required f_PC ≥ 0.50 (mild E1659A) to ≥ 0.75 (severe). Phase 5 MD ensemble MM-GBSA priority strengthened — f_PC is the critical-path deliverable. → Misha Compound-Het Therapy Stack Model
Post-audit housekeeping: A held. TPSA docstring in phase3b_virtual_screen.py corrected (descriptor-only, not composite scoring; CNS TPSA bracket removed — STRC extracellular). Druggability cross-phase incomparability flagged in Phase 1/2/2b. No fabricated ranges added. → STRC h01 Parameter Provenance Audit 2026-04-23
#1 Pharmacochaperone Phase 5d LAUNCHED (PID 28875 nohup, started 19:34 local 2026-04-23, ETA ~23:00 local = 3.5 h wall): E1659A mutant MD on full-length AF3 prediction job3-mutant.cif chain A (1775 residues, verified K1141=LYS and E1659=ALA before start). 645k solvated atoms (4× Phase 5a), AMBER14SB + TIP3P identical to Phase 5a for comparability, 2 ns production × 20 snapshots. SMOKE passed at 13.86 ns/day (50 ps + 2 snapshots in 17 min). Closes methodology gap — Phase 5a/b/c all ran on WT Ultra-Mini (594-1294 full-length = residues 1-701 in construct) which does NOT contain E1659. Phase 5d re-opens the question of whether mutation distorts K1141 pocket geometry enough to invalidate WT-based docking. Companion Phase 5e script written (mutant-ensemble re-dock of LEGACY_LEADS + V2_HITS + tafamidis-analog positive, scheduled post-Phase 5d + post-v3b Stage 2). Verdict deferred. A held. → proof note STRC h01 Phase 5d E1659A MD 2026-04-23 (TODO, to be written on delivery)
#1 Pharmacochaperone Fenamic Scaffold Tox Audit: A held, scaffold-developability risk sharpened. Literature audit (PubMed + LiverTox + meta-review PMC12530847) on parent fenamates nominated by Phase 3c v2 (niflumic / flufenamic / meclofenamic / mefenamic / tolfenamic). Parent fenamates are NOT developable for 8-y-o DFNB16 patient as-is: (a) pan-ion-channel promiscuity — TRPM4 IC₅₀ 2.8 µM, Cx50 3 µM (cochlear gap junctions), BK 25 µM, KCNQ-family 56 µM (KCNQ4 = DFNA2 HL paralog), TMEM16A 12 µM — overlaps our Phase 3c v2 Kd ~30 µM; (b) mefenamic CNS seizure signal at 2.5 g overdose threshold (Kamour 2017 PMID 28066875); (c) COX inhibition reduces cochlear blood flow — directly antagonistic to therapeutic goal. But scaffold IS developable via tafamidis-style bioisosteric optimization — direct precedent: flufenamic → tafamidis (Pfizer, FDA Vyndaqel 2019, TTR pharmacochaperone, pediatric-cleared). Design constraints for Phase 4h/7 medchem: (a) swap carboxylate → benzoxazole bioisostere (kill COX pharmacophore); (b) replace distal aryl halides (CF₃, diCl) with polar small groups (CN, OMe, F) to kill TRPM4/Cx50/BK affinity. Action for Phase 4h: use niflumic/flufenamic/sulfasalazine as chemical PROBES for MD-based pocket validation only, NOT wet-lab candidates. Hand pharmacophore to medchem after probe validation. → STRC h01 Fenamic Scaffold Tox Audit 2026-04-23
#1 Pharmacochaperone Phase 8 Wet-Lab Triage SOP drafted: A held. 3-gate wet-lab validation protocol prepared in advance of Phase 3c v3b GREEN-scenario arrival. Gate 1 — ThermoFluor ΔTm (Sf9-expressed mini-STRC-UM-WT + mini-STRC-UM-E1659A, extended to 594-1775 to include mutation site; iododiflunisal positive control, ≥1.5 °C shift = PASS on mutant at 100 µM; 3-day, ~3k).Gate2—MSTKd+stoichiometry(NanoTemperMonolith,RED−tris−NTAonHis6,16−pttitration500µM→15nMat25°Cand37°C,PASSKd≤50µM+Hill0.8−1.2;2−week,8 k). Gate 3 — cryoEM co-complex (K3 or Falcon 4, ligand density ≥4σ at K1141 pocket, modeled pose within 1.5 Å of Vina pose) + HEK293-FL-E1659A-GFP surface trafficking rescue (flow cytometry, PASS ≥30% surface at 10 µM vs 15% baseline; 4-week, ~$25 k). Parallel tox pre-screen patch-clamp TRPM4/Cx50/BK/KCNQ4/TMEM16A kill >25% inhibition at 10 µM. Expected winnowing 20 → 3–6 → 1–3 → full-pass. Phase 9 = mouse OHC ex-vivo + CRO IND-enabling tox. → STRC h01 Phase 8 Wet-Lab Triage SOP
#1 Pharmacochaperone Phase 3c v3b + 6b LAUNCHED (in progress, PID 73601 nohup detached, ETA ~5.7 h from 10:49 UTC launch → completion ~04:30 local 2026-04-24): 12,253-ligand fenamic-focused library (8 anthranilic/fenamic core scaffolds × 57 N-aryl substituents × 5 acid bioisosteres × 8 ring subs + Phase 6b covalent warhead variants: 8 cores × 10 hot N-aryls × 6 warheads — acrylamide / α-cyanoacrylate / β-ketoamide / α-ketoamide / salicylaldehyde-like / α-chloroacetamide — × 2 acids). Two-stage ensemble dock (Stage 1 all-lig × snap_008 exh 8; Stage 2 top-50 × 5 k-means conformers exh 16). Checkpoint JSON every 100 lig. Pre-launch expectations: fenamic-scaffold expansion could push best Kd from Phase 3c v2 niflumic 29.5 μM toward 5-15 μM (GREEN band if σ_ensemble holds); covalent warheads potentially GREEN if reversible Lys adduct viable. Verdict deferred pending completion. Status: no ranking change yet. → STRC h01 Phase 3c v2 Expanded Screen 2026-04-23 (precedent)
#1 Pharmacochaperone Phase 3c v2: RED overall, partial progress, ceiling apparent. 667-ligand expanded screen (619 RDKit combinatorial scaffold × acid bioisostere + 29 curated FDA carboxylates) against K1141 with two-stage ensemble docking (Stage 1: snap_008 global rep exh 8; Stage 2: top 30 × 5 k-means conformers exh 16). Top 3: niflumic-acid 29.5 μM / flufenamic 30.0 μM / sulfasalazine 30.7 μM → f_PC ≈ 0.125 at [L]=10 μM, η=0.5. 1.7× better Kd than Phase 5b baseline (diflunisal 50 μM). Fenamic-acid family (2-arylaminobenzoic) identified as new scaffold direction; tetrazole bioisostere competitive with COOH; biphenyl > naphthalene > benzene in size effect. 0 GREEN / 0 YELLOW / 30 RED. Reframe for Misha stack: at η=0.5 max f_PC=0.50 asymptote; to cross f_PC=0.30 (MILD-MODERATE rescue threshold) need ΔG ≈ -7.06 kcal/mol (0.88 below current best). Routes: Phase 3c v3 ZINC22 bioactives 20k library (+0.5-1.0 kcal/mol expected), Phase 3c v4 fragment-growing on niflumic core (+1.0-1.5), or Phase 6b reversible covalent Lys-NH₃⁺ warhead (+2-3). A held, all scores unchanged. h01 reframed from “NORMAL monotherapy path” to “MILD-MODERATE adjunct lever with conditional NORMAL”; h03 S-tier primary lever unchanged. → STRC h01 Phase 3c v2 Expanded Screen 2026-04-23
#1 Pharmacochaperone Phase 5c: GREEN-LIGHT on K1141 site stability. Custom grid-based cavity analysis + MD stability on Phase 5a 20-snapshot trajectory (fpocket 4.0 brew build broken on qhull Delaunay; pivoted to numpy/scipy grid + Kabsch alignment). K1141 pocket Cα RMSF 0.62 Å vs global 1.23 Å — pocket residues 2× more rigid than average; local void volume 719-850 ų across 20 frames (15% CV) — pocket does not collapse or open into alternative geometry. Global cavity scan (snap_010, 26-direction burial ≥16/26, voxels ≥70 ų): no alt cavity > 152 ų anywhere on the protein; nearest alt 18.7 Å from K1141 at 91 ų. Phase 5b RED-LIGHT interpretation nailed: chemistry-limited, not site-limited. Phase 3c v2 expanded virtual screen targeted at K1141 with ensemble receptor docking confirmed as correct next move. A held, all scores unchanged. → STRC h01 Phase 5c Cryptic Pocket Analysis 2026-04-23
#1 Pharmacochaperone Phase 5: RED-LIGHT from ensemble re-docking. Phase 5a OpenMM MD pipeline validated on local Mac (62 ns/day OpenCL, 2 ns production in 45 min, 20 snapshots). Phase 5b Vina re-docking of Phase 4b top-5 leads + diflunisal against 20 MD-relaxed snapshots → all f_PC at [L]=10 μM < 0.10 (best: diflunisal positive control 0.083; best lead naphthalene-2-COOH 0.047). Phase 4b single-structure rankings over-optimistic by +0.36 to +0.92 kcal/mol — ensemble averaging revealed K1141 pocket less accommodating of carboxylate ligands than static snapshot. None of six cleared NORMAL-rescue threshold f_PC ≥ 0.50. A held, all scores unchanged (hypothesis mechanism intact; only current shortlist insufficient). Next step reordered: Phase 5c HEK293 wet-lab → Phase 3c v2 expanded virtual screen (DrugBank FDA + ZINC22 carboxylate tranche + fragment-based, ensemble filter ΔG ≤ −7.5 kcal/mol). h03 primary Misha lever reinforced (MILD rescue via AAV remains short-term path). → STRC h01 Phase 5 MD Ensemble Rescoring 2026-04-23
Misha compound-het stack integration: ref-entry added (Misha Compound-Het Therapy Stack Model) integrating #1 + #3 for Misha’s specific genotype (paternal 98 kb del + maternal E1659A). h03 AAV monotherapy physically cannot reach NORMAL hearing (≤ 25 dB) — ceiling-capped at MILD/MODERATE because non-transduced OHCs inherit 0 paternal + 0.5 × f_mat < θ from maternal, functional fraction capped at ε ≤ 0.5 → max 50% functional OHCs → ABR ~38 dB. h01 PC monotherapy CAN reach NORMAL at f_PC ≥ 0.50-0.75 (mild/moderate/severe E1659A); stack unlocks low-burden NORMAL at mild (9 combinations ε≤0.3 × f_PC≤0.5). Conclusion robust across θ ∈ {0.25, 0.35, 0.45}. Reframes h01 as the only monotherapy route to full cure for Misha; h03 remains S-tier as primary “meaningful rescue” path; clinical plan = parallel stack. Phase 5 MD on h01 priority strengthened (f_PC is the critical-path parameter). #1 + #3 scores unchanged; this is strategic framing. → Misha Compound-Het Therapy Stack Model
Misha compound-het stack integration: ref-entry added (Misha Compound-Het Therapy Stack Model) integrating #1 + #3 for Misha’s specific genotype (paternal 98 kb del + maternal E1659A). h03 AAV monotherapy physically cannot reach NORMAL hearing (≤ 25 dB) — ceiling-capped at MILD/MODERATE because non-transduced OHCs inherit 0 paternal + 0.5 × f_mat < θ from maternal, functional fraction capped at ε ≤ 0.5 → max 50% functional OHCs → ABR ~38 dB. h01 PC monotherapy CAN reach NORMAL at f_PC ≥ 0.50-0.75 (mild/moderate/severe E1659A); stack unlocks low-burden NORMAL at mild (9 combinations ε≤0.3 × f_PC≤0.5). Conclusion robust across θ ∈ {0.25, 0.35, 0.45}. Reframes h01 as the only monotherapy route to full cure for Misha; h03 remains S-tier as primary “meaningful rescue” path; clinical plan = parallel stack. Phase 5 MD on h01 priority strengthened (f_PC is the critical-path parameter). #1 + #3 scores unchanged; this is strategic framing. → Misha Compound-Het Therapy Stack Model
#9 Hydrogel: A held, Mech 4 held, Deliv 3 held. Phase 4i Kd × Kd 5×5 sensitivity sweep on therapeutic window: f ≥ 0.3 reachable on 80% of plausible grid (PASS 56% single-dose ≤ 5 mg + MARGINAL 24% at 5-20 mg); FAIL 20% only at WH2 Kd ≥ 1 mM (Tβ4 analog floor) where G-actin toxicity ceiling hits before f=0.3 achievable. Strategic reorder: WH2×F-actin Kd dominates, TMEM145 Kd secondary. Phase 2c WH2 bundling (HEK293 GFP-actin confocal) now wet-lab gate #1; TMEM145 SPR/BLI → #2 (ipSAE already places in operable band). Verdict A_hold_multi_dose_ok — S promotion still requires WH2 Kd narrowing. → STRC h09 Phase 4i Kd Sensitivity Sweep 2026-04-23
#1 Pharmacochaperone post-audit housekeeping: A held. TPSA docstring corrected in phase3b_virtual_screen.py (descriptor-only, not scoring; STRC extracellular so CNS TPSA bracket irrelevant; no fabricated replacement range). Druggability cross-phase incomparability flagged in Phase 1/2/2b. → STRC h01 Parameter Provenance Audit 2026-04-23