STRC h01 Phase 8 Wet-Lab Triage SOP

Concrete protocol for wet-lab validation of pharmacochaperone hits from Phase 3c v3b / Phase 5e. Intended hand-off to Misha’s collaborator molecular biology lab (TBD). Must be executable from a GREEN compute hit within 6–8 weeks.

design philosophy

Three-gate triage, each stage kills most candidates so the next stage’s cost stays bounded. Gate thresholds are calibrated against iododiflunisal (known TTR pharmacochaperone at related ΔG range) as an internal positive control and DMSO as negative.

GATE 1 (1 wk, $3k, 96-well)  — ThermoFluor ΔTm on mini-STRC apo + mutant
  ├── ≥ 1.5 °C stabilization on mutant → pass
  └── fail → killed, feedback to medchem
GATE 2 (2 wk, $8k, n=3 replicates) — MST Kd + stoichiometry
  ├── Kd ≤ 50 µM AND Hill 0.8–1.2 → pass
  └── fail → killed
GATE 3 (4 wk, $25k, cryoEM co-complex, cell rescue) — structural + functional
  ├── co-complex density at K1141 pocket AND E1659A HEK293 rescue ≥ 30% → validated lead
  └── fail → de-prioritized

construct design (Gate 0 — pre-req)

Already in progress with AF3-validated Ultra-Mini boundary. Cloning target:

Construct	Residues (full-length)	Purpose	Tag
STRC-UM-WT	594–1294	WT Ultra-Mini, ThermoFluor + MST + cryoEM	N-His6-TEV
STRC-UM-E1659A	594–1775	extended to include mutation site; probe fold stability	N-His6-TEV
STRC-FL-WT	1–1775	full length for cell rescue	N-GFP (rescue readout)
STRC-FL-E1659A	1–1775	E1659A full length; Misha allele	N-GFP

Cloning: pcDNA3.4 mammalian expression for HEK293 rescue; pFastBac1 baculovirus/Sf9 for milligram-scale protein. SignalP-6 predicts N-terminal signal peptide at residues 1–26 — cleave for mature protein. C-term propeptide (1775–1809) not included in any construct (removed post-ER in vivo per Verpy 2001).

Protein production:

• Sf9 baculovirus: 1 L → 0.8–2 mg soluble mini-STRC after Ni-NTA + SEC (reference yield from STRC-related stereociliary proteins, see Grillet 2019 Nature Commun for stereocilin-CDH23 complex protocol)
• HEK293 GnTI⁻ for homogeneous glycosylation (stereocilin has 7 N-linked sites in UM boundary per NetNGlyc)
• pI ≈ 5.8 → buffer: 25 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 1 mM TCEP
• Stability check: SEC trace monodispersity + DLS PDI < 0.15 before any compound assay

GATE 1 — ThermoFluor ΔTm (differential scanning fluorimetry)

Purpose: first-pass stabilization readout. Chaperone binding raises Tm.

Reagents:

• Protein: 5 µM mini-STRC-UM-WT and mini-STRC-UM-E1659A
• SYPRO Orange 5× in assay buffer
• Compounds: 1 mM DMSO stock → final 100 µM (2% DMSO), also 30 µM and 10 µM
• Buffer: 25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP
• Positive control: iododiflunisal 100 µM
• Negative control: 2% DMSO vehicle

Plate layout: 96-well PCR plate, n=3 per compound per concentration per construct. 20 compounds × 3 conc × 2 constructs × 3 reps = 360 wells → 4 plates.

Instrument: QuantStudio 3 or Applied Biosystems StepOnePlus qPCR, melt 25 → 95 °C at 1 °C/min. Filter: TAMRA (SYPRO Orange).

Analysis: fit Boltzmann sigmoid to -dF/dT peak. ΔTm = Tm(compound) - Tm(DMSO). Calibrate:

• iododiflunisal positive control should give ≥ 2 °C shift on WT.
• Mutant baseline Tm expected 2–4 °C lower than WT (E1659A destabilizes).
• GATE 1 PASS: ΔTm ≥ 1.5 °C on E1659A at 100 µM, ≥ 0.5 °C at 30 µM, monotonic with dose.

Expected timing: 3 days (prep + plates + analysis), cost ≈ $3k compound + consumables.

Probes to include first pass:

• Phase 3c v2 parent probes (pocket validation only): niflumic acid, flufenamic acid, sulfasalazine, meclofenamic acid.
• Phase 3c v3b top compounds (once delivered): up to 16 hits ranked by f_PC.
• Positive: iododiflunisal, diflunisal, tafamidis.
• Negative: DMSO, aspirin (unrelated NSAID).

Kill criteria: ΔTm < 0.5 °C at 100 µM on E1659A OR destabilizing (ΔTm < -1 °C) on either construct. Destabilizers sent back to medchem as counter-probes (useful geometry info).

GATE 2 — MicroScale Thermophoresis (MST) Kd + stoichiometry

Purpose: precise Kd, cross-check ΔTm rank order.

Instrument: NanoTemper Monolith NT.115 (RED channel; use RED-tris-NTA dye on His6 tag, no covalent labeling needed).

Reagents:

• Labeled mini-STRC-UM-E1659A: 50 nM in assay buffer + 0.05 % Tween-20 (prevent aggregation)
• Compound titration: 16-point 1:1 serial from 500 µM → 15 nM (2% DMSO constant).
• Capillaries: standard treated.
• Temperature: 25 °C and 37 °C (human cochlear temp).

Data analysis: fit normalized F_norm vs [compound] with Hill equation. Require Kd ≤ 50 µM AND Hill slope 0.8–1.2 (no cooperativity / no aggregation artifacts). Compound-induced aggregation presents as U-shaped curve and is a kill.

Replicates: n ≥ 3 per compound × 2 temperatures. Report Kd ± SEM.

GATE 2 PASS criteria:

• Kd ≤ 50 µM (calibrated to phase5b ensemble expectations, 3× loosened for first wet-lab pass to catch soft hits).
• Hill 0.8–1.2.
• Kd on WT within 2-fold of mutant (selectivity is not required but extreme mutant > WT discrimination flags allostery).
• Reproducible 25 °C vs 37 °C (factor ≤ 2×).

Expected timing: 2 weeks post-G1, cost ≈ $8k (capillaries, dye, compound, instrument time).

Expected winnowing: 20 → 3–6 compounds pass G1; 3–6 → 1–3 pass G2.

GATE 3 — cryoEM co-complex + HEK293 E1659A functional rescue

3a cryoEM

Purpose: structural confirmation that compound binds K1141 pocket, not a decoy cryptic site.

Reagents: mini-STRC-UM-E1659A 5 mg/mL + compound 500 µM (2× saturation). Grids: Quantifoil R1.2/1.3 or gold-coated for anti-aggregation. Vitrify on Vitrobot Mark IV.

Collection: K3 or Falcon 4, 300 kV, 0.8 Å/px, 40 frames/movie, 40 e⁻/Ų. Target 4000 movies for 3–3.5 Å map.

Analysis: RELION-5 or cryoSPARC standard single-particle pipeline. Check ligand density in refined map at K1141 pocket. Bonus: unbiased difference map (apo − holo) at ≥ 4σ isolated at pocket coordinates.

GATE 3a PASS criteria:

• Ligand density at Phase 4a pocket coordinates (centered near full-length K1141 / residue 548 in chain-A numbering) at ≥ 4σ in difference map.
• Modeled pose within 1.5 Å RMSD of Phase 5b/5e best Vina pose.
• Protein conformation within 2 Å Cα RMSD of apo (confirms pocket doesn’t collapse on compound dissociation).

Expected timing: 6–8 weeks (grid optimization + 3 days collection + 2 weeks processing), cost ≈ $12kEMtime+$3k labor.

3b HEK293 E1659A rescue assay

Purpose: functional endpoint — does the compound rescue surface trafficking of E1659A stereocilin in a cell?

Reagents:

• HEK293-T stably transfected with STRC-FL-E1659A-GFP (or transient via Lipofectamine 3000).
• Compound pre-treatment: 10 µM (therapeutic intracochlear target conc), 100 µM (saturating), in complete medium for 24 h.
• Positive control: WT construct (full surface trafficking).
• Negative: E1659A + DMSO (baseline impaired trafficking).

Readouts:

1. Surface GFP vs total GFP (live-cell flow cytometry, non-permeabilized vs permeabilized).
   • WT: 65 ± 5 % surface.
   • E1659A + DMSO: 15 ± 5 % surface (impaired trafficking).
   • Rescue target: ≥ 30 % surface with compound.
2. ER retention (co-stain BiP/GRP78) — immunofluorescence. Rescued cells show reduced BiP co-localization with stereocilin.
3. Secretion into medium (Western blot supernatant) — confirm mature glycoform released.

Triage rank:

• GREEN: rescue ≥ 30 % surface at 10 µM → carry to mouse OHC ex-vivo and toxicology.
• YELLOW: rescue 15–30 % at 10 µM OR ≥ 30 % at 100 µM → medchem optimization.
• RED: < 15 % rescue at any dose → kill compound, feed pocket info back.

Expected timing: 4 weeks cell-culture + assay dev + triage, cost ≈ $10k.

Tox pre-screen (concurrent with Gate 3b)

Per STRC h01 Fenamic Scaffold Tox Audit 2026-04-23: any parent fenamate hit must be profiled against cochlear ion-channel panel BEFORE mouse OHC work:

• TRPM4 (patch-clamp in HEK293-TRPM4, 10 µM compound)
• Cx50 gap junction (HEK293-Cx50, dye transfer)
• BK channel (OHC-relevant, HEK293-BK)
• KCNQ4 (DFNA2 gene — critical — HEK293-KCNQ4, whole-cell patch)
• TMEM16A/ANO1 (stria vascularis)

Kill threshold: > 25 % inhibition at 10 µM on any cochlear-relevant channel.

Phase 8 output artifacts

For each candidate compound:

• ThermoFluor ΔTm table (3 conc × 2 constructs)
• MST Kd table (25 °C + 37 °C)
• cryoEM map + PDB deposition (if G3a passed)
• HEK293 rescue % surface (10/100 µM, n=6 biological replicates)
• Ion-channel panel IC₅₀s (at least TRPM4, KCNQ4, Cx50)

Reported in proof note **STRC h01 Phase 8 Wet-Lab Triage Results** (placeholder — to be written after wet-lab).

Decision tree into Phase 9

• 1–2 compounds full-pass G1+G2+G3 → Phase 9 mouse OHC ex-vivo + PK/PD + IND-enabling tox. Engage CRO (Charles River / Envigo).
• 0–1 compound passes → Phase 3c v4 fragment-growing or Phase 3c v5 de novo RFdiffusion pocket design. Feed G1 SAR back to medchem.
• All RED on G1 → pocket dead at chemistry; pivot to h01-alt (interface rescue at STRC dimer interface) or h11 (if open).

Connections

• [part-of] h01 hub
• [see-also] STRC h01 Fenamic Scaffold Tox Audit 2026-04-23
• [see-also] STRC Pharmacochaperone Phase 4 Plan
• [see-also] STRC h01 Phase 3c v2 Expanded Screen 2026-04-23
• Misha Compound-Het Therapy Stack Model
• [about] Misha