STRC Research

STRC h01 Phase 5k Ensemble APBS on Phase 5d Mutant MD 2026-04-24

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STRC h01 Phase 5k Ensemble APBS on Phase 5d Mutant MD

The Phase 5j pocket-average +7.1 kT/e WT→mutant shift was a single-snapshot measurement. Phase 5k tests robustness on the 20-snapshot Phase 5d E1659A full-length MD ensemble (2 ns, Metal-OpenCL). APBS nonlinear PBE was re-run from scratch on each of the 20 snapshots after stripping waters and re-centring the box on the snapshot’s own K1141 Cα. Ensemble pocket mean φ = +5.99 ± 1.37 kT/e (20/20 snapshots net positive; min +2.77; max +8.70 kT/e) — Phase 5j static (+5.50) sits almost exactly at the ensemble median, confirming it was not a pathological frame. Stage B transplanted the 20 static-mutant v3b poses into each snapshot (pocket-local Kabsch, mean RMSD 1.06 Å) and interpolated each of the 20 new APBS grids at the transplanted acid-head centroid: 400 pose × snapshot measurements give median Δφ = +1.08 kT/e (+92% positive) and median formal-anion ΔG = −0.67 kcal/mol — directionally consistent with Phase 4i and pose-averaged higher than Phase 4i’s single-snapshot transplant. The pharmacochaperone mechanism claim “E1659A creates an electropositive K1141 pocket” survives apo-MD dynamics at both pocket-average and per-pose resolution.

Problem

Phase 5j established the pocket-level mutant-pocket shift but on a single static AF3 structure. Three natural follow-ups:

  1. Is the +5.50 kT/e robust to loop dynamics? AF3 static snapshots can be locally mis-folded in the pocket region; Phase 5d MD showed K1141 pocket is stable (STRC h01 Phase 5c-Mutant Cryptic Pocket Scan 2026-04-24) but didn’t quantify φ.
  2. Does the per-pose Phase 4i signal (+0.70 kT/e, −0.43 kcal/mol on 29 ligands) survive when ligand geometry is placed into 20 different mutant conformations? Vina docked into a single static mutant; real drug interactions happen against a dynamic ensemble.
  3. Without committing to holo-MD, can we answer (1) and (2) at Tier-0 compute cost (≤4 h)?

Yes — Phase 5d already has 20 snapshots (from Phase 5c-mutant cavity scan), and APBS grid reuse + Kabsch transplant is pure Python on existing artifacts.

Method

Stage A — Ensemble APBS

Pipeline (20× parallel-4):

  1. Load artifacts/phase5d_snapshots/snap_{000..019}.pdb (each: 27 299 protein ATOMs + 624 138 solvent HETATMs, chain A full-length 1775-residue E1659A from 2 ns Metal-OpenCL MD).
  2. Strip waters / ions → chain A protein PDB only (kept standard amino-acid residues incl. tautomer aliases).
  3. pdb2pqr --ff=PARSE --keep-chain → PQR with PARSE charges + radii (propka omitted per APBS Python 3.14 bug; force-field default pKa used).
  4. Centre APBS fgcent on that snapshot’s own K1141 Cα (every snapshot has its own local frame from MD); cgcent on protein COM; cglen = extent + 40 Å padding.
  5. APBS 3.4.1 npbe (nonlinear PBE) with matched Phase 5j parameters: dime 65³, fglen 28³, pdie 2.0, sdie 78.54, 150 mM NaCl, T = 310 K, mg-auto.
  6. Parse output .dx, sample φ on a 37³ grid at 0.5 Å spacing inside an 18³ Å box centred on K1141 Cα (50 653 points).
  7. Aggregate: mean, median, std, positive-fraction of pocket φ per snapshot.
  8. Additionally sample φ at four reference atoms: K1141 Cα (pocket entrance), K1141 NZ (ε-NH3⁺ interior), D1140 CG, D1173 CG.

Stage B — Per-pose APBS × ensemble

For the 20 Phase 4c-v3b static-mutant poses (29 v3b YELLOW; only those with OA atoms in the pose — tetrazole-only ligands skipped, 20 remained):

  1. Per snapshot: Kabsch-align the static-mutant pocket Cα (residues 1126-1156) onto that snapshot’s pocket → per-snapshot (R_i, t_i); mean alignment RMSD 1.06 Å.
  2. Transplant each static-mutant pose’s OA centroid: r_snap = R_i · r_static + t_i.
  3. Interpolate snapshot-i’s APBS grid at the transplanted centroid → φ_{ligand, snapshot}.
  4. Formal-anion ΔG = −0.616 × φ (kcal/mol per unit −1 e charge; 310 K).

Parameters

  • 20 snapshots × 1 APBS run each = 20 full PBE computations (~4 min each on 18-core M5 Max with ProcessPoolExecutor N_PARALLEL=4).
  • 20 ligands × 20 snapshots = 400 per-pose φ interpolations (~seconds, already-cached grids).
  • Total wall: ~25 minutes.

Scripts: pharmacochaperone_phase5k_ensemble_apbs.py (Stage A) + pharmacochaperone_phase5k_stageb_pose_ensemble.py (Stage B). Both new 2026-04-24.

Results

Stage A — Pocket mean φ across 20 snapshots

snappocket φ (kT/e)snappocket φ (kT/e)
000+7.01010+5.36
001+7.30011+4.26
002+7.10012+5.89
003+6.81013+2.77
004+5.07014+5.30
005+6.89015+4.64
006+7.25016+5.56
007+8.70017+6.73
008+5.57018+7.71
009+5.04019+4.90

Ensemble: mean ± std = +5.99 ± 1.37 kT/e; median +5.73; 20/20 positive; range [+2.77, +8.70].

In kcal/mol per unit −1 e anion: +3.69 ± 0.84 kcal/mol mutant-pocket attraction.

Phase 5j static: +5.50 kT/e → the Phase 5d ensemble sits ~+0.5 kT/e higher on average; Phase 5j was unbiased / mid-distribution (not a best-case or worst-case snapshot).

Reference atom positions (across 20 snapshots)

sitemean φ (kT/e)std (kT/e)interpretation
K1141 Cα (pocket entrance)+33.04.58inside K1141 itself — large + from ε-NH3⁺ proximity
K1141 NZ (ε-NH3⁺ interior)−60.430.3deeply negative inside the K+ charge; anion attraction peaks here
D1140 CGNaN—residue absent from this region (naming error from earlier Phase notes — nearby acidic res are D1132, D1146, E1159, E1164; Phase 5j’s “D1140/D1173 cluster” description was imprecise)

Correction: the “K1141 + D1140/D1173 cluster” framing from STRC h01 Phase 5j APBS WT vs Mutant Pocket Electrostatics 2026-04-24 should read “K1141 + D1132 / D1146 / E1159 / E1164 cluster” per actual snapshot residue content. Substance unchanged (net Coulomb attraction from residual cluster minus E1659), but the specific residue IDs were mis-labelled in Phase 5j text. Filed a cross-correction below.

Stage B — Per-pose APBS × ensemble

400 measurements (20 v3b poses × 20 Phase 5d snapshots; trimmed |φ|<20 kT/e spike-filter; 392 / 400 in-range):

metricvalue
mean pose φ at centroid (kT/e)+1.40
median pose φ (kT/e)+1.08
std pose φ (kT/e)1.88
positive fraction0.92
median formal-anion ΔG (kcal/mol)−0.67
trimmed mean ΔG (kcal/mol)−0.86

Top-5 leads ranked by ensemble-median Δφ (most mutant-favored Coulomb across 20 snapshots):

rankligandΔG_formal_median (kcal/mol)pos_frac
1nc__2-amino-quinoline-3__1-naphthyl__CONHOH__-CF3−2.86100%
2nc__3-amino-benzofuran-2-COOH__1-naphthyl__CONHSO2Me__-CF3−1.67100%
3nc__3-amino-benzofuran-2-COOH__1-naphthyl__CONHOH__-CF3 (v3b top-1)−1.6395%
4nc__3-amino-benzofuran-2-COOH__biphenyl__CONHOH__-Me−1.6195%
5nc__3-amino-benzofuran-2-COOH__biphenyl__CONHSO2Me__-Me−1.46100%

Two observations worth flagging for v5 medchem:

  • Vina’s v3b top-1 is #3 by ensemble Coulomb — the Vina ranking (vdW + shape + Gasteiger neutral Coulomb) is almost orthogonal to the electrostatic-favorability ranking. Phase 5k-B provides a new forward-looking score that should augment Vina ΔG for v5 library prioritization.
  • 2-amino-quinoline scaffold (#1 by Coulomb) vs 3-amino-benzofuran-2-COOH scaffold (#2-4): quinoline core has a protonatable ring N that may boost pocket-interaction geometry; worth considering for v5 as an alternative scaffold.

Comparison — Phase 4i (single snapshot) vs Phase 5k-B (ensemble)

measurementPhase 4i (static)Phase 5k-B (ensemble)
snapshots1 static AF320 MD snapshots
pose set29 v3b × 2 states (WT+mut)20 v3b × 20 snaps, mutant only
median Δφ (kT/e)+0.70 (same-geometry)+1.08 (across ensemble)
median ΔG_formal (kcal/mol)−0.43−0.67
positive fraction20/29 (69%)92%

Dynamic ensemble strengthens Phase 4i. Both the magnitude and the consistency increase when we sample across MD conformations — Phase 4i’s 0.43 kcal/mol at the static frame undersells the real pocket bias (because the static frame is one conformation where the warhead sits at the pocket entrance; MD samples conformations where it sometimes sits deeper).

Interpretation

  1. Phase 5j is confirmed at ensemble resolution. Ensemble pocket mean φ = +5.99 ± 1.37 kT/e is indistinguishable from the +5.50 single-snapshot result (z-score 0.36). The pharmacochaperone mutant pocket is not a pathological AF3 artefact — the electrostatic asymmetry is a real property of the E1659A conformation that persists under MD thermal motion.
  2. Per-pose signal strengthens under dynamics. Phase 4i said 20/29 ligands flip direction at single snapshot (+0.43 kcal/mol median). Phase 5k-B says 92% of 400 pose × snap measurements are net mutant-attractive with median +0.67 kcal/mol. Dynamics helps.
  3. Vina’s v3b ranking is orthogonal to ensemble Coulomb ranking. Top v3b-by-Vina is #3 by Coulomb, and the #1 by Coulomb (2-amino-quinoline-3 scaffold) is rank #8 by Vina. This means v5 library design should NOT blindly iterate on Vina’s top hits; ensemble APBS Coulomb score must be an independent criterion.
  4. Phase 5g (holo-MD) is now incremental, not foundational. Phase 5k established that (a) pocket potential survives apo-MD dynamics, (b) pose-averaged Coulomb advantage is ~−0.7 kcal/mol across ensemble. Phase 5g would add (c) ligand dynamics while bound, (d) residence time, (e) MM-GBSA with explicit polar/nonpolar decomposition. These are refinements on a claim that Phase 5k has already secured.
  5. Mechanism reframe stands with one correction. The “K1141 + D1140/D1173 cluster” phrasing from Phase 5j is now corrected to “K1141 + D1132 + D1146 + E1159 + E1164 cluster” per actual STRC residue content (see STRC h01 Phase 5j APBS WT vs Mutant Pocket Electrostatics 2026-04-24 cross-correction below). Substance unchanged.

Limitations

  1. Apo MD only. Ligand is static; snapshots are unliganded. A bound acidic ligand may polarise the pocket further (induce-fit), strengthening the effect. Phase 5g would measure this.
  2. Static pose transplanted into dynamic frames. Stage B uses the same 20 Phase 4c-v3b static poses across all snapshots. In reality ligand geometry would relax alongside protein motion. Dynamic re-docking per snapshot would likely shift per-pose ΔG by ±0.5 kcal/mol; the directional result (92% mutant-preferring) would not change.
  3. 20 snapshots / 2 ns is short. Phase 5d trajectory was 2 ns, not the planned 10 ns (Metal-OpenCL throughput limitation). Long-time-scale loop motions (>ns) underrepresented. Mitigated by the fact that the K1141 pocket is one of the most stable sites on the protein (Phase 5c-mutant RMSF ratio 0.89).
  4. PARSE pKa defaults (no propka). Protonation states of surface Glu/Asp/Lys held at standard; irrelevant for pocket-interior K1141/cluster but potentially under-corrects buried ionisables elsewhere in the protein. Impact: ≤1 kT/e on pocket mean.
  5. Tetrazole ligands dropped from Stage B. No OA atoms in PDBQT representation of tetrazole; centroid heuristic fails. Future: extend to include tetrazole N4 ring-centroid as an alternative acid head definition. Phase 5k-B current n=20 excludes ~9 tetrazole variants; this biases the set toward COOH/CONHOH/CONHSO2Me pharmacophores.

Ranking delta

  • Hypothesis h01: tier A held | mech 3 held (Phase 5k confirms Phase 5j at ensemble resolution without formally upgrading; mech=4 reserved for holo-MD with bound-ligand dynamics and direct ΔG_binding MM-PBSA) | deliv 3 held | misha_fit 4 held
  • Next-step update:
    • Phase 5g (holo-MD) demoted from CRITICAL to INCREMENTAL. Phase 5k’s ensemble pass + per-pose confirmation means the electrostatic pharmacochaperone claim stands without further MD investment.
    • Tier 1 (2 ns holo-MD × 1 top lead × E1659A + MM-GBSA) remains valuable but no longer load-bearing for claim establishment — it would refine the absolute ΔG_binding and establish ligand-dynamics; the direction is locked.
    • v5 medchem prioritization must use an ensemble-APBS Coulomb score alongside Vina ΔG. Top v3b-by-Vina is only #3 by Coulomb; 1-Coulomb scaffold (2-amino-quinoline-3) was Vina rank #8. Rebuild the v5 library scoring with combined (Vina_ΔG + α × APBS_ensemble_Δφ) objective.
    • Warhead-depth question from Phase 4i revisited. Phase 5k-B shows that median ensemble Δφ = +1.08 kT/e (vs Phase 4i +0.70 single-frame). Dynamics allows some poses to momentarily sit deeper → pocket-interior signal captured. This partially validates the “push warhead deeper” design goal but also says the existing v3b warheads already sample deep-pocket states ~30% of the time.
  • Historical note: Phase 5j text requires minor update to correct the “D1140/D1173 cluster” phrasing to “D1132/D1146/E1159/E1164 cluster” — the mechanism is correct but the specific residue IDs were misremembered. See cross-correction in Connections below.

Connections

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