| # Free-PROTAC molecular dynamics |
|
|
| Explicit-solvent MD of a generated PROTAC on its own, with no target protein and |
| no E3 ligase. Runs in chloroform (a membrane-interior mimic) and in water, and |
| measures the conformational descriptors published work ties to cell |
| permeability: radius of gyration, 3D polar surface area, intramolecular |
| H-bonds, and conformational state populations. |
|
|
| The protocol follows "Linker-Dependent Folding Rationalizes PROTAC Cell |
| Permeability" (*J. Med. Chem.* 2022, DOI 10.1021/acs.jmedchem.2c00877) and takes |
| its permeability cutoffs from Atilaw et al., *ACS Med. Chem. Lett.* 2021, 12, |
| 107-114. `reference_protocol.md` has the settings and the target numbers. |
|
|
| ## Layout |
|
|
| ``` |
| protac_md.py MD driver: conformer -> parametrize -> solvate -> replicas |
| analyze_conformations.py Rg / RMSD / 3D PSA / H-bonds / clustering dashboard |
| rg_basins.py Rg free-energy profiles and basin populations |
| psa_ensemble.py 3D PSA over a finished ensemble |
| ternary_span.py warhead-to-E3 span distribution (water ensembles) |
| validate_psa.py PSA code path checked against grazoprevir |
| psa_method_probe.py element vs charge-threshold polar-atom selection |
| plot_results.py per-replica summary figure |
| render_3d.py extended and collapsed conformations from a trajectory |
| run_candidate.sh detached driver: chloroform + water + analysis |
| environment_md.yml conda environment |
| protacmd-lock-linux64.txt pinned environment, linux-64 |
| reference_protocol.md reference protocol and permeability yardsticks |
| linker_design.md linker rules read off this project's own MD runs |
| ``` |
|
|
| PROTAC SMILES are embedded in `protac_md.py` (`CANDIDATES`), so no input files |
| are needed; anything else runs through `--smiles`. |
|
|
| ## Environment |
|
|
| On linux-64 with a CUDA 12 capable driver (525 or newer), use the lockfile and |
| skip dependency solving: |
|
|
| ``` |
| conda create --name protacmd --file protacmd-lock-linux64.txt |
| conda activate protacmd |
| pip install "setuptools<81" |
| ``` |
|
|
| That reproduces the environment: openmm 8.5.2, ambertools 24.8, |
| rdkit 2025.09.5, openff-toolkit 0.16.10, python 3.10.20. The lockfile carries no |
| `packmol`, which `--solvent chloroform` needs, so add it afterwards |
| (`conda install -c conda-forge packmol`) or solve from the yml instead. |
|
|
| Elsewhere, solve from the yml. Check the driver's CUDA version with |
| `nvidia-smi` first and edit `cuda-version` in `environment_md.yml` to match |
| (remove it entirely for CPU-only): |
|
|
| ``` |
| conda env create -f environment_md.yml |
| conda activate protacmd |
| ``` |
|
|
| The solve takes around ten minutes: ambertools has a large dependency tree and |
| libmamba is single-threaded, so the spinner sits still while one core works. The |
| install itself takes seconds. Keep `setuptools<81` in the `pip:` section rather |
| than as a conda dependency; an upper bound on setuptools triples the solve time. |
|
|
| Activation is not optional. `sqm`, `antechamber` and `packmol` are only on PATH |
| inside the env, and calling `<env>/bin/python` directly is not enough. |
|
|
| ## Run |
|
|
| ``` |
| conda activate protacmd |
| |
| # chloroform: the membrane mimic, the permeability-relevant run |
| python protac_md.py --solvent chloroform --replicas 3 --prod-ns 50 --gpu 0 |
| # water: the polar reference; the pair shows chameleonic folding |
| python protac_md.py --solvent water --replicas 3 --prod-ns 50 --gpu 0 |
| |
| python analyze_conformations.py --dir egfrvhl_rank4/chloroform --name egfrvhl_rank4 |
| python analyze_conformations.py --dir egfrvhl_rank4/water --name egfrvhl_rank4 |
| ``` |
|
|
| Useful flags: `--candidate {egfrvhl_rank4,egfrvhl_rank5,brd9vhl_rank1,cdk6crbn_rank2}`, |
| `--smiles "<custom>"` with `--name ms39_ref` for a reference compound, |
| `--platform CPU`, `--prod-ns`, `--replicas`, `--gpu N`. |
|
|
| Output lands in `<name>/<solvent>/rep<k>/` (`*_prod.dcd`, `*_prod.csv`, PDBs), |
| with the dashboard and the cluster centroid PDBs in `<name>/<solvent>/`. |
|
|
| `run_candidate.sh` chains both solvents and both analyses for one candidate and |
| reuses the chloroform charge cache for water, so AM1-BCC is paid once. |
|
|
| Smoke test on a new machine first, about two minutes: |
|
|
| ``` |
| python protac_md.py --solvent chloroform --replicas 1 --prod-ns 0.05 \ |
| --heat-ps 5 --equil-ps 10 --report-ps 1 --min-iters 1000 --name smoke |
| python analyze_conformations.py --dir smoke/chloroform --name smoke --clusters 4 |
| ``` |
|
|
| ## Comparing finished runs |
|
|
| These take run directories, one per compound. `ms39_ref` is not bundled: run the |
| pipeline on MS39 yourself with `--smiles ... --name ms39_ref`. Its components |
| and exact linker are in `../src/protac/ErlotinibVHL.py`. |
|
|
| ``` |
| python rg_basins.py egfrvhl_rank4 ms39_ref --solvents chloroform water |
| python psa_ensemble.py egfrvhl_rank4 ms39_ref --solvent chloroform |
| python ternary_span.py egfrvhl_rank4 ms39_ref |
| ``` |
|
|
| ## Hardware and runtime |
|
|
| The system is small, 2500-2800 atoms, so it is GPU-latency-bound rather than |
| memory-bound and fits in under 2 GB of VRAM. |
|
|
| | Platform | Speed | 50 ns x 3 x 2 solvents | |
| |---|---|---| |
| | Free A100 | ~1100 ns/day | ~6.5 h | |
| | Shared GPU (~75% busy) | ~32 ns/day | ~9 days | |
| | CPU (16 threads) | ~28 ns/day | ~10 days | |
|
|
| A shared GPU is about 35x slower than a free one and no faster than CPU, so use |
| an idle card. Replicas run sequentially in one process; parallelize by launching |
| one process per GPU. |
|
|
| The reference converged within 5-10 ns; 50 ns x 3 leaves a wide margin. Confirm |
| with the RMSD plateau and the replica-mean spread in the dashboard, and extend |
| if either is still drifting. |
|
|
| ## Known failure modes |
|
|
| 1. `ModuleNotFoundError: No module named 'pkg_resources'` at `SystemGenerator`. |
| `openff.amber_ff_ports` imports `pkg_resources`, which setuptools 81 removed. |
| Install `setuptools<81`. |
| 2. `OSError: Packmol not found`, chloroform only. `pack_box` resolves `packmol` |
| through PATH, so the env has to be activated, and so does `sqm` / |
| `antechamber` for AM1-BCC charges. |
| 3. Charge assignment is slow, one to three minutes on a single CPU core, and |
| happens inside `create_system`. Wrapping the run in a short `timeout` kills it |
| mid-charge and leaves a 0-byte `ff_cache.json`. Charges are cached per |
| molecule in `<name>/<solvent>/ff_cache.json`; copying a populated cache in |
| skips the step. |
| 4. Never select the PROTAC by atom name or element. It carries its own Cl, and |
| counterions named `Na`/`Cl` slip past naive regexes, which inflated Rg by |
| about 13%. Every script here selects it by residue instead: the non-water |
| residue with more than 5 atoms. |
| 5. mdtraj mislabels DCD frame times, so `traj.time` is not trustworthy. Derive |
| time from the frame index times `--report-ps`, as the scripts do. |
|
|
| ## Scope |
|
|
| The simulations show that a generated PROTAC is physically sane and describe its |
| conformational ensemble, extended versus folded, along with the folding-linked |
| permeability descriptors. That is the same scope as the reference work, which |
| also simulates the free PROTAC only. |
|
|
| They do not show that a linker works as a degrader. Degradation needs a |
| productive ternary complex of target, PROTAC and E3, which is a different |
| simulation. `ternary_span.py` describes the span a linker can hold, not whether |
| a complex forms. |
|
|
| Folding propensity only means something against a baseline, so run the same |
| pipeline on a reference PROTAC (`--smiles ... --name ms39_ref`) alongside each |
| candidate. |
|
|
| Yardsticks, from the 2022 MD paper: a compact minimum at Rg 5.4-5.6 A, and 3D |
| PSA 190-265 A^2 for the permeable compounds against 290-330 A^2 for the poorly |
| permeable one. From Atilaw 2021: passive permeability below Rg 7 A, and |
| low-to-moderate permeability up to roughly 200-210 A^2. The 7 A cutoff on its |
| own does not separate the 2022 compounds; see `reference_protocol.md`. |
|
|
