Paul Robustelli
@paulrobustelli.bsky.social
Assistant Professor at Dartmouth College
Computational Biophysics / Disordered Proteins / Molecular Recognition
Computational Biophysics / Disordered Proteins / Molecular Recognition
We think that stabilizing R2:R2 interfaces (and other less populated R2:R3 and R3:R3 interafces) may also be how EPI-001 lowers the cloud temperature (Tc) of condensation, rigidifies AR condensates, and interferes with RNA pol II partitioning in cellls.
November 22, 2025 at 7:11 PM
We think that stabilizing R2:R2 interfaces (and other less populated R2:R3 and R3:R3 interafces) may also be how EPI-001 lowers the cloud temperature (Tc) of condensation, rigidifies AR condensates, and interferes with RNA pol II partitioning in cellls.
So MD suggests these ligands effectively act as "stickers" to bring aromatic residues together from different monomers together.
Good news: so does NMR! @borjaml.bsky.social and Stase found that EPI-001 increases the magnitude of intermolecular NOEs of aromatic protons in Tau-5.
Good news: so does NMR! @borjaml.bsky.social and Stase found that EPI-001 increases the magnitude of intermolecular NOEs of aromatic protons in Tau-5.
November 22, 2025 at 7:11 PM
So MD suggests these ligands effectively act as "stickers" to bring aromatic residues together from different monomers together.
Good news: so does NMR! @borjaml.bsky.social and Stase found that EPI-001 increases the magnitude of intermolecular NOEs of aromatic protons in Tau-5.
Good news: so does NMR! @borjaml.bsky.social and Stase found that EPI-001 increases the magnitude of intermolecular NOEs of aromatic protons in Tau-5.
Lets zoom back out to ensemble averages. They key result is that the alkyne linker of 1aa pushes the phenyl groups further apart and gives more rotational freedom. This allows 1aa to simultaneously intercalate into both R2 monomers and form aromatic contacts that stabilize ternary complexes.
November 22, 2025 at 7:11 PM
Lets zoom back out to ensemble averages. They key result is that the alkyne linker of 1aa pushes the phenyl groups further apart and gives more rotational freedom. This allows 1aa to simultaneously intercalate into both R2 monomers and form aromatic contacts that stabilize ternary complexes.
Lets take a closer look at the heterogeneity of binding poses and interactions in each kinetically metastable state in the SI.
Maybe one day we can try to design ligands to stablize specific substates?
Maybe one day we can try to design ligands to stablize specific substates?
November 22, 2025 at 7:11 PM
Lets take a closer look at the heterogeneity of binding poses and interactions in each kinetically metastable state in the SI.
Maybe one day we can try to design ligands to stablize specific substates?
Maybe one day we can try to design ligands to stablize specific substates?
Lets look at 1aa ternary complexes to see what we're talking about.
Ligands tend to bind at one dominant interface, engage aromatic side chains and induce helix folding, but do so with a collection of fuzzy(ish) binding modes. We feel this matches the magnitude of NMR CSPs in experiments well
Ligands tend to bind at one dominant interface, engage aromatic side chains and induce helix folding, but do so with a collection of fuzzy(ish) binding modes. We feel this matches the magnitude of NMR CSPs in experiments well
November 22, 2025 at 7:11 PM
Lets look at 1aa ternary complexes to see what we're talking about.
Ligands tend to bind at one dominant interface, engage aromatic side chains and induce helix folding, but do so with a collection of fuzzy(ish) binding modes. We feel this matches the magnitude of NMR CSPs in experiments well
Ligands tend to bind at one dominant interface, engage aromatic side chains and induce helix folding, but do so with a collection of fuzzy(ish) binding modes. We feel this matches the magnitude of NMR CSPs in experiments well
We get valuable insight by looking at ensemble-averaged statistics of these states too.
We can see that in apo simulations, residues AWAAAAAQ in R2 associate in a fuzzy manner. Adding ligands extends the apo interaction interfaces and stabilizes and rigidify specific pairs of aromatic contacts.
We can see that in apo simulations, residues AWAAAAAQ in R2 associate in a fuzzy manner. Adding ligands extends the apo interaction interfaces and stabilizes and rigidify specific pairs of aromatic contacts.
November 22, 2025 at 7:11 PM
We get valuable insight by looking at ensemble-averaged statistics of these states too.
We can see that in apo simulations, residues AWAAAAAQ in R2 associate in a fuzzy manner. Adding ligands extends the apo interaction interfaces and stabilizes and rigidify specific pairs of aromatic contacts.
We can see that in apo simulations, residues AWAAAAAQ in R2 associate in a fuzzy manner. Adding ligands extends the apo interaction interfaces and stabilizes and rigidify specific pairs of aromatic contacts.
Here are some snapshots of metastable substates identified by projecting simulation frames onto a kinetically meaningful 2D tCCA latent space.
Dig into the SI if you want to see detailed characterizations of each state.
Dig into the SI if you want to see detailed characterizations of each state.
November 22, 2025 at 7:11 PM
Here are some snapshots of metastable substates identified by projecting simulation frames onto a kinetically meaningful 2D tCCA latent space.
Dig into the SI if you want to see detailed characterizations of each state.
Dig into the SI if you want to see detailed characterizations of each state.
We see 1000s of dimerization events. Lets go back to our NMR autocorrelation function (ACF) fitting days, and show that if we compute an ACF on time series of dimer contacts - you get a double exponential decay, showing two timescales of dissociation, with ligands slowing down both timescales.
November 22, 2025 at 7:11 PM
We see 1000s of dimerization events. Lets go back to our NMR autocorrelation function (ACF) fitting days, and show that if we compute an ACF on time series of dimer contacts - you get a double exponential decay, showing two timescales of dissociation, with ligands slowing down both timescales.
We ran simulations of two monomers of the AR AD R2 fragment (apo), and in the presence of EPI-002 or 1aa.
We see that adding ligands substantially stabilizes the intermolecular association of R2 domains and helicity increases in ternary complexes, in perfect agreement with experiments.
We see that adding ligands substantially stabilizes the intermolecular association of R2 domains and helicity increases in ternary complexes, in perfect agreement with experiments.
November 22, 2025 at 7:11 PM
We ran simulations of two monomers of the AR AD R2 fragment (apo), and in the presence of EPI-002 or 1aa.
We see that adding ligands substantially stabilizes the intermolecular association of R2 domains and helicity increases in ternary complexes, in perfect agreement with experiments.
We see that adding ligands substantially stabilizes the intermolecular association of R2 domains and helicity increases in ternary complexes, in perfect agreement with experiments.
Earlier this year they released a beautiful preprint with a trove of NMR, DLS and condensation assay data showing that EPI-001 stabilizes oligomers of the AR AD, predominantly by strengthening intermolecular interactions formed by the R2 helix of the AR AD.
www.biorxiv.org/content/10.1...
www.biorxiv.org/content/10.1...
November 22, 2025 at 7:11 PM
Earlier this year they released a beautiful preprint with a trove of NMR, DLS and condensation assay data showing that EPI-001 stabilizes oligomers of the AR AD, predominantly by strengthening intermolecular interactions formed by the R2 helix of the AR AD.
www.biorxiv.org/content/10.1...
www.biorxiv.org/content/10.1...
First the IRB Barcelona team get some important experimental results.
In 2021 Stase and co. showed the the R2 helix of the AR AD shows the largest NMR chemical shift perturbations (CSPs) with increasing AR concentration - identifying it as the dominant oligomerization interface of the AR AD.
In 2021 Stase and co. showed the the R2 helix of the AR AD shows the largest NMR chemical shift perturbations (CSPs) with increasing AR concentration - identifying it as the dominant oligomerization interface of the AR AD.
November 22, 2025 at 7:11 PM
First the IRB Barcelona team get some important experimental results.
In 2021 Stase and co. showed the the R2 helix of the AR AD shows the largest NMR chemical shift perturbations (CSPs) with increasing AR concentration - identifying it as the dominant oligomerization interface of the AR AD.
In 2021 Stase and co. showed the the R2 helix of the AR AD shows the largest NMR chemical shift perturbations (CSPs) with increasing AR concentration - identifying it as the dominant oligomerization interface of the AR AD.
In our 2023 NSMB paper, @xsalvatella1.bsky.social and Denes Hnisz led an effort to identify new AR inhibitors
www.nature.com/articles/s41..., and Jiaqi found that that MD correctly predicts that more potent ligands with diphenylacetylene moieties have higher affinity to AR AD monomers
www.nature.com/articles/s41..., and Jiaqi found that that MD correctly predicts that more potent ligands with diphenylacetylene moieties have higher affinity to AR AD monomers
November 22, 2025 at 7:11 PM
In our 2023 NSMB paper, @xsalvatella1.bsky.social and Denes Hnisz led an effort to identify new AR inhibitors
www.nature.com/articles/s41..., and Jiaqi found that that MD correctly predicts that more potent ligands with diphenylacetylene moieties have higher affinity to AR AD monomers
www.nature.com/articles/s41..., and Jiaqi found that that MD correctly predicts that more potent ligands with diphenylacetylene moieties have higher affinity to AR AD monomers
In 2016, @xsalvatella1.bsky.social used #NMR to identify the binding site of EPI-001:
pubs.acs.org/doi/full/10....
In 2022, Jiaqi used MD simulations to model monomer binding modes of EPI-002 (the highest affinity stereoisomer of EPI-001):
www.nature.com/articles/s41...
pubs.acs.org/doi/full/10....
In 2022, Jiaqi used MD simulations to model monomer binding modes of EPI-002 (the highest affinity stereoisomer of EPI-001):
www.nature.com/articles/s41...
November 22, 2025 at 7:11 PM
In 2016, @xsalvatella1.bsky.social used #NMR to identify the binding site of EPI-001:
pubs.acs.org/doi/full/10....
In 2022, Jiaqi used MD simulations to model monomer binding modes of EPI-002 (the highest affinity stereoisomer of EPI-001):
www.nature.com/articles/s41...
pubs.acs.org/doi/full/10....
In 2022, Jiaqi used MD simulations to model monomer binding modes of EPI-002 (the highest affinity stereoisomer of EPI-001):
www.nature.com/articles/s41...
Small molecules that target the disordered androgen receptor activation domain (AR AD) have shown promise for treating castration resistant prostate cancer (CPCR) in mouse models - but have shown insufficient potency in human trials.
If only we understood their mechanisms better....
If only we understood their mechanisms better....
November 22, 2025 at 7:11 PM
Small molecules that target the disordered androgen receptor activation domain (AR AD) have shown promise for treating castration resistant prostate cancer (CPCR) in mouse models - but have shown insufficient potency in human trials.
If only we understood their mechanisms better....
If only we understood their mechanisms better....
Proud to share our manuscript:
"How small molecules stabilize oligomers of a phase-separating disordered protein"
www.biorxiv.org/content/10.1...
by Jiaqi Zhu and Tommy Sisk, in collaboration with @borjaml.bsky.social , Stase Bielskute-Garcia & @xsalvatella1.bsky.social
"How small molecules stabilize oligomers of a phase-separating disordered protein"
www.biorxiv.org/content/10.1...
by Jiaqi Zhu and Tommy Sisk, in collaboration with @borjaml.bsky.social , Stase Bielskute-Garcia & @xsalvatella1.bsky.social
November 22, 2025 at 7:11 PM
Proud to share our manuscript:
"How small molecules stabilize oligomers of a phase-separating disordered protein"
www.biorxiv.org/content/10.1...
by Jiaqi Zhu and Tommy Sisk, in collaboration with @borjaml.bsky.social , Stase Bielskute-Garcia & @xsalvatella1.bsky.social
"How small molecules stabilize oligomers of a phase-separating disordered protein"
www.biorxiv.org/content/10.1...
by Jiaqi Zhu and Tommy Sisk, in collaboration with @borjaml.bsky.social , Stase Bielskute-Garcia & @xsalvatella1.bsky.social
Today Writhe, our favorite knot theory descriptor for characterizing IDP dynamics, is the star of the lab pumpkin for the annual department carving contest.
If you can make sense of the design— I’ll buy you a coffee, bao bun, or wheatgrass shot at the next conference. No substitutions.
If you can make sense of the design— I’ll buy you a coffee, bao bun, or wheatgrass shot at the next conference. No substitutions.
October 23, 2025 at 1:05 AM
Today Writhe, our favorite knot theory descriptor for characterizing IDP dynamics, is the star of the lab pumpkin for the annual department carving contest.
If you can make sense of the design— I’ll buy you a coffee, bao bun, or wheatgrass shot at the next conference. No substitutions.
If you can make sense of the design— I’ll buy you a coffee, bao bun, or wheatgrass shot at the next conference. No substitutions.
Fun day at @dartmouthchem.bsky.social
serving buldak ramen and explaining the biophysics and structural biology of heat and capsaicin activation of the ion channels to celebrate @acs.org #NationalChemistryWeek.
serving buldak ramen and explaining the biophysics and structural biology of heat and capsaicin activation of the ion channels to celebrate @acs.org #NationalChemistryWeek.
October 20, 2025 at 6:52 PM
Fun day at @dartmouthchem.bsky.social
serving buldak ramen and explaining the biophysics and structural biology of heat and capsaicin activation of the ion channels to celebrate @acs.org #NationalChemistryWeek.
serving buldak ramen and explaining the biophysics and structural biology of heat and capsaicin activation of the ion channels to celebrate @acs.org #NationalChemistryWeek.
Also, take note of our 15 (!) exp. refined all-atom IDP ensembles deposited in the protein ensemble database:
proteinensemble.org/entries/PED0...
If you think you have good method / force field for generating IDP ensembles, you can benchmark your agreement with exp. against these ensembles.
proteinensemble.org/entries/PED0...
If you think you have good method / force field for generating IDP ensembles, you can benchmark your agreement with exp. against these ensembles.
October 10, 2025 at 3:47 PM
Also, take note of our 15 (!) exp. refined all-atom IDP ensembles deposited in the protein ensemble database:
proteinensemble.org/entries/PED0...
If you think you have good method / force field for generating IDP ensembles, you can benchmark your agreement with exp. against these ensembles.
proteinensemble.org/entries/PED0...
If you think you have good method / force field for generating IDP ensembles, you can benchmark your agreement with exp. against these ensembles.
While they're only rarely populated at the same time - these multisite binding mode give us a better understanding of how a network of hydrogen bond donors and acceptors confer pronounced affinity to residues 7_CATQRLANFLV_17.
September 27, 2025 at 1:45 PM
While they're only rarely populated at the same time - these multisite binding mode give us a better understanding of how a network of hydrogen bond donors and acceptors confer pronounced affinity to residues 7_CATQRLANFLV_17.
To get more insight into the diversity of binding modes and search for more structured modes - we looked at binding poses where at least 15 residues of hIAPP were in contact with each ligand. This represented 12.9% of bound frames for YX-I-1 but only 3.9% of YX-A-1.
September 27, 2025 at 1:45 PM
To get more insight into the diversity of binding modes and search for more structured modes - we looked at binding poses where at least 15 residues of hIAPP were in contact with each ligand. This represented 12.9% of bound frames for YX-I-1 but only 3.9% of YX-A-1.
Interestingly, the exposed regions of YX-A-1 are quite hydrophobic (cylcohexane and benzene) - and we think this could be part of how it accelerates aggregation into higher order oligomers and protofilaments
September 27, 2025 at 1:45 PM
Interestingly, the exposed regions of YX-A-1 are quite hydrophobic (cylcohexane and benzene) - and we think this could be part of how it accelerates aggregation into higher order oligomers and protofilaments
We see that each ligand has moieties that are consistently buried and others that are consistently exposed across binding modes. We think that buried moieties might confer monomer affinity - while exposed moieties could affect rates of oligomerization into higher order species.
September 27, 2025 at 1:45 PM
We see that each ligand has moieties that are consistently buried and others that are consistently exposed across binding modes. We think that buried moieties might confer monomer affinity - while exposed moieties could affect rates of oligomerization into higher order species.
Comparing populations of intermolecular interactions we found something unique about this pair: the largest difference is elevated populations of hydrogen bonds with YX-I-1, not increased populations of aromatic stacking interactions -which we usually see in tighter IDP ligands
September 27, 2025 at 1:45 PM
Comparing populations of intermolecular interactions we found something unique about this pair: the largest difference is elevated populations of hydrogen bonds with YX-I-1, not increased populations of aromatic stacking interactions -which we usually see in tighter IDP ligands
We have a detailed comparison of contact profiles and helicity changes with NMR CSPs from in the SI. We don't see perfect agreement, but observe that the average magnitude of CSPs correlate pretty well with average contact populations and changes in helicity upon and binding.
September 27, 2025 at 1:45 PM
We have a detailed comparison of contact profiles and helicity changes with NMR CSPs from in the SI. We don't see perfect agreement, but observe that the average magnitude of CSPs correlate pretty well with average contact populations and changes in helicity upon and binding.
In ligand binding simulations, we see heterogenous ensembles of binding modes. We see YX-I-1 is a tighter binder than YX-A-1, and produces larger conformational changes upon binding, consistent with larger NMR chemical shift perturbations (CSPs) and other assays from @radford-lab.bsky.social
September 27, 2025 at 1:45 PM
In ligand binding simulations, we see heterogenous ensembles of binding modes. We see YX-I-1 is a tighter binder than YX-A-1, and produces larger conformational changes upon binding, consistent with larger NMR chemical shift perturbations (CSPs) and other assays from @radford-lab.bsky.social