How small molecules stabilize oligomers of a phase-separating disordered protein

How small molecules stabilize oligomers of a phase-separating disordered protein | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results How small molecules stabilize oligomers of a phase-separating disordered protein View ORCID Profile Jiaqi Zhu , View ORCID Profile Thomas R. Sisk , View ORCID Profile Borja Mateos , View ORCID Profile Stasė Bielskutė-Garcia , View ORCID Profile Xavier Salvatella , View ORCID Profile Paul Robustelli doi: https://doi.org/10.1101/2025.11.20.689623 Jiaqi Zhu † Department of Chemistry , Dartmouth College, Hanover, NH, 03755, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jiaqi Zhu Thomas R. Sisk † Department of Chemistry , Dartmouth College, Hanover, NH, 03755, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas R. Sisk Borja Mateos ‡ Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology , Baldiri Reixac 10, 08028 Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Borja Mateos Stasė Bielskutė-Garcia ‡ Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology , Baldiri Reixac 10, 08028 Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stasė Bielskutė-Garcia Xavier Salvatella ‡ Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology , Baldiri Reixac 10, 08028 Barcelona, Spain ¶ ICREA , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xavier Salvatella Paul Robustelli † Department of Chemistry , Dartmouth College, Hanover, NH, 03755, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paul Robustelli For correspondence: paul.j.robustelli{at}dartmouth.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Small molecule inhibitors of the intrinsically disordered androgen receptor activation domain have been tested in clinical trials for the treatment of castration-resistant prostate cancer. These compounds have been shown to stabilize oligomeric forms of the androgen receptor activation domain in solution and alter the properties of androgen receptor condensates. The molecular mechanisms by which small molecules modulate these processes have been poorly understood in atomic detail. Here, we use long-timescale all-atom molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy to determine how small molecules stabilize highly dynamic, heterogeneous intermolecular interfaces that mediate oligomerization of the androgen receptor activation domain. The mechanisms determined here explain the relative potencies of androgen receptor activation domain inhibitors and suggest general strategies for designing small molecules that target oligomeric and, potentially, condensed forms of intrinsically disordered proteins. Intrinsically disordered proteins (IDPs) play central roles in cellular signaling and regulation and are increasingly being pursued as drug targets 1 – 3 . The structural plasticity of IDPs enables them to form transient multivalent interactions that drive the assembly of biomolecular condensates 1 , 4 . This plasticity also makes IDPs extremely challenging drug targets. Several small molecules have been discovered that bind and inhibit specific IDPs and IDP-targeting compounds have entered human trials 3 . Biophysical experiments 5 – 12 and atomistic molecular dynamics (MD) computer simulations 10 – 18 have shown that IDPs can remain disordered upon binding small molecules, and that the selectivity and affinity of IDP ligands discovered thus far is conferred through dynamic networks of transient interactions that may only subtly shift their conformational ensembles. The androgen receptor (AR) is a transcription factor containing an intrinsically disordered activation domain (AD) 19 . Small molecules targeting the disordered AR AD have shown promise for the treatment of castration-resistant prostate cancer (CRPC), as they inhibit AR splice variants lacking a ligand-binding domain that confer resistance to FDA-approved anti-androgen drugs 20 , 21 . The AR AD mediates the formation of transcriptional AR condensates in cells 12 , 22 – 24 . Nuclear magnetic resonance (NMR) spectroscopy, biophysical characterization of AR biomolecular condensates and cellular assays have identified aromatic residues and short transient helices in transactivation unit 5 (Tau-5) of the AR AD as the primary drivers of AR oligomerization and phase separation 12 , 24 , 25 . Tau-5 contains the interaction site of EPI-001 5 , 13 , 24 , a small molecule previously tested in human clinical trials to treat CRPC 3 , 20 , 21 . In recent studies, it was observed that EPI-001 binds oligomeric states of Tau-5 with higher affinity than monomeric states, reduces the condensation cloud point ( T C ) of Tau-5, rigidifies AR condensates in biophysical assays and reduces the ability of AR condensates to recruit RNA polymerase II in live cells 12 , 24 . We previously employed all-atom MD simulations to determine atomic resolution binding mechanisms of EPI-002 (the highest affinity stereoisomer of EPI-001 5 ) and related inhibitors to monomeric Tau-5 12 , 13 , 16 . In these simulations, which are in excellent agreement with NMR data, AR AD inhibitors predominantly bind at the interface between the partially helical R2 and R3 regions of Tau-5 and stabilize the formation of collapsed yet highly dynamic, molten-globule-like helical states. Subsequently, we observed that the AR AD inhibitor 1aa, which replaces the gem-dimethyl linker of EPI-002 with a linear alkyne ( Figure 1 ), has a higher affinity to Tau-5 and more potently inhibits AR transcriptional activity in androgen-induced PSA-luciferase reporter assays 12 . We further observed that 1ae, a derivative of 1aa with an additional phenyl substituent on the diphenylacetylene moiety, more effectively partitions into AR condensates, produces a larger reduction in T C and has improved potency in cellular and human xenograft mouse models of CRPC relative to EPI-001 12 . Download figure Open in new tab Figure 1: Molecular dynamics simulations of the intermolecular association of Tau-5 R2 monomers in the presence of EPI-002 and 1aa. (A) Amino acid composition of Tau-5 R2 and chemical structures of EPI-002 and 1aa. (B) The presence of ligands drives the equilibrium of Tau-5 R2 towards the formation of dynamic and heterogeneous dimers. (C) Tau-5 R2 dimer dissociation constants ( K D ) observed in unbiased 85–100 µ s MD simulations. (D) Helical propensities observed in MD simulations of Tau-5 R2 dimerization. (E) Auto-correlation functions ( C(t) ) of the persistence of intermolecular contacts between Tau-5 R2 monomers represent the probability that monomers in contact at time t=0 remain associated at a given lag time. Each C(t) was fit to a double exponential decay, revealing two timescales of intermolecular dissociation with correlation times τ fast and τ slow . To elucidate the molecular mechanisms by which EPI-001 and 1aa stabilize the inter-molecular association of Tau-5 in AR oligomers and condensates, we performed unbiased long-timescale (85–100 µ s) all-atom MD simulations of the reversible intermolecular association of Tau-5 R2 , the primary oligomerization interface of Tau-5 24 , 25 , in the presence and absence of EPI-002 and 1aa ( Figure 1 ). Simulations were performed with the a99SB- disp protein force field and water model on the Anton 2 supercomputer 26 , 27 (“Molecular Dynamics Simulations” in Supporting Information). We observe that the simulated K D of Tau-5 R2 dimerization decreases from 1.8 ± 0.3 mM in the absence of ligands (“apo”) to 1.0 ± 0.3 mM and 0.30 ± 0.03 mM in the presence of EPI-002 and 1aa, respectively, and that ligand binding substantially increases the population of helices in Tau-5 R2 dimers. In each MD simulation, we observe both transient, short-lived Tau-5 R2 dimer species with residence times of 10–300 ns and more stable dimer complexes with residence times of up to 38 µ s. Distributions of Tau-5 R2 dimer and ternary complex residence times (Supporting Figure S1) reflect the formation of both relatively non-specific, transient monomer–monomer encounters and metastable complexes with more structured intermolecular interfaces. To quantify the timescales of Tau-5 R2 dimer dissociation, we computed autocorrelation functions from time series indicating the presence of intermolecular protein contacts ( Figure 1E ). These correlation functions, which represent the probability that Tau-5 R2 monomers that are in contact at time t = 0 remain associated after a given lag time, are well-described by a double-exponential decay, suggesting the presence of fast ( τ fast =50–250 ns) and slow ( τ slow =0.5–5.0 µ s) Tau-5 R2 complex dissociation processes. EPI-002 and 1aa binding increases the survival times of both species, with 1aa producing the most pronounced stabilization of long-lived Tau-5 R2 dimers and ternary complexes. These simulations suggest that EPI-002 and 1aa enhance Tau-5 R2 self-association by stabilizing dynamic multivalent oligomerization interfaces, providing a molecular basis by which these ligands and related compounds lower the T C of AR condensation and rigidify AR condensates 12 , 24 . We characterized the conformational heterogeneity and kinetic stability of Tau-5 R2 dimers and ternary complexes observed in each MD simulation by clustering conformational states based on fluctuations of chain writhe 28 – 30 , a geometric descriptor from knot theory that quantifies the crossing of curves in three-dimensional space (“Kinetic clustering of conformational states from writhe” in Supporting Information). We observe heterogeneous conformational ensembles of interconverting Tau-5 R2 dimer and ternary complex structures rather than a single well-defined rigid intermolecular interface ( Figure 2 , Supporting Figures S2–S14, Supporting Tables S1-S3). We observe several common intermolecular interfaces and ligand binding modes across conformational states and find that, on average, Tau-5 R2 ternary complexes with ligands have higher populations of helical elements and exhibit a greater degree of structural ordering compared to apo dimers, in agreement with biophysical experiments 24 . Download figure Open in new tab Figure 2: Conformational heterogeneity of Tau-5 R2 dimers and Tau-5 R2 ternary complexes observed in molecular dynamics simulations. Free energy surfaces of Tau-5 R2 conformational ensembles derived from fluctuations of chain writhe identify multiple structurally and kinetically distinct conformational states of Tau-5 R2 dimers and Tau-5 R2 ternary complexes in the absence of ligands (A) and in the presence of EPI-002 (B) . Representative structural snapshots are shown for selected clusters, illustrating the conformational heterogeneity of intermolecular oligomerization interfaces and ternary complex ligand binding modes. Detailed analyses of the structural properties of all conformational states are provided in Supporting Information. To elucidate the driving forces of Tau-5 R2 dimerization and ternary complex formation we analyzed the populations of intermolecular contacts formed between Tau-5 R2 monomers ( Figure 3 ) and the populations of specific protein-ligand interactions ( Figure 4 ). In the absence of ligands, the most populated intermolecular interactions occur between aromatic residues and the alanine-rich, transiently helical 396 AWAAAAAAQ 403 region. The addition of ligands strengthens these interactions, promoting tighter and more specific contacts between aromatic residues and polyalanine regions and stabilizing more rigid, but still dynamic, partially helical intermolecular interfaces. These results are highly consistent with NMR chemical shift perturbations and intermolecular paramagnetic relaxation enhancements (PREs) observed in Tau-5 upon addition of EPI-001 24 . Download figure Open in new tab Figure 3: Ligand binding stabilizes aromatic contacts between Tau-5 R2 monomers. (A) Populations of intermolecular contacts between Tau-5 R2 monomers in unbiased long-timescale MD simulations in both the presence and absence of ligands. Interactions of aromatic residues are highlighted with dashed white lines. Average helical propensities are shown for each monomer. (B) Free energy surfaces of Tau-5 R2 dimerization in each MD simulation as a function of the total α -helical content (S α ) and the number of intermolecular protein-protein contacts, calculated with a smoothed switching function. (C) Schematic illustration of intermolecular contacts between an NMR-detected aromatic side chain proton of an 13 C-labeled Tau-5 monomer (red) and protons of an unlabeled 12 C Tau-5 monomer (blue). (D) Comparison of average NMR intermolecular nuclear Overhauser effect (NOE) intensities of Tau-5 aromatic side chain protons with the average number of intermolecular proton contacts observed in MD simulations of Tau-5 R2 dimerization. Download figure Open in new tab Figure 4: Populations of protein–ligand interactions in molecular dynamics simulations of the intermolecular association of Tau-5 R2 monomers in the presence of EPI-002 and 1aa. (A) Probability that ligands simultaneously form contacts with pairs of residues from opposing Tau-5 R2 monomers. (B) Average populations of protein-ligand interactions observed between EPI-002 and 1aa and Tau-5 R2 . Populations are reported as mean values ± statistical error estimates from block averaging. (C) Representative structures of 1aa forming ternary complexes with two Tau-5 R2 monomers. We measured residue-type specific intermolecular 13 C-filtered/ 13 C-edited nuclear Over-hauser effects (NOEs) 31 in aromatic side chain protons of Tau-5 in the presence and absence of EPI-001, demonstrating that EPI-001 increases the probability of forming intermolecular aromatic contacts. ( Figure 3 ; Supporting Figures S15–S16; Supporting Information section “NMR Spectroscopy”). Changes in intermolecular NOE intensities of aromatic side chain protons in Tau-5 observed upon adding EPI-001 are in qualitative agreement with differences in the populations of intermolecular proton contacts made by Tau-5 R2 aromatic side chain protons in MD simulations. Due to limited solubility, it was not possible to record intermolecular NOE measurements in the presence of 1aa at equivalent concentrations to EPI-001. Lastly, we characterized the molecular mechanisms of ligand-mediated Tau-5 R2 oligomerization by quantifying the populations of hydrophobic contacts, hydrogen bonds, and aromatic stacking interactions formed between EPI-002 and 1aa and each residue of Tau-5 R2 ( Figure 4 , Supporting Figure S9, Supporting Figure S14). We observe that both ligands interact most strongly with aromatic residues of Tau-5 R2 , and that 1aa forms substantially higher populations of hydrophobic and aromatic stacking interactions than EPI-002. To assess the spatial organization of ligand binding in ternary Tau-5 R2 -ligand complexes, we compared the probabilities of simultaneously forming ligand contacts with pairs of residues from opposing Tau-5 R2 monomers. EPI-002 primarily binds at interfaces formed by residues 396 AWAAAAAAQ 403 of both monomers, consistent with previously reported NMR measurements 5 , 12 , 24 . In contrast, 1aa forms a broader, more distributed binding interface. We observe that the extended linear alkyne linker of 1aa increases the distance between phenyl rings and provides greater rotational flexibility compared to the more rigid gem-dimethyl linker of EPI-002. The geometry and dihedral flexibility of the diphenylacetylene group enables 1aa to intercalate into both Tau-5 R2 monomers and form stacking interactions with aromatic residues in each monomer simultaneously, more effectively stabilizing partially helical AR AD oligomerization interfaces ( Figure 4C ). In this work, we have integrated molecular simulations and NMR spectroscopy to understand the molecular basis by which small molecules stabilize the intermolecular association of the AR AD in atomic detail. Our results, which are consistent with biophysical data from several previous studies 5 , 12 , 24 , 25 , provide an atomistic description of how small molecules stabilize AR AD oligomerization interfaces through networks of transient aromatic and hydrophobic interactions that promote the formation of partially helical oligomers with increased structural order. The ternary complexes described here identify ligand features and binding modes that stabilize AR AD oligomers and provide an atomic-level explanation for the increased potency of 1aa and other diphenylacetylene derivatives in AR transcriptional inhibition assays compared to EPI-001 12 . Recent work has shown that EPI-001 binds the AR AD with greater affinity than four other steroid-receptor ADs and selectively stabilizes AR AD oligomers without stabilizing oligomers of the other ADs 24 . This suggests that, in some systems, ligand recognition of oligomeric interfaces—rather than monomeric states—may be a major determinant of IDP ligand selectivity. In such cases, designing ligands that preferentially bind and stabilize specific oligomeric conformations could provide a general strategy for enhancing IDP ligand affinity and selectivity. Mechanistic insight from all-atom MD simulations of ligand-mediated IDP oligomerization, as provided here, may prove valuable for guiding this emerging structure-based drug design paradigm for IDPs. Lastly, we speculate that the mechanisms of ligand-mediated oligomerization of Tau-5 R2 reported here, together with interactions involving additional segments of Tau-5, may underlie the increased rigidity of AR condensates observed in the presence of EPI-001 24 . In future studies, we will investigate this hypothesis by attempting to leverage mechanistic insights from all-atom MD simulations of ligand-mediated IDP oligomerization to design small molecules that rationally tune the material properties of biomolecular condensates. In summary, the atomic-resolution mechanistic framework presented here will inform ongoing efforts to develop therapeutics for the treatment of CRPC and other currently untreatable diseases associated with IDP and condensate dysfunction 32 , 33 . Data and Code Availability All molecular dynamics simulation input files, trajectories and analysis code are freely available from https://github.com/paulrobustelli/Zhu_Sisk_AR_oligomers_2025 . Competing Interest Statement Xavier Salvatella is a cofounder and scientific advisor of Nuage Therapeutics. Paul Robustelli’s laboratory has previously received funding through a sponsored research agreement with Nuage Therapeutics. The authors declare that they have no other competing interests. Acknowledgments P.R., J.Z. and T.R.S. acknowledge the support of NIGMS award R35GM142750. T.R.S. additionally acknowledges the support of the U.S. Department of Education award GAANN P200A240037. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health and Anton award MCB200087P. The Anton 2 machine at PSC was made available by D.E. Shaw Research. B.M. acknowledges the support of the AECC (Spanish Cancer Association) postdoctoral grant. S.B.G. acknowledges support from a PhD fellowship awarded by IRB in the 2020 call and an FPI fellowship awarded by MINECO in the 2021 call (PID2019-110198RV-100). X.S. was supported by AGAUR (2021 SGR 476), the AEI (PID2019-110198RB-I00 and PID2022-141816OB-I00), the Fundació La Caixa (CI20-00098), the AECC (INNO20010FRIG) and the Mark Foundation. Funder Information Declared National Institute of General Medical Sciences, https://ror.org/04q48ey07 , R35GM142750 United States Department of Education, https://ror.org/021adze67 , GAANN P200A240037 Asociación Española Contra el Cáncer , Postdoctoral Grant Ministry of Economy, Industry and Competitiveness, https://ror.org/034900433 , PID2019-110198RV-100 Institute for Research in Biomedicine, https://ror.org/01z1gye03 , PhD fellowship Agència de Gestió d’Ajuts Universitaris i de Recerca , 2021 SGR 476 Agencia Estatal de Investigación , PID2019-110198RB-I00 , PID2022- 141816OB-I00 Fundación Bancaria Caixa dEstalvis i Pensions de Barcelona, https://ror.org/05r3yzw86 , CI20-00098 The Mark Foundation for Cancer Research, https://ror.org/00v7th354 , ASPIRE award Asociación Española Contra el Cáncer, https://ror.org/00gxct719 , INNO20010FRIG Footnotes https://github.com/paulrobustelli/Zhu_Sisk_AR_oligomers_2025/ References (1). ↵ Holehouse , A. S. ; Kragelund , B. B. 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