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Review for: Oligomerization enables the selective targeting of intrinsically disordered regions by small molecules.

https://www.biorxiv.org/content/10.1101/2025.03.21.644603v1.full

Summary:

This study aims to expand our knowledge on intrinsically disordered regions (IDRs) of proteins and to identify potential strategies to selectively target them with small molecules. The paper focuses on EPI-001, the first small molecule in a clinical trial targeting IDRs,  and its target, the activation domain of the androgen receptor (AR). The goal of the paper is to determine how EPI-001 inhibits the AR and why this inhibition is relatively specific to the AR. Using nuclear magnetic resonance (NMR), dynamic light scattering, and mutational analysis, the authors demonstrate that EPI-001 alters the conformational ensemble of the AR activation domain and interpret this as causing  changes to the physical properties of condensates it forms. While much of the data in this paper is interesting, the observed selectivity of EPI-001 for AR and high concentrations of EPI-001 used in assays weakens this paper’s conclusions that specific interactions between EPI-001 and transiently structured regions of the AR are responsible for the shift in AR behavior. Additionally, the correlation between physicochemical properties such as lipophilicity/aromaticity as drivers may suggest that the condensate properties themselves are the result of non-specific interactions that could layer on top of the (weak) specific interactions. The manuscript would benefit from a discussion of the role of nonspecific hydrophobic and also the covalent interactions in the AR-EPI-001 system and their implications for targeting IDRs more broadly.

The authors demonstrate the importance of aromatic-rich motifs and transient helical structures in assembling into a higher-order structure that EPI-001 can bind to and stabilize. Mutations disrupting aromaticity or helical propensity decreases chemical shift perturbation upon EPI-001 binding and reduces ligand partitioning into phase-separated condensates. Additionally, the authors find that  EPI-001 partitions relatively selectively into AR activation domain condensates and rigidifies them, reducing their intramolecular dynamics and hindering recruitment of RNA Polymerase II. Similar to the oligomeric states, the authors show that phase-separated condensate behavior is coupled to the aromatic and secondary structural features of the AR activation domain. However, we think the conclusion that this is the result of specific interactions is not sufficiently supported, given it is also possible that nonspecific interactions between the AR and high concentrations of EPI-001 can result in a clustering of hydrophobic regions which is further complicated by the formation of covalent adducts.

Overall, this work proposes that oligomerization of IDRs can transiently create structured, druggable sites, offering a strategy to overcome the long-standing challenges of targeting disordered proteins with small molecules. However, since the paper studies an IDR that has hydrophobic regions that form ordered structure upon homoligomerization and a ligand that reacts promiscuously with thiols, its strategy may not generalize to systems that lack these properties.

Major points:

• In section 1. the authors use CSP and DLS to demonstrate EPI-001 is selective for the activation domain of AR compared to other nuclear receptors. As a reader, it is unclear what the authors mean by “selective” because the data does not appear to reflect that claim. We recommend the authors discuss alternative interpretations of the data and encourage them to provide further evidence or commentary to justify this claim.

  • From CSP the authors show that AR had the largest CSP but other nuclear receptors such as MR (Figure 1f) also showed a significant shift with the addition of ligand. The magnitude of CSP alone is insufficient to comment on selectivity of a ligand. We encourage the authors to discuss alternative interpretations and to acknowledge the limitations of this technique.

  • The authors state: “EPI-001 selectively stabilizes the oligomers formed by the AR AD”(line 79) from their DLS data (Extended Data Figure 2d). Nuclear receptor ERα shows a noteworthy decrease of monomers with increasing concentration of EPI-001 and MR already exists as a higher order oligomer prior to addition of drug. The authors have demonstrated sufficiently that EPI-001 stabilizes AR oligomers but as a reader it still remains unclear if this is selective to AR AD. Further discussion from the author on this point could provide the reader with some clarity. 

• In section 1.5, the authors make a a disulfide linked version of Tau-5* called 2Tau-5* which they demonstrated to have a much higher propensity to form ligand-independent  oligomers compared to Tau-5* (Figure 2g). The authors hypothesize that “EPI-001 has a higher affinity for the oligomeric than for the monomeric form of the target” (line 104) and use CSP to confirm this. We suggest the authors discuss limitations or more fully justify  their current interpretation of this data if MST binding data is not included (see comment below - or just show the data!). A higher CSP does not directly translate to a higher binding affinity. We found these statements to be distracting and weaken the authors’ claims rather than strengthen them. An alternative interpretation that aligns better with the limitations of the technique used (e.g., EPI-001 induces larger conformational change to oligomeric form compared to the monomeric form of target), would remedy this concern.

• Authors show MST binding curve for 2Tau-5* with ligand but not Tau-5* (Extended Data Figure. 2m) with a calculated Kd. Can the authors comment on why they do not include data on Tau-5*? If EPI-001 is more potent for the oligomeric form of AR than the assay should demonstrate that and be moved to the main figure rather than supplemental. 

• Can the authors comment on why they had to treat with up to 400µM to induce oligomerization of the target (Figure 2). Earlier work (https://www.cell.com/cancer-cell/fulltext/S1535-6108(10)00200-X) determined the IC50 of EPI-001 for AR to be 6µM in a cell-based screen. One could assume that the drug would be equally or, potentially, more potent in a biochemical assay (e.g., DLS) than in a cell-based one. As a reader, it remains unclear if EPI-001 induced oligomerization of  AR is mechanistically relevant to ligand binding or merely a consequence of high drug concentrations. There may be both (weak) specific interactions (measured by CSP) and highly cooperative non-specific interactions (measured by condensate properties) at play.

• We found the role of covalent binding in the interaction between EPI-001 and the AR AD unclear. The formation of a covalent adduct between EPI-001 and the AR AD is not mentioned until Figure 4. We think it would benefit to mention the formation of this covalent adduct earlier and explain when it is and is not important for experiments of the AR AD / EPI-001 interaction. Experiments in Figure 2f/g/h/i, 3c, 4, and 5a feature mutants of the AR AD of cysteine residues involved in covalent adduct formations. We can infer that adduct formation is slow since adduct forms on the scale of hours in figure 4h, and that a significant amount of adduct would not form during NMR experiments. We think it would help if the authors could explicitly state their reasoning for why inhibited covalent adduct formation is not a factor in analysis of mutants with mutated cysteine residues. Additionally, we think covalent bond formation should be mentioned when discussing the mechanism of EPI-001 interaction with the AR since covalent bonding has been demonstrated to be important for this interaction by the increased effectiveness of EPI-001 over bisphenol A bis [2,3-dihydroxypropyl] ether.

Minor points:

• Can the authors state their reasoning for choosing the C404S mutant to make 2Tau*. Since 2Tau* is formed by a disulfide at C448, it is not clear to us why a C404S mutation was chosen to make 2Tau* over wild type.

• On first read, it was not immediately clear that Tau referred to a part of the AR, given that Tau usually refers to microtubule associated protein Tau in papers on intrinsically disordered regions. It would be helpful to specify that Tau stands for Transactivation unit in this text. This is currently only stated in the references.

• Figure 6f/g have their labels swapped

• The subcategories of Figure 3c (top/bottom/left/right) do not match their positions in the figure well.

• The Figure 4 legend has a typo in line 395 and Figure 1 legend missing space between amino acid in line 339.

Competing interests

The authors declare that they have no competing interests.