Catalytic Proximal Protein Oligomerization as an Anti-Tumor Strategy Targeting WDR5

Domain: chemistry
Published: 2026-03-13
Editorial outcome: major_revision

Abstract
Inducing protein oligomerization holds therapeutic promise, yet identifying or rationally designing effective inducers remains challenging. In this study, we leverage nanopore technology to screen small molecules capable of modulating WD repeat domain 5 (WDR5) protein oligomerization and uncover a unique oligomerization mechanism which we term Catalytic Proximal Protein Oligomerization (CaPPO). Nanopore sensing enabled rapid identification of WZ-1, a selective WDR5 oligomerization inducer, from our in-house compound library. Biochemical and cryo-EM analyses reveal that WZ-1 binds the WBM site of WDR5 through its 2-aminothiazole moiety, reacts with Cys248 to form an intramolecular disulfide, and, through N-terminal tail-induced proximity of WDR5 molecules, triggers oligomerization via thio–disulfide exchange. Structure–activity relationship studies indicate that the disulfide motif within WZ-1 acts as a molecular “scout” initiating oligomerization. In cellular assays, WZ-1 exhibits potent anti-tumor activity by disrupting WDR5 interactions at both WIN and WBM sites, leading to transcriptional downregulation of target genes. Collectively, this work not only establishes a nanopore-based screening platform for identifying protein oligomerization inducers, but also validates CaPPO as a promising small-molecule design strategy for therapeutic development.

Introduction
Proteins can self-assemble or hetero-assemble to form various higher-order complexes with diverse biological functions (1,2). In response to environmental stimuli or mutations, they often oligomerize or aggregate, exhibiting a range of material properties from solid-like amyloid fibers to dynamic liquid droplets (3-6). These aggregates can be nonfunctional and disease-causing. Historically, protein aggregates were primarily associated with diseases such as neurodegenerative disorders (7-10). However, growing evidence suggests that they can also play non-pathogenic and functional roles in regulating cell activities (3,11). This has led to the intriguing concept of therapeutically exploiting protein oligomerization , whereby intentional modulation of protein aggregation could be leveraged for clinical benefit (7). A compelling example supporting this perspective is the recent discovery of LBI-3802, a small molecule that degrades B-cell lymphoma 6 (BCL6) protein. BI-3802 promotes the aggregation of BCL6, leading to its ubiquitination and degradation (12). Despite several reports highlighting the therapeutic potential of small molecules in controlling protein oligomerization (12-15), most such compounds have been identified serendipitously, often via phenotypic screening. Therefore, developing effective in vitro screening methods for targeted protein aggregation regulation remains a challenge, hindering rational drug design.

Nanopore sensing, a rapidly advancing technology, enables label-free examination of native proteins in a liquid-phase setting, making it ideal for studying protein response to changes in buffer composition or specific environmental stimuli (16-21). When an external electric field is applied, analytes are driven electrophoretically or electroosmotically through nanoscale channels. Each translocation event, induced by a single analyte, results in a characteristic temporal alteration in the measured ionic current within the nanoscale channel. Analysis of these current signatures can reveal key properties of the analyte, including charge, size, conformational state, and interactions with other biomolecules (18,22,23). These capabilities position nanopore sensing as a powerful platform for protein analysis (22,24). In particular, quartz nanopores, known for their cost-efficiency and ease of fabrication (22,25-28), overcome the cost barriers associated with conventional nanopore technologies, enabling high-throughput drug screening.

WD repeat domain 5 (WDR5) is an essential scaffolding protein that participates in multiple protein complexes and regulates a broad range of cellular processes (29-31). Notably, it is a key component of the mixed-lineage leukemia (MLL/SET) complex and promotes Myc recruitment to chromatin at tumor-associated gene loci (32,33). Intriguingly, both interaction events between WDR5 and MLL or Myc have been observed at the single molecule level using engineered FhuA nanopores (34,35). Given WDR5’s central role in solid and hematologic malignancies and its ability to engage multiple partners to form higher-order complexes (36,37), it raises the question of whether inducing its controlled oligomerization could represent a effective strategy for functional modulation.

In this study, we utilize quartz nanopores to screen our in-house library of symmetric small molecules for their capacity to selectively induce WDR5 oligomerization. This approach lead to the identification of WZ-1 as a potent and selective inducer of WDR5 aggregation. Mechanistic studies reveal that WZ-1 targets the WDR5 protein, promoting its oligomerization through a catalytic proximity thio-disulfide bond exchange (a process we term Catalytic Proximal Protein Oligomerization (CaPPO)), ultimately hindering the biological functions of WDR5 in cells. Our findings not only establish an efficient nanopore-based platform for discovering protein oligomerization inducers but also introduce CaPPO as a design paradigm for targeting WDR5 and other therapeutically relevant proteins.

Results
Identifying WZ-1 as a WDR5 oligomerization-inducing molecule using nanopore sensing.
To facilitate the discovery of protein oligomerization inducers, we selected WDR5 as the model protein for several reasons. First, WDR5 is a well-characterized scaffold protein within the WD40 repeat (WDR) domain family, one of the most abundant protein interaction domains in the human proteome (38). Second, several drug-like protein-protein interaction (PPI) inhibitors targeting WDR5 have been identified, providing a reference point to compare the effects of oligomerization inducers with those of conventional PPI inhibitors (39). Third, prior studies have identified at least two functional binding pockets associated with WDR5: the WDR5 interaction WIN site (40-43) and the WDTR5-binding motif WBM site (42,44), which provides an opportunity to explore whether oligomerization induction can disrupt WDR5 function.

To reliably detect WDR5 proteins in various oligomerization states, quartz nanopipettes were fabricated into nanopores with an average diameter of 22.2 ± 5.0 nm (n = 5), as confirmed by scanning electron microscopy (SEM) (Fig. S1). Linear I-V curves were observed in the protein buffer, with the nanopore conductance measured at 35.70 ± 4.87 nS (Fig. S2a). A thermal shift assay confirmed that WDR5 maintained its binding affinity for known inhibitors under high-salt conditions (Fig. S3), ruling out potential salt-induced alterations in protein functionality.

Our screening strategy leverages the sensitivity of quartz nanopores to different oligomeric states of proteins (22). In all nanopore sensing experiments, the reference/ground Ag/AgCl electrode and the patch/working Ag/AgCl electrode were separately placed in the external reservoir (cis) and the pipette (trans), both filled with the same protein buffer. Analytes, including proteins and molecules, were introduced into the external reservoir (Fig. 1a). The consistent translocation characteristics of pure WDR5 across multiple nanopores, fabricated with the same parameters, were observed at an applied voltage of 250 mV, with a mean peak current of 76.33 ± 28.8 pA (Fig. 1b and S2b). This value represents the ion excluded volume caused by the translocation of monomeric WDR5. Non-uniform translocation events of monomeric WDR5, along with atypical translocation events exhibiting significantly higher peak currents and longer dwell times, were ascribed to either impurities compromising protein purity or transient oligomerization, unfolding and surface adsorption during the translocation process (22,45,46).

Next, we employed our established nanopore sensing platform to screen for molecules capable of inducing WDR5 oligomerization (Fig. 1a). Bivalent compounds, composed of two fragments that each bind the protein of interest (POI) and connected by a suitable linker, can simultaneously engage two POI molecules, thereby promoting dimerization (47). Inspired by this principle, we screened an in-house library of 436 small molecule compounds, most of which possess bivalent-like structures. To improve screening efficiency, we categorized the 436 compounds into nine groups (46-50 compounds per group, designated 1st-#1 to 1st-#9) based on structural similarity , and each group was evaluated using nanopore sensing (Fig. 2a-b). The mean peak current under identical conditions was determined using Gaussian fitting, and the rate of change was calculated by comparing the values in the mean before and after the introduction of the candidate compounds (Fig. S4). The rate of change served as the key metric for identifying groups containing potential oligomerization inducers. In the first screening round, group 1st-#9 produced the most pronounced effect, showing a ~45.1% increase in average peak current (Fig. 2a-2d). These 46 compounds were then divided into five subgroups for a second-round screen, in which subgroup 2nd-#5 produced an even larger increase of 60.5% in mean peak current. Finally, the 10 candidate compounds from subgroup 2nd-#5 were tested individually, leading to the identification of WZ-1 (Fig. 2e) as the most potent inducer of WDR5 oligomerization.

To exclude potential confounding factors, such as the change in translocation events, being solely caused by the binding of molecules to protein sites, we conducted real-time monitoring of the WDR5 translocation in the presence of high-affinity inhibitors OICR-9429 (39,48) and SADS-1 (49), which target WIN and WBM sites respectively. In this experiment, we recorded translocation events in the presence of these two compounds and compared them to the behavior of the WDR5 monomer. As expected, neither OICR-9429 nor SADS-1 produced any discernible change in WDR5 translocation characteristics (Fig. S5a). In addition, control experiments confirmed that the observed signals were not due to micelles or precipitates formed by high concentrations of WZ-1 (Fig. S6). Together, these observations confirm that the changes in WDR5 translocation behavior are indeed induced by WZ-1 through its promotion of WDR5 oligomerization.

Next, we monitored the real-time oligomerization of WDR5 to assess its proteodynamics. To enable more stable and long-term detection of oligomerized WDR5 translocation events, we utilized a larger-diameter quartz nanopore. These pores generated smaller protein peak signals, reducing the risk of clogging and allowing efficient translocation of oligomeric species. Nanopore sensing was conducted over three hours at a WZ-1 concentration of 5 μM, with translocation events recorded in 5-minute intervals every 30 minutes and visualized as scatter plots (Fig. 2f-g). Following the addition of WZ-1, the mean peak current gradually increased, reaching approximately twice its initial value. The aggregation behavior of WDR5 displayed a time-dependent pattern but did not follow a distinct sigmoidal trajectory (Fig. 2h). After the peak current plateauing at the 10× WZ-1 concentration, we increased the concentration of WZ-1 to 100×, which resulted in a broader peak-current distribution and a concurrent decrease in capture rate (Fig. S7), consistent with a concentration-dependent induction of WDR5 aggregation by WZ-1. Notably, this study represents the first successful use of nanopore sensing for in vitro screening of protein oligomerization inducers, highlighting its potential as a platform for drug discovery.

The N-terminal domain and Cys248 residue of WDR5 underlie the specificity of WZ-1.
To determine whether WZ-1 is indeed a WDR5-selective oligomerization-inducing small molecule, we conducted classical biochemical experiments. First, we confirmed the ability of WZ-1 to induce recombinant WDR5 protein oligomerization in vitro using immunoblotting. Interestingly, oligomer formation was observed only when the reducing agent dithiothreitol (DTT) was omitted from the loading buffer (Fig. 3a). Inspired by this clue, we hypothesized that non-reducing conditions are required for WZ-1-induced WDR5 oligomerization. To test this, we introduced the reducing agents DTT (50) and Glutathione (GSH) (51) in our in vitro assays. As shown in Fig. 3b-3d, both immunoblotting and nano sensing assays demonstrated that these reducing agents completely abolished the WZ-1-induced WDR5 oligomerization.

To explore the selectivity of WZ-1 for WDR5, we obtained all 10 commercially available WDR proteins (WDR16, WDR21B, WDR21C, WDR23, WDR24, WDR30, WDR31, WDR40A, WDR77, and WDR86) to assess whether WZ-1 could induce their oligomerization. As shown in Fig. S5b, WZ-1 induced oligomerization exclusively in WDR5, with no detectable oligomerization in any of the other tested WDR proteins. These findings indicate that WZ-1 selectively induces aggregation of WDR5.

Given that WZ-1 contains a disulfide motif, we hypothesized that its disulfide bond might function as a latent covalent warhead, reacting with cysteine residues in WDR5. To test this, we pretreated WDR5 with iodoacetamide (IAM) to block free cysteine residues before incubation with WZ-1 (52) (Fig. 3e). As shown in Fig. 3e, cysteine blocking completely abolished WDR5 oligomerization, supporting the involvement of cysteine residues in the WZ-1-mediated process.

To determine which cysteine residue is involved in WZ-1-triggered oligomerization, we individually mutated each of WDR5’s ten cysteines to serine (CS mutation). Seven single-mutant recombinant WDR5 proteins (C163S, C195S, C205S, C248S, C261S, C309S, and C334S) were successfully purified. Following WZ-1 treatment, most mutants still exhibited oligomer formation. However, several mutations, particularly C205S, modestly reduced oligomerization, whereas the C248S mutant—despite displaying minimal background oligomerization—completely lost the ability to form WZ-1-induced oligomers (Fig. 3f). This result suggests that cys248 is the key residue involved in WZ-1-induced WDR5 oligomerization and other cysteine residues facilitate the progress of oligomerization. Similar results were observed using the SEC-MALS assay. As shown in Fig. 3g, wildtype WDR5 protein appeared mostly as a monomer in solution and WZ-1 resulted in the formation of dimers, while the C248S mutation remained as monomers.

It has been observed that the N-terminal residues of WDR5 can bind to the WIN site of a neighboring molecule in the crystal lattices (38,53), suggesting a potential role in WDR5 oligomerization. Consistently, AlphaFold structure prediction of a WDR5 dimer produced a symmetric homodimer stabilized by WIN-site interactions (Fig. 4a). Notably, the Cys248 residues of each monomer are positioned at the dimer interface, approximately 7.6 Å apart (Fig. 4b), providing a structural rationale for why Cys248 is the preferred site for disulfide bond formation. Supporting this model, SEM-MALS analysis, immunoblotting analysis and Cryo-EM analysis demonstrate that N-terminal tail markedly enhances WZ-1-induced aggregation (Fig. 3g, Fig. S5d and Fig. S8), consistent with the transient interactions mediated by WIN sites and N-terminal tail bring cys248 or other cysteine residues in close proximity for WZ-1 catalyzed reaction. Cryo-EM analysis of WDR5 treated with WZ-1 further demonstrated the coexistence of monomers, dimers, and higher-order oligomers (Fig. 4c).

WZ-1 induces WDR5 oligomerization through a disulfide bond switch mechanism.
Having identified the N-terminal tail and Cys248 as key elements in regulating WDR5 dimerization, we next investigated the binding mechanism between WZ-1 and WDR5. We focused on whether the 2-aminothiazole core and the disulfide motif were essential for WZ-1 to function as an oligomerization inducer (54). To this end, we conducted a Structure-Activity Relationship(SAR) study WZ-1: WZ-2, WZ-3, and WZ-4, in which the 2-aminothiazole core was simplified, and WZ-5 and WZ-6, which lacked the disulfide motif (Fig. 5a). We then evaluated their binding affinity to WDR5 alongside their ability to induce oligomerization. As shown in Fig. 5b and Fig. S9, only symmetric compounds containing the intact 2-aminothiazole core (WZ-1, WZ-4 and WZ-6) exhibited low-micromolar dissociation constants (Kd) compared with WZ-2, WZ-3, and WZ-5, underscoring the importance of the 2-aminothiazole core in WDR5 binding. Interestingly, replacing the disulfide motif in WZ-1 with a hydrazine group (WZ-6) completely abolished oligomerization, even though the compound retained both symmetry and the 2-aminothiazole core. These findings outline a strategy for triggering protein oligomerization that differs from conventional bivalent compound design (Fig. 5c-d and Fig. S5c). Our preliminary structure-activity relationship analysis suggests that the 2-aminothiazole core mediates stable binding to WDR5, while the disulfide motif acts as a “scout” element, initiating WDR5 oligomerization.

Unlike many other covalent warheads, a disulfide bond can be highly dynamic under physiological conditions due to a series of reversible reactions collectively known as thio-disulfide exchanges (55). We therefore hypothesized that the disulfide bond in WZ-1 could exchange with Cys248 of WDR5, initiating the formation of a new intermolecular disulfide bond between WDR5 molecules and thereby promoting oligomerization. We further speculated that WZ-1 might function catalytically-being released after the exchange to trigger subsequent thio-disulfide reactions. To test this hypothesis, we carried out a model reaction between WZ-1 and N-acetylcysteine (NAC). HPLC-HRMS analysis revealed that the thiol group of NAC initially reacted with WZ-1 via a thio-disulfide exchange, generating a disulfide intermediate (A2) and releasing intermediate A1. Intermediate A1 was subsequently trapped by ethyl bromide to form A3. Meanwhile, A2 could undergo a second reaction with NAC, yielding the corresponding NAC dimer (dNAC) (Fig. 5e-f and Fig. S10).

Based on these results, and considering that Cys248 is positioned next to the WBM site while the 2-aminothiazole core of WZ-1 is critical for binding and aggregation, we propose a two-step mechanism for WZ-1-induced WDR5 oligomerization (Fig. 4d). In the first step, the 2-aminothiazole core of WZ-1 binds the WBM site of WDR5, positioning its disulfide bond for reaction with the surface-exposed Cys248. In the second step, the N-terminal tail promotes further proximity between WDR5 molecules, enabling protein-to-protein disulfide exchange between the WZ-1-Cys248 adduct and cysteine residues on adjacent WDR5 molecules, thereby driving oligomer growth. Whether the cysteine residue engaged in this exchange is Cys248 itself or another surface cysteine likely determines whether the reaction terminates or continues, thus controlling the extent of oligomerization.

WZ-1 inhibits cancer cell proliferation by disrupting the interaction between WDR5 and its partner proteins.
Next, we investigated the cellular effects of WZ-1. Using the sulforhodamine B (SRB) assay, we assessed the anti-cancer activity of WZ-1 against four colon cancer cell lines: CT26, RKO, DLD1, and SW620. As shown in Fig. 6a, WZ-1 effectively inhibited proliferation in all four cell lines, with IC50 values ranging from 1.1 to 6.2 μM, proving more potent than the reported WDR5 inhibitor SADS-1 (IC50 > 30 μM). In contrast, WZ-1 did not substantially inhibit proliferation in normal colon cell lines NCM460 and immortalized human colon fibroblasts, indicating a degree of selectivity toward cancer cells (Fig. S11a). Although we didn’t detect endogenous WDR5 oligomerization in CT26 and RKO cells, we observed dimerization of exogenously expressed WDR5 only after treatment with WZ-1 (Fig. 6b), suggesting that WZ-1 can also induce WDR5 oligomerization in cells.

To understand the link between WZ-1-induced WDR5 oligomerization and cell proliferation inhibition, we performed RNA-Seq on CT26 and RKO cells after WZ-1 treatment and conducted GSEA analysis on the significantly changed genes (Fig. 6c). Interestingly, we observed a significant negative enrichment of E2F and G2M checkpoint signaling pathways in WZ-1 treated cells compared to controls (Fig. 6d-e and Fig. S11d-e). Consistent with these findings, we also discovered that WZ-1 caused G0/G1 phase cell cycle arrest (Fig. S11b). These results closely parallel previous findings that WDR5 WIN-site inhibitors cause a decrease in gene expression associated with the E2F and G2M checkpoint signaling pathways (42,56).

Notably, we noticed that WZ-1 treatment also inhibits the expression of Myc target genes (Fig. 6d-e). Given that WDR5 directly interacts with c-Myc via its WBM site (33), and that Cys248 is positioned adjacent to this site (Fig. S11c), these results further suggest that WZ-1-induced WDR5 oligomerization disrupts its full functionality in both the WIN and WBM interactions. To test this hypothesis, we examined whether WZ-1-induced WDR5 oligomerization affects its interaction with MLL1 or c-Myc. As shown in Fig. 6f and 6g, WZ-1 treatment markedly disrupted WDR5 binding to both MLL1 and c-Myc. Collectively, these results suggest that WZ-1 inhibits cancer cell proliferation by impairing both WIN- and WBM-mediated functions of WDR5, thereby compromising its role in these critical oncogenic pathways.

Methods
Reagents
Primary antibodies against WDR5 (db4107), MLL1 (db12766), c-Myc (db1667), HA (db2603), FLAG (db1435), and GARPDH (db106) were purchased from Diagnostic Biosystems (Hangzhou, China). The antibody against ASH2L (#5019) was obtained from Cell Signaling Technology (Boston, MA, USA).

OICR-9429 (HY-16993), DTT (HY-15917), GSH (HY-D0187), IAM (HY-34477), and buthionine sulfoximine (BSO, HY-106376) were obtained from MedChemExpress (MCE, New Jersey, USA). N-acetylcysteine (NAC, A7250) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound SADS-1 was synthesized in Dr. Chen’s laboratory.

Synthesis of compounds
All solvents were purified according to standard methods prior to use. NMR spectra were recorded for 1H NMR at 500 MHz and for 13C NMR at 125 MHz. For 1H NMR, tetramethylsilane (TMS) (δ = 0), DMSO-d6 (δ = 2.500) or CDCl3 (δ = 7.260) served as internal standard and data were reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and coupling constant (s) in Hz. For 13C NMR, TMS (δ = 0), DMSO-d6 (δ = 39.52) or CDCl3 (δ = 77.16) was used as internal standard and spectra were obtained with complete proton decoupling. LC-MS of all final products were confirmed on an Agilent 1290 HPLC-6224 Time of Fight Mass Spectrometer using PhenomenexLuna 5μ C18, 100 Å, 150 × 4.60 mm 5-micron column at a flow rate of 0.5 mL/min using liner gradients buffer B in A (B: CH3OH containing 0.1 % formic acid, A: H2O containing 0.1% formic acid). Mobile phase B was increased linearly from 5% to 95% over 7 min and 95% over the next 2 min, after which the column was equilibrated to 5% for 1 min. The optical rotation was conducted at 25 °C using JASCO P-1030/1010 polarimeter, with a wavelength of 589 nm. The WZ-2 was commercially obtained.

5,5'-disulfanediylbis(4-(4-chlorophenyl) thiazol-2-amine) (WZ-1)
4-(4-chlorophenyl)-5-thiocyanatothiazol-2-amine (200 mg, 0.75 mmol, 1.0 equiv) and glutathione (300 mg, 0.98 mmol, 1.3 equiv) were dissolved in a mixture of DMF, MeCN and distilled water (V/V/V = 4/4/1). Then the reaction mixture was stirred at room temperature. Upon the completion of the reaction via TLC (PET: EA, 1/1), the mixture was concentrated in vacuo and diluted with H2O (3 mL). The resulting precipitate was filtered and purified via silica gel chromatography to give the yellow compound. (227 mg, 63 %) 1H NMR (500 MHz, DMSO-d6) δ 7.63 (s, 4H), 7.59 (d, J = 8.5 Hz, 4H), 7.34 (d, J = 8.5 Hz, 4H). 13C NMR (125 MHz, DMSO-d6) δ 171.0, 157.0, 132.8, 132.3, 130.3, 127.7, 107.4. HRMS (ESI): m/z calcd for C18H13Cl2N4S4 [M+H]+: 482.9395, found: 482.9394.

2,2'-disulfanediyldianiline (WZ-3)
2-Mercaptoaniline (250 mg) was added to a 25 mL round-bottom flask, followed by the addition of 5 mL of anhydrous ethanol to dissolve the compound. Subsequently, 508 mg of iodine was introduced, and the mixture was stirred at room temperature. After an overnight reaction, the solution turned colorless, and TLC indicated the complete disappearance of the starting material. Then, 100 mL of EA was added, and the mixture was washed three times with 50 mL of saturated saline solution. The organic phase was collected and dried over anhydrous sodium sulfate. The residue was purified via silica gel chromatography to yield a white solid (190 mg, 76%). 1H NMR (500 MHz, DMSO-d6) δ 7.08 (ddd, J = 8.0, 7.1, 1.5 Hz, 2H), 7.00 (dd, J = 7.5, 1.5 Hz, 2H), 6.73 (dd, J = 8.0, 1.5 Hz, 2H), 6.43 (td, J = 7.5, 1.5 Hz, 2H), 5.45 (s, 4H). HRMS (ESI) m/z calcd for C12H13N2S2 [M+H]+: 249.0515. Found: 249.0510.

5,5'-disulfanediylbis(4-methylthiazol-2-amine) (WZ-4)
4-methyl-5-thiocyanatothiazol-2-amine (68 mg, 0.4 mmol, 1.0 equiv) and glutathione (152 mg, 0.52 mmol, 1.3 equiv) were dissolved in a mixture of DMF, MeCN and distilled water (V/V/V = 4/4/1). Then the reaction mixture was stirred at room temperature. Upon the completion of the reaction via TLC (PET: EA, 1/1), the mixture was concentrated in vacuo and diluted with H2O (3 mL). The resulting precipitate was filtered and purified via silica gel chromatography to give the white compound (69 mg, 60 %). 1H NMR (500 MHz, DMSO-d6) δ 7.70 (s, 4H), 2.23 (s, 6H). HRMS (ESI) m/z calcd for C8H11N4S4 [M+H]+: 290.9861. Found: 290.9862.

4-(4-chlorophenyl)-5-(methylthio) thiazol-2-amine (WZ-5)
To a solution of 4'-chloroacetophenone (6.1 g, 39.5 mmol) in a mixture of 48% HBr (20 mL) and dimethylsulfoxide (20 mL) in a sealed flask. This mixture was heated at 60 °C for 12 h and then cooled. After the addition of isopropanol (15 mL) and ethyl ether (15 mL), the solution was stirred for another 0.5 h and allowed to stand overnight in the ice box. The precipitate was filtered and washed with ethyl ether to afford the desired sulfonium salt as white crystals. To a solution of the crude product (3.7 g, 10 mmol) in ethyl alcohol (20 mL), thiourea (912 mg, 12 mmol, 1.2 equiv) was added at room temperature and then refluxed for 2 hours. The precipitated product was collected by filtration, washed with water (3 x 10 mL) and dichloromethane (3 x 10 mL). The resulting residue was purified via silica gel chromatography (PET/EA = 10:1) to afford a yellow solid (1.8 g, 75% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.91-7.88 (m, 2H), 7.48-7.44 (m, 2H), 7.33 (s, 2H), 2.32 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.1, 150.8, 133.3, 132.2, 130.0, 128.0, 111.0, 21.7. HRMS (ESI) m/z: [M + H]+ calcd for C10H10ClN2S2 :256.9968; found: 256.9964.

5,5'-(hydrazine-1,2-diyl)bis(4-(4-chlorophenyl)thiazol-2-amine) (WZ-6)
287 mg of 2-amino-4-(4-chlorophenyl)-5-bromothiazole was added to a 50 mL sealed tube, followed by the addition of 5 mL acetonitrile and 5 mL hydrazine hydrate. The mixture was heated to 80 °C and allowed to react overnight. Afterward, 50 mL water was added, and the mixture was extracted three times with 50 mL ethyl acetate. The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, and concentrated. The residue was purified via silica gel chromatography to yield a white solid (85 mg, 30%). 1H NMR (500 MHz, DMSO-d6) δ 7.87-7.86 (m, 2H), 7.84-7.80 (m, 2H), 7.47-7.43 (m, 2H), 7.28-7.23 (m, 2H), 7.17 (s, 2H), 5.14 (s, 2H). HRMS (ESI) m/z calcd for C18H15Cl2N6S2 [M+H]+: 449.0171. Found: 449.0169.

Expression and purification of WDR5
WDR5 and its corresponding mutants were cloned into the pET-28a vector (#69864-3, Addgene) to produce an N-terminal 6×His-tagged fusion protein and expressed in Escherichia coli Rosetta 2 (DE3) cells (the primers are listed in Table. S2). A single transformed colony was inoculated into LB medium containing kanamycin and grown at 37 °C, 220 rpm for 8 h. The culture was expanded in fresh LB medium with kanamycin and grown for an additional 12 h. This secondary culture was then inoculated at a 1:100 ratio into 400 mL LB medium containing kanamycin and grown at 37 °C, 220 rpm until the optical density at 600 nm (OD600) reached 0.4–0.6. Protein expression was induced by adding 500 µM isopropyl β-D-thiogalactopyranoside (IPTG) and continuing incubation under the same conditions.

Cells were harvested by centrifugation at 4,000 × g for 5 min at 4 °C, resuspended in 80–100 mL lysis buffer (20 mM HEPES, 300 mM NaCl, 10% glycerol, 0.1 mM PMSF, pH 7.5), and passed through a 70-mesh strainer for dispersion. Cell disruption was performed using a pre-chilled high-pressure homogenizer at 750 bar. The lysate was clarified by centrifugation at 8,000 × g for 30 min at 4 °C, followed by filtration through a 0.45 µm membrane.

The supernatant was loaded onto a Ni-NTA affinity column (AKTA Purifier system) pre-equilibrated with lysis buffer. Bound protein was eluted with a gradient of increasing imidazole concentration, and protein elution was monitored at 280 nm. Fractions containing WDR5 were pooled, aliquoted into 1.5 mL tubes, and further purified by size-exclusion chromatography (SEC) on a GE column using buffer containing 20 mM HEPES, 200 mM NaCl, and 0.3 mM TCEP, pH 7.5.

Plasmid construction and lentivirus transduction
WDR5 and c-Myc were subcloned into pCDH-CMV-MCS-EF1-Puro plasmids (#CD510B-1, System Biosciences) with an indicated tag for transient expression using the primers listed in Table. S2). Lentivirus was produced by HEK293 cells with pCMV-dR8.91 (packaging vector, P0259, miaolingbio.Inc), pMD2.G-VSVG (envelope vector, #12259, Addgene), and targeted plasmids co-transfected using Lipofectamine 2000 (#11668019, Invitrogen). Medium containing virus was harvested 48 h after transfection and filtered through a 0.45-μm Millipore filter. For infection, cells were grown to 20-30% confluency in six-well plate, and 1 mL of each virus was added with 2 μL polybrene (6 mg/mL). After the cells were infected for 12-16 h, the medium was changed for fresh medium.

MicroScale Thermophoresis (MST) assay
Recombinant His-tagged WDR5 protein and fluorescently labeled RED-tris-NTA dye (NanoTemper Technologies, MO-L018) were diluted to final concentrations of 800 nM and 100 nM, respectively, in 1× PBS-T buffer. A total of 90 µL of diluted WDR5 protein was mixed with 90 µL of RED-tris-NTA dye and incubated at room temperature in the dark for 30 min to allow labeling.

Small-molecule compounds were prepared as a 50 µM stock solution in 1× PBS-T buffer containing 10% DMSO. Sixteen consecutive two-fold serial dilutions of the compound were prepared in the same buffer, with a final volume of 10 µL per dilution. For interaction measurements, 10 µL of labeled WDR5 protein (400 nM in 1× PBS-T buffer) was added to each 10 µL aliquot of serially diluted compound.

The resulting mixtures were loaded into MST capillaries (NanoTemper Technologies, MOK022) and analyzed using a Monolith NT.115 instrument controlled by MO.Control software (NanoTemper Technologies). Binding affinities (Kd) were determined using MO.Affinity Analysis software.

Nanopore fabrication
All the nanopores were fabricated using a laser-assisted pipette puller (Sutter Instrument, P-2000, USA) by pulling quartz capillaries (Sutter Instrument, QF100-50-7.5). One capillary was then positioned onto the holder of the puller by aligning and securing it in the groove. The capillary was subsequently pulled to generate two similar nanopipettes using an optimised two-line pulling parameter: (1) HEAT = 775, FIL = 4, VEL = 30, DEL = 170, PUL = 80; (2) HEAT = 805, FIL = 3, VEL = 20, DEL = 145, PUL = 180. It is worth noting that the pore geometry is significantly influenced by the instrument’s state and ambient conditions, including laser intensity, temperature and humidity. Under our experimental conditions (20°C and 34% humidity), the average size of our nanopores with this protocol is 22.2 ± 5.0 nm (n=5) unless stated otherwise, based on the SEM estimation.

Conductance measurement of nanopores
Before each experiment, nanopore conductance measurement was conducted in a solution of 1 × PBS and 1 M KCl (pH = 8.6~9.4) by measuring the current ramping from −400 to +400 mV at a rate of 50 mV. Subsequently, the current–voltage plots were linearly fitted, and the conductance was estimated by subtracted the slope of the line to ensure that the consistency of nanopore sizes used in each experiment.

Translocation experiments and drug screening
All translocation experiments through nanopores were conducted in a solution of 1 × PBS and 1 M KCl (pH = 8) at room temperature. A nanopipette filled with electrolyte (≈ 10 µl) was immersed into the aforementioned solution (≈ 200 µl). Two freshly prepared Ag/AgCl electrodes were inserted into the nanopipette and the bath, respectively, serving as working and reference electrodes. Measurements were performed using an Axopatch 700B patch clamp amplifier (Molecular Devices, USA). The signal was digitized with a Digidata 1440B at a sampling rate of 250 kHz. For protein measurement experiments, WDR5 proteins at a final concentration of 500 nM were added to the bath. A bias, typically 250 mV in this study unless stated otherwise, was applied to drive the translocation of WDR5 in an outside-to-inside direction. Peak currents of pure WDR5 were detected and recorded during each translocation experiment, ensuring consistency in nanopore geometries by using freshly pulled nanopores. For oligomerization inducer detection from our in-house compounds library, molecular mixture at selected concentrations (single molecule: protein= 2.5 : 1 or 10 : 1) were added to the bath and incubated for half an hour. After incubation, all relevant peaks were detected and recorded for subsequent data analysis.

SEC-MALS Analysis
Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) were performed using a Superdex 200 increase column (Cytiva) combined with multi-angle laser light scattering using a Wyatt HELEOS-II 18-angle photometer coupled to a Wyatt Optilab rEX differential refractometer (Wyatt Technology Corp). Experiments were conducted at room temperature using tag-free WDR5 wild-type, C248S mutant, and N-terminal truncation mutant proteins at a concentration of 1.0 mg/mL (≈27 µM), and a flow rate of 0.5 mL/minute in 20 mM Hepes pH 7.5, 200 mM NaCl. Before analysis, the proteins were pre-incubated with 200 µM WZ-1 or DMSO control for 10 minutes and then centrifugated at 4℃, 12,000rpm for 2 minutes. The data were analyzed using the ASTRA 6.1 software (Wyatt) and GraphPad Prism 8.0. The molecular mass was determined across the protein elution peak(66).

Nano-sensing data acquisition and analysis
A custom-written program in MATLAB (R2022b) was employed to analyze the translocation events of WDR5. In summary, the following steps were performed: (1) The I-t signal containing the peak segments was exported using the Clamfit and loaded by MATLAB for subsequent analysis. (2) The trace was filtered using a 10 MHz low-pass filter and subtracted to compensate for any baseline fluctuations. (3) Peak signals were distinguished from background noise using a threshold method , allowing for the identification of the start and end points of each peak. Peaks amplitude >45 pA; dwell time >10 μs were selected and the current values within the pulse plateau were averaged to obtain mean values as peak currents(67). (4) Each peak was idealized to extract relevant features. Dwell time is calculated from the width of the peak, and Peak current is determined by the average points within the signal of each peak, respectively. (5) Finally, peak statistics are then extracted (e.g., number of peaks, Dwell time, Peak current)

Defining the rate of change
All Peak currents and Dwell times from a single experiment were Gaussianized and averaged. The change in the mean value of the WDR5 current signal before and after each addition of compound mixtures was compared, and the rate of change was defined as follows:
Rate of change = (Mean value of Peak current in experimental group - Mean value of Peak current in control group) / Mean value of Peak current in control group.
A change of more than 50 percent was considered positive. The Gaussian fitting and figure plotting were performed using Originlab (2023) and Excel (Microsoft Office 365), and the results were further imported into Adobe Illustrator (Adobe CC) for visualization.

Cell culture
The CT26, RKO, DLD1, and SW620, 293T, NCM460 cell lines were purchased from Chinese Academy of Sciences National Collection of Authenticated Cell Cultures (Shanghai, China). Immortalized human colon fibroblasts were obtained from Hunan Fenghui Biotechnology Co., Ltd (Changsha, China). All cell lines were examined by STR profiling and were monitored for mycoplasma contamination semiannually. CT26, DLD1 and NCM460 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Cytiva, Cat#SH30396.03). RKO cells were cultured in Eagle's minimum essential medium supplemented with 10% FBS. SW620 cells were cultured in L-15 media supplemented with 10% FBS. 293T cells were cultured in DMEM medium supplemented with 10% FBS. Immortalized human colon fibroblasts were cultured with special culture medium (Fenghui, FH1118-21). All cell lines were cultured in a humidified 5% CO2 incubator at 37℃.

Co-immunoprecipitation
Twenty-four hours after transfection with pCDH or tagged target plasmids, the culture medium was replaced with Opti-MEM supplemented with 100 µM BSO and incubated for 8 h. Cells were then treated with WZ-1 for an additional 3 h, harvested, and lysed in ice-cold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, pH 7.4, 0.1 mM sodium orthovanadate, 5 µg/mL leupeptin, and 0.1 mM PMSF) at 4 °C for 30 min. Lysates were clarified by centrifugation at 15,000 × g for 30 min at 4 °C. An aliquot of the supernatant was mixed with 6 × loading buffer, heated at 95 °C for 30 min, and used as the input sample. For immunoprecipitation, FLAG beads (Smart-Lifesciences, Cat# SA042005) were washed twice with 1% NP-40 buffer (25 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 1% NP-40, pH 7.4) before incubation with the remaining supernatant in a 1.5 mL microcentrifuge tube overnight at 4 °C on a 360° rotator. After incubation, beads were pelleted by centrifugation and washed 3–5 times with 0.2% NP-40 buffer (25 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 0.2% NP-40, pH 7.4). The final bead pellet was resuspended in 2 × loading buffer, heated at 95 °C for 30 min, and subjected to downstream analysis.

Western blot analysis
An equal amount of protein samples (20–40 µg) was pipetted and loaded into 10% Tris-glycine gels filled with Tris-glycine SDS running buffer. After the proteins were resolved, they were transferred onto methanol-activated PVDF membrane using a Trans-Blot Electrophoretic Transfer Cell. Upon completion of membrane transfer, the membrane was blocked with the blocking buffer (1 × TBS-T containing 5% skim milk) and incubated for 30 min at room temperature on a horizontal shaker. Subsequently, the membranes were washed three times for 5 minutes each with 1 × Tris-buffered saline Tween 20 (TBS-T) for subsequent blue staining or immunoblotting. The solution containing primary antibodies was then added and placed on a horizontal shaker at 4°C for overnight incubation. Following this, the PVDF membranes were washed with 1 × TBST on a shaking shaker, repeating the wash three times for durations of 15 minutes, 5 minutes, and 5 minutes, respectively. Next, the membranes were incubated in the solution containing HRP-labeled secondary antibodies corresponding to the species of the primary antibodies. This solution was diluted to 1:5,000 in 1 × TBS-T buffer containing 5% skimmed milk and incubated on a horizontal shaker at room temperature for 1 hour. After the completion of the incubation period, the membranes were washed three times with 1 × TBS-T. Finally, the PVDF membrane was uniformly covered with an ECL exposure solution and placed into the AI680 imager for exposure and imaging. For all Western blot experiments mentioned, each experiment was independently repeated at least twice with similar results.

SRB assay
All cells were cultured in Opti-MEM medium supplemented with 100 µM BSO and maintained in a humidified incubator at 37 °C with 5% CO₂. Cell lines were tested for mycoplasma contamination every three months. For the SRB assay, cells seeded in 96-well plates were fixed with 10% (w/v) trichloroacetic acid at 4 °C for 2 h. The cells were then rinsed thoroughly with deionized water and dried at 65 °C. Next, cells were stained with SRB solution at room temperature for 25 min, washed with 1% glacial acetic acid to remove unbound dye, and dried again at 65 °C. Finally, bound dye was solubilized in 10 mM Tris-base, and absorbance was measured at 515 nm using a microplate reader.

RNA seq analysis
After treatment with WZ-1 for 12 h in Opti-MEM medium, RKO and CT26 cells were harvested and immediately subjected to rapid freezing in liquid nitrogen before storage at -80°C. Subsequently, the samples were sent to LC-Bio Technologies (China, Hangzhou) Co for detection and analysis.

Cell cycle analysis
Cell cycle distribution analysis was conducted using the Cell Cycle Analysis Kit (Beyotime Biotechnology, cat#C1052). After seeding cells in 6-well plates, they were treated with indicated compounds. The cells were then harvested and fixed in 70% ethanol at 4℃ overnight. Then cells were washed twice with PBS and incubated in the dark with propidium iodide (PI) and RNase A for 30 min at 37℃. Finally, the cell cycle phases were analyzed using a flow cytometer (BD FACscanta™ II).

Cryo-EM data acquisition and analysis
Initially, 1 mg/mL tag-free WDR5 was incubated with 200 µM WZ-1 on ice for 30 min before vitrification. Aliquotes of 3 µl of the reaction mixture were applied to glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3). The grids were blotted for 3.5 s and flash-frozen in liquid ethane with Vitrobot (Mark IV, Thermo Fisher Scientific). The prepared grids were transferred to a Titan Krios operating at 300 kV equipped with a Falcon 4 detector. Movie stacks were automatically collected using EPU software (Thermo Fisher Scientific) with a defocus range from −0.8 to −1.6 µm at a nominal magnification of × 130,000, yielding a pixel size of 0.93 Å on images. Movies were recorded with a 3.42 s exposure to give each stack a total dose of 52 e/Å2. A total of 929 movie stacks were imported and processed with cryoSPARC version 4.3.1. 1,150,237 particles were auto picked with the Blob picker program and subjected to 2 rounds of 2D classification to remove non-protein particles. Later, four additional datasets were collected with a protein concentration of 0.1 mg/mL to compare the oligomerization states of WT WDR5 (561 movies), WT WDR5 with WZ-1 (588 movies), WDR5 C248S with WZ-1 (667 movies) and WDR5 31-334 with WZ-1 (537 movies). All datasets were subjected to the same data processing protocol. After 2 rounds of 2D classification, the number of particles representing monomer, dimer and oligomer WDR5 classes were used for analysis.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0 software. The number of biological replicates for each experiment is indicated in the figure legends. Differences between means were determined using one-way analysis of variance (ANOVA) and were considered significant at P < 0.05.

Chemical probe consideration
WZ-1 was evaluated against community guidelines for chemical probes68 and shows reproducible, target-relevant cellular activity consistent with WDR5 biology. As it does not yet meet all probe validation criteria, including extensive proteome-wide selectivity profiling, WZ-1 is used here as a target-directed small-molecule research tool.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_2": "The “Catalytic Proximal Proteins Oligomerization (CaPPO) as a Novel anti-Tumor Strategy Targeting WDR5” by Fang Et al. is an intriguing work focused on identifying a small molecule capable of inducing WDR5 aggregation. Initial testing utilizes a quartz solid-state nanopore to conduct three rounds of screening of a 436-compound library from their lab. These single-molecule studies concluded that WZ-1 was most effective at promoting oligomerization. The authors have conducted bulk studies to identify that WZ-1 selectively targets WDR5 and not other common WD repeat proteins. They also deduce that WZ-1 utilizes C248 to bridge dimers via disulfate bonds. This work does a nice job of studying the influence WZ-1 has on WDR5 aggregation and function at various scales, from single molecules to cellular. Despite these strengths, the work lacks a direct mechanistic demonstration that the WDR5 oligomerization and aggregation observed in a cell-free environment are therapeutically necessary. Furthermore, the rationale and justification for WDR5 oligomerization screenings are not supported by prior studies, specifically those involving WDR5. Aside from these concerns, as mentioned by the authors, the N-terminal tail is likely to catalyze the WDR5 oligomerization and aggregation. Therefore, additional work is needed to disentangle the contributions of the N-terminal disordered tail and WZ-1 response. In the absence of these tests, the current outcomes lead to many questions that can weaken the solidity of the claims made. Additionally, a more in-depth discussion of the results is needed. Please see my comments for more specific details.\nAdditional comments:\n1. In the introduction, the authors completely ignore the role of biological nanopores in this field. Biological nanopores are also more cost-effective and can enable high-throughput drug screening. Specifically, they have also already been used to study both the WIN and WBM sites of WDR5. These citations should be added to the introduction of nanopore sensing in the context of WDR5.\na. Mayse, L. A., A. Imran, et al. (2022). \"Disentangling the recognition complexity of a protein hub using a nanopore.\" Nat Commun 13(1): 022-28465.\nb. Mayse, L. A., Y. Wang, et al. (2024). \"Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision.\" ACS Nano 18(31): 20468-20481.\n2. There is an unnecessary “)” on line 107 after the reference.\n3. In Figure 1b, there are three different current peaks shown for the WDR5 monomer. Is there a reason monomeric WDR5 doesn’t yield a uniform blockage of the nanopore? WDR5 is a structural ridge protein that the 22 nM nanopore diameter should not heavily constrain. Not to mention that the amplitude histogram supporting the numerical claim of the average current blockade is missing.\n4. In most traces, WZ-1 treatment also reduces the number of events, and as shown in Figure S8, after 10 minutes of treatment, no translocation events occur. This indicates that WZ-1 triggers the formation of huge aggregates that are no longer able to enter the nanopore. However, the authors do not discuss what causes this reduction in the number of events. They only state that they expect the results in Figure S8 to be based on previously published work from another group. This discussion is critical and would strengthen the conclusion that WZ-1 promotes significant WDR5 aggregation. Additionally, TEM images can show the size of these oligomers, supporting this discussion.\n5. There is a grammatical error in line 137, “ecorded” should be “recorded”.\n6. In Figure 2a, I expect the number of events to decrease as oligomerization will reduce the effective concentration in the bath. If this is a time-dependent condition, the authors should include this information in the text or figure legend. Why does the number of events stay the same?\n7. In Figure 2c, the standard deviations for the controls vs WZ-1 peak current overlap for all rounds.\n8. In Figure 3e, it appears that in the presence of DMSO, dimers of IAM start to form. The authors need to discuss this. I assume this is because dimerization is happening between the N-terminal region and the WIN site of monomers. This dimerization occurs independently of WZ-1.\n9. It is known that dimerization occurs between monomers when the N-terminal regions of a neighboring WDR5 interact with the WIN site of another (see the authors’ citations). The authors mention this, but it is unclear whether they are proposing that those interactions occur first, and then WZ-1 stabilizes that formation. Or if the WZ-1 brings two monomers together via C248.\nThe testing of a truncation WDR5 mutant that lacks the N-terminal tail will make it very clear if WZ-1 itself can initiate dimerization or if the Win site-mediated dimerization needs to be present for this response to be observed (see my comments above).\n10. It is unclear what mechanism would enable WZ-1 to impact the Win site itself. If these dimers were primarily occurring by the N-terminal tail interacting with the WIN site, then that is why the MLL1 interaction is being influenced. The Win site is being occupied by the neighboring monomer, which is being held in proximity by the thiol-sulfide bonds created by WZ-1. Again, this conclusion requires that the truncation of the WDR5 mutant lacking the N-terminal tail be identified to determine whether WZ-1 itself influences the WIN site binders.\n11. I would like to know if the authors removed the histidine tag if the results remain the same.\n12. In nano-sensing data acquisition and analysis, there is no reference to similar data analysis, and a precise threshold definition is lacking. Without this information, it's very challenging to understand how they identified peaks versus noise. This threshold must be clearly defined, as the current presentation is not convincing.\n13. In Figure S2, it is labeled as 34 nanopipettes, and it should be 34 nanopores.\n14. The figure legend in Figure S3 is cut off and incomplete.\n15. In Figure S4, it would be far more informative to include the histograms from mixture 1, 2, and WZ-1.\n16. Figure S8: “Typical Current-time,” there is unnecessary capitalization of letters in this sentence.\n17. Figure S7abc, Figure S9 are unreadable. All fonts and legends are way too small. Please look and rectify other figures with the same characteristics.", "reviewer_3": "This article is a collaborative work across multiple groups that takes an initial hit molecule through validation and subsequent cancer cell line testing, including a degree of SAR work. A key part of this is the initial screening technology, which employs nanopore-based detection to differentiate molecules capable of inducing WDR5 oligomerisation from those that cannot. Multiple molecules are tested in a ‘cocktail’ approach in a single experiment and molecule groups are successively narrowed down until the active molecule is identified. The authors validate a covalent binding/oligomerization mechanism and explore how this mechanism disrupts binding of WDR5 with its partners c-myc and MLL1. The authors argue that this mechanism is responsible for the cell death of cancer lines reliant on this pathway.\nThis is a very well presented work, combining an advanced detection approach with a novel method of disrupting cancer cell growth through induced protein oligomerization. The authors have to address the following points (including additional experiments):\nMajor\n1. Molecules such as these could plausibly form micelles or precipitate, especially at high ionic strength. Please show data in the nanopore with WZ-1 alone under the same conditions to remove any possibility that the aggregate signal arises from this.\n2. There is a discrepancy between Figure 3g and Figure 3f. Figure 3g shows no dimer formation at all in SEC for C248S in the absence of WZ-1, whereas the Coomassie stain in Figure 3f shows a strong dimer band. What is the cause of this? It’s peculiar in the Coomassie stain that the dimer band is then gone in the presence of WZ-1, given this molecule otherwise enhances oligomerisation.\n3. C261S actually seems to enhance WZ-1 activity given the complete depletion of the monomer band in Figure 3f, do the authors have an explanation for this?\n4. The behaviour of C205S is as expected for a key residue that disrupts WZ-1 induced oligomerization, showing essentially the same behaviour between the negative control and WZ-1 lanes in Figure 3f, while C248S shows the odd behaviour mentioned above. For that reason it seems an overreach to pin the mechanism initiation on C248S and not C205S.\n5. Figure 5 states dissociation constants for the various WZ molecules in binding to WDR5, but I don’t see any representations of the actual data supporting these values. What method was used? Microscale thermophoresis is mentioned in the main text, and the reader is directed to figure S3, but this contains thermal shift data, not MST data, for only the control drugs rather than the WZ derivatives. Where is the WZ data for binding to WDR5? Furthermore, how did the authors obtain Kds given that the mechanism of binding for these molecules is largely mediated via a disulphide, unless DTT was incorporated in the buffer to allow reversible binding without reaction?\n6. Do the authors have a good sense for why WZ-1 is specific to WDR5 and does not impact any other homologues as shown in Figure S6? The molecular scaffold is an interesting one but does not present obvious mechanisms of specificity.\n7. This point would be expanding the scope and so is optional, but the impact of the work would be emphasized by demonstrating some kind of control cell work that shows these molecules are able to impact cancer cell growth to a greater extent than normal cell growth. For instance these cell lines are presumably heavily reliant on c-myc, and so one would expect normal cell lines to be less affected by the activity of WZ-1. This would then fit in with the ‘synthetic lethality’ paradigm that is currently dominating drug discovery in oncology.\nMinor\n1. It would be good to clearly explain the 1st-#9 labelling used\n2. Figure S5 has a solid line underneath it that should be removed\n3. There is a mismatch between figure S7 and its caption\n4. Supply figure s7 at higher res, can’t confirm the mass spec\n5. Supply figure s8 at higher res, can’t confirm nmr shifts\n6. There are a number of instances of incorrect grammar in the main text and elsewhere e.g. Xiaoyang Dai for his assistant on HPLC, it would be good to correct these to ensure the authors’ work is viewed purely on the merit of the science presented.", "reviewer_4": "This work by Fang, Jiang and co-workers describes a new method to screen compounds with the ability to induce selective oligomerization of the protein of interest based in the use of nanopores. By using quartz nanopores and measuring changes in its ionic current, the authors tested a library of compounds for the oligomerization of WD repeat domain 5 (WDR5), a protein involved in epigenetic regulation and oncogenesis, where participates in the MLL/SET complex and directs c-Myc to chromatin at tumor-associated genes.\nThe authors successfully find one molecule, WZ-1, that induces formation of dimers and higher oligomers of WDR5, and their data suggest that WZ-1 promotes disulfide bond exchanges in WDR5 inducing its association. This WZ-1 compound was also tested in cells and it inhibits cancer cell proliferation by interfering in the interaction of WDR5 with MLL1 or c-Myc, and resulting in reduced expression of c-Myc target genes or others involved in E2F and G2M checkpoint signalling pathways.\nThe approach is innovative and the results with WDR5 are promising. The results are clearly presented, and the manuscript is well written.\nA.-I just have a concern regarding the cryoEM results depicted in Figure 4C.\nIn the work about the effect of BI-3802 on the aggregation of B-cell lymphoma 6 (BCL6) protein (Ref #12), the cryoEM of BCL6 oligomers made a lot of sense since the protein constructed helical structures and the authors presented a 3.7 Å resolution cryoEM map. The cryoEM results with WDR5 in the presence of WZ-1 are not so successful. It is understandable, since the associations of WDR5 are not regular and are difficult to average (even at 2D). Thus, the cryoEM analysis is of limited value and only clusters of nearby WDR5 copies are seen. My concern relates to the crowding of electron micrographs (or movies), where WDR5 particles can be in contact without an actual oligomerization event.\nLines 756-757:\n“A total of 929 movie stacks were imported and processed with cryoSPARC version 4.3.1. 1,150,237 particles were auto picked …”\nThis means that are more than 1,200 particles per movie initially, and this is a very high concentration. It is not clear to me how can the authors discriminate between real dimers or larger oligomers and spurious groups of particles that are together in such crowded area. If the author would have shown high quality 2D averages, the real association could have been inferred, but this is not the case.\nI would recommend the use of negative stained samples with lower concentration, and by comparing WDR5 alone and WDR5+WZ-1 the distribution of particles in the filed of view needs to be really different.\nIn any case, the authors have shown the oligomerization effect by WZ-1 on WDR5 by other means (gel electrophoresis and SEC-MALS), so this is not under discussion, but the cryoEM data presented does not add very much.\nOther minor points:\ni.-The authors have clearly stablished that Cys248 preferentially participates in the disulfate bond formation. Their model postulates a C248-C248 bridge between WDR5 subunits. Other possibilities might be considered, for instance a C248-C121 bridge, since the C121 mutant was not obtained, and such an association would just require a 180 degrees flip of one of the WDR5 subunits. Just a suggestion about an interaction that cannot be ruled out.\nBTW, on lines 214-215\n“To determine which cysteine residue is involved in WZ-1-triggered oligomerization, we individually mutated the 11 cysteine residues of WDR5 to serine”\nI have checked the sequence of WDR5 several times and I have seen just 10 cysteine residues. Is this an isoform or is there a Cys in the tag?\nii.-Line 295. First time that “SRB assay” is introduced. Please, spell it out.\niii.-Line 168. Move “fascilitated” to “facilitated”.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_2": "Overall, this revision is a significant improvement; the authors have addressed most of my previous concerns and added a reasonable amount of new data. This new data is critical for the conclusions made (e.g., WDR5 NTD). The authors need to add more data and explanation about the agglomerates now discussed in the revision. Here, the primary concern is that this surface buildup on the nanopore would introduce oligomeric detection that derives from aggregates dissociating. Once the agglomerate discussion has been addressed, this manuscript is ready for publication. Please see the comments below.\nComment 3: Adding the histogram in Fig. S2 and additional text with reference was critical.\nComment 4: The discussion of agglomerates raises a few concerns. In Fig. S13, I assume the “untreated” or “control” SEM images are of the nanopore without WDR5. Without showing the nanopore with WDR5 alone, I question if these agglomerates form from WDR5 itself. Also, could these agglomerates be dissociating and entering the pore, representing oligomer formation? If this is true, can this event be separated from true oligomerization that is happening freely in solution? I understand that the authors have addressed my original question, but now that these agglomerates have been shown, there is a need for a more in-depth inquiry into how they can affect the key findings of this paper.\nComment 9: I applaud the authors for adding the WDR5 NTD experiments. These results show that the dominant factor in oligomerization is the interaction of the N-terminus with the Win site, as Fig. S8a shows that no oligomers form with that mutant.\nNew notes: Please add the full name of “SAR” study\nFig. S9 should have a figure legend.", "reviewer_3": "The authors provided a revised manuscript However the new figures and data contain issues.\nFigure S4 C right histogram the x-axis is wrong (50-10-15...)\nFigure S6 the right hand plot probably means 5 \\muM molar not micrometers!\nFigure S9 the axis labels are barely readable and need to be increased in font size - also the colours for the legend in WZ-2 do not match with the colours in the main plot. This also needs to be corrected. The authors also give two (WZ-1, WZ-3, WZ-6) or three (WZ-2, WZ-5) Kd values for the compounds. Why? There is no explanation of the different balues that can differ by a factor of three!\nThe authors need to provide a more in depth explanation of the supplementary data figures, make sure that the data is accurate and the labels are correct. In the current form the manuscript and supplement should not be accepted.", "reviewer_4": "The authors have properly addressed my concern regarding some cryoEM data presented in the original manuscript with new experiments at lower concentration of the protein. In my opinion the work is now ready for publication.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response letter\n(NCOMMS-25-26811-T)\nWe sincerely thank all five reviewers for their insightful comments and\nconstructive suggestions. We have carefully addressed each point in the\nrevised manuscript.\nA detailed point-by-point response is provided below.\nCOMMENTS TO AUTHOR:\nReviewer #1 & #2:\nThe “Catalytic Proximal Proteins Oligomerization (CaPPO) as a Novel anti-\nTumor Strategy Targeting WDR5” by Fang et al. is an intriguing work focused\non identifying a small molecule capable of inducing WDR5 aggregation. Initial\ntesting utilizes a quartz solid-state nanopore to conduct three rounds of\nscreening of a 436-compound library from their lab. These single-molecule\nstudies concluded that WZ-1 was most effective at promoting oligomerization.\nThe authors have conducted bulk studies to identify that WZ-1 selectively\ntargets WDR5 and not other common WD repeat proteins. They also deduce\nthat WZ-1 utilizes C248 to bridge dimers via disulfate bonds. This work does a\nnice job of studying the influence WZ-1 has on WDR5 aggregation and function\nat various scales, from single molecules to cellular. Despite these strengths, the\nwork lacks a direct mechanistic demonstration that the WDR5 oligomerization\nand aggregation observed in a cell-free environment are therapeutically\nnecessary. Furthermore, the rationale and justification for WDR5\noligomerization screenings are not supported by prior studies, specifically those\ninvolving WDR5. Aside from these concerns, as mentioned by the authors, the\nN-terminal tail is likely to catalyze the WDR5 oligomerization and aggregation.\nTherefore, additional work is needed to disentangle the contributions of the N-\nterminal disordered tail and WZ-1 response. In the absence of these tests, the\ncurrent outcomes lead to many questions that can weaken the solidity of the\nclaims made. Additionally, a more in-depth discussion of the results is needed.\nPlease see my comments for more specific details.\nResponse: We want to thank the reviewer for the positive feedback. We also\nappreciate that the reviewer raised several specific issues regarding the\nstandard of nanopore sensing, which is very helpful to improve the manuscript.\nAs suggested, we have tried our best to address the concerns and revised the\nmanuscript accordingly.\n1. In the introduction, the authors completely ignore the role of biological\nnanopores in this field. Biological nanopores are also more cost-effective and\ncan enable high-throughput drug screening. Specifically, they have also already\nbeen used to study both the WIN and WBM sites of WDR5. These citations\nshould be added to the introduction of nanopore sensing in the context of\nWDR5.\na. Mayse, L. A., A. Imran, et al. (2022). \"Disentangling the recognition\ncomplexity of a protein hub using a nanopore.\" Nat Commun 13(1): 022-28465.\nb. Mayse, L. A., Y. Wang, et al. (2024). \"Real-Time Measurement of a Weak\nInteraction of a Transcription Factor Motif with a Protein Hub at Single-Molecule\nPrecision.\" ACS Nano 18(31): 20468-20481\nResponse: We sincerely thank the reviewer for the suggestion. These two\nrecommended articles are highly relevant to our work and offer substantial\ninsights. We have included them as references 34 and 35 (lines 83-85), with\nthe following added description: \" Intriguingly, both interaction events between\nWDR5 and MLL or Myc have been observed at the single molecule level using\nengineered tFhuA nanopores (34,35).\"\n2. There is an unnecessary “)” on line 107 after the reference.\nResponse: We sincerely apologize for this typo. We have deleted unnecessary\n“)” in the revised manuscript.\n3. In Figure 1b, there are three different current peaks shown for the WDR5\nmonomer. Is there a reason monomeric WDR5 doesn’t yield a uniform blockage\nof the nanopore? WDR5 is a structural ridge protein that the 22 nM nanopore\ndiameter should not heavily constrain. Not to mention that the amplitude\nhistogram supporting the numerical claim of the average current blockade is\nmissing.\nResponse: We appreciate the constructive critique that helps to improve our\nwork. Unlike biological nanopores, translocation events from solid-state\nnanopores are subject to more interference and signals are more prone to be\nnou-uniform (Chem. Sci., 2020, 11, 970). However, the amplitude histograms\nof our obtained signals show a Gaussian distribution, indicating that the\ndifference of signals is acceptable and the average current blockade value can\nbe reliably derived from these data. We have incorporated the amplitude\nhistogram of mono WDR5 in Fig.S2 for supporting the numerical claim of the\naverage current blocking.\nFig S2: Detecting conditions for WDR5 characterization via quartz nanopore sensing. All of\nthe measurements were performed in 1 × PBS buffer with 1M KCl (pH= 8.6~ 9.4). (a) Current-\nCurrent-\nvoltage curves obtained from 34 individual nanopores used in quartz nanopore measurements. (b)\nAll-point current amplitude histogram of quartz nanopore sensor for monomeric WDR5.\nIn lines 124-127, we have revised our description to: “Non-uniform\ntranslocation events of monomeric WDR5, along with atypical translocation\nevents exhibiting significantly higher peak currents and longer dwell times, were\nascribed to either impurities compromising protein purity or transient\noligomerization, unfolding and surface adsorption during the translocation\nprocess (22,45,46).” Additional reference 46 has been cited too.\n.\n4. In most traces, WZ-1 treatment also reduces the number of events, and as\nshown in Figure S8, after 10 minutes of treatment, no translocation events occur.\nThis indicates that WZ-1 triggers the formation of huge aggregates that are no\nlonger able to enter the nanopore. However, the authors do not discuss what\ncauses this reduction in the number of events. They only state that they expect\nthe results in Figure S8 to be based on previously published work from another\ngroup. This discussion is critical and would strengthen the conclusion that WZ-\n1 promotes significant WDR5 aggregation. Additionally, TEM images can show\nthe size of these oligomers, supporting this discussion.\nResponse: We sincerely thank the reviewer for this suggestion. Our cryo-EM\nanalysis showed that no huge aggregations were observed after incubating\nWDR5 with WZ-1 for 10 min.\n(a). A representative micrograph image of WT WDR5 incubated with WZ-1, low passed at 10\nÅ to increase contrast.\nSEM imaging and SEM-EDS analysis of the nanopores after WDR5\naggregation detection revealed that irregularly shaped protein agglomerates\nsurrounded the nanopore, obstructing their surfaces. It can be supposed that\nunder electrophoretic force, a fraction of WDR5 will not translocate but instead\nform excessive aggregates in the presence of WZ-1, creating a cap-like\nstructure that blocks further translocation of free WDR5s. This may explain the\nrapid signal disappearance observed upon WZ-1 addition.\nWe have addressed this point in line 369-374 and supplemented Fig.S13\nas follows: “SEM imaging of nanopores following WDR5 aggregation detection\n(Fig. S13a-b) revealed irregular agglomerates near the pore surface, which\nSEM-EDS analysis confirmed to be protein aggregates (Fig. S13c). These\nfindings suggest that WDR5 molecules, concentrated near the nanopore by\nelectrophoretic forces, undergo hyper-polymerization in the presence of WZ-1,\nthereby hindering the translocation of unbound WDR5.”\nFig S13: SEM imaging and SEM‑EDS analysis of quartz nanopores following WDR5\naggregation detection induced by WZ‑1. (a) Representative SEM image of untreated quartz\nnanopores (control). (b) Representative SEM image of quartz nanopores after WDR5 aggregation\ndetection induced by WZ‑1. (c) SEM‑EDS spectra of untreated nanopores and nanopores exposed\nto WDR5 in the presence of WZ‑1. A distinct accumulation of phosphorus (P) was detected at the\nnanopipette tip surface, consistent with the presence of protein material.\n5. There is a grammatical error in line 137, “ecorded” should be “recorded”.\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript.\n6. In Figure 2a, I expect the number of events to decrease as oligomerization\nwill reduce the effective concentration in the bath. If this is a time-dependent\ncondition, the authors should include this information in the text or figure legend.\nWhy does the number of events stay the same?\nResponse: We sincerely appreciate the reviewer’s suggestion and\napologize for the insufficient explanation provided in our original submission.\nFor a clearer expression, the figure legend of Fig. 2a is revised as:\n“Representative ionic current traces depicting the translocation of WDR5 in the\npresence of Mixture 1st-#9, Mixture 2nd-#5, and WZ‑1 during screening Rounds\n1, 2, and 3, respectively. ”\nIn addition, during the initial phase of oligomerized WDR5 signals emerging,\nno significant decrease in signal density was observed. ZETA potentials of\nWDR5 before and after WZ-1 treatment have been supplemented to Table S1\nin the revised manuscript. The WZ-1-induced oligomerization of WDR5 resulted\nin a multiplicative increase in surface charge density. This enhanced\nelectrophoretic mobility likely facilitates faster capture of the oligomers by the\nnanopore, thereby maintaining signal density without significant reduction.\nTable S1: ZETA potential for WDR5 before and after WZ-1 treatment.\nZeta potential measurements were performed in 1× PBS (pH 7.4) containing 0.1 mg/mL WDR5.\nThe zeta potential of WDR5+WZ-1 was measured after 30 minutes incubation with 10×\nconcentrated WZ-1 (27.3 μM).\n7. In Figure 2c, the standard deviations for the controls vs WZ-1 peak current\noverlap for all rounds.\nResponse: Thanks for the reviewer’s suggestion. The peak currents of Mixture\n1st-#9, Mixture 2nd-#5 and WZ-1 that we collected contain all peak currents in\ntraces. Both oligomerized WDR5 and monomeric WDR5 that have not yet been\ninduced by WZ-1 are analyzed for standard deviations, so the standard\ndeviations for the controls vs WZ-1 peak current may overlap.\n8. In Figure 3e, it appears that in the presence of DMSO, dimers of IAM start to\nform. The authors need to discuss this. I assume this is because dimerization\nis happening between the N-terminal region and the WIN site of monomers.\nThis dimerization occurs independently of WZ-1.\nResponse: Thanks for the reviewer’s suggestion. We concur that during the\nincubation of WDR5 with IAM, the N-terminus mediates WDR5 dimerization,\nwhich brings cysteine residues (particularly C248) into spatial proximity, thereby\nfacilitating unexpected oxidation. To obtain the Western blot results presented\nin Fig. 3e, WDR5 native structures were disrupted by SDS-PAGE and boiling.\nHowever, unlike standard Western blot protocols, no reducing agent was added\nin order to preserve the formation of disulfide bonds. The absence of the\nreducing agent may retain the unexpected oxidation, potentially resulting in\noligomeric bands. We have addressed this possibility in our revised manuscript.\n9. It is known that dimerization occurs between monomers when the N-terminal\nregions of a neighboring WDR5 interact with the WIN site of another (see the\nauthors’ citations). The authors mention this, but it is unclear whether they are\nproposing that those interactions occur first, and then WZ-1 stabilizes that\nformation. Or if the WZ-1 brings two monomers together via C248.\nThe testing of a truncation WDR5 mutant that lacks the N-terminal tail will make\nit very clear if WZ-1 itself can initiate dimerization or if the Win site-mediated\ndimerization needs to be present for this response to be observed (see my\ncomments above).\nResponse: It’s a good point! As shown in Fig.3g and Fig.S5d, both SEC-MALS\nanalysis and Western blot results demonstrated that deletion of the N-terminal\ntail markedly reduced WZ-1-induced oligomerization, indicating that the N-\nterminal tail significantly enhances this process.\nTo further probe the mechanism of WZ-1 mediated oligomerization, we\nperformed the Cryo-EM analysis of WDR5 WT, WDR5 WT with WZ-1, WDR5\nC248S with WZ-1 and WDR5 NTD (N-terminal domain, 31-334) truncation with\nWZ-1. All four dataset were subjected to the sample processing procedure, as\nshown in Figure S8, the lack of the NTD led to fewer oligomers induced by WZ-\n1.\nIn line 254-257, we have included these new data:\n“Supporting this model, SEM-MALS analysis, immunoblotting analysis and\nCryo-EM analysis demonstrate that N-terminal tail markedly enhances WZ-1-\ninduced aggregation (Fig. 3g, Fig. S5d and Fig.S8), consistent with the transient\ninteractions mediated by WIN sites and N-terminal tail bring C248 or other\ncysteine residues in close proximity for WZ-1 catalyzed reaction. ”\nFig 3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (g) SEC-\nMALS analysis of 15 µM wild-type tag-free WDR5 and variants (C248S, 31–334, 31–334_C248S)\ntreated with 200 µM WZ‑1. Calculated molecular weight of WDR5: 41.2 kDa.\nFig S5: WZ-1 acts as a specific WDR5 oligomerization inducer. (d) Immunoblot analysis of\n6 µM WDR5 (either untagged or with 31-334 mutant) treated with 100 µM WZ‑1 under\nnon‑reducing loading conditions.\nFig S8: 2D classification analysis of WDR5 particles obtained by cryo-EM. (a) 2D classification\nanalysis of WDR5 WT, WDR5 WT with WZ-1, WDR5 C248S with WZ-1 and WDR5 31-334\nmutant with WZ-1 at a protein concentration of 0.1 mg/ml. (b) 2D images for statistics in (a).\n10. It is unclear what mechanism would enable WZ-1 to impact the Win site\nitself. If these dimers were primarily occurring by the N-terminal tail interacting\nwith the WIN site, then that is why the MLL1 interaction is being influenced. The\nWin site is being occupied by the neighboring monomer, which is being held in\nproximity by the thiol-sulfide bonds created by WZ-1. Again, this conclusion\nrequires that the truncation of the WDR5 mutant lacking the N-terminal tail be\nidentified to determine whether WZ-1 itself influences the WIN site binders.\nResponse: Thanks for the reviewer’s suggestion. Our latest results\ndemonstrate that the NTD serves as one of the critical components for WDR5\noligomerization induced by WZ-1. The NTD-truncated WDR5 exhibited limited\nresponsiveness to WZ-1 treatment, failing to form substantial oligomers. Thus,\nthe experimental validation about whether NTD truncation affects the\ninteraction between WDR5 oligomers induced by WZ-1 and WIN site binders is\ncurrently unfeasible. Theoretically, the WIN site intrinsically possesses higher\nbinding affinity for its natural partners (e.g., MLL1), making it particularly\nchallenging to achieve WIN site inhibition solely through N-terminal modulation.\n11. I would like to know if the authors removed the histidine tag if the results\nremain the same.\nResponse: We apologize for the lack of explanation. The WDR5s used in SEC-\nMALS and Cryo-EM analysis are all without tags. The missing information is\nadded in the figure legends and methods. In addition, immunoblotting analysis\nin Fig S5d showed that WZ-1 induced the oligomerization of WDR5 after\nremoval of its histidine tag.\nFig S5: WZ-1 acts as a specific WDR5 oligomerization inducer. (d) Immunoblot analysis of\n6 µM WDR5 (either untagged or with 31-334 mutant) treated with 100 µM WZ‑1 under\nnon‑reducing loading conditions.\n12. In nano-sensing data acquisition and analysis, there is no reference to\nsimilar data analysis, and a precise threshold definition is lacking. Without this\ninformation, it's very challenging to understand how they identified peaks\nversus noise. This threshold must be clearly defined, as the current\npresentation is not convincing.\nResponse: The reviewer is right, and we sincerely apologize for the omission.\nWe supplemented the threshold definition in line 597-600 and reference 67 for\na more clear data analysis understanding: “Peak signals were distinguished\nfrom background noise using a threshold method, allowing for the identification\nof the start and end points of each peak. Peaks amplitude >45 pA; dwell\ntime >10 μs were selected and the current values within the pulse plateau were\naveraged to obtain mean values as peak currents”.\n13. In Figure S2, it is labeled as 34 nanopipettes, and it should be 34 nanopores.\nResponse: Thanks for reviewer’s suggestion, we have modified it in the revised\nmanuscript.\n14. The figure legend in Figure S3 is cut off and incomplete.\nResponse: Thanks for reviewer’s suggestion, we have supplemented correct\ncontent to the figure legend in Fig.S3.\n15. In Figure S4, it would be far more informative to include the histograms from\nmixture 1, 2, and WZ-1.\nResponse: Thanks for reviewer’s suggestion, we have supplemented\nhistograms from mixture 1,2 and WZ-1 in Fig.S4:\nFig S4. Schematic of Gaussian fitting for mean value determination. The left three\nplots show the frequency distributions of WDR5 peak current amplitudes for the control\ngroups, and the right three plots show the corresponding distributions after incubation\nwith (a) Mixture 1, (b) Mixture 2, and (c) WZ‑1. The rate of change was calculated by\ndividing the mean value after incubation by the corresponding control mean value, as\nmeasured within the same nanopore.\n16. Figure S8: “Typical Current-time,” there is unnecessary capitalization of\nletters in this sentence.\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript.\n17. Figure S7abc, Figure S9 are unreadable. All fonts and legends are way too\nsmall. Please look and rectify other figures with the same characteristics.\nResponse: We sincerely apologize for this mistake. Besides the issues\nmentioned, we externally identified similar issue in Fig.6. We have\nsubsequently updated them with higher-resolution versions on Fig.6, Fig.S10,\nS11 and S14-20 in the revised manuscript.\nFig S10: The mass spectrometry of dNAC, A2 and A3.\nFig S11. Functional regulation of WDR5 by WZ‑1. (a) NCM460 cells, immortalized human colon\nfibroblasts, and CT26 cells treated with varying concentrations of WZ‑1 for 72 h, followed by\nviability assessment using the SRB assay. (b) HCT15 cells treated with 10 µM WZ‑1 for 24 h,\nfollowed by cell‑cycle analysis via PI staining. (c) Schematic diagram showing the WBM binding\nsite and the MYC peptide interaction interface in WDR5, based on the crystal structure of the\nWDR5–MYC complex (PDB ID: 4Y7R). The WBM site is located in close proximity to residue\nC248. (d) Bar plot of the top 30 enriched GSEA terms for CT26 cells treated with WZ‑1. (e) GSEA\nenrichment plots for WBM site– and WIN site–related pathways in CT26 cells (WIN site: E2F\ntargets, G2M checkpoint; WBM site: Myc targets v1, Myc targets v2).\nReviewer #3:\nThis article is a collaborative work across multiple groups that takes an initial\nhit molecule through validation and subsequent cancer cell line testing,\nincluding a degree of SAR work. A key part of this is the initial screening\ntechnology, which employs nanopore-based detection to differentiate\nmolecules capable of inducing WDR5 oligomerisation from those that cannot.\nMultiple molecules are tested in a ‘cocktail’ approach in a single experiment and\nmolecule groups are successively narrowed down until the active molecule is\nidentified. The authors validate a covalent binding/oligomerization mechanism\nand explore how this mechanism disrupts binding of WDR5 with its partners c-\nmyc and MLL1. The authors argue that this mechanism is responsible for the\ncell death of cancer lines reliant on this pathway.\nThis is a very well presented work, combining an advanced detection approach\nwith a novel method of disrupting cancer cell growth through induced protein\noligomerization. The authors have to address the following points (including\nadditional experiments):\nResponse: We thank the reviewer for acknowledging that our experiments were\nwell‑presented and our findings significant. We also appreciate the reviewer’s\nconstructive comments highlighting areas for improvement. In accordance with\nthe reviewer’s suggestions, we have performed additional experiments to better\nclarify the mechanism by which WZ‑1 induces WDR5 aggregation.\nMajor\n1. Molecules such as these could plausibly form micelles or precipitate,\nespecially at high ionic strength. Please show data in the nanopore with WZ-1\nalone under the same conditions to remove any possibility that the aggregate\nsignal arises from this.\nResponse: Thanks for reviewer’s suggestion. The current-time trace of WZ-1\nunder the same condition of nanopore detection have been supplemented in\nFig. S6, which shows no aggregate signals arose by WZ-1 alone. Relative\ndescription is added in line 163-165: “In addition, control experiments confirmed\nthat the observed signals were not due to micelles or precipitates formed by\nhigh concentrations of WZ-1 (Fig. S6).”\nFig S6. Current–time traces for WZ‑1 alone. Representative ionic current–time traces for 5 µM\nWZ‑1 (10× the concentration of WDR5 used in nanopore detection experiments).\n2. There is a discrepancy between Figure 3g and Figure 3f. Figure 3g shows\nno dimer formation at all in SEC for C248S in the absence of WZ-1, whereas\nthe Coomassie stain in Figure 3f shows a strong dimer band. What is the cause\nof this? It’s peculiar in the Coomassie stain that the dimer band is then gone in\nthe presence of WZ-1, given this molecule otherwise enhances oligomerisation.\nResponse: We thank the reviewer for pointing out the apparent discrepancy\nbetween the Coomassie staining results (Fig. 3f) and the SEC results (Fig. 3g)\nfor the C248S mutant. We believe several factors contributed to this difference:\n(1) Reducing conditions: For the Coomassie staining experiment, native\nWDR5 protein structures were denatured by SDS–PAGE and boiling. Unlike\nstandard Western blot protocols, no reducing agent was included in order to\npreserve any disulfide bonds. Under these non-reducing conditions,\nspontaneous oxidation during sample incubation could lead to nonspecific\ndisulfide-mediated dimers or oligomers. In contrast, for the SEC experiment,\nthe WDR5 protein was purified in the presence of 0.1 mM TCEP, which prevents\nnonspecific disulfide bond formation.\n(2) Methodological differences: Oligomerization of WDR5 appears to be\ndynamic. During SEC, such weak non-covalent oligomers may readily\ndissociate as the protein is separated into the pores of the column matrix and\neluted in the mobile phase. This dissociation is commonly observed in weak\nprotein complexes, which often elute as distinct monomeric peaks in SEC.\n(3) Protein purity: The recombinant protein used for the initial Coomassie\nstaining was purified only by affinity chromatography, which may have allowed\nresidual impurities to contribute to background staining.\nTo address this concern, in the revised manuscript, we repeated the\nexperiment using further-purified protein (affinity followed by SEC) and\nmonitored the dimeric/oligomeric state by Western blot rather than Coomassie\nstaining. As shown in the revised Fig. 3f, the background dimeric/oligomeric\nsignal for C248S is greatly reduced, and WZ-1 does not further cause an\nabnormal reduction in dimer band.\nFig.3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (f)\nImmunoblot analysis of 6 µM recombinant single-cysteine-mutant WDR5 proteins (C163S, C195S,\nC205S, C248S, C261S, C309S, C334S) treated with 100 µM WZ‑1.\n3. C261S actually seems to enhance WZ-1 activity given the complete depletion\nof the monomer band in Figure 3f, do the authors have an explanation for this?\nResponse: Thank you for pointing this out. As mentioned above, In the original\nCoomassie-stained gel, the apparent complete depletion of the monomer band\nfor C261S after WZ-1 treatment may have been exaggerated by limited protein\npurity. In the revised manuscript, we re-performed the experiment using\nfurther-purified protein (affinity chromatography followed by SEC) and\nmonitored the dimeric/oligomeric state by Western blot under non-reducing\nconditions. As shown in the revised Fig. 3f, the C261S mutant does show an\nincrease in higher-order species upon WZ-1 treatment.\nFig.3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (f)\nImmunoblot analysis of 6 µM recombinant single-cysteine-mutant WDR5 proteins (C163S, C195S,\nC205S, C248S, C261S, C309S, C334S) treated with 100 µM WZ‑1.\n4. The behaviour of C205S is as expected for a key residue that disrupts WZ-1\ninduced oligomerization, showing essentially the same behaviour between the\nnegative control and WZ-1 lanes in Figure 3f, while C248S shows the odd\nbehaviour mentioned above. For that reason it seems an overreach to pin the\nmechanism initiation on C248S and not C205S.\nResponse: We sincerely thank the reviewer for this insightful comment. As\nshown in the immunoblot analysis in the revised Fig. 3f, the C205S mutant can\nstill be induced by WZ-1 to form oligomers, although the extent of\noligomerization is noticeably lower than that observed for most other cysteine\nmutants. In contrast, the C248S mutant completely lost the ability to form\nWZ-1–induced oligomers, despite exhibiting only minimal background\noligomerization. Consistent with this, our SEC-MALS analysis (Fig. 3g) further\nsupports the critical role of C248 in mediating WZ-1-induced oligomerization.\nTo better reflect these findings, we have revised the text on lines 221-224 as\nfollows:“Following WZ-1 treatment, most mutants still exhibited oligomer\nformation. However, several mutations, particularly C205S, modestly reduced\noligomerization, whereas the C248S mutant—despite displaying minimal\nbackground oligomerization—completely lost the ability to form WZ-1–induced\noligomers (Fig. 3f).”\nFig.3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (f)\nImmunoblot analysis of 6 µM recombinant single-cysteine-mutant WDR5 proteins (C163S, C195S,\nC205S, C248S, C261S, C309S, C334S) treated with 100 µM WZ‑1.\nFig 3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (g) SEC-\nMALS analysis of 15 µM wild-type tag-free WDR5 and variants (C248S, 31–334, 31–334_C248S)\ntreated with 200 µM WZ‑1. Calculated molecular weight of WDR5: 41.2 kDa.\n5. Figure 5 states dissociation constants for the various WZ molecules in\nbinding to WDR5, but I don’t see any representations of the actual data\nsupporting these values. What method was used? Microscale thermophoresis\nis mentioned in the main text, and the reader is directed to figure S3, but this\ncontains thermal shift data, not MST data, for only the control drugs rather than\nthe WZ derivatives. Where is the WZ data for binding to WDR5? Furthermore,\nhow did the authors obtain Kds given that the mechanism of binding for these\nmolecules is largely mediated via a disulphide, unless DTT was incorporated in\nthe buffer to allow reversible binding without reaction?\nResponse: We sincerely apologize for the confusion. The dissociation\nconstants (K ) for the WZ series compounds were determined using microscale\nd\nthermophoresis (MST). In the revised manuscript, we have included the\ncomplete MST binding data for the WZ series with WDR5 in Fig. S9.\nRegarding the feasibility of obtaining K values despite the disulfide-mediated\nd\nmechanism, we note that the 2-aminothiazole core of WZ-1 functions as a\n“warhead” that specifically targets the WBM site on WDR5, enabling\nsubsequent thio-disulfide exchange and aggregation. MST experiments were\ncarried out at low concentrations of WZ compounds, which substantially limits\nWZ-1-induced WDR5 aggregation under assay conditions. Therefore, under\nMST conditions, the measured signal largely reflects the non-covalent binding\nstep to the WBM site, rather than aggregation, allowing us to determine Kd\nvalues for the WZ series.\nFig S9. MicroScale Thermophoresis (MST) assay for WZ-1, WZ-2, WZ-3, WZ-4, WZ-5 and\nWZ-6.\n6. Do the authors have a good sense for why WZ-1 is specific to WDR5 and\ndoes not impact any other homologues as shown in Figure S6? The molecular\nscaffold is an interesting one but does not present obvious mechanisms of\nspecificity.\nResponse: We thank the reviewer for this excellent question. Based on insights\nraised by reviewers #1 and #2, we believe that the specificity of WZ-1-induced\noligomerization toward WDR5 is likely driven by two synergistic factors: (1)\nbinding of the molecular core of WZ-1 to WDR5 followed by thio–disulfide\nexchange, and (2) the intrinsic propensity of the WDR5 N-terminal tail to\nmediate dimerization, which may compensate for the relatively modest\n(low-micromolar) affinity of the molecular core.\nWe have obtained additional experimental evidence to support this hypothesis.\nAs shown in Fig.3g and Fig.S5d, both SEC-MALS analysis and Western blot\nresults demonstrated that deletion of the N-terminal tail markedly reduced\nWZ-1–induced oligomerization, indicating that the N-terminal tail significantly\nenhances this process.\nFurthermore, we performed cryo-EM analyses of WDR5 WT alone, WDR5 WT\nwith WZ-1, WDR5 C248S with WZ-1, and WDR5 N-terminal domain (NTD)\ntruncation with WZ-1. All four datasets were processed under identical\nconditions. As shown in Fig. S8, either removal of the NTD or mutation of C248\nsubstantially reduced WZ-1–induced oligomer formation. Taken together, these\nresults strongly suggest that both the N-terminal tail and the molecular core\nbinding of WZ-1, followed by thio-disulfide exchange with C248, underlie the\nspecificity of WZ-1 for WDR5 compared with its homologues.\nFig 3. Discovery of a thiol-dependent specific oligomerization inducer for WDR5. (g) SEC-\nMALS analysis of 15 µM wild-type tag-free WDR5 and variants (C248S, 31–334, 31–334_C248S)\ntreated with 200 µM WZ‑1. Calculated molecular weight of WDR5: 41.2 kDa.\nFig S5. WZ-1 acts as a specific WDR5 oligomerization inducer. (d) Immunoblot analysis of\n6 µM WDR5 (either untagged or with 31-334 mutant) treated with 100 µM WZ‑1 under\nnon‑reducing loading conditions.\nFig S8. 2D classification analysis of WDR5 particles obtained by cryo-EM. (a) 2D classification\nanalysis of WDR5 WT, WDR5 WT with WZ-1, WDR5 C248S with WZ-1 and WDR5 31-334\nmutant with WZ-1 at a protein concentration of 0.1 mg/mL. (b) 2D images for statistics in (a).\n7. This point would be expanding the scope and so is optional, but the impact\nof the work would be emphasized by demonstrating some kind of control cell\nwork that shows these molecules are able to impact cancer cell growth to a\ngreater extent than normal cell growth. For instance these cell lines are\npresumably heavily reliant on c-myc, and so one would expect normal cell lines\nto be less affected by the activity of WZ-1. This would then fit in with the\n‘synthetic lethality’ paradigm that is currently dominating drug discovery in\noncology.\nResponse: Thanks for reviewer’s suggestion. We performed a comparative\nanalysis between the c-Myc overexpressing cancer cell line CT26 and the\nnormal colon cell lines NCM460 and Immortalized human colon fibroblasts. As\nshown in Fig S11a., WZ-1 did not substantially inhibit proliferation in the normal\ncolon cell lines NCM460 and immortalized human colon fibroblasts, indicating\na degree of selectivity toward cancer cells. We have revised the manuscript\naccordingly.\nFig S11. Functional regulation of WDR5 by WZ‑1. (a) NCM460 cells, immortalized human colon\nfibroblasts, and CT26 cells treated with varying concentrations of WZ‑1 for 72 h, followed by\nviability assessment using the SRB assay.\nMinor\n1. It would be good to clearly explain the 1st-#9 labelling used\nResponse: Thanks for reviewer’s suggestion, we have supplemented\nexplanation in line 145-146:\n“…436 compounds into nine groups (46-50 compounds per group, labeled 1st-\n#1 to 1st-#9)…”\n2. Figure S5 has a solid line underneath it that should be removed\nResponse: Thanks for reviewer point out the issue. But in our submitted\nmanuscript, there is no solid line can be found in original figure S5. The mistake\nlikely stems from file format conversion during the submission process and\nwon’t be observed in our revised vision.\n3. There is a mismatch between figure S7 and its caption\nResponse: Thanks for reviewer point out the issue. we have divided the original\nFig. S7 into Fig. S10 and S11 and corrected the caption:\nFig S10. The mass spectrometry of dNAC, A2 and A3.\nFig S11. Functional regulation of WDR5 by WZ‑1. (a) NCM460 cells, immortalized human colon\nfibroblasts, and CT26 cells treated with varying concentrations of WZ‑1 for 72 h, followed by\nviability assessment using the SRB assay. (b) HCT15 cells treated with 10 µM WZ‑1 for 24 h,\nfollowed by cell‑cycle analysis via PI staining. (c) Schematic diagram showing the WBM binding\nsite and the MYC peptide interaction interface in WDR5, based on the crystal structure of the\nWDR5–MYC complex (PDB ID: 4Y7R). The WBM site is located in close proximity to residue\nC248. (d) Bar plot of the top 30 enriched GSEA terms for CT26 cells treated with WZ‑1. (e) GSEA\nenrichment plots for WBM site– and WIN site–related pathways in CT26 cells (WIN site: E2F\ntargets, G2M checkpoint; WBM site: Myc targets v1, Myc targets v2).\n4. Supply figure s7 at higher res, can’t confirm the mass spec\nResponse: We sincerely apologize for this mistake. We have subsequently\nupdate them with higher-resolution versions in Fig. S10 and S11\n5. Supply figure s8 at higher res, can’t confirm nmr shifts\nResponse: We sincerely apologize for this mistake. We have subsequently\nupdate them with higher-resolution versions in Fig. S14-20.\n6. There are a number of instances of incorrect grammar in the main text and\nelsewhere e.g. Xiaoyang Dai for his assistant on HPLC, it would be good to\ncorrect these to ensure the authors’ work is viewed purely on the merit of the\nscience presented.\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript and thoroughly re-examined our manuscript to enhance its\nclarity and accuracy.\nReviewer #4&#5:\nThis work by Fang, Jiang and co-workers describes a new method to screen\ncompounds with the ability to induce selective oligomerization of the protein of\ninterest based in the use of nanopores. By using quartz nanopores and\nmeasuring changes in its ionic current, the authors tested a library of\ncompounds for the oligomerization of WD repeat domain 5 (WDR5), a protein\ninvolved in epigenetic regulation and oncogenesis, where participates in the\nMLL/SET complex and directs c-Myc to chromatin at tumor-associated genes.\nThe authors successfully find one molecule, WZ-1, that induces formation of\ndimers and higher oligomers of WDR5, and their data suggest that WZ-1\npromotes disulfide bond exchanges in WDR5 inducing its association. This WZ-\n1 compound was also tested in cells and it inhibits cancer cell proliferation by\ninterfering in the interaction of WDR5 with MLL1 or c-Myc, and resulting in\nreduced expression of c-Myc target genes or others involved in E2F and G2M\ncheckpoint signalling pathways.\nThe approach is innovative and the results with WDR5 are promising. The\nresults are clearly presented, and the manuscript is well written.\nResponse: We are grateful to the reviewer #4 and #5 for both their positive\nassessment and insightful comments. Each point has been thoroughly\nconsidered, and we have implemented all suggested improvements in the\nrevised manuscript.\nA.-I just have a concern regarding the cryoEM results depicted in Figure 4C.\nIn the work about the effect of BI-3802 on the aggregation of B-cell lymphoma\n6 (BCL6) protein (Ref #12), the cryoEM of BCL6 oligomers made a lot of sense\nsince the protein constructed helical structures and the authors presented a 3.7\nÅ resolution cryoEM map. The cryoEM results with WDR5 in the presence of\nWZ-1 are not so successful. It is understandable, since the associations of\nWDR5 are not regular and are difficult to average (even at 2D). Thus, the\ncryoEM analysis is of limited value and only clusters of nearby WDR5 copies\nare seen. My concern relates to the crowding of electron micrographs (or\nmovies), where WDR5 particles can be in contact without an actual\noligomerization event.\nLines 756-757:\n“A total of 929 movie stacks were imported and processed with cryoSPARC\nversion 4.3.1. 1,150,237 particles were auto picked …”\nThis means that are more than 1,200 particles per movie initially, and this is a\nvery high concentration. It is not clear to me how can the authors discriminate\nbetween real dimers or larger oligomers and spurious groups of particles that\nare together in such crowded area. If the author would have shown high quality\n2D averages, the real association could have been inferred, but this is not the\ncase.\nI would recommend the use of negative stained samples with lower\nconcentration, and by comparing WDR5 alone and WDR5+WZ-1 the\ndistribution of particles in the filed of view needs to be really different.\nIn any case, the authors have shown the oligomerization effect by WZ-1 on\nWDR5 by other means (gel electrophoresis and SEC-MALS), so this is not\nunder discussion, but the cryoEM data presented does not add very much.\nResponse: We thank the reviewer for the insightful suggestion. We initially used\nCryo-EM to direct visualized WZ-1 induced oligomerization. We used the\nAutopicker in Cryosparc to exhaustively pick all possible particles to prevent\nbias, thus a large number of particles were picked and subjected to subsequent\n2D classification analysis. We initially used a protein concentration of 1 mg/mL\nfor sample freezing. Following the reviewer’s suggestion, we carried out new\nCryo-EM experiments WDR5 WT, WDR5 WT with WZ-1, WDR5 C248S with\nWZ-1 and WDR5 NTD truncation with WZ-1 at a protein concentration of 0.1\nmg/mL. All four dataset were subjected to the sample processing procedure, as\nshown in Figure S8, the lack of the NTD or C248S mutation led to fewer\noligomers induced by WZ-1.\nFig S8. 2D classification analysis of WDR5 particles obtained by cryo-EM. (a) 2D classification\nanalysis of WDR5 WT, WDR5 WT with WZ-1, WDR5 C248S with WZ-1 and WDR5 31-334\nmutant with WZ-1 at a protein concentration of 0.1 mg/mL. (b) 2D images for statistics in (a).\nWe also extensively tried negative staining. However, we couldn’t obtain clear\nimages with good contrast for analysis (Figure below).\nOther minor points:\ni.-The authors have clearly stablished that Cys248 preferentially participates in\nthe disulfate bond formation. Their model postulates a C248-C248 bridge\nbetween WDR5 subunits. Other possibilities might be considered, for instance\na C248-C121 bridge, since the C121 mutant was not obtained, and such an\nassociation would just require a 180 degrees flip of one of the WDR5 subunits.\nJust a suggestion about an interaction that cannot be ruled out.\nBTW, on lines 214-215\n“To determine which cysteine residue is involved in WZ-1-triggered\noligomerization, we individually mutated the 11 cysteine residues of WDR5 to\nserine”\nI have checked the sequence of WDR5 several times and I have seen just 10\ncysteine residues. Is this an isoform or is there a Cys in the tag?\nResponse: Firstly, we sincerely apologize for our mistake of the number of\ncysteine residues. We have corrected into the right number in line 218-219:\n“To determine which cysteine residue is involved in WZ-1-triggered\noligomerization, we individually mutated each of WDR5’s ten cysteines to\nserine (CS mutation).”\nSecondly, we agree that while our current data support a C248–C248 disulfide\nlinkage model, alternative possibilities such as a C248–C121 bridge cannot be\nconclusively ruled out, especially given that the C121 mutant was not included\nin our current mutagenesis panel. Cys121 is surface-exposed in certain\norientations, and as the reviewer noted, a 180° rotation of one subunit could\npotentially bring Cys121 into proximity with Cys248 for intermolecular disulfide\nformation.\nTo address this uncertainty, we have revised our mechanistic description in the\nmanuscript to explicitly reflect that the cysteine residue involved in the thio-\ndisulfide exchange may be Cys248 itself or another accessible surface cysteine.\nThe updated statement now reads: “Whether the cysteine residue engaged in\nthis exchange is Cys248 itself or another surface cysteine likely determines\nwhether the reaction terminates or continues, thus controlling the extent of\noligomerization.”\nii.-Line 295. First time that “SRB assay” is introduced. Please, spell it out.\nResponse: We sincerely apologize for this omission. We have supplemented it\nin the revised manuscript\niii.-Line 168. Move “fascilitated” to “facilitated”.\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript\nResponse letter\n(NCOMMS-25-26811A)\nWe sincerely thank all five reviewers for their insightful comments and\nconstructive suggestions. We have carefully addressed each point in the\nrevised manuscript.\nA detailed point-by-point response is provided below.\nCOMMENTS TO AUTHOR:\nReviewer #1 & #2:\nOverall, this revision is a significant improvement; the authors have\naddressed most of my previous concerns and added a reasonable amount of\nnew data. This new data is critical for the conclusions made (e.g., WDR5 NTD).\nThe authors need to add more data and explanation about the agglomerates\nnow discussed in the revision. Here, the primary concern is that this surface\nbuildup on the nanopore would introduce oligomeric detection that derives from\naggregates dissociating. Once the agglomerate discussion has been\naddressed, this manuscript is ready for publication. Please see the comments\nbelow.\nResponse: We sincerely thank the reviewer for the positive comment and\nconstructive suggestions. These suggestions previous were highly\nmeaningful, helping us to identify potential area of the manuscript and further\nimprove its quality. The new concern about the surface buildup is also\nreasonable. We have performed new experiment data and revised the\nmanuscript to address the issue.\n1. Comment 3: Adding the histogram in Fig. S2 and additional text with\nreference was critical.\nResponse: We sincerely thank the reviewer's valuable comment. We added the\nhistogram to Fig. S2 and supplement the corresponding text with relevant\nreferences as suggested.\n2. Comment 4: The discussion of agglomerates raises a few concerns. In Fig.\nS13, I assume the “untreated” or “control” SEM images are of the nanopore\nwithout WDR5. Without showing the nanopore with WDR5 alone, I question if\nthese agglomerates form from WDR5 itself. Also, could these agglomerates be\ndissociating and entering the pore, representing oligomer formation? If this is\ntrue, can this event be separated from true oligomerization that is happening\nfreely in solution? I understand that the authors have addressed my original\nquestion, but now that these agglomerates have been shown, there is a need\nfor a more in-depth inquiry into how they can affect the key findings of this paper.\nResponse: We appreciate the constructive critique that helps to improve our\nwork. To clarify if WDR5 alone forms agglomerates, we have supplemented\nSEM and SEM-EDS data of nanopores treated with buffer only and WDR5 only\n(corresponding to Fig. S13a). Specifically, Fig. S13a includes four groups:\nuntreated nanopores (Untreated), nanopores in buffer (1M KCl, 1×PBS)\nwithout WDR5 (Buffer), nanopores with WDR5 alone (WDR5), and nanopores\nwith WDR5 + WZ-1 (WDR5 + WZ-1). The Buffer group shows tip occlusion from\ncrystalline deposits (due to solvent evaporation), with the WDR5 alone group\nshowing slight surface adsorption but no obvious agglomeration. SEM-EDS\nanalysis (Fig. S13b) further confirms that distinct phosphorus (P) accumulation\n(indicating protein aggregates) only appears in the WDR5+WZ-1 group. These\nresults clearly confirm that WZ-1, rather than WDR5 itself, is the key factor\ninducing agglomeration.\nFig S13. SEM imaging and SEM‑EDS analysis of quartz nanopores following\nWDR5 aggregation detection induced by WZ‑1. (a) Representative SEM image of\nuntreated quartz nanopore (Untreated), quartz nanopore after detection in 1M KCl,\n1×PBS buffer solution without WDR5 (Buffer), quartz nanopore after WDR5 detection\nwithout WZ-1 (WDR5) and quartz nanopore after WDR5 aggregation detection\ninduced by WZ‑1 (WDR5 + WZ-1). Nanopores exposed to buffer solution exhibited\ntip occlusion due to crystalline deposits resulting from solvent evaporation. (b)\nSEM‑EDS spectra of untreated nanopores and nanopores exposed to WDR5 in the\npresence of WZ‑1. A distinct accumulation of phosphorus (P) was detected at the\nnanopipette tip surface, consistent with the presence of protein material. Images in (a)\ndemonstrate a progressive formation of irregular aggregates upon the addition of\nWDR5 and WZ-1. Correspondingly, SEM-EDS reveals a concurrent accumulation of\nP, thereby validating that the aggregates are composed of WDR5s and induced by WZ-\n1.\nRegarding the concern about agglomerate dissociation and potential\ninterference with oligomerization signals, we address this through both\nexperimental evidence and data analysis. First, the WZ-1-induced\nagglomerates are relatively stable and not easily dissociated, primarily due to\nthe stability of disulfide bonds in the aggregates. Second, if dissociation\noccurred, large aggregates would generate abnormally high blocking peaks\nduring nanopore detection. However, we observed no frequent occurrence of\nsuch peaks in our experiments; occasional individual peaks were attributed to\ntrue oligomerization events. Third, we excluded these infrequent, discrete data\npoints via Gaussian distribution fitting during statistical analysis, ensuring they\ndid not affect the overall data trend.\nTo reflect this, we have added the following content in Lines 375–377 of the\nmanuscript: :\n“Given that these aggregates are stabilized by disulfide bonds, it is\nreasoned that the nanopore test is unlikely to produce false oligomerization\nsignals caused by agglomerates dissociation. The absence of over high\nblocking peaks supports this speculation.”\n3. Comment 9: I applaud the authors for adding the WDR5 NTD experiments.\nThese results show that the dominant factor in oligomerization is the interaction\nof the N-terminus with the Win site, as Fig. S8a shows that no oligomers form\nwith that mutant.\nResponse: We sincerely appreciate the reviewer's recognition of our WDR5\nNTD experiments. Your previous valuable suggestions not only directed us to\nexplore this critical research area but also significantly improved the\ncompleteness of our work, particularly in confirming that the N-terminus-Win\nsite interaction dominates oligomerization (as reflected by the absence of\noligomers in the mutant, Fig. S8a).\n4. New notes: Please add the full name of “SAR” study\nResponse: Thanks for the reminder. The full name of SAR is supplied on line\n265-266: “Structure-Activity Relationship(SAR)”\n5. Fig. S9 should have a figure legend.\nResponse: We apologize for the lack of explanation. The detailed legend has\nbeen added and the image is regulated for clearer interpretation and accuracy.\nFig S9: MicroScale Thermophoresis (MST) assay for WZ-1, WZ-2, WZ-3, WZ-4,\nWZ-5 and WZ-6\nMST Dose-Response Curve of WZ-1, WZ-2, WZ-3, WZ-4, WZ-5, WZ-6. The K value\nd\nwas derived from the inflection point of the fluorescence intensity curve plotted against\nthe logarithm of the small molecule concentration. For compounds WZ-2 and WZ-5,\nwhich showed higher initial variability, the binding affinities were confirmed with three\nindependent replicate experiments.\nReviewer #3:\nThe authors provided a revised manuscript However the new figures and data\ncontain issues.\nResponse: We sincerely apologize for these typos. We have addressed the\nreviewer's comments and re-examined the new figures and data to ensure a\nmore accurate representation.\n1. Figure S4 C right histogram the x-axis is wrong (50-10-15...)\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript.\n2. Figure S6 the right hand plot probably means 5 \\muM molar not micrometers!\nResponse: We sincerely apologize for this typo. We have corrected it in the\nrevised manuscript.\n3. Figure S9 the axis labels are barely readable and need to be increased in\nfont size - also the colours for the legend in WZ-2 do not match with the colours\nin the main plot. This also needs to be corrected. The authors also give two\n(WZ-1, WZ-3, WZ-6) or three (WZ-2, WZ-5) Kd values for the compounds. Why?\nThere is no explanation of the different balues that can differ by a factor of three!\nResponse: We sincerely apologize for these mistakes and the absence of\nexplanation. We have addressed each concern specifically as follows:\nTo improve readability, we have increased the font size of all axis labels in\nFigure S9. The adjusted font size ensures that labels are clearly visible without\ncompromising the overall layout of the figure.\nWe have corrected the color mismatch in the legend for compound WZ-2 to\nalign with the data plot. We have also double-checked all other plots in the\nfigure to ensure their legends and data series are consistent.\nWe apologize for the lack of clarity regarding the number of replicates. Our\nstandard procedure was to perform two independent experiments for each\ncompound. For compounds WZ-2 and WZ-5, the initial two replicates showed\ngreater variability. To ensure the accuracy and reliability of the binding affinity,\nwe proactively performed a third independent experiment for these specific\ncompounds. We now present all collected data for full transparency. This\nexplanation has been added to the revised legend for Figure S9 to clarify our\nmethodology.\nFig S9: MicroScale Thermophoresis (MST) assay for WZ-1, WZ-2, WZ-3, WZ-4,\nWZ-5 and WZ-6\nMST Dose-Response Curve of WZ-1, WZ-2, WZ-3, WZ-4, WZ-5, WZ-6. The K value\nd\nwas derived from the inflection point of the fluorescence intensity curve plotted against\nthe logarithm of the small molecule concentration. For compounds WZ-2 and WZ-5,\nwhich showed higher initial variability, the binding affinities were confirmed with three\nindependent replicate experiments.\n4. The authors need to provide a more in depth explanation of the\nsupplementary data figures, make sure that the data is accurate and the labels\nare correct. In the current form the manuscript and supplement should not be\naccepted.\nResponse: Thanks for reviewer’s suggestion, we have recheck our new data\nfigures and provide detailed explanations as follows.\n(1) Additional explanation of Fig S9 have been added in the legend: “MST\nDose-Response Curve of WZ-1, WZ-2, WZ-3, WZ-4, WZ-5, WZ-6. The\nKd value was derived from the inflection point of the fluorescence\nintensity curve plotted against the logarithm of the small molecule\nconcentration. For compounds WZ-2 and WZ-5, which showed higher\ninitial variability, the binding affinities were confirmed with three\nindependent replicate experiments.”\n(2) Additional explanation of Fig S2b have been added in the legend: “A\nGaussian fit was used to determine the mean peak current of\nmonomeric WDR5.”\n(3) Additional explanation of Fig S4 have been added in the legend:\n“Gaussian fit was applied to determine the mean peak current……The\nextent of WDR5 polymerization induced by the small molecule was\nquantified by the rate of change.”\n(4) Additional explanation of Fig S6 have been added in the legend: “In\nthe absence of WDR5, no distinct blockage peaks were observed in the\ncurrent trace. All non-specific signals were below 15 pA and were\nexcluded during data analysis, thus not affecting the statistic of WDR5\npeaks.”\n(5) Additional explanation of Fig S8b have been added in the legend:\n“……showing both the native oligomerization of WDR5 and the WZ-1-\ninduced oligomerization under different conditions.”\n(6) Additional explanation of Fig S13a have been added in the legend:\n“Representative SEM image of untreated quartz nanopore (Untreated),\nquartz nanopore after detection in 1M KCl, 1×PBS buffer solution\nwithout WDR5 (Buffer), quartz nanopore after WDR5 detection without\nWZ-1 (WDR5) and quartz nanopore after WDR5 aggregation detection\ninduced by WZ‑1 (WDR5 + WZ-1). Nanopores exposed to buffer\nsolution exhibited tip occlusion due to crystalline deposits resulting from\nsolvent evaporation.”\n(7) Additional explanation of Fig S13b have been added in the legend:\n“Images in (a) demonstrate a progressive formation of irregular\naggregates upon the addition of WDR5 and WZ-1. Correspondingly,\nSEM-EDS reveals a concurrent accumulation of P, thereby validating\nthat the aggregates are composed of WDR5s and induced by WZ-1.”\n(8) Additional explanation of Table S1 have been added in the legend:\n“Treatment with WZ-1 resulted in a significant increase in Zeta\npotential, indicating that WZ-1 induced oligomerization enhances the\nsurface charge ,which benefits the electrophoretic force.”\nReviewer #4:\nThe authors have properly addressed my concern regarding some cryoEM data\npresented in the original manuscript with new experiments at lower\nconcentration of the protein. In my opinion the work is now ready for publication.\nResponse: We sincerely appreciate the reviewer’s recognition of our study. The\nconstructive suggestion has helped us refine our work and improve its quality.\nReviewer #5:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nResponse: We sincerely appreciate the reviewer’s constructive feedback on our\nwork, which has improved its quality a lot."}, {"round": 3, "reviewer_comments": {"reviewer_2": "We do not have anything further to add and feel this manuscript is ready for publication in Nature Communications."}, "author_response": ""}]

Decision letter