Identification of a ligand-binding site on tubulin mediating the tubulin–RB3 interaction

Significance

Microtubules are essential for cell division, making them prime targets for cancer chemotherapy. While eight tubulin-binding sites for ligands are known, we have identified a ninth site, the Tumabulin site, located at the interface of α- and β-tubulin and the stathmin-like protein B3 (RB3). This site is unique because its binding is entirely dependent on RB3. We demonstrate that small molecules binding to this site don’t directly disrupt microtubule polymerization but rather function as “molecular glues”, strengthening the RB3–tubulin interaction and significantly enhancing RB3’s inherent tubulin depolymerizing activity. This represents a unique mechanism of action for tubulin inhibition and the finding of the Tumabulin site opens avenues for designing selective cancer drugs targeting cancers with elevated RB3 expression.

Abstract

For decades, microtubules—composed of αβ-tubulin dimers—have been primary targets for cancer chemotherapy. While eight binding sites on the tubulin dimer have been structurally characterized, this study reveals a ninth. We found that the tubulin inhibitor Tumabulin-1 (TM1, a BML284 derivative) binds simultaneously to the well-known colchicine site and a previously unknown site, designated as Tumabulin site. This site resides at the interface of α1-tubulin, β1-tubulin, and RB3 within the tubulin–RB3–tubulintyrosine ligase complex. Remarkably, two TM1 molecules bind cooperatively to this relatively large pocket, interacting with all three proteins. Crucially, this binding is dependent on RB3; it is absent when RB3 is missing or the key residue H71 is mutated (H71Q). We further designed and synthesized Tumabulin-2 (TM2) that selectively binds the Tumabulin site, excluding binding the colchicine site. TM2 acts as a molecular glue, strengthening the interaction between RB3 and the tubulin dimer and consequently enhancing RB3’s tubulin-depolymerizing activity. In conclusion, our findings confirm the existence of a ninth tubulin-binding site and offer a promising foundation for developing Tubulin–RB3 molecular glues as a next generation of anticancer therapeutics.

αβ–Tubulin heterodimers, the main components of microtubules that are essential for cancer cell mitosis, are primary targets for cancer chemotherapeutics (1). Drugs such as paclitaxel and vinca alkaloids, which inhibit tubulin, have shown significant success in inducing antimitotic effects in clinical settings (2). Tubulin inhibitors can be functionally categorized into three groups: Microtubule destabilizing agents (MDAs) that inhibit microtubule polymerization, Microtubule stabilization agents (MSAs) that promote microtubule polymerization, and the recently discovered Microtubule-Targeting Degraders (MTDs) that facilitate tubulin degradation (1). The recently discovered MTDs also work as well as MDAs. They inhibit tubulin assembly and induce tubulin degradation and it is unclear where more MDA do induce degradation. Structural biology has identified eight binding sites on tubulin dimers, including sites for paclitaxel (3), colchicine (4), vinblastine (5), laulimalide (6), maytansine (7), pironetin (8), gatorbulin (9, 10), and todalam (11). MSAs act as inhibitors at the paclitaxel- and laulimalide-binding sites, while MDAs act as inhibitors at the colchicine-, vinblastine-, maytansine-, pironetin-, gatorbulin-, and todalam-binding sites (1). MTDs primarily target the gatorbulin site and also include some colchicine-site inhibitors (1). However, additional research utilizing molecular dynamics and crystallographic fragment screening suggests the possible existence of 10 more binding sites on tubulin (12). These sites not only mediate tubulin polymerization or degradation but might also potentially regulate tubulin’s interaction with microtubule-associated proteins (MAPs) (12). These findings are captivating and encourage further exploration of additional binding sites on tubulin.

RB3, also known as Stathmin-4, is a member of the Stathmin family of proteins, which also includes Stathmin-1, Stathmin-2, and Stathmin-3 (13). These proteins belong to the MAPs family, with each member being capable of binding to two molecules of tubulin dimers and thereby inhibiting the further polymerization of tubulin into microtubes (14). Among the Stathmin family, Stathmin-1 has received the most research attention, being highly expressed in various tumor cells and significantly associated with the development and progression of tumors (13). In contrast, the study of other family members, such as Stathmin-4, is relatively limited. For instance, Stathmin-4 was only found to be expressed in a subset of neurons and is associated with nervous system development (15). Furthermore, a few studies have suggested a potential link between Stathmin-4 and the differentiation of neuroblastoma (16). No further reports have explored the relationship between Stathmin-4 and tumors. The Stathmin family of proteins has a simple structure, consisting of just a secondary structure α-helix without a complex tertiary structure and lacks a compound-binding pocket, making it an undruggable target. In structure biology, Stathmin-4 is commonly used in X-ray crystallography experiments to maintain tubulin in an unpolymerized state, which has been invaluable for investigating the details of tubulin–inhibitor interactions (17). However, to date, no tubulin inhibitors have been reported to modulate the interaction between tubulin and Stathmin-4, and this could be a potential direction for future research on tubulin inhibitors.

In this study, we found a ninth binding site (Tumabulin site) on tubulin located at the interface between α1-tubulin, β1-tubulin, and RB3. Small molecules binding to this site enhance the interaction between RB3 and tubulin to inhibit tubulin polymerization. This finding represents the first identified site that mediates the interaction between tubulin and MAPs. This breakthrough opens avenues for the rational design of analogs aimed at developing a next generation of anticancer drugs.

Results

Tumabulin-1 (TM1) Binds to a Ninth Site on Tubulin.

Our previous study revealed that BML284 (Fig. 1A) functions as a tubulin inhibitor by binding to the colchicine site (18). In our ongoing research aimed at developing more potent tubulin inhibitors, we synthesized numerous derivatives of BML284 (19). Among these derivatives, TM1 (Fig. 1A and SI Appendix, Scheme S1) displayed superior activity compared to BML284, exhibiting antiproliferation IC₅₀ values ranging from 17.89 to 46.01 nM on cancer cells, whereas BML284 had IC₅₀ values of 90.11 to 346.78 nM (SI Appendix, Table S1). Also, TM1 was more potent to inhibit in vitro tubulin polymerization than BML284 (SI Appendix, Fig. S1). To gain further insights, we utilized the widely employed Tubulin–RB3–tubulintyrosine ligase (TTL) complex crystal (17), which consists of two tubulin dimers, one RB3 protein stathmin-like domain (RB3) and one TTL. By analyzing the crystal structure of the Tubulin–RB3–TTL complex bound to TM1 (Fig. 1B and SI Appendix, Table S2), we observed that TM1 interacts with the colchicine site in a similar manner to BML284 as expected (SI Appendix, Fig. S2). Surprisingly, we also found that TM1 simultaneously binds to a previously unidentified site, as confirmed by the 2mFo-DFc electron density (Fig. 1C). This site could be named as Tumabulin site, is located within a region surrounded by α1-tubulin, β1-tubulin, and RB3 in the Tubulin–RB3–TTL complex. Notably, two TM1 molecules stack together within this site (Fig. 1 C and D), filling the binding pocket and forming strong intermolecular interactions. These interactions include five hydrogen bonds, as depicted in Fig. 1E, as well as π–π stacking interactions between three aromatic groups, which guaranteed a stable TM1–TM1 complex. For clarity, we will refer to the TM1 molecule closer to RB3 as L-TM1 and the other as U-TM1. U-TM1 forms hydrogen bonds through its amino group with the main chain carbonyl oxygen of αH406 and the main chain nitrogen of αG410, the 3′-oxygen atom of U-TM1 forms a hydrogen bond with the main chain nitrogen of αE411, and the 1′-oxygen atom of U-TM1 forms a hydrogen bond with the side chain of RB3’s R61(Fig. 1 F and G). L-TM1 forms a hydrogen bond with the side chain of R61 via its 1′-oxygen atom, and its 3′-oxygen atom forms a hydrogen bond with the main chain nitrogen of βD161. The amino group of L-TM1 interacts with the side chain of βR156 and the main chain carbonyl oxygen of βE194, the 1″-nitrogen atom of L-TM1 also engages in a hydrogen bond with the side chain of RB3’s H71(Fig. 1 F and G).

Fig. 1.

TM1 interaction with tubulin revealed in structural biology. (A) The chemical structures of BML284 and TM1 are displayed. (B) An overall structure of the TM1–Tubulin–RB3–TTL complex is shown, with each component colored differently for clarity: TTL is depicted in red, RB3 in blue, α-tubulin in gray, and β-tubulin in black. TM1 molecules are presented as spheres colored yellow. (C) A cross-sectional view of the binding pocket occupied by TM1 at the Tumabulin site. The Tubulin–RB3–TTL complex is rendered in surface representation, with two TM1 molecules shown in yellow sticks. (D) The electron density map of TM1 at the Tumabulin site is visualized. The 2mFo-DFc omit map, colored light blue, is contoured at 1δ, 2δ, and 3δ. (E) The hydrogen bond interactions between the two TM1 molecules are illustrated, with the bonds depicted as yellow dotted lines. (F and G) Depictions of hydrogen bond interactions involving TM1 with the Tubulin–RB3–TTL complex are shown from two distinct perspectives. The Tubulin–RB3–TTL complex is represented in cartoon form, with hydrogen bond interactions shown as yellow dotted lines.

Direct Biochemical Assay Confirms the Binding of TM1 to Colchicine Site and Tumabulin Site of Tubulin.

The crystal structure reveals that the tubulin–RB3 complex possesses two colchicine sites (one on each tubulin dimer) and one tumabulin site. Therefore, a 4:1 binding stoichiometry of TM1 to tubulin–RB3 was anticipated—two TM1 molecules binding to the tumabulin site and another two TM1 binding to two colchicine sites. To ensure that the interaction between TM1 and the Tumabulin site is not an artifact caused by the highly concentrated environment of the crystal, we conducted additional experiments to measure the binding of TM1 to the Tubulin–RB3 complex or tubulin in solution. We incubated the Tubulin–RB3 complex (10 μM) or tubulin (20 μM) in solution with varying concentrations of TM1 or BML284 (used as a reference compound). The amount of bound TM1 or BML284 was then collected and quantified using liquid chromatography–tandem mass spectrometry (LC-MS/MS). As depicted in Fig. 2A, at concentrations of 64, 128, and 256 μM of TM1, the stoichiometry was determined to be 2.70, 3.42, and 4.04 TM1 molecules per tubulin–RB3 complex, respectively, indicating the formation of a 4:1 TM1/Tubulin–RB3 complex. In contrast, the stoichiometry was 1.78, 2.10, and 2.39 for BML284 per Tubulin–RB3 complex, for concentrations of 64, 128, and 256 μM of BML284, respectively, indicating a 2:1 BML284/Tubulin–RB3 complex. These results confirmed that only TM1, but not BML284, could bind to the Tumabulin site. When we examined the binding stoichiometry of TM1 and BML284 to tubulin dimer alone (without RB3), we observed a 1:1 binding ratio (compound: tubulin dimer) for both TM1 and BML284 (Fig. 2B), indicating that RB3 is a prerequisite for TM1’s binding to the Tumabulin site. We attempted to soak both TM1 and colchicine into the Tubulin–RB3–TTL complex crystal and aim to inhibit the binding of TM1 to the colchicine site. However, it was found that colchicine could not displace TM1 at the colchicine site, and we obtained a Tubulin–RB3–TTL–TM1 complex similar to the one shown in Fig. 1B. To block the colchicine site, we used a covalent colchicine-site ligand called EBI [N,N-ethylene bis(iodoacetamide)] (20, 21). Subsequently, we determined the binding stoichiometry of TM1 to the EBI–Tubulin–RB3 complex, revealing a 2:1 binding ratio (TM1/EBI–Tubulin–RB3 complex) (Fig. 2C), further confirming the binding of TM1 to the Tumabulin site. Additionally, using the same EBI–Tubulin–RB3 complex, we assessed the binding affinity of TM1 to the Tumabulin site, obtaining a dissociation constant (Kd) of 19.01 ± 2.41 μM (Mean ± SEM, n = 3) through a microscale thermophoresis (MST) assay (Fig. 2D).

Fig. 2.

Quantification of TM1 binding to tubulin in solution. (A and B) The binding of TM1 to the Tumabulin site was evaluated in solution by incubating various concentrations of TM1 or the positive control BML284 with a Tubulin–RB3 complex at 10 μM (A) or tubulin at 20 μM (B) for 10 min. The bound compounds were then quantified using LC-MS/MS. The results are depicted as the molecular ratio of the compounds to tubulin–RB3 complex (A) or tubulin dimer (B). The data are presented as the mean ± SEM of three independent experiments. (C) The binding of TM1 to the EBI–Tubulin–RB3 complex was assessed at different TM1 concentrations. This was also evaluated by incubating the complex with 10 μM EBI–Tubulin–RB3 for 10 min, followed by quantification of the bound TM1 using LC-MS/MS. The outcome is shown as the molecular ratio of the TM1 to the EBI–tubulin–RB3 complex. These data are also given as the mean ± SEM of three independent experiments. (D) The binding affinity of TM1 to the EBI–Tubulin–RB3 complex was determined using the MST method. The data points reflect the mean ± SEM of three technical replicates for each experiment.

H71 of RB3 Is Important for TM1 Binding to Tumabulin Site.

Based on the structures of BML284 and TM1, we understand that the indol-4-yl group of TM1 is essential for its binding to the Tumabulin site. In the crystal structure, the nitrogen of the indol-4-yl group forms a hydrogen bond with the side chain of RB3’s H71, and there is also a π–π stacking interaction between the indol-4-yl group and H71. Moreover, TM1 does not bind to the Tumabulin site in the absence of RB3. These findings suggest that the interaction between the indol-4-yl group of TM1 and the side chain of RB3’s H71 is crucial for TM1’s binding to the Tumabulin site. RB3, also known as stathmin-4, belongs to the stathmin-family proteins, which includes stathmin 1-4 (13). Notably, at position 71, only stathmin-4 has a histidine residue, whereas stathmin 1-3 have a glutamine residue instead (Fig. 3A). We examined the binding stoichiometry of TM1 to all four tubulin–stathmin complexes. As shown in Fig. 3B, TM1 binds to the tubulin–stathmin4 complex with a 4:1 ratio (TM1/tubulin–stathmin4 complex), while it binds to the other three tubulin–stathmin complexes with a 2:1 ratio (TM1/tubulin–stathmin 1,2 or 3 complex). This indicates that TM1 specifically binds to the Tumabulin site on the tubulin–stathmin4 (RB3) complex but not on the other tubulin–stathmin complexes. We further investigated the binding of TM1 to the mutant RB3(H71Q)–Tubulin complex and observed a 2:1 ratio [TM1/tubulin–RB3 (H71Q) complex] binding (Fig. 3C). The crystal structure of the mutant RB3(H71Q)–Tubulin complex with TM1 showed that TM1 was only present at the colchicine sites and not at the Tumabulin site (Fig. 3D). Instead, a MES molecule occupied the Tumabulin site in the mutant crystal (SI Appendix, Fig. S3). These results further confirmed the binding of TM1 to the Tumabulin site and implied that the binding is dependent on the interactions between the indol-4-yl group of TM1 and RB3’s H71.

Fig. 3.

The essential role of RB3’s H71 in TM1 binding to the Tumabulin site. (A) The amino acid sequence of stathmin1, 2, 3, and 4 at the ID-βV site. (B) Various concentrations of TM1 were incubated with a tubulin–stathmin complex at 10 μM, including stathmin 1, 2, 3, and 4, for 10 min. The bound TM1 was then quantified using LC-MS/MS, and the results are presented as the molecular ratio of TM1 to the tubulin–stathmin 1, 2, 3, or 4 complex. These data are given as the mean ± SEM of three independent experiments. (C) The binding of TM1 to the tubulin-stathmin4 complex, where the H71 residue is mutated to glutamine (H71Q), was assessed with the same experimental procedure as in panel (B). The molecular ratio of TM1 to the tubulin–RB3(H71Q) complex is shown in this graph, which also features the mean ± SEM of three independent experiments. (D) An overall view of the TM1–Tubulin–RB3(H71Q)-TTL complex is presented. TTL is depicted in yellow, RB3(H71Q), is colored blue, α-tubulin is gray, and β-tubulin is black. The TM1 molecules are shown as spheres in yellow.

Finding of Tumabulin-2 (TM2), a TM1 Derivative That Binds Exclusively to the Tumabulin Site but Not Colchicine Site.

Since TM1 binds to both the colchicine site and the Tumabulin site, potential interference between these two sites may hinder accurate analysis of the specific biochemical function of TM1 binding to the Tumabulin site. To overcome this limitation, we aimed to develop a modified TM1 derivative that binds exclusively to the Tumabulin site without interacting with the colchicine site. By carefully comparing the details of TM1’s interaction with tubulin at both sites, we observed that the indole ring of TM1 is positioned within the β-tubulin at the colchicine site, leaving limited space for further modifications or additions of chemical groups. However, at the Tumabulin site, the indole ring is located on the outer side, offering more opportunity for structural adjustments (Fig. 4A). Therefore, we introduced a cyano-substitution at the para position of the benzene ring in the indole moiety, resulting in the synthesis of compound TM2 (Fig. 4B and SI Appendix, Scheme S1). In vitro activity assays demonstrated that TM2 lost its antiproliferative activity on HeLa cells (Fig. 4B), suggesting that it no longer binds to the colchicine site. Additionally, TM2 failed to compete with a fluorescent probe of the colchicine site, [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC)] (22) (Fig. 4C). In contrast, TM1 successfully competed with MTC with a Kd value of 0.60 ± 0.13 μM (Mean ± SEM, n = 3) (Fig. 4C). This confirmed that TM2 does not bind to the colchicine site. We determined a 2:1 binding stoichiometry of TM2 to the tubulin–RB3 complex (Fig. 4D). TM2 effectively competed with TM1 for binding to the Tumabulin site on the tubulin–RB3 complex (Fig. 4E). Furthermore, in the EBI–tubulin–RB3 complex, TM1 also competed with TM2 for binding to the Tumabulin site (Fig. 4F). Through MST experiments, we accurately measured the Kd value of TM2 binding to the Tumabulin site of the tubulin–RB3 complex as 36.48 ± 7.46 μM (Mean ± SEM, n = 3) (Fig. 4G), while TM2 did not bind to tubulin in the absence of RB3 (Fig. 4H). These findings collectively confirm that TM2 exhibits selectivity for the Tumabulin site on the tubulin–RB3 complex, without interacting with the colchicine site. Unfortunately, we were unable to obtain a crystal structure of the TM2–tubulin–RB3 complex using either soaking or cocrystallization methods.

Fig. 4.

TM2, a derivative of TM1, binds exclusively to the Tumabulin site but not colchicine site. (A) Schematic showing the cavity around the colchicine site and the Tumabulin site for TM1. (B) Structures of TM1 and TM2, and their antiproliferation IC₅₀ values against HeLa cancer cells. (C) Displacement isotherm of MTC by TM1 and TM2. Line is best fit of the TM1 binding equilibrium constant, assuming 0.8 sites per tubulin dimer. (D) Binding stoichiometry detection of TM2 to tubulin–RB3 complex. (E) Indicated concentrations of TM2 were incubated with TM1 (125 μM) preincubated tubulin–RB3 complex (10 μM) for 10 min and then the bound TM1 and TM2 were quantified by LC-MS/MS, and then the binding stoichiometries were calculated. (F) Indicated concentrations of TM1 were incubated with TM2 (125 μM) preincubated EBI–tubulin–RB3 complex (10 μM) for 10 min, and then the bound TM2 and TM1 were quantified by LC-MS/MS, and then, the binding stoichiometries were calculated. (G and H) MST experiments determining the binding affinity of TM2 to Tubulin–RB3 complex (G) or tubulin (H). All data were shown as means ± SEM of three independent experiments.

TM2 Enhances RB3’s Binding to Tubulin to Inhibit Tubulin Polymerization.

The crystal structure analysis of the TM1–tubulin–RB3 complex revealed intriguing details about their interaction. Upon TM1 binding to the Tumabulin site, E194 in β-tubulin shifted downward, forming a salt bridge with H71 of RB3 (Fig. 5 A and B). Notably, no interaction between H71 of RB3 and α-tubulin was observed in the Apo-tubulin crystal (Fig. 5A). Additionally, R61 of RB3 underwent a conformational change, establishing two hydrogen bonds with TM1 and a salt bridge with E411 of α-tubulin (Fig. 5 C and D). In contrast, in the Apo-tubulin crystal, R61 only formed two hydrogen bonds, one with the side chain of αT109 and the other with the main chain of αE411 (Fig. 5C). These specific binding interactions suggest that the Tumabulin site ligand might enhance the binding affinity between RB3 and the tubulin dimer. To investigate the impact of TM2 on RB3–tubulin binding, we employed an MST assay. The results presented in Fig. 5E demonstrate that RB3 binds to tubulin with a Kd value of 127 ± 9 nM (Mean ± SEM, n = 3). Upon the addition of 100 μM TM2, the binding Kd value of RB3 to tubulin significantly increase to 11 ± 3 nM (Mean ± SEM, n = 3), signifying a substantial enhancement effect. We further examined whether TM2 could amplify the tubulin depolymerization effect of RB3 on tubulin. As shown in Fig. 5F, TM2 did not exhibit any depolymerization effect on tubulin. However, RB3 significantly inhibited tubulin polymerization, an effect that was significantly enhanced by TM2. This suggests that the binding of TM2 to the Tumabulin site augments the tubulin depolymerization activity of RB3. Since the tubulin depolymerization effect of TM2 was contingent upon the presence of RB3, we evaluated the antiproliferative effect of TM2 on cancer cells overexpressing RB3. HeLa cells stably expressing RB3 (HeLa-RB3) and H1385 cancer cells with high RB3 expression levels were chosen for this study (SI Appendix, Fig. S4). TM2 exhibited antiproliferative IC₅₀ values of 15.39 ± 2.94 μM and 28.78 ± 4.95 μM in HeLa-RB3 and H1385 cell lines, respectively, while showing no effect on HeLa cells (Fig. 5G). Immunofluorescence experiments employing α-tubulin antibodies revealed that TM2 did not alter microtubule morphology in HeLa cells. However, in HeLa–RB3 and H1385 cells, which exhibit high RB3 expression, TM2 significantly inhibited tubulin polymerization (Fig. 5H). Collectively, these findings demonstrate that TM2 binds to the Tumabulin site to enhance the interaction between tubulin and RB3, ultimately leading to the inhibition of tubulin polymerization in cancer cells with high RB3 expression levels.

Fig. 5.

TM2 enhances RB3’s binding to tubulin to inhibit tubulin polymerization. (A) A magnified view of the Tumabulin site within an apo-Tubulin-RB3-TTL structure (PDB code:5JQG) highlights the positions of βE194 and RB3’s H71. The proteins are depicted in cartoon form, with βE194 and H71 of RB3 illustrated in stick representation. (B) In TM1–Tubulin–RB3–TTL structure, a close-up of the Tumabulin site presents the salt bridge interaction between βE194 and H71 of RB3, as well as the hydrogen bond between H71 of RB3 and TM1. The proteins are shown in cartoon form, while βE194, H71 of RB3, and TM1 are depicted in stick representation. (C) A zoomed-in view of the Tumabulin site from the apo-Tubulin-RB3-TTL structure (PDB code:5JQG) displays two hydrogen bonds: R61 of RB3 interacting with the side chain of αT109, and R61 of RB3 bonding to the main chain of αE411. The proteins are shown in cartoon form, with R61 of RB3, αT109, and αE411 shown in stick representation. (D) The same close-up view of the Tumabulin site in the TM1–Tubulin–RB3–TTL structure reveals two hydrogen bonds between R61 of RB3 and two TM1 molecules, respectively, and a salt bridge between R61 of RB3 and αE411. The proteins are depicted in cartoon form, with R61 of RB3, αT109, αE411, and TM1 shown in stick representation. (E) A MST assay to evaluate the impact of 100 μM TM2 on the RB3–tubulin interaction. The data points represent the mean ± SEM of three technical replicates. (F) The effect of TM2 (100 μM) on in vitro tubulin polymerization assay of tubulin or Tubulin–RB3 complex. These data are given as the mean ± SEM of three independent experiments. A t test was used to calculate the P-value for the 60-min time point data. (G) Antiproliferative effects of TM2 in HeLa, HeLa-RB3, and H1385. (H) HeLa, HeLa-RB3, and H1385 cells were treated with or without TM2 (50 μM) for 16 h and then microtubule morphology was monitored by immunofluorescence using α-tubulin antibody. (Scale bar: 10 μm.)

Discussion

In this study, through crystallographic and stoichiometric analyses, we found that TM1, a BML284 derivative, binds simultaneously to both the colchicine site and a ninth site (Tumabulin site) on the tubulin–RB3–TTL complex. Leveraging this binding information, we designed and synthesized TM2, a TM1 derivative, which selectively binds only to the Tumabulin site, excluding the colchicine site. We demonstrate that TM2, by binding to the Tumabulin site, acts as a molecular glue, significantly strengthening the interaction between RB3 and tubulin, thereby enhancing RB3’s tubulin depolymerization activity. This finding identifies a binding site mediating the interaction between tubulin and MAPs, providing a foundation for the development of a next class of tubulin inhibitors targeting tubulin–MAP interactions.

Tubulin is expressed widely across various tissues without exhibiting selectivity, which implies that directly targeting tubulin would inevitably lead to toxicity (23). However, by focusing on the interaction between tubulin and MAPs (12, 24, 25), it becomes possible to reduce toxicity while maintaining high efficacy. Our study demonstrated that TM1 and TM2 bind to the Tumabulin site on tubulin only in the presence of RB3, a finding not observed in tubulin–stathmin 1, 2, or 3 complexes. Given the limited research on RB3 expression in various cancers, we consulted the Human Protein Atlas (www.proteinatlas.org) and identified several cancer cell lines with relatively high RB3 expression, including lung cancer cell lines and neuroblastoma cell lines. We expected that TM2 might exhibit selective antiproliferative effects in these cell lines. Indeed, while inactive in parental HeLa cells, TM2 demonstrated significant antiproliferative activity and tubulin depolymerization in RB3-overexpressing HeLa and H1385 cells. These findings support the potential of developing less toxic tubulin inhibitors by targeting the Tumabulin site, exploiting its RB3 dependence for improved selectivity.

Mechanism studies suggest that TM2 functions as tubulin–RB3 molecular glues, specifically inhibiting tubulin polymerization in RB3-overexpressing cancers. Although we were unable to obtain a crystal structure of the TM2–tubulin–TTL-RB3 complex, MTC competition assays, binding stoichiometry measurements, and MST experiments confirmed its specific binding to the Tumabulin site and not the colchicine site. Given the relatively low binding affinity to the Tumabulin site and antiproliferative activity of TM2, further optimization is needed to identify more potent lead compounds for the continued exploration of the Tumabulin site and development of tubulin–RB3 molecular glue inhibitors.

Collectively, we have identified a novel agent binding site on tubulin that exhibits a unique function in mediating the interaction between tubulin and RB3. Our findings lay a crucial foundation for the discovery of a next generation of anticancer tubulin inhibitors.

Materials and Methods

SI Appendix provides detailed descriptions of the synthesis of TM1 and TM2 (SI Appendix, Scheme S1), biological methods, cell lines, proteins, and reagents. Specifically, SI Appendix includes complete descriptions of protein expression and purification, crystal structure determination, cell culture, CCK8 assay, in vitro tubulin polymerization assay, binding stoichiometry determination, MTC competition assay, immunofluorescence, and MST assay. Supporting data are also provided, including the antiproliferation activity of TM1 (SI Appendix, Table S1), crystallographic data collection and refinement statistics (SI Appendix, Table S2), and supplementary figures illustrating in vitro tubulin polymerization (SI Appendix, Fig. S1), TM1 binding to the colchicine site (SI Appendix, Fig. S2), a close-up view of the Tumabulin site in the TM1–Tubulin–RB3(H71Q)–TTL structure (SI Appendix, Fig. S3), RB3 protein levels in HeLa, H1385, and HeLa-RB3 cell lines (SI Appendix, Fig. S4), and NMR and MS spectra of TM1 and TM2 (SI Appendix, Figs. S5–S9).

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

The crystallographic data, comprising both the coordinates and the structure factors, have been submitted to the Protein Data Bank under the accession code: 9IMO (Tubulin–RB3–TTL–TM1) (26), 9IM5 [Tubulin–RB3(H71Q)–TTL–TM1] (27). All other data are included in the manuscript and/or SI Appendix.