# Microtubule-targeting agents for cancer treatment:
Seven binding sites and three strategies

Xingyu Wang, Benoît Gigant, Xi Zheng, Qiang Chen

► To cite this version:

Xingyu Wang, Benoît Gigant, Xi Zheng, Qiang Chen. Microtubule-targeting agents for cancer treatment: Seven binding sites and three strategies. MedComm – Oncology, 2023, 2 (3), pp.e46. 10.1002/mog2.46. hal-04193641

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Submitted on 1 Sep 2023

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# Microtubule-targeting agents for cancer treatment: Seven binding sites and three strategies

Xingyu Wang<fnref n="1" />

Benoît Gigant<fnref n="2" />

<figure><img src="image_3.png" /></figure>

Xi Zheng<fnref n="3" />,<fnref n="4" />

Qiang Chen<fnref n="1" />

<figure><img src="image_4.png" /></figure>

Correspondence

Xi Zheng, Department of Thoracic
Surgery, West China Hospital, Sichuan
University, Chengdu 610041, China.

Email: zhengxi@scu.edu.cn

Qiang Chen, Department of Biotherapy,
Cancer Center and State Key Laboratory of
Biotherapy, West China Hospital, Sichuan
University, Chengdu 610041, China.

Email: qiang_chen@scu.edu.cn

Funding information

1-3-5 Project for Disciplines of Excellence,
West China Hospital, Sichuan University,
Grant/Award Number: ZYJC21073;
National Natural Science Foundation of
China, Grant/Award Number: 32270761;
Agence Nationale de la Recherche,
Grant/Award Number: ANR-22-CE11-
0002-01; Fondation ARC pour la
Recherche sur le Cancer,
Grant/Award Number:
ARCPJA2021050003651; Natural Science
Foundation of Sichuan Province,
Grant/Award Number: 2023NSFSC1890

Abstract

Microtubules are pivotal in diverse cellular functions encompassing cell signaling, morphology, intracellular trafficking, and cell mitosis/division. They are validated targets for disease treatment, notably hematological cancers and solid tumors. Microtubule-targeting agents (MTAs) exert their effects by modulating microtubule dynamics, impeding cell proliferation, and promoting cell death. Recent advances in structural biology have unveiled novel perspectives for investigating multiple binding sites and mechanisms of action used by MTAs. In this review, we first provide an overview of the intricate structure and dynamics of microtubules. Then we explore the seven binding sites and the three primary strategies (stabilization, destabilization, and degradation) harnessed by MTAs. Furthermore, we introduce the emerging domain of microtubule-targeting degraders, exemplified by PROteolysis TTargeting Chimeras and small-molecule degraders, which enable precise degradation of specific microtubule-associated proteins implicated in cancer pathogenesis. Additionally, we discuss the promising realm of precision-targeted approaches, including antibody-drug conjugates and the utilization of photopharmacology in MTAs. Lastly, we provide a comprehensive overview of the clinical applications of microtubule-targeting therapies, assessing their efficacy and current challenges. We aim to provide a global picture of MTAs development as well as insights into the microtubule-targeting drug discovery for cancer treatment.

KEYWORDS

cancer therapy, microtubule, microtubule-targeting agents, tubulin, tubulin-targeting sites

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *MedComm – Oncology* published by John Wiley & Sons Australia, Ltd on behalf of Sichuan International Medical Exchange & Promotion Association (SCIMEA).

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<footnote n="1">¹Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China</footnote>

<footnote n="2">²Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France</footnote>

<footnote n="3">³Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu, China</footnote>

<footnote n="4">⁴Lung Cancer Center, West China Hospital, Sichuan University, Chengdu, China</footnote>
# 1 | INTRODUCTION

Cancer continues to be a global health challenge, necessitating the development of innovative therapeutic strategies. Among the diverse therapeutic approaches, microtubule-targeting agents (MTAs) have emerged as prominent candidates for cancer treatment.<sup>1-4</sup> Microtubules are integral components of the cell cytoskeleton. They are dynamic filaments that play a pivotal role in various cellular processes, including cell division, intra-cellular trafficking, and maintenance of cell shape and structure.<sup>5-7</sup> Their crucial involvement in these vital cellular functions has rendered microtubules an attractive target for anticancer interventions.

MTAs are regarded as highly promising drugs for treating diverse cancers, including lung, breast, ovarian, and prostate cancers.<sup>8</sup> By disrupting the normal function of microtubules, MTAs have demonstrated their effectiveness in halting cell cycle progression and inducing programmed cell death.<sup>9,10</sup> These agents can be classified into three primary categories based on their mechanisms of action: microtubule-stabilizing agents (MSAs), microtubule-destabilizing agents (MDAs), and microtubule-targeting degraders (MTGs). MSAs, exemplified by taxanes and laulimalide/peloruside-A, promote microtubule polymerization and stabilization. Conversely, MDAs, such as vinca

alkaloids and colchicine, inhibit microtubule polymerization and promote depolymerization. The third category, MTGs, represents a novel class of MTAs that induce microtubule denaturation and degradation. Despite different molecular mechanisms, all the three types of MTAs lead to microtubules abnormality and subsequent cell death.

Recent advances in structural biology have greatly contributed to our understanding of the mechanisms of MTAs at the atomic level. In particular, seven distinct binding sites for MTAs have been identified: those of taxanes, of peloruside-A/laulimalide, of colchicine, of vinca alkaloids, of maytansine, and of pironetin, and the recently discovered gatorbulin site (Figure 1).

In this review, we aim to provide a detailed and up-to-date overview of MTAs for cancer treatment. We summarize the existing knowledge regarding the dynamics and structure of microtubules and highlight the well-established binding sites for MTAs, offering a comprehensive overview of the current status and challenges associated with the clinical use of these agents. Furthermore, we will delve into the challenges associated with traditional MTA development and introduce emerging precision-targeted approaches that hold promise for enhancing therapeutic efficacy and minimizing adverse effects. This review aims to contribute to the advancement of microtubule-targeted

<figure><img src="image_6.png" /><figcaption>**FIGURE 1** Seven binding sites of microtubule-targeting agents (MTAs). (A) Seven MTA binding sites of tubulin. Two tubulin heterodimers are shown in cartoon (α-subunits, sky blue; β-subunits, light green). The representative agents bound to the seven sites are shown in sphere representation: vinca site (vinblastine, purple, PDB ID 5J2T); maytansine site (olive, PDB ID 4TV8); peloruside A/laulimalide site (orange, PDB ID 4O4J); colchicine site (dark blue, PDB ID 4O2B); the seventh site/gatorbulin site (red, PDB ID 7ALR); pironetin site (gray, PDB ID 5LA6); and taxane site (paclitaxel, green, PDB ID 6WVR). Cevipabulin (pink, PDB ID 7CLD) binds to the vinca site as a microtubule stabilizer and to the seventh site as a tubulin degrader, respectively. (B) The chemical structures of representative agents, colored according to Panel A. MDA, microtubule-destabilizing agent; MSA, microtubule-stabilizing agent; MTG, microtubule-targeting degrader.</figcaption></figure>
therapies, hoping to provide valuable insights that can be
used to discover more effective treatments for cancer.

2 | MICROTUBULE STRUCTURE AND DYNAMICS

Microtubules are essential components of the eukaryotic cell cytoskeleton and play a crucial role in various cellular processes, including cell division, intracellular transport, cell shape maintenance, and cell motility.⁶,¹¹ They are hollow cylinders of about 25 nm in diameter formed by 13 parallel protofilaments, themselves consisting of αβ-tubulin (50 kDa each in size) heterodimers assembled into a head-to-tail fashion.¹²⁻¹⁴ Thus, microtubules possess polarity. The α-tubulin subunits are exposed at one end, known as the minus end, while the β-tubulin subunits are exposed at the other end, known as the plus end (Figure 2A). This polarity is essential for microtubule dynamics and their involvement in cellular processes such as cell division and intracellular transport.

The sophisticated dynamics of microtubules is exquisitely regulated both spatially and temporally in response to varied cellular requirements, primarily driven by guanosine triphosphate (GTP) hydrolysis

and guanosine diphosphate (GDP)-GTP exchange
cycles.<sup>15,16</sup> Specifically, whereas GTP bound to the
α-tubulin subunit, at the N site, is nonhydrolyzable
and nonexchangeable, the one bound to β-tubulin, at
the E site, can be hydrolyzed and exchanged with free
nucleotide.<sup>17</sup> The GTP-bound form promotes micro-
tubule growth, while GDP-bound form does not
polymerize. The continuous polymerization process
requires the presence of a GTP-bound tubulin cap
at the growing end of the microtubule.<sup>11</sup> The
stochastic loss of this cap triggers a phenomenon
known as catastrophe, characterized by a switch into a
depolymerization phase.<sup>11,18</sup> The transition from the
stable GTP-tubulin lattice to the metastable GDP-
tubulin lattice lies at the core of the intricate dynamics
observed in microtubule behavior.<sup>11,18</sup> Nonequilibrium
dynamics of microtubules encompasses two distinct
behaviors: dynamic instability and treadmilling.
Dynamic instability, the most characteristic dynamic
behavior, refers to individual microtubule ends
switching between growing and shrinking periods,
the transition between these two phases being
called catastrophes and rescues (Figure 2B).<sup>19</sup> Whereas
dynamic instability concerns both minus and plus ends
in vitro, the plus end grows faster and undergoes
more catastrophe and rescue events, that is, is more

<figure><img src="image_7.png" /><figcaption>FIGURE 2 Microtubule structure and dynamics. (A) Microtubule structure. Tubulin heterodimers consisting of α- (blue) and β-subunits (green) assemble into protofilaments in a head-to-tail fashion. Thirteen parallel protofilaments further polymerize to generate the hollow cylinder about 25 nm in diameter with the plus end exposing the β-tubulin and the minus end exposing the α-tubulin. (B) Cartoon illustrating structural intermediates of microtubule dynamic instability. The polymerization–depolymerization cycle is powered by GTP hydrolysis and GDP/GTP exchange, during which the microtubule undergoes catastrophe and rescue events. The GTP bound to α-tubulin subunit (called N site) is nonhydrolyzable and nonexchangeable, whereas the one bound to β-tubulin (called E site) can be hydrolyzed and exchanged with free GDP. Due to the delay of GTP hydrolysis, a GTP heterodimer-rich region (β-tubulin bound GTP showed in purple), named GTP cap, will form during tubulin polymerization. GDP, guanosine diphosphate; GTP, guanosine triphosphate.</figcaption></figure>
dynamic, than the minus end.¹² By comparison, treadmillking is characterized by net growth at one end of the microtubule and balanced net shortening at the other end.¹² The exquisite dynamic behavior allows microtubules to rapidly remodel their structure and participate in cellular processes. Disruption of microtubule dynamics, such as through the use of antimitotic drugs that suppress dynamic instability, can result in cell cycle arrest, abnormal formation of mitotic spindles, and apoptotic cell death.²⁰

Microtubule dynamics is modulated by tubulin posttranslational modifications (e.g., acetylation, phosphorylation, and detyrosination),²¹⁻²⁴ and by the expression of diverse tubulin isotypes.²⁵⁻²⁷ It is also tightly regulated through the coordination of microtubule-associated proteins (MAPs).²⁸,²⁹ MAPs, encompassing structural MAPs and motor MAPs, are involved in the regulation of microtubule dynamics, organization, intra-cellular transport, and cell division.³⁰ Structural MAPs, exemplified by MAP4, MAP2, and Tau, contribute to microtubule polymerization, spacing regulation, and the formation of intricate microtubule architectures within specific cellular compartments.²⁹ Motor MAPs, including dyneins and kinesins, bind to microtubules and generate directed movements along them and facilitate the transportation of cargos along microtubules, enabling crucial cellular functions such as protein trafficking, neurotransmitter release, and organelle positioning.²⁹

Increasing evidence has suggested that dysregulation of MAPs plays a role in cancer pathogenesis and is associated with tumor growth, metastasis, and resistance to cancer treatments.³¹,³² For instance, the overexpression of MAPs, such as MAP4, can enhance microtubule stability, resulting in resistance to microtubule-targeting anticancer drugs and promoting tumor cell survival.³³,³⁴ Dysregulation of motor MAPs involved in spindle formation, such as Eg5 (a kinesin-5 family member), can lead to chromosome missegregation and aneuploidy, commonly observed in cancer cells.³⁵⁻³⁷ Moreover, MAPs have emerged as potential diagnostic and prognostic markers in diverse cancer types. Notably, in urothelial carcinoma, MAP1B shows significant upregulation associated with cancer progression.³⁸ Similarly, elevated expression of MAP4 is significantly correlated with enhanced metastatic potential of cancer cells and unfavorable long-term survival outcomes among patients diagnosed with lung adenocarcinoma.³⁴ In the context of non-small-cell lung cancer (NSCLC), the enriched expression of MAP2, MAP4, MAP6, MAP7, and MAP7D3 is associated with a favorable prognosis, improved patient survival outcomes, and a reduced likelihood of disease recurrence.³⁹

The understanding of microtubule structure and dynamics establishes the groundwork for exploring

precise microtubule-targeting sites, while also yielding valuable insights into cancer biology and the identification of therapeutic targets.

# 3 | TUBULIN-BINDING SITES AND MECHANISMS OF ACTION OF MSAs

## 3.1 | Taxane site

Paclitaxel (Taxol®), the first discovered MSA, was initially isolated from *Taxus brevifolia* tree bark in 1966.⁴⁰ It was found to possess antileukemic and antitumor properties. Due to its potent antiproliferative activity, paclitaxel was approved by the US Food and Drug Administration (FDA) in 1992 for treating metastatic ovarian cancer. Docetaxel (Taxotere®), a semisynthetic analog of paclitaxel, emerged as a second-generation broad-spectrum anticancer drug and is widely employed for solid tumor malignancies, including breast cancer, ovarian cancer, and NSCLC.⁴¹,⁴²

Early investigations revealed that paclitaxel binds specifically and reversibly to microtubules, suggesting the existence of a distinct binding site.⁴³ The atomic-level structure of tubulin–paclitaxel was elucidated using electron crystallography in 1998.⁴⁴ Subsequent advances in X-ray crystallography provided high-resolution insights into taxane-site interaction. This is exemplified by the binding of taxane-site MSAs to tubulin, including epothilone, zampanolide, and taccalonolide.⁴⁵⁻⁴⁷

The taxane site, recognized as the initially identified microtubule-stabilizing site, plays a crucial role in the mechanism of action of MSAs.⁴⁰,⁴⁸ These agents exhibit specific and reversible binding to microtubules, effectively inhibiting depolymerization and promoting stabilization.⁴⁹ The taxane-binding site agents, interact specifically with β-tubulin at the interface between neighboring subunits and form hydrogen bonds with specific residues on M-loop that a key element involved in establishing lateral contacts (between protofilaments) in microtubules.⁵⁰,⁵¹

Binding of taxane-site agents induces conformational changes in the β-tubulin subunit, leading to structural alterations in the microtubule itself.⁵² These conformational changes are crucial in preventing the curvature of protofilaments, a critical step in microtubule depolymerization.⁴⁹ By promoting the stability of protofilaments, taxane-binding site agents facilitate interactions between the M-loop of one β-monomer and a β-monomer of an adjacent protofilament.⁵³,⁵⁴ Recently, Prota et al.⁵⁵ conducted a study that elucidated the preferential binding of taxanes to microtubules over tubulin, as well as the mechanism underlying the longitudinal expansion of microtubule lattices
driven by the accommodation of the taxane core. These findings provide new molecular insights into taxane-mediated microtubule stabilization.

## 3.2 | Laulimalide/peloruside-A site

Laulimalide and peloruside A, nontaxane MSAs derived from marine sponges,⁵⁶,⁵⁷ have demonstrated potent antiproliferative activity against P-glycoprotein-mediated multidrug resistance (MDR) cells and paclitaxel-resistant cells.⁵⁸⁻⁶⁰ These compounds offer improved aqueous solubility and enhanced tolerability compared to taxol. However, the clinical development of laulimalide and peloruside A have been hindered by their narrow therapeutic index, severe toxicity, and limited tumor inhibition.⁶¹ Combination therapy involving nontaxane site and taxane-site agents shows promise in overcoming these challenges and has emerged as an important strategy in cancer treatment.⁶¹,⁶²

The laulimalide/peloruside-A site, representing an alternative MSAs binding site, serves as a target for microtubule stabilization. Elucidation of the molecular model of tubulin-bound laulimalide and peloruside A has been achieved through structural biology, revealing that this site is located on the external surface of the microtubule.⁵²,⁶³ Upon binding to the microtubule, laulimalide/peloruside-A deeply insert their side chain and macrocycle into a specific pocket formed by β-tubulin residues, inducing allosteric stabilization of the M-loop region without any regular secondary structure and establishing lateral contacts between adjacent tubulin subunits across protofilaments.⁶³ These interactions do not significantly alter the overall conformation of the αβ-tubulin dimer but effectively impede microtubule disassembly. Furthermore, an intriguing phenomenon of allosteric crosstalk between the laulimalide/peloruside A-binding site and the taxane pocket has been observed in both unassembled and assembled tubulin states. This discovery suggests a potential synergistic effect between laulimalide/peloruside and taxane-site ligands in modulating tubulin assembly and effectively inhibiting cancer cell growth.⁶³

# 4 | TUBULIN-BINDING SITES AND MECHANISMS OF ACTION OF MDAs

## 4.1 | Colchicine site

Colchicine, initially extracted from the autumn crocus *Colchicum autumnale* in 1820,⁶⁴ has a long history for

the treatment of diseases such as gout and familiar Mediterranean fever.⁶⁵ However, neither colchicine nor colchicine-binding site inhibitors (CBSIs) have been approved as antitumor drugs due to their high toxicity and narrow therapeutic index in disrupting microtubule function across different cell types.⁶⁶,⁶⁷

In 2004, Ravelli et al.⁶⁸ characterized the X-ray structure of tubulin in complex with colchicine, providing structural insights into its binding mechanism. In subsequent studies, the structure of many CBSIs bound to tubulin has been determined.⁶⁹⁻⁷¹ Zhang et al.⁷⁰ elucidated the crystal structures of tubulin complexed with a diverse set of CBSIs, including lexibulin, nocodazole, plinabulin, and tivantinib. The colchicine binding site is mainly contributed by the tubulin β subunit, near the interface with the α subunit and adjacent to the GTP N-site of α-tubulin. This binding pocket comprises two hydrophilic centers suitable for hydrogen bond formation and three hydrophobic centers: (i) the α/β tubulin interface, (ii) the major hydrophobic core, and (iii) the “deep binding site” of some ligands.⁶⁹ Collectively, ligands targeting the colchicine site interact with unassembled tubulin and inhibit tubulin polymerization. This inhibition occurs by preventing curved (unassembled) tubulin from adopting a straight structure through steric clash between colchicine and α-tubulin or by inhibiting the subdomain movements of tubulin during the curved-to-straight structural conversion.⁷² Notably, a few compounds such as KXO-1, nocodazole, and tivantinib exhibit inhibitory effects on kinase signaling pathways as well as tubulin polymerization, suggesting a novel multitarget therapeutic approach.⁷³⁻⁷⁵

## 4.2 | Vinca alkaloids site

Vinca alkaloids, initially extracted from the Madagascar periwinkle *Catharanthus roseus* in the 1950s, were originally recognized for their antidiabetic and antimalarial properties.⁷⁶⁻⁷⁸ Subsequent studies demonstrated the potent antileukemic effects of vinblastine, the prototype of vinca alkaloids, leading to its development as a promising chemotherapeutic agent against cancer. The vinca-site family of MDAs has since exhibited clinical success in the treatment of hematologic malignancies, lymphatic neoplasms, and solid tumors, including breast cancer, NSCLC, and small-cell lung cancer (SCLC).⁷⁹⁻⁸³

The binding site of vinca alkaloids was initially elucidated in 2005 when the structure of vinblastine bound to tubulin was determined.⁸⁴ Further structural studies revealed that various other drugs also bind to the same site, known as the vinca domain.⁸⁴,⁸⁵ This domain is located at
the interdimer interface of a curved protofilament, proximal to the exchangeable GTP site. It consists of a core pocket that extends toward the GTP-binding site on β-tubulin at the plus end of the microtubule. The residues within the inner lumen of microtubules associated with tubulin stabilization through longitudinal protofilament contacts contribute to the vinca domain. Binding of vinca alkaloids at this site destabilizes microtubules by introducing a wedge at the interdimer interface between α- and β-tubulin, thereby preventing the conformational transition of protofilaments from curved to straight which occurs during microtubule assembly.⁸⁶ Interestingly, the effects of both vinca alkaloids and colchicine on tubulin are concentration-dependent.⁸⁷,⁸⁸ At high concentrations (e.g., 10–100 nmol/L in Hela cells), tubulin depolymerization is promoted, mitotic progress is blocked, and mitotic spindles are disrupted.⁸⁹ Conversely, at low but clinically relevant concentrations (e.g., IC₅₀ 0.8 nmol/L in Hela cells), they do not induce tubulin depolymerization but instead suppress microtubule dynamics, leading to apoptotic cell death.⁸⁹

## 4.3 | Maytansine site

Maytansine, initially isolated from the bark of the African shrubs *Maytenus serrata* and *Maytenus ovatus* in 1972, has exhibited remarkable cytotoxicity against a diverse range of cancer cell lines.⁹⁰ However, its clinical application as a standalone therapeutic agent has encountered challenges due to dose-limiting toxicity and limited tumor specificity.⁹¹ Nonetheless, the advent of antibody-drug conjugates (ADCs) has revived interest in maytansinoids for cancer chemotherapy. Notably, the FDA approval of mertansine (DM1) in 2013, which incorporates maytansine as a payload into trastuzumab emtansine (Kadcyla®), has proven successful in the treatment of advanced breast cancer.⁹² Additionally, various analogs of maytansine, including DM4 (ravtansine/soravtansine), have shown promising potential as ADC payloads in clinical trials.⁹³,⁹⁴

In 2014, Prota et al.⁹⁵ determined the X-ray crystallography structure of maytansine bound to tubulin. Their findings unveiled a distinct binding site for maytansine, adjacent to the vinca alkaloids binding site.⁹⁶,⁹⁷ This unique site is located at an exposed pocket of the β-tubulin subunit, at the longitudinal interdimer interface corresponding to the plus end of microtubules. By sterically hindering the normal formation of αβ-tubulin protofilaments, maytansine disrupts microtubule assembly.⁹⁵ The agents targeting the maytansine binding site exert their influence on microtubules through two distinct mechanisms. At high concentrations, they sequester tubulin subunits, forming complexes that are

incompetent for assembly. At lower concentrations, they impede the addition of further tubulin dimers to the plus ends of growing microtubules.⁹⁵

## 4.4 | Pironetin site

Pironetin, originally isolated from the fermentation broth of *Streptomyces* species in the 1990s,⁹⁸ represents a unique natural compound that exclusively targets the α-tubulin subunit. Initially characterized as a plant growth regulator,⁹⁹ pironetin and its derivatives were subsequently recognized as potent antiproliferative cytotoxic agents.¹⁰⁰,¹⁰¹ Notably, pironetin demonstrated remarkable activity against cell lines both sensitive and resistant to first-generation MTAs, including taxol, thereby highlighting its significant potential for the development of a new generation of anticancer drugs.¹⁰²

Initial investigations into the binding mode of pironetin suggested its interaction with Lys-352 of α-tubulin through systematic alanine scanning.¹⁰³ However, two recent X-ray crystallographic studies have provided valuable insights, unveiling a distinct binding mechanism wherein pironetin forms a covalent interaction with Cys-316 of α-tubulin via a Michael-type addition reaction.¹⁰⁴,¹⁰⁵ The crystal structure of the tubulin–pironetin complex has further revealed that the binding site is fully buried within the α-tubulin subunit, positioned in close proximity to the interdimer interface crucial for the regulation of tubulin assembly. By perturbing the longitudinal tubulin contacts within microtubules, pironetin exerts its disruptive effect on tubulin assembly, employing a mechanism akin to that of vinca-site agents. Specifically, pironetin introduces a wedge at the interdimer interface between α- and β-tubulin, effectively impeding the conformational transition from curved to straight.

The absence of GTPase activity and limited conformational changes in the α subunit make agents targeting the pironetin-binding site on α-tubulin potentially unaffected by mutations in β-tubulin and the overexpression of βIII-tubulin in drug-resistant cells. The development of drugs that selectively target α-tubulin holds significant promise in overcoming chemotherapeutic drug resistance and represents an area of substantial potential advancement.

## 4.5 | Gatorbulin-1 site or the seventh site

In addition to the six distinct MTA binding sites previously identified, a noteworthy discovery of a seventh binding site was made by Matthew et al.¹⁰⁶ using X-ray crystallography. This newly discovered site demonstrated specific affinity for a cyclic depsipeptide known as
gatorbulin-1 (GB1), which was derived from the marine cyanobacterium *Lyngbya cf. confervoides*.¹⁰⁶

The structure of GB1 bound to tubulin clearly demonstrates the proximity and distinction between this seventh binding site and the colchicine binding site.¹⁰⁶ Similar to the colchicine site, the GB1 binding pocket is situated at the intra-dimer interface between α- and β-tubulin, but with α-tubulin contributing a greater buried area. This similarity implies that the seventh site and the colchicine site share a same mechanism of action. Specifically, GB1 prevents the critical curve-to-straight structural conversion of tubulin during assembly, effectively creating a wedge between the α and β subunits within the tubulin heterodimer, hence inhibiting tubulin polymerization.¹⁰⁶

The recently discovered seventh binding site of tubulin has demonstrated notable variability in the effects of compound binding. In a study conducted by Yang et al.,¹⁰⁷ it was demonstrated that cevipabulin (also known as TTI-237) exhibits binding affinity to this specific site. However, in contrast to GB1, the binding of cevipabulin to the seventh site elicits distinct consequences. It disrupts the critical hydrogen-bonding interaction between the nonexchangeable GTP and the T5 loop of α-tubulin, leading to the conversion of the nonexchangeable GTP into an exchangeable state. This conversion event ultimately triggers the denaturation and degradation of tubulin.¹⁰⁶,¹⁰⁷ The binding of cevipabulin to the seventh site demonstrates its potential as a tubulin degradation agent, providing a foundation for the development of future agents that specifically target tubulin degradation.

## 5 | MICROTUBULE-TARGETING DEGRADERS

Traditional MTAs primarily act by stabilizing or destabilizing microtubules, leading to mitotic arrest and subsequent cell death. Despite their initial efficacy, the emergence of resistance to these agents and the occurrence of associated side effects have underscored the need for alternative strategies. One such strategy involves targeted protein degradation by MTGs, which offers a promising avenue for overcoming these limitations.

### 5.1 | PROteolysis TTargeting Chimeras

Targeted protein degradation, specifically PROteolysis TTargeting Chimeras (PROTACs), has emerged as a promising strategy in cancer therapy.¹⁰⁸,¹⁰⁹ This approach works by promoting the degradation of target proteins instead of solely inhibiting their activity holds

potential for overcoming resistance and improving therapeutic outcomes.¹¹⁰⁻¹¹² PROTACs are designed as heterobifunctional molecules comprising three essential components: a ligand that specifically binds to the protein of interest (POI), a ligand that recruits an E3 ubiquitin ligase, and a carefully designed linker that connects these components. By bridging the POI and the E3 ubiquitin ligase, PROTACs facilitate target protein degradation through the ubiquitin-proteasome system, a highly regulated cellular degradation pathway.¹¹³,¹¹⁴ This unique mechanism of action addresses the limitations commonly associated with traditional small-molecule inhibitors, such as the emergence of drug resistance, which is particularly relevant in the context of MTAs.¹¹⁵ Despite the promising outcomes observed in preclinical and clinical trials, the expanding application of PROTACs is confronted with several challenges. A pivotal concern lies in the assessment of drug resistance that may arise from the prolonged administration of PROTAC drugs in clinical settings.¹¹⁶ Furthermore, the underlying mechanism of action employed by PROTACs involves intricate cellular entry, which limits its application to cell membrane proteins and extracellular proteins.¹¹⁶

Recently, Gasic et al.,¹¹⁷ in a study conducted, explored the development of the first tubulin-targeting PROTACs that incorporate an E3 ubiquitin ligase cereblon (CRBN) moiety. Several compounds previously reported to induce tubulin degradation were incorporated into the design.¹¹⁸,¹¹⁹ However, the study surprisingly revealed that none of the designed PROTACs were capable of inducing tubulin degradation.¹¹⁷ These findings raise concerns regarding the potential limitations of CRBN-recruiting PROTAC degraders in facilitating tubulin degradation. Consequently, despite the considerable promise of PROTAC technology in overcoming drug resistance associated with MTAs, it is evident that there are remaining challenges and complexities that necessitate further investigation and refinement. Ongoing research efforts should primarily focus on optimizing PROTAC design by exploring alternative ligands, linkers, and E3 ubiquitin ligases to enhance the specificity and efficacy of tubulin-targeting PROTACs.¹²⁰⁻¹²² Additionally, comprehensive investigations into factors influencing tubulin degradation efficiency, including the cellular microenvironment and signaling pathways, are imperative for advancing the field.

### 5.2 | Small-molecule degraders

Small-molecule degraders are a promising class of MTAs that induce the degradation of tubulin through a distinct mechanism of action. Unlike other targeted protein
degradation strategies, such as PROTACs, these degraders have the advantage of not requiring a bulky bifunctional structure.¹⁰⁹ They directly bind to the target protein and autonomously recruit the E3 ubiquitin ligase or interact with other components of the ubiquitin-proteasome system to initiate degradation.¹⁰⁷,¹¹⁹,¹²³⁻¹²⁶ Small-molecule degraders possess favorable characteristics, including reduced molecular weight, advantageous physicochemical properties, and improved bioavailability, making them a compelling approach for targeted protein degradation. However, it is worth noting that small-molecule degraders were initially discovered as microtubule modulators but were fortuitously discovered as degraders later on. Hence, small-molecule degraders also inherit the drawbacks associated with conventional small-molecule inhibitors, including a lack of target specificity and an increased risk of off-target effects.

Recent studies have identified several MTAs, such as isothiocyanates,¹²³ thymoquinone,¹²⁴ withaferin A,¹²⁵ T0070907,¹¹⁹ 3-(3-phenoxybenzyl)amino-β-carboline (PAC),¹²⁶ and cevipabulin,¹⁰⁷ that exhibit protein-degrading properties. These findings highlight the potential application of these MTAs as small-molecule degraders in cancer therapy. Structural studies of ligand-tubulin complexes have provided insights into the mechanisms of tubulin degradation. For instance, T0070907 and T007-1 covalently bind to Cys-239 of β-tubulin, resulting in tubulin unfolding and

degradation.¹¹⁸ Cevipabulin, on the other hand, interacts with a specific site near the nonexchangeable GTP, destabilizing tubulin and promoting its degradation.¹⁰⁷ PAC induces tubulin denaturation by forming a low-barrier hydrogen bond with β-tubulin, leading to tubulin aggregation and subsequent ubiquitin-mediated degradation.¹²⁶ These mechanisms disrupt tubulin stability and induce aggregation, ultimately causing cell cycle arrest and apoptosis through the ubiquitin-proteasome pathway. The understanding of these processes underscores the potential of small-molecule degraders as promising agents for targeted protein degradation in cancer treatment.

## 6 | AntiCANCER MECHANISMS OF MTAs

The diverse mechanisms of action employed by MTAs contribute to their potent anticancer effects, including disruption of cell division, induction of apoptosis, interference with signaling pathways, inhibition of angiogenesis, and modulation of tumor suppressor proteins.¹²⁷,¹²⁸

Primarily, MTAs perturb microtubule assembly and spindle formation, leading to impaired cell division and cell cycle arrest at the G2/M phase, resulting in abnormal chromosome segregation (Figure 3).¹²⁹ Moreover, MTAs induce apoptosis through intricate pathways involving

<figure><img src="image_8.png" /><figcaption>FIGURE 3 Major mechanism of action of MTAs involved in cancer cell apoptosis. The interaction of chemotherapeutic agents that stabilize, destabilize, and denature microtubules leads to an increase in microtubule polymerization, depolymerization, and degradation. These actions subsequently trigger cellular apoptosis by the formation of an abnormal mitotic spindle or impaired microtubule-related functions. MDA, microtubule-destabilizing agent; MSA, microtubule-stabilizing agent; MTA, microtubule-targeting agent; MTG, microtubule-targeting degrader.</figcaption></figure>
the phosphorylation of Bcl-2 and Bcl-xL, activation of E2F1, and subsequent release of cytochrome c.<sup>130,131</sup> Additionally, MTAs interfere with the protein kinase B/mammalian target of the rapamycin signaling pathway, inducing autophagy and impeding tumor cell proliferation.<sup>132</sup> Their antiangiogenic potential involves selective destruction of tumor blood vessels, potentially mediated by alterations in vascular endothelial growth factor expression.<sup>133</sup> Importantly, the damage inflicted to tumor vasculature by MTAs is reversible, making them suitable as vascular inhibitors.<sup>134</sup> Furthermore, some MTAs, such as paclitaxel, stimulate the production of the tumor suppressor protein p53, which is intricately linked to microtubule dynamics and the expression of diverse tubulin isotypes.<sup>135-137</sup> These multifaceted mechanisms collectively contribute to the therapeutic efficacy of MTAs for cancer therapy.

## 7 | PRECISION-TARGETED THERAPY APPROACHES

Conventional MTAs, such as taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine and vincristine), have demonstrated remarkable efficacy in cancer therapy. However, their clinical application is hindered by the emergence of off-target effects as they nonspecifically bind to tubulin in both normal and cancer cells, thereby compromising their therapeutic effectiveness. To overcome these limitations, researchers have developed and investigated various precision-targeted approaches. One successful example is the utilization of ADCs, which have shown promising results in clinical settings. Additionally, preclinical investigations have revealed the potential of light-triggered analogs of MTAs. Further exploration of these precision-targeted approaches may contribute to optimizing the clinical utility of MTAs and improving outcomes in cancer therapy.

### 7.1 | Antibody-drug conjugates

MTA-based ADCs represent a promising and rapidly advancing class of oncology therapeutics with therapeutic potential.<sup>138-140</sup> These innovative bioconjugates combine the potent cytotoxicity of MTAs with the specificity and selectivity of monoclonal antibodies (mAbs). This combination offers several advantages, including the reduction of off-target effects and the enhancement of cytotoxicity that specifically targets malignant cells. However, notable challenges of ADCs encompass the occurrence of off-target toxicity arising from the

premature release of cytotoxic small molecules into the blood circulation, and tumor-associated versus tumor-specific antigens and consequent unintended biodistribution and toxicity.<sup>141,142</sup> Addressing these challenges is critical for the successful application of ADCs in clinical settings.

In recent years, potent MTAs with well-established cytotoxic properties, have undergone extensive validation and exploration as payloads in ADCs.<sup>143</sup> These MTAs, including auristatin derivatives (MMAE, MMAF), maytansinoid derivatives (DM1, DM2, DM4), and tubulysins, disrupt microtubule assembly and affect mitosis, showing promising effects in preclinical and clinical studies.<sup>144-146</sup> Notably, ADCs based on MMAE, MMAF, DM4, and DM1 have achieved market approval, leading to huge commercial success.<sup>147,148</sup> MMAE and MMAF, known for their exceptional stability and potent antiproliferative activity by binding to vinca alkaloid sites on microtubules,<sup>149</sup> have demonstrated efficacy across various cancer types, including lymphoma, leukemia, and solid tumors (e.g., lung, gastric, and breast cancers).<sup>147,150,151</sup> Additionally, Maytansine-based ADCs, exemplified by trastuzumab emtansine (T-DM1), have exhibited promising results in treating HER2-positive metastatic breast cancer.<sup>152</sup> The diverse range of MTA-based payloads expands the opportunities for the development of ADCs and other targeted therapies in the realm of microtubule-targeting approaches.

### 7.2 | Photopharmacology of MTAs

Photopharmacology, as a swiftly advancing research field, integrates the classical pharmacological approach with photochemical light control, enabling precise regulation of drug activity to mitigate systemic toxicity, minimize off-target effects, and enhance treatment precision.<sup>153,154</sup> Hence, extensive efforts have been dedicated to the development of light-triggered MTAs, aiming to achieve precise modulation of microtubule dynamics.<sup>155</sup> Among these efforts, analogs based on combretastatin A-4, paclitaxel, epothilone, and colchicinoid have garnered attention.<sup>156-160</sup> These photoswitchable MTAs have demonstrated potential for elucidating the intricate mechanisms underlying microtubule-related processes and have proven to be useful tools in the study of various fields such as embryology, neuroscience, and cytoskeleton research.<sup>161</sup> Importantly, these photopharmaceuticals also show potential for targeted therapeutic interventions by precisely modulating microtubule dynamics in cancer cells or disease-specific microtubule networks.<sup>155</sup> However, the field of photopharmacology is still in its nascent stage of development. There is still a
considerable distance to traverse before they progress
towards clinical application. It is important to note that
further research is still necessary to optimize their
pharmacokinetic properties, enhance their selectivity
towards specific microtubule networks, and refine their
photoswitching capabilities to achieve a more precise and
effective modulation of microtubule function in disease
contexts.

8 | CLINIC APPLICATION

Microtubules, critical components involved in numerous
cellular processes, have attracted significant attention in
cancer therapy. A breakthrough in the development of
microtubule-targeting drugs occurred in 1963 with the
FDA approval of vincristine sulfate (Oncovin®) for the
treatment of leukemia and lymphoma.¹⁶² Subsequent
extensive research and drug discovery endeavors have
further highlighted the importance of MTAs as crucial
components in the development of effective chemo-
therapeutic agents against cancer.

By 2023, extensive clinical investigation has been
conducted on MTAs candidates (Figure 4), and the market
provides a wide range of over 20 tubulin-targeting drugs
for treating diverse diseases. Approved MTAs are broadly
categorized into four classes: taxanes, vinca alkaloids,
colchicine, and MT-ADCs (Table 1 and Figure 5). Taxanes,
such as paclitaxel and docetaxel, are widely recognized as
potent MSAs and have demonstrated significant clinical
efficacy in the treatment of diverse cancers, including
breast, ovarian, lung, and prostate cancers.¹⁶³⁻¹⁶⁵ Vinca
alkaloids, including vincristine and vinblastine, are
commonly acknowledged as MDAs, and are extensively

employed in the treatment of hematological malignancies,
such as lymphoma and leukemia, as well as solid tumors
like SCLC.⁸³,¹⁶⁶ Apart from their significant role in cancer
treatment, MTAs have also demonstrated therapeutic
potential in other disease areas. For instance, colchicine,
traditionally employed for gouty arthritis and Familial
Mediterranean fever, has expanded its therapeutic appli-
cation to encompass conditions like osteoarthritis, peri-
carditis, and atherosclerosis.¹⁶⁷⁻¹⁶⁹ The success of MTAs in
treating different diseases underscores their significant
contributions to the field of medicine.

However, the clinical application of conventional MTAs
is hindered by a number of limitations, including MDR,
high toxicity such as peripheral neuropathy and myelo-
suppression, and poor solubility.¹⁶³,¹⁷⁰,¹⁷¹ To overcome
these limitations, researchers are actively investigating
more targeted therapies, including nanoparticle-based drug
delivery systems,¹⁷²,¹⁷³ ADCs,¹³⁸,¹⁷⁴,¹⁷⁵ and combination
therapies with targeted agents.¹⁷⁶⁻¹⁷⁸ For example,
albumin-bound paclitaxel nanoparticles (Abraxane®) have
gained clinical approval in 2005 for the treatment of
metastatic breast cancer, due to their remarkable activity
and tolerability compared to conventional paclitaxel
formulations.¹⁷⁹,¹⁸⁰ Moreover, researchers have developed
nanotechnology-based delivery systems for a diverse array
of MTAs, including colchicine, LY293, etoposide, and
others.¹⁸¹⁻¹⁸³ These advancements have demonstrated
effects such as increased drug release, enhanced cyto-
toxicity, and precise targeting of tumor cells in vitro.¹⁸¹⁻¹⁸³

The combination of MTAs with other anticancer
agents is widely recognized as the most applied strategy
to enhance therapeutic effects, reduce toxicities, and
overcome MDR. Now, combination therapies become
standard clinical protocols. Several approaches have been
explored to achieve synergistic effects and improve
treatment outcomes. For instance, the combination of
MTAs with receptor tyrosine kinase inhibitors (such as
KX2-391),¹⁸⁴ histone deacetylase inhibitors,¹⁸⁵ DNA
damage agents,¹⁸⁶ or topoisomerase inhibitors,¹⁸⁷ has
been developed and has shown promising results in
preclinical and clinical studies.¹⁸⁸,¹⁸⁹

Notably, MT-ADCs have emerged as successful
therapeutic options in clinical settings.¹⁷⁴ Out of the
commercially introduced microtubule-targeting drugs, a
specific subgroup of eight drugs stands out as antibody-
conjugated formulations (Table 1). They have shown
great efficacy in hematological tumors (e.g., Brentuximab
vedotin [BV], Polatuzumab vedotin [PV], Belantamab
mafodotin [Bm]) and solid tumors (e.g., Enfortumab
vedotin, Tisotumab vedotin, Trastuzumab emtansine,
Mirvetuximab soravtansine, Disitamab vedotin).

BV (Adcetris®) and PV (Poliivy®) are effective for
the treatment of hematological tumors, especially

<figure><img src="image_9.png" /><figcaption>FIGURE 4 Tubulin modulators in clinical trial phases up to April 2023. Data are obtained from https://clinicaltrials.gov. Total of 4406 trials, either active or completed. Active: The study is ongoing, and participants are receiving an intervention or being examined, but potential participants are not currently being recruited or enrolled. Completed: The study has ended normally, and participants are no longer being examined or treated (i.e., the last participant's last visit has occurred).</figcaption></figure>
<table><caption>TABLE 1 Tubulin-targeting ADCs and small-molecule drugs approved by FDA, EMA, or NMPA up to April 2023.</caption><thead><tr><th>Drug</th><th>Brand name</th><th>Type</th><th>Mechanism</th><th>Condition</th><th>Time</th><th>Payload/site</th></tr></thead><tbody><tr><td>Brentuximab vedotin</td><td>Adcetris®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Lymphoma, Hodgkin lymphoma</td><td>2011</td><td>Monomethylauristatin E (MMAE)</td></tr><tr><td>Trastuzumab emtansine</td><td>Kadycya®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Cancer, breast metastatic</td><td>2013</td><td>Maytansinoid Mertansine (DM1)</td></tr><tr><td>Enfortumab vedotin</td><td>Padcev®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Cancer, urothelial carcinoma</td><td>2019</td><td>Monomethylauristatin E (MMAE)</td></tr><tr><td>Polatuzumab vedotin</td><td>Polivy®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Lymphoma, diffuse large B-cell</td><td>2019</td><td>Monomethylauristatin E (MMAE)</td></tr><tr><td>Belantamab mafodotin</td><td>Blenrep®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Multiple myeloma</td><td>2020</td><td>Monomethylauristatin F (MMAF)</td></tr><tr><td>Tisotumab vedotin</td><td>Tivdak®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Cancer, cervix</td><td>2021</td><td>Monomethylauristatin E (MMAE)</td></tr><tr><td>Disitamab vedotin</td><td>Aidixi®</td><td>ADC</td><td>Microtubule destabilizers</td><td>Cancer, stomach; urothelial</td><td>2021</td><td>Monomethylauristatin E (MMAE)</td></tr><tr><td>Mirvetuximab soravtansine</td><td>Elahere</td><td>ADC</td><td>Microtubule destabilizers</td><td>Cancer, ovarian</td><td>2022</td><td>Maytansinoid Ravtansine (DM4)</td></tr><tr><td>Vinblastine sulfate</td><td>Velban®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Cancer, breast</td><td>1961</td><td>Vinca alkaloids site</td></tr><tr><td>Vincristine sulfate</td><td>Oncovin®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Acute lymphocytic leukemia</td><td>1963</td><td>Vinca alkaloids site</td></tr><tr><td>Vindesine</td><td>Eldisine®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Acute lymphoblastic leukemia</td><td>1979</td><td>Vinca alkaloids site</td></tr><tr><td>Vinorelbine</td><td>Navelbine®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Cancer, lung (NSCLC)</td><td>1989</td><td>Vinca alkaloids site</td></tr><tr><td>Eribulin mesylate</td><td>Halaven®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Cancer, breast metastatic</td><td>2010</td><td>Vinca alkaloids site</td></tr><tr><td>Vinflunine</td><td>Javlor®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Cancer, lung (NSCLC/SCLC), prostate</td><td>2010</td><td>Vinca alkaloids site</td></tr><tr><td>Noscapine</td><td>Narcotussin®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Antitussives, lymphoma, multiple myeloma</td><td>1963</td><td>Colchicine site</td></tr><tr><td>Albendazole</td><td>Albenza®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Infection, helminthic</td><td>1996</td><td>Colchicine site</td></tr><tr><td>Colchicine</td><td>Colcrys®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Gout, Familial Mediterranean fever</td><td>2002</td><td>Colchicine site</td></tr><tr><td>Flubendazole</td><td>Fluvermal®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Filariasis</td><td>2006</td><td>Colchicine site</td></tr><tr><td>Tirbanibulin</td><td>Klisyri®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Keratosis, actinic</td><td>2021</td><td>Colchicine site</td></tr><tr><td>Griseofulvin</td><td>Fulvicin®</td><td>Small molecule</td><td>Microtubule destabilizers</td><td>Infection, fungal</td><td>1958</td><td>Taxane site</td></tr><tr><td>Paclitaxel</td><td>Taxol®</td><td>Small molecule</td><td>Microtubule stabilizers</td><td>Cancer, breast</td><td>1992</td><td>Taxane site</td></tr><tr><td>Docetaxel</td><td>Taxotere®</td><td>Small molecule</td><td>Microtubule stabilizers</td><td>Cancer, breast</td><td>1996</td><td>Taxane site</td></tr><tr><td>Ixabepilone</td><td>Ixempra®</td><td>Small molecule</td><td>Microtubule stabilizers</td><td>Cancer, breast metastatic</td><td>2007</td><td>Taxane site</td></tr><tr><td>Cabazitaxel</td><td>Jevtana®</td><td>Small molecule</td><td>Microtubule stabilizers</td><td>Cancer, prostate metastatic</td><td>2010</td><td>Taxane site</td></tr><tr><td>Utitidelone</td><td>Youtidi®</td><td>Small molecule</td><td>Microtubule stabilizers</td><td>Cancer, breast metastatic</td><td>2021</td><td>Taxane site</td></tr></tbody></table>

Abbreviations: ADCs, antibody-drug conjugates; EMA European Medicine Agency; FDA, Food and Drug Administration; NMPA, National Medical Products Administration; NSCLC, non-small-cell lung cancer; SCLC small-cell lung cancer.
<figure><img src="image_10.png" /><figcaption>### Microtubule-stabilizing drugs</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>### ADC drug payloads</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**FIGURE 5** Chemical structures of a set of clinical small-molecule and ADC drugs targeting microtubules. ADC, antibody-drug conjugate.</figcaption></figure>

lymphoma. They use MMAE as the payload, targeting CD30-positive lymphocytes and CD79b in malignant lymphoma, respectively.¹⁹⁰,¹⁹¹ Through disruption of mitosis, cell cycle arrest, and induction of apoptosis, BV and PV demonstrate direct cytotoxicity and exhibit antitumor effects through various mechanisms. BV received FDA approval in 2011 for Hodgkin lymphoma

and systemic anaplastic large cell lymphoma after treatment failure,¹⁹² while PV gained approval in 2019 for refractory or relapsed diffuse large B-cell lymphoma in combination with bendamustine and rituximab.¹⁹³ Besides, BM employs MMAF as the payload to target BCMA in multiple myeloma.¹⁹⁴ Following binding, BM internalizes and releases MMAF, resulting in the
<table><caption>TABLE 2 Clinical trials of MTA-based antibody-drug conjugates.</caption><thead><tr><td>ADCs</td><td>Status</td><td>Indication</td><td>Target</td><td>mAb</td><td>Payload</td><td>Reference</td></tr></thead><tbody><tr><td>IMGN901</td><td>Phase 2</td><td>Hematologic-blood cancer, sarcoma, neuroblastoma, SCLC, MM.</td><td>CD56</td><td>huN901</td><td>DM1<sup>a</sup></td><td>NCT02420873</td></tr><tr><td>IMGN529</td><td>Phase 1</td><td>Lymphoma, NHL, CLL.</td><td>CD37</td><td>K7153A</td><td>DM1</td><td>NCT01534715</td></tr><tr><td>IMGN289</td><td>Phase 1</td><td>EGFR-positive solid tumors.</td><td>EGFR</td><td>J2898A</td><td>DM1</td><td>NCT01963715</td></tr><tr><td>AMG 172</td><td>Phase 1</td><td>RCC.</td><td>CD27L</td><td>Anti-CD27L</td><td>DM1</td><td>NCT01497821</td></tr><tr><td>AMG 595</td><td>Phase 1</td><td>Glioma, AA.</td><td>EFGRvIII</td><td>Anti-EGFRvIII</td><td>DM1</td><td>NCT01475006</td></tr><tr><td>BAY94-9343</td><td>Phase 2</td><td>NSCLC, mesothelioma, solid tumors.</td><td>Mesothelin</td><td>Anti-mesothelin</td><td>DM4<sup>a</sup></td><td>NCT03926143</td></tr><tr><td>SAR3419</td><td>Phase 2</td><td>LBCL.</td><td>CD19</td><td>huB4</td><td>DM4</td><td>NCT01472887</td></tr><tr><td>BT062</td><td>Phase 1/2</td><td>MM.</td><td>CD138</td><td>nBT062</td><td>DM4</td><td>NCT01001442</td></tr><tr><td>SAR428926</td><td>Phase 1</td><td>Solid tumors.</td><td>LAMP-1</td><td>853K3</td><td>DM4</td><td>NCT02575781</td></tr><tr><td>SAR566658</td><td>Phase 1</td><td>Solid tumors.</td><td>CA6</td><td>huDS6</td><td>DM4</td><td>NCT01156870</td></tr><tr><td>CDX-011</td><td>Phase 2</td><td>Breast cancer, melanoma, osteosarcoma, NSCLC.</td><td>GPNMB</td><td>CR-011</td><td>MMAE<sup>b</sup></td><td>NCT01997333</td></tr><tr><td>DCDT2980S</td><td>Phase 2</td><td>NHL, DLBCL.</td><td>CD22</td><td>Anti-CD22</td><td>MMAE</td><td>NCT01691898</td></tr><tr><td>DCDS4501A</td><td>Phase 3</td><td>NHL, DLBCL, CLL.</td><td>CD79b</td><td>Anti-CD79b</td><td>MMAE</td><td>NCT03274492</td></tr><tr><td>DNIB0600A</td><td>Phase 2</td><td>Ovarian cancer, NSCLC.</td><td>NaPi2b</td><td>Anti-NaPi2b</td><td>MMAE</td><td>NCT01363947</td></tr><tr><td>PSMA ADC</td><td>Phase 2</td><td>Prostate cancer.</td><td>PSMA</td><td>Anti-PSMA</td><td>MMAE</td><td>NCT01695044</td></tr><tr><td>DMOT-4039A</td><td>Phase 1</td><td>Pancreatic cancer, ovarian cancer.</td><td>MSLN</td><td>MMOT-0530A</td><td>MMAE</td><td>NCT01469793</td></tr><tr><td>AGS-22M6E</td><td>Phase 1</td><td>Genitourinary cancer, solid tumors.</td><td>Nectin-4</td><td>Anti-Nectin-4</td><td>MMAE</td><td>NCT01409135</td></tr><tr><td>SGN-LIV1A</td><td>Phase 1</td><td>Breast cancer.</td><td>LIV1</td><td>Anti-LIV1</td><td>MMAE</td><td>NCT01969643</td></tr><tr><td>HuMax-TF-ADC</td><td>Phase 1/2</td><td>Solid tumors.</td><td>Tissue factor</td><td>TF-011</td><td>MMAE</td><td>NCT02001623</td></tr><tr><td>AGS-15E</td><td>Phase 1</td><td>Urothelial.</td><td>SLITRK6</td><td>AGS15</td><td>MMAE</td><td>NCT01963052</td></tr><tr><td>DLYE-5953A</td><td>Phase 1</td><td>breast cancer, NSCLC, solid tumors.</td><td>LY6E</td><td>Anti-Ly6E</td><td>MMAE</td><td>NCT02092792</td></tr><tr><td>DEDN-6526A</td><td>Phase 1</td><td>Melanoma.</td><td>EDNRB</td><td>Anti-EDNRB</td><td>MMAE</td><td>NCT01522664</td></tr><tr><td>AGS-67E</td><td>Phase 1</td><td>Hematologic-blood cancer, AML.</td><td>CD37</td><td>Anti-CD37</td><td>MMAE</td><td>NCT02175433</td></tr><tr><td>AGS-16M8F</td><td>Phase 1</td><td>RCC.</td><td>ENPP3</td><td>Anti-AGS-16</td><td>MMAF<sup>b</sup></td><td>NCT01114230</td></tr><tr><td>ABT-414</td><td>Phase 3</td><td>Glioblastoma multiforme, NSCLC, brain cancer, solid tumors.</td><td>EGFRvIII</td><td>ABT-806</td><td>MMAF</td><td>NCT02573324</td></tr><tr><td>ARX-788</td><td>Phase 2</td><td>Breast cancer.</td><td>Her2</td><td>Anti-Her2</td><td>MMAF</td><td>NCT04829604</td></tr><tr><td>GSK-2857916</td><td>Phase 2</td><td>MM.</td><td>BCMA</td><td>J6M0</td><td>MMAF</td><td>NCT03525678</td></tr><tr><td>MEDI4276</td><td>Phase 1</td><td>Breast cancer, gastric cancer.</td><td>HER2</td><td>39S</td><td>AZ13599185<sup>c</sup></td><td>NCT02576548</td></tr><tr><td>M1231</td><td>Phase 1</td><td>Metastatic solid tumors, esophageal cancer, NSCLC.</td><td>EGFR</td><td>Anti-EGFR</td><td>Hemiasterlin<sup>d</sup></td><td>NCT04695847</td></tr></tbody></table>

Note: Payload based on <sup>a</sup>maytansinoid; <sup>b</sup>dolastatin; <sup>c</sup>tubulysin; <sup>d</sup>hemiasterlin.

Abbreviations: AA, anaplastic astrocytomas; ADCs, antibody-drug conjugates; AML, acute myelogenous leukemia; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B cell lymphoma; LBCL, large B-cell lymphoma; mAb, monoclonal antibody; MM, multiple myeloma; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; MTA, microtubule-targeting agent; NHL, non-Hodgkin's lymphoma; NSCLC, non-small-cell lung cancer; PSMA, prostate-specific membrane antigen; RCC, renal cell carcinoma; SCLC, small-cell lung cancer.

Source: https://clinicaltrials.gov.
disruption of the intracellular microtubule network, cell cycle arrest, and apoptosis. BM demonstrates potent antitumor activity through MMAF-induced apoptosis, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis.¹⁹⁵ In 2020, it received FDA approval for the treatment of relapsed or refractory multiple myeloma.¹⁹⁴

Furthermore, MT-ADCs have demonstrated promising results in the treatment of various solid tumors, including urothelial cancer, cervical cancer, breast cancer, and epithelial ovarian cancer.¹³⁸ Enfortumab vedotin (Padcev®) is an FDA-approved ADC comprising a human mAb (AGS-22C3) that selectively targets nectin-4, a transmembrane protein highly expressed in urothelial carcinoma.¹⁹⁶ The antibody is conjugated to the cytotoxic agent MMAE through a protease-cleavable linker. It received accelerated approval in December 2019 for the treatment of locally advanced or metastatic urothelial cancer.¹⁹⁷ Tisotumab vedotin (Tivdak®) is a recently approved ADC containing a fully humanized antibody against tissue factor, which is overexpressed in various solid tumors. The antibody is linked to MMAE via a cleavable linker.¹⁴⁷ FDA approval was granted in September 2021 for recurrent or metastatic cervical cancer.¹⁹⁸ Ado-trastuzumab emtansine (Kadcyla®) is an ADC targeting HER2-positive breast cancer.¹⁹⁹ It consists of a humanized antibody linked to DM1, a potent cytotoxic agent, through a noncleavable linker. Kadcyla® was first approved in 2013 for HER2-positive metastatic breast cancer and subsequently gained expanded approval in May 2019 for adjuvant treatment of HER2-positive early breast cancer.⁹²,¹⁹⁹ Mirvetuximab soravtansine-gynx (IMGN853) is an ADC designed to target folate receptor alpha (FRα), which is overexpressed in various epithelial tumors. It incorporates a cleavable disulfide linker that connects the antibody (M9346A) to the genotoxic compound DM4.²⁰⁰ In November 2022, IMGN853 received accelerated approval for the treatment of FRα-positive, platinum-resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer based on positive outcomes from the phase 3 SORAYA trial (NCT04296890).²⁰⁰,²⁰¹ Moreover, a number of MT-ADC candidates are currently undergoing active clinical trials. Their corresponding status, therapeutic indications, targets, mAbs, payloads, and NCT numbers are summarized in Table 2. Data were generated from the ClinicalTrials.gov. The development of microtubule-targeting drugs and MT-ADCs provides more promising options for various types of cancers. However, further studies are needed to develop ADCs with tumor-restricted mAbs and effective linkers for successful clinical application.

9 | CONCLUSIONS AND PERSPECTIVES

Recent breakthroughs in the field of tubulin structural biology have led to compelling discoveries of novel tubulin-targeting sites. The exploration of the seven binding sites within microtubules, along with the understanding of the three primary strategies employed by MTAs (stabilization, destabilization, and degradation) have not only fostered drug discovery endeavors targeted at effective cancer treatment but also provided more promising options for therapeutic intervention in various disease contexts.

Notably, tubulin encompasses seven distinct binding sites, with five primarily localized on β-tubulin. However, the presence of the E-site within β-subunits, which possesses GTP hydrolysis ability, renders them vulnerable to drug resistance mediated by β-tubulin mutations and heightened βIII-tubulin expression. Consequently, targeting α-subunits MTAs offers a promising strategy to surmount drug resistance. Furthermore, it is important to mention that conventional tubulin inhibitors necessitate high systemic drug exposures, increasing the likelihood of undesirable off-target effects. The microtubule-targeting degradation strategy shows promise as a third novel class of tubulin inhibitors. As we look to the future, further investigations into microtubule sites and their functional roles will continue to inspire discoveries for cancer treatment.

AUTHOR CONTRIBUTIONS

**Xingyu Wang**: Investigation (lead); visualization (lead); writing—original draft (lead). **Benoît Gigant**: Writing—review and editing (equal). **Xi Zheng**: Funding acquisition (supporting); project administration (supporting); validation (supporting). **Qiang Chen**: Conceptualization (lead); funding acquisition (lead); project administration (lead); writing—review and editing (lead). All authors have read and approved the final manuscript.

ACKNOWLEDGMENTS

The authors thank Dr. Yuquan Wei for the critical discussion. Financial support for this work was provided by the National Natural Science Foundation of China (32270761), 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21073), and Natural Science Foundation of Sichuan Province (2023NSFSC1890). Benoît Gigant acknowledges support from the Fondation ARC pour la Recherche sur le Cancer (ARCPJA2021050003651) and from the Agence Nationale de la Recherche (ANR-22-CE11-0002-01).
## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

Not applicable.

## ETHICS STATEMENT

Not applicable.

## ORCID

Benoît Gigant http://orcid.org/0000-0002-5946-6759

Qiang Chen http://orcid.org/0000-0002-2398-4482

## REFERENCES

1. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. *Nat Rev Drug Discov.* 2010;9(10):790-803.

2. Čermák V, Dostál V, Jelínek M, et al. Microtubule-targeting agents and their impact on cancer treatment. *Eur J Cell Biol.* 2020;99(4):151075.

3. Wordeman L, Vicente JJ. Microtubule targeting agents in disease: classic drugs, novel roles. *Cancers*. 2021;13(22):5650.

4. Stanton RA, Gernert KM, Nettles JH, Aneja R. Drugs that target dynamic microtubules: a new molecular perspective. *Med Res Rev.* 2011;31(3):443-481.

5. Tang DD, Gerlach BD. The roles and regulation of the actin cytoskeleton, intermediate filaments and microtubules in smooth muscle cell migration. *Respir Res.* 2017;18(1):54.

6. Logan CM, Menko AS. Microtubules: evolving roles and critical cellular interactions. *Exp Biol Med.* 2019;244(15):1240-1254.

7. Huang L, Peng Y, Tao X, et al. Microtubule organization is essential for maintaining cellular morphology and function. *Oxid Med Cell Longev.* 2022;2022:1-15.

8. Karahalil B, Yardım-Akaydin S, Nacak Baytas S. An overview of microtubule targeting agents for cancer therapy. *Arch Ind Hyg Toxicol.* 2019;70(3):160-172.

9. Mukhtar E, Adhami VM, Mukhtar H. Targeting microtubules by natural agents for cancer therapy. *Mol Cancer Ther.* 2014;13(2):275-284.

10. Steinmetz MO, Prota AE. Microtubule-targeting agents: strategies to hijack the cytoskeleton. *Trends Cell Biol.* 2018;28(10):776-792.

11. Brouhard GJ, Rice LM. Microtubule dynamics: an interplay of biochemistry and mechanics. *Nat Rev Mol Cell Biol.* 2018;19(7):451-463.

12. Gudimchuk NB, McIntosh JR. Regulation of microtubule dynamics, mechanics and function through the growing tip. *Nat Rev Mol Cell Biol.* 2021;22(12):777-795.

13. Tilney LG, Bryan J, Bush DJ, et al. Microtubules: evidence for 13 protofilaments. *J Cell Biol.* 1973;59(2 Pt 1):267-275.

14. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. *Nat Rev Cancer.* 2004;4(4):253-265.

15. Horio T, Murata T. The role of dynamic instability in microtubule organization. *Front Plant Sci.* 2014;5:111.

16. Heald R, Nogales E. Microtubule dynamics. *J Cell Sci.* 2002;115(Pt 1):3-4.

17. Roostalu J, Thomas C, Cade NI, Kunzelmann S, Taylor IA, Surrey T. The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability. *eLife.* 2020; 9:e51992.

18. LaFrance BJ, Roostalu J, Henkin G, et al. Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. *Proc Natl Acad Sci.* 2022;119(2):e2114994119.

19. Bowne-Anderson H, Hibbel A, Howard J. Regulation of microtubule growth and catastrophe: unifying theory and experiment. *Trends Cell Biol.* 2015;25(12):769-779.

20. Kaur R, Kaur G, Gill RK, Soni R, Bariwal J. Recent developments in tubulin polymerization inhibitors: an overview. *Eur J Med Chem.* 2014;87:89-124.

21. Carmona B, Marinho HS, Matos CL, Nolasco S, Soares H. Tubulin post-translational modifications: the elusive roles of acetylation. *Biology.* 2023;12(4):561.

22. Bär J, Popp Y, Bucher M, Mikhaylova M. Direct and indirect effects of tubulin post-translational modifications on microtubule stability: insights and regulations. *Biochim Biophys Acta Mol Cell Res.* 2022;1869(6):119241.

23. Lopes D, Maiato H. The tubulin code in mitosis and cancer. *Cells.* 2020;9(11):2356.

24. Castro TG, Ferreira T, Matamá T, Munteanu FD, Cavaco-Paulo A. Acetylation and phosphorylation processes modulate Tau's binding to microtubules: a molecular dynamics study. *Biochim Biophys Acta Gen Subj.* 2023;1867(2):130276.

25. Nsamba ET, Gupta ML. Tubulin isotypes—functional insights from model organisms. *J Cell Sci.* 2022;135(9):jcs259539.

26. Hausrat TJ, Radwitz J, Lombino FL, Breiden P, Kneussel M. Alpha- and beta-tubulin isotypes are differentially expressed during brain development. *Dev Neurobiol.* 2021;81(3):333-350.

27. Ti SC, Alushin GM, Kapoor TM. Human β-tubulin isotypes can regulate microtubule protofilament number and stability. *Dev Cell.* 2018;47(2):175-190 e5.

28. Janke C, Magiera MM. The tubulin code and its role in controlling microtubule properties and functions. *Nat Rev Mol Cell Biol.* 2020;21(6):307-326.

29. Bodakuntla S, Jijumon AS, Villablanca C, Gonzalez-Billault C, Janke C. Microtubule-associated proteins: structuring the cytoskeleton. *Trends Cell Biol.* 2019;29(10):804-819.

30. Goodson HV, Jonasson EM. Microtubules and microtubule-associated proteins. *Cold Spring Harb Perspect Biol.* 2018;10(6):a022608.

31. Wattanathamsan O, Pongrakhananon V. Emerging role of microtubule-associated proteins on cancer metastasis. *Front Pharmacol.* 2022;13:935493.

32. Torrino S, Grasset EM, Audebert S, et al. Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis. *Cell Metab.* 2021;33(7):1342-1357.e10.

33. Xie S, Ogden A, Aneja R, Zhou J. Microtubule-binding proteins as promising biomarkers of paclitaxel sensitivity in cancer chemotherapy. *Med Res Rev.* 2016;36(2):300-312.

34. Xia X, He C, Wu A, Zhou J, Wu J. Microtubule-associated protein 4 is a prognostic factor and promotes tumor progression in lung adenocarcinoma. *Dis Markers.* 2018;2018:1-8.

35. Waitzman JS, Rice SE. Mechanism and regulation of kinesin-5, an essential motor for the mitotic spindle. *Biol Cell.* 2014;106(1):1-12.
36. Rincon SA, Lamson A, Blackwell R, et al. Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast. *Nat Commun.* 2017;8:15286.

37. Hassan Ibrahim I, Balah A, Gomaa Abd Elfattah Hassan A, Gamal Abd El-Aziz H. Role of motor proteins in human cancers. *Saudi J Biol Sci.* 2022;29(12):103436.

38. Chien TM, Chan TC, Huang SKH, et al. Role of microtubule-associated protein 1b in urothelial carcinoma: overexpression predicts poor prognosis. *Cancers*. 2020;12(3):630.

39. Luo J, Hu Q, Gou M, et al. Expression of microtubule-associated proteins in relation to prognosis and efficacy of immunotherapy in non-small cell lung cancer. *Front Oncol.* 2021;11:680402.

40. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. isolation and structure of taxol, a novel antileukemic and antitumor agent from *Taxus brevifolia*. *J Am Chem Soc.* 1971;93(9):2325-2327.

41. Yared JA, Tkaczuk KH. Update on taxane development: new analogs and new formulations. *Drug Des Devel Ther.* 2012;6: 371-384.

42. Yang CP, Horwitz S. Taxol®: the first microtubule stabilizing agent. *Int J Mol Sci.* 2017;18(8):1733.

43. Schiff PB, Horwitz SB. Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins. *Biochemistry.* 1981;20(11):3247-3252.

44. Nogales E, Wolf SG, Downing KH. Structure of the αβ tubulin dimer by electron crystallography. *Nature*. 1998; 391(6663):199-203.

45. Xiao Q, Xue T, Shuai W, et al. High-resolution X-ray structure of three microtubule-stabilizing agents in complex with tubulin provide a rationale for drug design. *Biochem Biophys Res Commun.* 2021;534:330-336.

46. Prota AE, Bargsten K, Zurwerra D, et al. Molecular mechanism of action of microtubule-stabilizing anticancer agents. *Science*. 2013;339(6119):587-590.

47. Sánchez-Murcia PA, Mills A, Cortés-Cabrera Á, Gago F. Unravelling the covalent binding of zampanolide and taccalonolide AJ to a minimalist representation of a human microtubule. *J Comput Aided Mol Des.* 2019;33(7):627-644.

48. Rowinsky EK, Onetto N, Canetta RM, Arbuck SG. Taxol: the first of the taxanes, an important new class of antitumor agents. *Semin Oncol.* 1992;19(6):646-662.

49. Debs GE, Cha M, Liu X, Huehn AR, Sindelar CV. Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy. *Proc Natl Acad Sci USA.* 2020;117(29):16976-16984.

50. Nogales E, Whittaker M, Milligan RA, Downing KH. High-resolution model of the microtubule. *Cell*. 1999;96(1):79-88.

51. Lowe J, Li H, Downing KH, Nogales E. Refined structure of alpha beta-tubulin at 3.5 Å resolution. *J Mol Biol.* 2001; 313(5):1045-1057.

52. Kellogg EH, Hejab NMA, Howes S, et al. Insights into the distinct mechanisms of action of taxane and non-taxane microtubule stabilizers from Cryo-EM structures. *J Mol Biol.* 2017;429(5):633-646.

53. Knossow M, Campanacci V, Khodja LA, Gigant B. The mechanism of tubulin assembly into microtubules: insights from structural studies. *iScience*. 2020;23(9):101511.

54. Elie-Caille C, Severin F, Helenius J, Howard J, Muller DJ, Hyman AA. Straight GDP-tubulin protofilaments form in the presence of taxol. *Curr Biol.* 2007;17(20):1765-1770.

55. Prota AE, Lucena-Agell D, Ma Y, et al. Structural insight into the stabilization of microtubules by taxanes. *eLife*. 2023; 12:e84791.

56. West LM, Northcote PT, Battershill CN. Peloruside A: a potent cytotoxic macrolide isolated from the New Zealand marine sponge *Mycale* sp. *J Org Chem.* 2000;65(2):445-449.

57. Corley DG, Herb R, Moore RE, Scheuer PJ, Paul VJ. Laulimalides. New potent cytotoxic macrolides from a marine sponge and a nudibranch predator. *J Org Chem.* 1988;53(15):3644-3646.

58. Gaitanos TN, Buey M, Díaz JF, et al. Peloruside A does not bind to the taxoid site on β-tubulin and retains its activity in multidrug-resistant cell lines. *Cancer Res.* 2004;64(15): 5063-5067.

59. Kumbhar BV, Bhandare VV. Exploring the interaction of peloruside-A with drug resistant αβII and αβIII tubulin isotypes in human ovarian carcinoma using a molecular modeling approach. *J Biomol Struct Dyn.* 2021;39(6):1990-2002.

60. Kanakkanthara A, Northcote PT, Miller JH. βII-tubulin and βIII-tubulin mediate sensitivity to peloruside A and laulimalide, but not paclitaxel or vinblastine, in human ovarian carcinoma cells. *Mol Cancer Ther.* 2012;11(2):393-404.

61. Liu J, Towle MJ, Cheng H, et al. In vitro and in vivo anticancer activities of synthetic (–)-laulimalide, a marine natural product microtubule stabilizing agent. *Anticancer Res.* 2007;27(3B):1509-1518.

62. Hamel E, Day BW, Miller JH, et al. Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly. *Mol Pharmacol.* 2006;70(5):1555-1564.

63. Prota AE, Bargsten K, Northcote PT, et al. Structural basis of microtubule stabilization by laulimalide and peloruside A. *Angew Chem Int Ed.* 2014;53(6):1621-1625.

64. Hartung EF. History of the use of colchicum and related medicaments in gout; with suggestions for further research. *Ann Rheum Dis.* 1954;13(3):190-200.

65. Leung YY, Yao Hui LL, Kraus VB. Colchicine—update on mechanisms of action and therapeutic uses. *Semin Arthritis Rheum.* 2015;45(3):341-350.

66. Kumar A, Sharma PR, Mondhe DM. Potential anticancer role of colchicine-based derivatives: an overview. *Anticancer Drugs.* 2017;28(3):250-262.

67. Lin ZY, Kuo CH, Wu DC, Chuang WL. Anticancer effects of clinically acceptable colchicine concentrations on human gastric cancer cell lines. *Kaohsiung J Med Sci.* 2016;32(2):68-73.

68. Ravelli RBG, Gigant B, Curmi PA, et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. *Nature.* 2004;428(6979):198-202.

69. Wang Y, Zhang H, Gigant B, et al. Structures of a diverse set of colchicine binding site inhibitors in complex with tubulin provide a rationale for drug discovery. *FEBS J.* 2016;283(1): 102-111.

70. Zhang Z, Wang C, Ma L, et al. Molecular mechanism of crolibulin in complex with tubulin provides a rationale for drug design. *Biochem Biophys Res Commun.* 2019;511(2): 381-386.
71. Tan L, Wu C, Zhang J, et al. Design, synthesis, and biological evaluation of heterocyclic-fused pyrimidine chemotypes guided by X-ray crystal structure with potential antitumor and anti-multidrug resistance efficacy targeting the colchicine binding site. *J Med Chem.* 2023;66(5):3588-3620.

72. Wang J, Miller DD, Li W. Molecular interactions at the colchicine binding site in tubulin: an X-ray crystallography perspective. *Drug Discov Today.* 2022;27(3):759-776.

73. Niu L, Yang J, Yan W, et al. Reversible binding of the anticancer drug KXO1 (tirbanibulin) to the colchicine-binding site of β-tubulin explains KXO1's low clinical toxicity. *J Biol Chem.* 2019;294(48):18099-18108.

74. Katayama R, Aoyama A, Yamori T, et al. Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition. *Cancer Res.* 2013;73(10):3087-3096.

75. Park H, Hong S, Hong S. Nocodazole is a high-affinity ligand for the cancer-related kinases ABL, c-KIT, BRAF, and MEK. *ChemMedChem.* 2012;7(1):53-56.

76. Warwick OH, Darte JM, Brown TC, Beer CT, Cutts JH, Noble RL. Some biological effects of vincaleukoblastine, an alkaloid in *Vinca rosea* Linn in patients with malignant disease. *Cancer Res.* 1960;20:1032-1040.

77. Efferth T, Li PCH, Konkimalla VSB, Kaina B. From traditional Chinese medicine to rational cancer therapy. *Trends Mol Med.* 2007;13(8):353-361.

78. Loh K. Know the medicinal herb: *Catharanthus roseus* (*Vinca rosea*). *Malays Fam Physician.* 2008;3(2):123.

79. Gerullis H, Wawroschek F, Köhne CH, Ecke TH. Vinflunine in the treatment of advanced urothelial cancer: clinical evidence and experience. *Ther Adv Urol.* 2017;9(1):28-35.

80. Madsen ML, Due H, Ejskjær N, Jensen P, Madsen J, Dybkær K. Aspects of vincristine-induced neuropathy in hematologic malignancies: a systematic review. *Cancer Chemother Pharmacol.* 2019;84(3):471-485.

81. Gourmelon C, Bourrien H, Augereau P, Patsouris A, Frenel JS, Campone M. Vinflunine for the treatment of breast cancer. *Expert Opin Pharmacother.* 2016;17(13):1817-1823.

82. Piccirillo MC, Daniele G, Di Maio M, et al. Vinorelbine for non-small cell lung cancer. *Expert Opin Drug Saf.* 2010;9(3): 493-510.

83. Banyal A, Tiwari S, Sharma A, et al. Vinca alkaloids as a potential cancer therapeutics: recent update and future challenges. *3 Biotech.* 2023;13(6):211.

84. Gigant B, Wang C, Ravelli RBG, et al. Structural basis for the regulation of tubulin by vinblastine. *Nature.* 2005;435(7041): 519-522.

85. Cormier A, Knossow M, Wang C, Gigant B. The binding of vinca domain agents to tubulin: structural and biochemical studies. *Methods Cell Biol.* 2010;95:373-390.

86. Cormier A, Marchand M, Ravelli RBG, Knossow M, Gigant B. Structural insight into the inhibition of tubulin by vinca domain peptide ligands. *EMBO Rep.* 2008;9(11):1101-1106.

87. Owellen RJ, Hartke CA, Dickerson RM, Hains FO. Inhibition of tubulin-microtubule polymerization by drugs of the *Vinca* alkaloid class. *Cancer Res.* 1976;36(4):1499-1502.

88. Panda D, Daijo JE, Jordan MA, Wilson L. Kinetic stabilization of microtubule dynamics at steady state in vitro by substoichiometric concentrations of tubulin-colchicine complex. *Biochemistry.* 1995;34(31):9921-9929.

89. Panda D, DeLuca K, Williams D, Jordan MA, Wilson L. Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends. *Proc Natl Acad Sci USA.* 1998;95(16):9313-9318.

90. Kupchan SM, Komoda Y, Court WA, et al. Tumor inhibitors. LXXIII. Maytansine, a novel antileukemic ansa macrolide from *Maytenus ovatus*. *J Am Chem Soc.* 1972;94(4):1354-1356.

91. Cassady JM, Chan KK, Floss HG, Leistner E. Recent developments in the maytansinoid antitumor agents. *Chem Pharm Bull.* 2004;52(1):1-26.

92. Verma S, Miles D, Gianni L, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. *N Engl J Med.* 2012;367(19):1783-1791.

93. Moore KN, Martin LP, O'Malley DM, et al. A review of mirvetuximab soravtansine in the treatment of platinum-resistant ovarian cancer. *Future Oncol.* 2018;14(2):123-136.

94. Martín-Sabroso C, Lozza I, Torres-Suárez AI, Fraguas-Sánchez AI. Antibody-antineoplastic conjugates in gynecological malignancies: current status and future perspectives. *Pharmaceutics.* 2021;13(10):1705.

95. Prota AE, Bargsten K, Diaz JF, et al. A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs. *Proc Natl Acad Sci USA.* 2014;111(38):13817-13821.

96. Marzullo P, Boiarska Z, Pérez-Peña H, et al. Maytansinol derivatives: side reactions as a chance for new tubulin binders. *Chemistry.* 2022;28(2):202103520.

97. Li W, Huang M, Li Y, et al. C3 ester side chain plays a pivotal role in the antitumor activity of maytansinoids. *Biochem Biophys Res Commun.* 2021;566:197-203.

98. Kobayashi S, Tsuchiya K, Nishide M, Nishikiori T, Nakagawa T, Shimada N. Azalomycin F complex from streptomyces hygroscopicus, MSU/MN-4-75B. *J Antibiot.* 1995;48(8):893-895.

99. Kobayashi S, Tsuchiya K, Harada T, et al. Pironetin, a novel plant growth regulator produced by *Streptomyces* sp. NK10958. I. Taxonomy, production, isolation and preliminary characterization. *J Antibiot.* 1994;47(6):697-702.

100. Coulup SK, Georg GI. Revisiting microtubule targeting agents: α-Tubulin and the pironetin binding site as unexplored targets for cancer therapeutics. *Bioorg Med Chem Lett.* 2019;29(15):1865-1873.

101. Noman MAA, Huang DS, Coulup SK, et al. Cytotoxicity of phenylpironetin analogs and the metabolic fate of pironetin and phenylpironetin. *Bioorg Chem.* 2022;125:105915.

102. Marco JA, García-Pla J, Carda M, et al. Design and synthesis of pironetin analogues with simplified structure and study of their interactions with microtubules. *Eur J Med Chem.* 2011;46(5):1630-1637.

103. Usui T, Watanabe H, Nakayama H, et al. The anticancer natural product pironetin selectively targets Lys352 of α-tubulin. *Chem Biol.* 2004;11(6):799-806.

104. Yang J, Wang Y, Wang T, et al. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule. *Nat Commun.* 2016;7:12103.

105. Prota AE, Setter J, Waight AB, et al. Pironetin binds covalently to αCys316 and perturbs a major loop and helix of α-tubulin to inhibit microtubule formation. *J Mol Biol.* 2016;428(15):2981-2988.
106. Matthew S, Chen QY, Ratnayake R, et al. Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site. *Proc Natl Acad Sci USA*. 2021;118(9):e2021847118.

107. Yang J, Yu Y, Li Y, et al. Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect. *Sci Adv*. 2021;7(21):eabg4168.

108. Li X, Pu W, Zheng Q, Ai M, Chen S, Peng Y. Proteolysis-targeting chimeras (PROTACs) in cancer therapy. *Mol Cancer*. 2022;21(1):99.

109. Békés M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. *Nat Rev Drug Discov*. 2022; 21(3):181-200.

110. Bond MJ, Crews CM. Proteolysis targeting chimeras (PROTACs) come of age: entering the third decade of targeted protein degradation. *RSC Chem Biol*. 2021;2(3):725-742.

111. Tamatam R, Shin D. Emerging strategies in Proteolysis-Targeting Chimeras (PROTACs): highlights from 2022. *Int J Mol Sci*. 2023;24(6):5190.

112. Zhao L, Zhao J, Zhong K, Tong A, Jia D. Targeted protein degradation: mechanisms, strategies and application. *Signal Transduct Target Ther*. 2022;7(1):113.

113. Khan S, He Y, Zhang X, et al. PROteolysis TArgeting chimeras (PROTACs) as emerging anticancer therapeutics. *Oncogene*. 2020;39(26):4909-4924.

114. Xi JY, Zhang RY, Chen K, et al. Advances and perspectives of proteolysis targeting chimeras (PROTACs) in drug discovery. *Bioorg Chem*. 2022;125:105848.

115. Eli S, Castagna R, Mapelli M, Parisini E. Recent approaches to the identification of novel microtubule-targeting agents. *Front Mol Biosci*. 2022;9:841777.

116. Chirnomas D, Hornberger KR, Crews CM. Protein degraders enter the clinic—a new approach to cancer therapy. *Nat Rev Clin Oncol*. 2023;20(4):265-278.

117. Gasic I, Groendyke BJ, Nowak RP, et al. Tubulin resists degradation by cereblon-recruiting PROTACs. *Cells*. 2020;9(5): 1083.

118. Yang J, Li Y, Yan W, et al. Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation. *J Biol Chem*. 2019;294(20):8161-8170.

119. Harris G, Schaefer KL. The microtubule-targeting agent T0070907 induces proteasomal degradation of tubulin. *Biochem Biophys Res Commun*. 2009;388(2):345-349.

120. García Jiménez D, Rossi Sebastiano M, Vallaro M, et al. Designing soluble PROTACs: strategies and preliminary guidelines. *J Med Chem*. 2022;65(19):12639-12649.

121. Pettersson M, Crews CM. PROteolysis TArgeting Chimeras (PROTACs)—past, present and future. *Drug Discov Today Technol*. 2019;31:15-27.

122. He S, Dong G, Cheng J, Wu Y, Sheng C. Strategies for designing proteolysis targeting chimaeras (PROTACs). *Med Res Rev*. 2022;42(3):1280-1342.

123. Mi L, Gan N, Cheema A, et al. Cancer preventive isothiocyanates induce selective degradation of cellular α- and β-tubulins by proteasomes. *J Biol Chem*. 2009;284(25):17039-17051.

124. Alhosin M, Ibrahim A, Boukhari A, et al. Anti-neoplastic agent thymoquinone induces degradation of α and β tubulin proteins in human cancer cells without affecting their level

in normal human fibroblasts. *Invest New Drugs*. 2012;30(5): 1813-1819.

125. Antony ML, Lee J, Hahm ER, et al. Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of β-tubulin. *J Biol Chem*. 2014; 289(3):1852-1865.

126. Yang J, Li Y, Qiu Q, et al. Small molecules promote selective denaturation and degradation of tubulin heterodimers through a low-barrier hydrogen bond. *J Med Chem*. 2022;65(13): 9159-9173.

127. Checchi PM, Nettles JH, Zhou J, Snyder JP, Joshi HC. Microtubule-interacting drugs for cancer treatment. *Trends Pharmacol Sci*. 2003;24(7):361-365.

128. Qi F, Zhou J, Liu M. Microtubule-interfering agents, spindle defects, and interkinetochore tension. *J Cell Physiol*. 2020; 235(1):26-30.

129. Wang TH, Wang HS, Ichijo H, et al. Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. *J Biol Chem*. 1998;273(9):4928-4936.

130. Whitaker RH, Placzek WJ. Regulating the BCL2 family to improve sensitivity to microtubule targeting agents. *Cells*. 2019;8(4):346.

131. Chen JG, Yang CP, Cammer M, Horwitz SB. Gene expression and mitotic exit induced by microtubule-stabilizing drugs. *Cancer Res*. 2003;63(22):7891-7899.

132. Clippinger AJ, Alwine JC. Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells. *Genes Dev*. 2012;26(18):2015-2026.

133. Meissner M, Pinter A, Michailidou D, et al. Microtubule-targeted drugs inhibit VEGF receptor-2 expression by both transcriptional and post-transcriptional mechanisms. *J Invest Dermatol*. 2008;128(8):2084-2091.

134. Chan A, Singh AJ, Northcote PT, Miller JH. Inhibition of human vascular endothelial cell migration and capillary-like tube formation by the microtubule-stabilizing agent peloruside A. *Invest New Drugs*. 2015;33(3):564-574.

135. Haq R, Zanke B. Inhibition of apoptotic signaling pathways in cancer cells as a mechanism of chemotherapy resistance. *Cancer Metastasis Rev*. 1998;17(2):233-239.

136. Arai K, Matsumoto Y, Nagashima Y, Yagasaki K. Regulation of class II β-tubulin expression by tumor suppressor p53 protein in mouse melanoma cells in response to Vinca alkaloid. *Mol Cancer Res*. 2006;4(4):247-255.

137. Lanni JS, Lowe SW, Licitra EJ, Liu JO, Jacks T. p53-Independent apoptosis induced by paclitaxel through an indirect mechanism. *Proc Natl Acad Sci USA*. 1997;94(18): 9679-9683.

138. Chen H, Lin Z, Arnst K, Miller D, Li W. Tubulin inhibitor-based antibody-drug conjugates for cancer therapy. *Molecules*. 2017; 22(8):1281.

139. Klute K, Nackos E, Tasaki S, Nguyen DP, Bander NH, Tagawa ST. Microtubule inhibitor-based antibody-drug conjugates for cancer therapy. *Onco Targets Ther*. 2014;7:2227-2236.

140. Joubert N, Beck A, Dumontet C, Denevault-Sabourin C. Antibody-drug conjugates: the last decade. *Pharmaceuticals*. 2020;13(9):245.
141. McKertish CM, Kayser V. Advances and limitations of antibody drug conjugates for cancer. *Biomedicines*. 2021;9(8): 872.

142. Tolcher AW. Antibody drug conjugates: lessons from 20 years of clinical experience. *Ann Oncol*. 2016;27(12):2168-2172.

143. Yaghoubi S, Karimi MH, Lotfinia M, et al. Potential drugs used in the antibody-drug conjugate (ADC) architecture for cancer therapy. *J Cell Physiol*. 2020;235(1):31-64.

144. Best RL, LaPointe NE, Azarenko O, et al. Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): mechanistic insights into MMAE ADC peripheral neuropathy. *Toxicol Appl Pharmacol*. 2021;421:115534.

145. Cao S, Dong YH, Wang DF, Liu ZP. Tubulin maytansine site binding ligands and their applications as MTAs and ADCs for cancer therapy. *Curr Med Chem*. 2020;27(27):4567-4576.

146. Tumey LN, Leverett CA, Vetelino B, et al. Optimization of tubulysin antibody-drug conjugates: a case study in addressing ADC metabolism. *ACS Med Chem Lett*. 2016;7(11):977-982.

147. Tong JTW, Harris PWR, Brimble MA, Kavianinia I. An insight into FDA approved antibody-drug conjugates for cancer therapy. *Molecules*. 2021;26(19):5847.

148. Saber H, Leighton JK. An FDA oncology analysis of antibody-drug conjugates. *Regul Toxicol Pharmacol*. 2015; 71(3):444-452.

149. Waight AB, Bargsten K, Doronina S, Steinmetz MO, Sussman D, Prota AE. Structural basis of microtubule destabilization by potent auristatin anti-mitotics. *PLoS One*. 2016;11(8):e0160890.

150. Katz J, Janik JE, Younes A. Brentuximab vedotin (SGN-35). *Clin Cancer Res*. 2011;17(20):6428-6436.

151. Chu Y, Zhou X, Wang X. Antibody-drug conjugates for the treatment of lymphoma: clinical advances and latest progress. *J Hematol Oncol*. 2021;14(1):88.

152. Baron JM, Boster BL, Barnett CM. Ado-trastuzumab emtansine (T-DM1): a novel antibody-drug conjugate for the treatment of HER2-positive metastatic breast cancer. *J Oncol Pharm Pract*. 2015;21(2):132-142.

153. Fuchter MJ. On the promise of photopharmacology using photoswitches: a medicinal chemist's perspective. *J Med Chem*. 2020;63(20):11436-11447.

154. Lerch MM, Hansen MJ, van Dam GM, Szymanski W, Feringa BL. Emerging targets in photopharmacology. *Angew Chem Int Ed*. 2016;55(37):10978-10999.

155. Kirchner S, Pianowski Z. Photopharmacology of antimitotic agents. *Int J Mol Sci*. 2022;23(10):5657.

156. Sheldon JE, Dcona MM, Lyons CE, Hackett JC, Hartman MCT. Photoswitchable anticancer activity via *trans-cis* isomerization of a combretastatin A-4 analog. *Org Biomol Chem*. 2016;14(1): 40-49.

157. Engdahl AJ, Torres EA, Lock SE, Engdahl TB, Mertz PS, Streu CN. Synthesis, characterization, and bioactivity of the photoisomerizable tubulin polymerization inhibitor azo-combretastatin A4. *Org Lett*. 2015;17(18):4546-4549.

158. Gao L, Meiring JCM, Heise C, et al. Photoswitchable epothilone-based microtubule stabilisers allow GFP-imaging-compatible, optical control over the microtubule cytoskeleton. *Angew Chem Int Ed.* 2022;61(10):e202114614.

159. Rastogi SK, Zhao Z, Barrett SL, et al. Photoresponsive azo-combretastatin A-4 analogues. *Eur J Med Chem*. 2018;143:1-7.

160. Müller-Deku A, Meiring JCM, Loy K, et al. Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton. *Nat Commun*. 2020;11(1): 4640.

161. Gao L, Meiring JCM, Varady A, et al. In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M. *J Am Chem Soc*. 2022;144(12): 5614-5628.

162. Bates D, Eastman A. Microtubule destabilising agents: far more than just antimitotic anticancer drugs. *Br J Clin Pharmacol*. 2017;83(2):255-268.

163. Mosca L, Ilari A, Fazi F, Assaraf YG, Colotti G. Taxanes in cancer treatment: activity, chemoresistance and its overcoming. *Drug Resist Updates*. 2021;54:100742.

164. Vergote I, Macarulla T, Hirsch FR, Hagemann C, Miller DS. Tumor treating fields (TTFields) therapy concomitant with taxanes for cancer treatment. *Cancers*. 2023;15(3):636.

165. Wang C, Aguilar A, Ojima I. Strategies for the drug discovery and development of taxane anticancer therapeutics. *Expert Opin Drug Discov*. 2022;17(11):1193-1207.

166. Dhyani P, Quispe C, Sharma E, et al. Anticancer potential of alkaloids: a key emphasis to colchicine, vinblastine, vincristine, vindesine, vinorelbine and vincamine. *Cancer Cell Int*. 2022;22(1):206.

167. Dossing A, Henriksen M, Ellegaard K, et al. Colchicine twice a day for hand osteoarthritis (COLOR): a double-blind, randomised, placebo-controlled trial. *Lancet Rheumatol*. 2023;9(7 Pt 2):1060-1066.

168. Ahmed AS, Miller J, Foreman J, et al. Prophylactic colchicine after radiofrequency ablation of atrial fibrillation: post ablation pericarditis reduction study (PAPERS). *JACC Clin Electrophysiol*. 2023;9(7 Pt 2):1060-1066.

169. Deftereros SG, Beerkens FJ, Shah B, et al. Colchicine in cardiovascular disease: in-depth review. *Circulation*. 2022; 145(1):61-78.

170. Poruchynsky MS, Komlodi-Pasztor E, Trostel S, et al. Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins. *Proc Natl Acad Sci USA*. 2015;112(5):1571-1576.

171. Krause W. Resistance to anti-tubulin agents: from vinca alkaloids to epothilones. *Cancer Drug Resist*. 2019;2(1):82-106.

172. Kim J, Lee J, Lee J, et al. Tubulin-based nanotubes as delivery platform for microtubule-targeting agents. *Adv Mater*. 2020; 32(33):2002902.

173. Banerjee S, Hwang DJ, Li W, Miller D. Current advances of tubulin inhibitors in nanoparticle drug delivery and vascular disruption/angiogenesis. *Molecules*. 2016;21(11):1468.

174. Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. *Signal Transduct Target Ther*. 2022;7(1):93.

175. Professional Committee on Clinical Research of Oncology Drugs, Chinese Anti-Cancer Association, Expert Committee for Monitoring the Clinical Application of Antitumor Drugs, Breast Cancer Expert Committee of National Cancer Quality Control Center, Cancer Chemotherapy Quality Control Expert Committee of Beijing Cancer Treatment Quality
Control and Improvement Center. Expert consensus on the clinical application of antibody drug conjugates in the treatment of malignant tumors (2020 edition). Chin J Oncol. 2021;43(1):78-91.

176. Liang TT, Lu L, Song X, Qi J, Wang J. Combination of microtubule targeting agents with other antineoplastics for cancer treatment. *Biochim Biophys Acta Rev Cancer*. 2022; 1877(5):188777.

177. Shuai W, Wang G, Zhang Y, et al. Recent progress on tubulin inhibitors with dual targeting capabilities for cancer therapy. *J Med Chem*. 2021;64(12):7963-7990.

178. Sarantis P, Bokas A, Papadimitropoulou A, et al. Combinatorial treatment of tinzaparin and chemotherapy can induce a significant antitumor effect in pancreatic cancer. *Int J Mol Sci*. 2021;22(13):7053.

179. Ma P, Mumper RJ. Paclitaxel nano-delivery systems: a comprehensive review. *J Nanomed Nanotechnol*. 2013;4(2): 1000164.

180. Mahtani RL, Parisi M, Gluck S, et al. Comparative effectiveness of early-line nab-paclitaxel vs. paclitaxel in patients with metastatic breast cancer: a US community-based real-world analysis. *Cancer Manag Res*. 2018;10:249-256.

181. Wang L, Peng Y, Song L, et al. Colchicine-containing nanoparticles attenuates acute myocardial infarction injury by inhibiting inflammation. *Cardiovasc Drugs Ther*. 2022;36(6): 1075-1089.

182. Mundra V, Peng Y, Kumar V, Li W, Miller DD, Mahato RI. Systemic delivery of nanoparticle formulation of novel tubulin inhibitor for treating metastatic melanoma. *Drug Deliv Transl Res*. 2015;5(3):199-208.

183. Kovshova T, Mantrov S, Boiko S, et al. Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells. *J Microencapsul*. 2023;40(4):246-262.

184. Wang P, Xiao X, Zhang Y, et al. A dual inhibitor overcomes drug-resistant FLT3-ITD acute myeloid leukemia. *J Hematol Oncol*. 2021;14(1):105.

185. Peng X, Chen J, Li L, et al. Efficient synthesis and bioevaluation of novel dual tubulin/histone deacetylase 3 inhibitors as potential anticancer agents. *J Med Chem*. 2021; 64(12):8447-8473.

186. Nilchan N, Li X, Pedzisa L, Nanna AR, Roush WR, Rader C. Dual-mechanistic antibody-drug conjugate via site-specific selenocysteine/cysteine conjugation. *Antib Ther*. 2019;2(4):71-78.

187. Ceramella J, Caruso A, Occhiuzzi MA, et al. Benzothienoquinazolinones as new multi-target scaffolds: dual inhibition of human topoisomerase I and tubulin polymerization. *Eur J Med Chem*. 2019;181:111583.

188. Jänne PA, van den Heuvel MM, Barlesi F, et al. Selumetinib plus docetaxel compared with docetaxel alone and progression-free survival in patients with KRAS-mutant advanced non-small cell lung cancer: the SELECT-1 randomized clinical trial. *JAMA*. 2017;317(18):1844-1853.

189. Zhang Y, Wang ZX, Shen L, et al. A phase Ib/II study of fruquintinib in combination with paclitaxel as the second-line therapy for advanced gastric cancer. *Cancer Commun*. 2023; 43(1):150-153.

190. Ansell SM, Radford J, Connors JM, et al. Overall survival with Brentuximab vedotin in Stage III or IV Hodgkin's lymphoma. *N Engl J Med*. 2022;387(4):310-320.

191. Tilly H, Morschhauser F, Sehn LH, et al. Polatuzumab vedotin in previously untreated diffuse large B-cell lymphoma. *N Engl J Med*. 2022;386(4):351-363.

192. Koga Y, Sekimizu M, Iguchi A, et al. Phase I study of Brentuximab vedotin (SGN-35) in Japanese children with relapsed or refractory CD30-positive Hodgkin's lymphoma or systemic anaplastic large cell lymphoma. *Int J Hematol*. 2020;111(5):711-718.

193. Sehn LH, Herrera AF, Flowers CR, et al. Polatuzumab vedotin in relapsed or refractory diffuse large B-cell lymphoma. *J Clin Oncol*. 2020;38(2):155-165.

194. Lonial S, Lee HC, Badros A, et al. Belantamab mafodotin for relapsed or refractory multiple myeloma (DREAMM-2): a two-arm, randomised, open-label, phase 2 study. *Lancet Oncol*. 2020;21(2):207-221.

195. Romano A, Storti P, Marchica V, et al. Mechanisms of action of the new antibodies in use in multiple myeloma. *Front Oncol*. 2021;11:684561.

196. Challita-Eid PM, Satpayev D, Yang P, et al. Enfortumab vedotin antibody-drug conjugate targeting Nectin-4 is a highly potent therapeutic agent in multiple preclinical cancer models. *Cancer Res*. 2016;76(10):3003-3013.

197. Hoffman-Censits J, Maldonado L. Targeted treatment of locally advanced and metastatic urothelial cancer: enfortumab vedotin in context. *Onco Targets Ther*. 2022;15:1519-1529.

198. Colombo N, Dubot C, Lorusso D, et al. Pembrolizumab for persistent, recurrent, or metastatic cervical cancer. *N Engl J Med*. 2021;385(20):1856-1867.

199. von Minckwitz G, Huang CS, Mano MS, et al. Trastuzumab emtansine for residual invasive HER2-positive breast cancer. *N Engl J Med*. 2019;380(7):617-628.

200. Heo YA. Mirvetuximab soravtansine: first approval. *Drugs*. 2023;83(3):265-273.

201. Matulonis UA, Lorusso D, Oaknin A, et al. Efficacy and safety of mirvetuximab soravtansine in patients with platinum-resistant ovarian cancer with high folate receptor alpha expression: results from the SORAYA study. *J Clin Oncol*. 2023;41(13):2436-2445.

**How to cite this article:** Wang X, Gigant B, Zheng X, Chen Q. Microtubule-targeting agents for cancer treatment: seven binding sites and three strategies. *MedComm – Oncology*. 2023;2:e46. doi:10.1002/mog2.46