Microtubules, as fundamental components of the cytoskeleton in eukaryocyte cells, play a key role in a variety of cellular functions such as cell division, signal transduction, secretion, intracellular macromolecular assemblies, maintenance of cell shape and vesicle trafficking [[1], [2], [3], [4], [5]]. Microtubules, consisted of α- and β-subunit heterodimers, are constantly in a dynamic equilibrium between polymerization and depolymerization, which is fundamental to the microtubules’ ability to perform their essential role [6,7]. The disruption of this dynamic process disturbs the cell division machinery at mitosis and leads to cell death [8,9]. Therefore, microtubules have become one of the most vital and attractive targets in anticancer therapy. According to the different dynamic influence modes, classical tubulin-targeting agents are mainly divided into two classes: microtubule stabilizers (paclitaxel and laulimalide) and tubulin polymerization inhibitors (colchicine, vinblastine, maytansine and pironetin) [10,11].

Targeted protein degradation agents represent a burgeoning frontier in drug development, leveraging the ubiquitin-proteasome system (UPS) to selectively degrade proteins of interest (POIs), thereby offering a novel approach to therapeutic intervention. These degradation agents primarily consist of three classes: proteolysis-targeting chimeras (PROTACs), hydrophobic taggings (HyTs) and molecular glues [12,13]. Thymoquinone (1) [14], WIT (2) [15] and compound 3 [16] have been discovered to possess the ability to degrade tubulin proteins (Fig. 1), adding a new dimension to the field of tubulin-targeting agents. These compounds represent a novel third class of tubulin-targeting agents, and their potential in the realm of anticancer therapy is increasingly being recognized [17]. The recent findings suggest that the application of PROTAC technology (PROTACs with CA-4 scaffold, Fig. 1) to the targeted degradation of tubulin protein may be subject to constraints, as evidenced by the unsuccessful attempt [18].

The HyT strategy emerges as a new avenue in protein degradation therapeutics, offering a promising alternative for the targeted degradation of tubulin. The HyT-based degrader is composed of a target-binding moiety, a linker and a hydrophobic tag (HTg) [19]. HTg attached to the surface of the protein mimics the partial misfolding state of the protein, leading to the POI degradation by the proteasome [20]. Tirbanibulin (4, Fig. 2C) is a potent tubulin polymerization inhibitor, which was approved by the FDA for the treatment of actinic keratosis in 2020 [21]. As shown in Fig. 2A, tirbanibulin was positioned in colchicine-binding site which was located at the intradimer interface of the α- and β-tubulin, by analyzing the docking model (PDB: 6KNZ) [22]. The molecular scaffold of tirbanibulin was inserted into hydrophobic pocket formed by Val238, Tyr202, Glu200, Leu252, Leu255 and Ala316 within β-tubulin, and the morpholine ethyl group extended out of the pocket, approaching the α-tubulin. The phenol oxygen atom of molecular scaffold was exposed to the solvent region between α- and β-tubulin, which made the oxygen atom to be the most appropriate site to introduce HTgs (Fig. 2B).

Based on the above analysis, we determined to develop novel tubulin-targeting agents that induced tubulin degradation and simultaneously maintained polymerization inhibition activity. By applying the hydrophobic tagging technology, the molecular scaffold of tirbanibulin was selected as the target-binding moiety which was predicted to occupy the colchicine-binding pocket. The HTg was introduced by a linker, connected to the phenol oxygen atom on the target-binding moiety. The choice of linker with optimal length was necessary, to ensure HTg attaching on the tubulin surface to mimic a partial misfolding state of tubulin. The predicted distance (including linker and hydrophobic group) between tubulin surface and phenol oxygen atom was about 13.8 Å (Fig. 2A); thus, the flexibility amide-containing alkyl chains of eight to ten atoms were required. In order to explore the optimum combination with the HTg and linker, twelve varieties of amines having diverse hydrophobic functional groups and four kinds of amide-containing alkyl chains with three different lengths were selected. A series of novel dual-mechanism tubulin-targeting agents (Fig. 2C) were designed and synthesized. Subsequently, the majority of them were proved to have potent antiproliferative activity against tumor cells.