Are the metal identity and stoichiometry of metal complexes important for colchicine site binding and inhibition of tubulin polymerization?

Quite recently we discovered that copper(II) complexes with isomeric morpholine-thiosemicarbazone hybrid ligands show good cytotoxicity in cancer cells and that the molecular target responsible for this activity might be tubulin. In order to obtain better lead drug candidates, we opted to exploit the power of coordination chemistry to (i) assemble structures with globular shape to better fit the colchicine pocket and (ii) vary the metal ion. We report the synthesis and full characterization of bis-ligand cobalt(III) and iron(III) complexes with 6-morpholinomethyl-2-formylpyridine 4N-(4-hydroxy-3,5-dimethylphenyl)-3-thiosemicarbazone (HL1), 6-morpholinomethyl-2-acetylpyridine 4N-(4-hydroxy-3,5-dimethylphenyl)-3-thiosemicarbazone (HL2), and 6-morpholinomethyl-2-formylpyridine 4N-phenyl-3-thiosemicarbazone (HL3), and mono-ligand nickel(II), zinc(II) and palladium(II) complexes with HL1, namely [CoIII(HL1)(L1)](NO3)2 (1), [CoIII(HL2)(L2)](NO3)2 (2), [CoIII(HL3)(L3)](NO3)2 (3), [FeIII(L2)2]NO3 (4), [FeIII(HL3)(L3)](NO3)2 (5), [NiII(L1)]Cl (6), [Zn(L1)Cl] (7) and [PdII(HL1)Cl]Cl (8). We discuss the effect of the metal identity and metal complex stoichiometry on in vitro cytotoxicity and antitubulin activity. The high antiproliferative activity of complex 4 correlated well with inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity were supported by experimental results and molecular docking calculations.

Dual-target inhibitors of colchicine binding site for cancer treatment.

Colchicine binding site inhibitors (CBSIs) have attracted much attention due to their antitumor efficacies and the advantages of inhibiting angiogenesis and overcoming multidrug resistance. However, no CBSI has been currently approved for cancer treatment due to the insufficient efficacies, serious toxicities and poor pharmacokinetic properties. Design of dual-target inhibitors is becoming a potential strategy for cancer treatment to improve anticancer efficacy, decrease adverse events and overcome drug resistance. Therefore, we reviewed dual-target inhibitors of colchicine binding site (CBS), summarized the design strategies and the biological activities of these dual-target inhibitors, expecting to provide inspiration for developing novel dual inhibitors based on CBS.

The cytotoxic natural compound erianin binds to colchicine site of ß-tubulin and overcomes taxane resistance.

Erianin, a natural compound derived from Dendrobium, has shown significant anticancer properties against a wide range of cancer cells. Despite the identification of multiple mechanisms of action for erianin, none of these mechanisms fully account for its broad-spectrum effect. In this study, we aimed to identify the cellular target and underlying mechanism responsible for the broad-spectrum antitumor effects of erianin. We found that erianin effectively inhibited tubulin polymerization in cancer cells and purified tubulin. Through competition binding assays and X-ray crystallography, it was revealed that erianin bound to the colchicine site of ß-tubulin. Importantly, the X-ray crystal structure of the tubulin-erianin complex was solved, providing clear insight into the orientation and position of erianin in the colchicine-binding site. Erianin showed activity against paclitaxel-resistant cells, evidenced by G2/M cell cycle arrest, apoptosis-related PARP and Caspase-3 cleavage, and in vivo xenograft studies. The study concluded that erianin bound reversibly to the colchicine site of ß-tubulin, inhibited tubulin polymerization, and displayed anticancer activity against paclitaxel-resistant cells, offering valuable insights for further exploration as potential anticancer agents.

Discovery of colchicine aryne cycloadduct as a potent molecule for the abrogation of epithelial to mesenchymal transition via modulating cell cycle regulatory CDK-2 and CDK-4 kinases in breast cancer cells.

In this study, we synthesized a new-generation library of colchicine derivatives via cycloaddition of colchicine utilizing position C-8 and C-12 diene system regioselectivity with aryne precursor to generate a small, focused library of derivatives. We assessed their anticancer activity against various cancer cell lines like MCF-7, MDA-MB-231, MDA-MB-453, and PC-3. Normal human embryonic kidney cell line HEK-293 was used to determine the toxicity. Among these derivatives, silicon-tethered compound B-4a demonstrated the highest potency against breast cancer cells. Subsequent mechanistic studies revealed that B-4a effectively modulates cell cycle regulatory kinases (CDK-2 and CDK-4) and their associated cyclins (cyclin-B1, cyclin-D1), inducing apoptosis. Additionally, B-4a displayed a noteworthy impact on tubulin polymerization, compared to positive control flavopiridol hydrochloride in a dose-dependent manner, and significantly disrupted the vimentin cytoskeleton, contributing to G1 arrest in breast cancer cells. Moreover, B-4a exhibited substantial anti-metastatic properties by inhibiting breast cancer cell migration and invasion. These effects are attributed to the down-regulation of major epithelial to mesenchymal transition (EMT) factors, including vimentin and Twist-1, and the upregulation of the epithelial marker E-cadherin in an apoptosis-dependent manner.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Molecular Dynamics and Machine Learning reveal distinguishing mechanisms of Competitive Ligands to perturb α,ß-Tubulin.

The mechanisms of action of ligands competing for the Colchicine Binding Site (CBS) of the α,ß-Tubulin are non-standard compared to the commonly witnessed ligand-induced inhibition of proteins. This is because their potencies are not solely judged by the binding affinity itself, but also by their capacity to bias the conformational states of the dimer. Regarding the latter requirement, it is observed that ligands competing for the same pocket that binds colchicine exhibit divergence in potential clinical outcomes. Molecular dynamics-based ∼5.2 µs sampling of α,ß-Tubulin complexed with four different ligands has revealed that each ligand has its customized way of influencing the complex. Primarily, it is the proportion of twisting and/or bending characteristic of modes of the intrinsic dynamics which is revealed to be 'fundamental' to tune this variation in the mechanism. The milder influence of 'bending' makes a ligand (TUB092), better classifiable under the group of vascular disrupting agents (VDAs), which are phenotypically effective on cytoskeletons; whereas a stronger impact of 'bending' makes the classical ligand Colchicine (COL) a better Anti-Mitotic agent (AMA). Two other ligands BAL27862 (2RR) and Nocodazole (NZO) fall in the intermediate zone as they fail to explicitly induce bending modes. Random Forest Classification method and K-means Clustering is applied to reveal the efficiency of Machine Learning methods in classifying the Tubulin conformations according to their ligand-specific perturbations and to highlight the significance of specific amino acid residues, mostly positioned in the α-ß and ß-ß interfaces involved in the mechanism. These key residues responsible to yield discriminative actions of the ligands are likely to be highly useful in future endeavours to design more precise inhibitors.

Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition.

Antimitotic agents are one of the more successful types of anticancer drugs, but they suffer from toxicity and resistance. The application of approved drugs to new indications (i.e., drug repurposing) is a promising strategy for the development of new drugs. It relies on finding pattern similarities: drug effects to other drugs or conditions, similar toxicities, or structural similarity. Here, we recursively searched a database of approved drugs for structural similarity to several antimitotic agents binding to a specific site of tubulin, with the expectation of finding structures that could fit in it. These searches repeatedly retrieved frentizole, an approved nontoxic anti-inflammatory drug, thus indicating that it might behave as an antimitotic drug devoid of the undesired toxic effects. We also show that the usual repurposing approach to searching for targets of frentizole failed in most cases to find such a relationship. We synthesized frentizole and a series of analogs to assay them as antimitotic agents and found antiproliferative activity against HeLa tumor cells, inhibition of microtubule formation within cells, and arrest at the G2/M phases of the cell cycle, phenotypes that agree with binding to tubulin as the mechanism of action. The docking studies suggest binding at the colchicine site in different modes. These results support the repurposing of frentizole for cancer treatment, especially for glioblastoma.

Identification of Novel ß-Tubulin Inhibitors Using a Combined In Silico/In Vitro Approach.

Due to their potential as leads for various therapeutic applications, including as antimitotic and antiparasitic agents, the development of tubulin inhibitors offers promise for drug discovery. In this study, an in silico pharmacophore-based virtual screening approach targeting the colchicine binding site of ß-tubulin was employed. Several structure- and ligand-based models for known tubulin inhibitors were generated. Compound databases were virtually screened against the models, and prioritized hits from the SPECS compound library were tested in an in vitro tubulin polymerization inhibition assay for their experimental validation. Out of the 41 SPECS compounds tested, 11 were active tubulin polymerization inhibitors, leading to a prospective true positive hit rate of 26.8%. Two novel inhibitors displayed IC50 values in the range of colchicine. The most potent of which was a novel acetamide-bridged benzodiazepine/benzimidazole derivative with an IC50 = 2.9 µM. The screening workflow led to the identification of diverse inhibitors active at the tubulin colchicine binding site. Thus, the pharmacophore models show promise as valuable tools for the discovery of compounds and as potential leads for the development of cancer therapeutic agents.

Synthesis, modeling, and biological evaluation of anti-tubulin indole-substituted furanones.

Due to the central role of tubulin in various cellular functions, it is a validated target for anti-cancer therapeutics. However, many of the current tubulin inhibitors are derived from complex natural products and suffer from multidrug resistance, low solubility, toxicity issues, and/or the lack of multi-cancer efficacy. As such, there is a continued need for the discovery and development of new anti-tubulin drugs to enter the pipeline. Herein we report on a group of indole-substituted furanones that were prepared and tested for anti-cancer activity. Molecular docking studies showed positive correlations between favorable binding in the colchicine binding site (CBS) of tubulin and anti-proliferative activity, and the most potent compound was found to inhibit tubulin polymerization. These compounds represent a promising new structural motif in the search for small heterocyclic CBS cancer inhibitors.

Systematic Studies on Anti-Cancer Evaluation of Stilbene and Dibenzo[b,f]oxepine Derivatives.

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle-a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells-leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and ß-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines-HCT116 and MCF-7-and two normal cell lines-HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a, 1c, 1d, 1i, 2i, 2j, and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton.

Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

Docking experiments suggest that gloriosine has microtubule-targeting properties similar to colchicine.

Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at the colchicine binding site (CBS) using an in silico-in vivo approach. The In silico docking of gloriosine showed a binding score of (-) 7.5 kcal/Mol towards ß-tubulin at CBS and was validated by overlapping the coupling pose of the docked ligand with co-crystallized colchicine. 2D plots (Ligplot +) showed > 85% overlap between gloriosine and colchicine. The ADMET profile of gloriosine was in accordance with Lipinski's rule of five. Gloriosine belongs to class II toxicity with anLD50 value of 6 mg/kg. In vivo and transmission electron microscopy studies revealed that gloriosine induces abnormalities in cell division such as condensed chromosomes in C-metaphase and enlarged nucleus with increased nuclear material. Gloriosine treated cells exhibited mitotic index of about 14% compared to control of 24% and high anti-proliferative activity i.e. 63.94% cell viability at a low concentration (0.0004 mg/ml). We conclude that gloriosine has a strong affinity for ß-tubulin at CBS and thus can be used as a colchicine alternative in cytology and other clinical conditions.

Design, synthesis and biological evaluation of novel tubulin inhibitors targeting colchicine sites.

Tubulin, a potential target for antitumor drug discovery, contains three main binding sites for clinical inhibitors: colchicine, vinblastine, and paclitaxel. CA-4 has been reported to be a classic tubulin inhibitor targeting the colchicine site. Herein, based on the structural modification of CA-4, 48 novel compounds were designed and synthesized by selecting structural fragments with various biological activities to replace the cis double bond of CA-4. Among these compounds, compound 8p was the most effective tubulin inhibitor (IC50 = 65 nM aganist HepG2 cells). Immunofluorescence experiment confirmed the anti-tumor effect of 8p by destroying the network structure of microtubules. Further studies showed that 8p induced tumor cell apoptosis, arrested cell cycle, inhibited tumor cell migration and invasion.

Replacing the tropolonic methoxyl group of colchicine with methylamino increases tubulin binding affinity with improved therapeutic index and overcomes paclitaxel cross-resistance.

AIMS: Microtubule inhibitors are widely used in first line cancer therapy, though drug resistance often develops and causes treatment failure. Colchicine binds to tubulins and inhibits tumor growth, but is not approved for cancer therapy due to systemic toxicity. In this study, we aim to improve the therapeutic index of colchicine through structural modification. METHODS: The methoxyl group of the tropolonic ring in colchicine was replaced with amino groups. The cross-resistance of the derivatives with paclitaxel and vincristine was tested. Antitumor effects of target compounds were tested in vivo in A549 and paclitaxel-resistant A549/T xenografts. The interaction of target compounds with tubulins was measured using biological and chemical methods. RESULTS: Methylamino replacement of the tropolonic methoxyl group of colchicine increases, while demethylation loses, selective tubulin binding affinity, G2/M arrest and antiproliferation activity. Methylaminocolchicine is more potent than paclitaxel and vincristine to inhibit tumor growth in vitro and in vivo without showing cross-resistance to paclitaxel. Methylaminocolchicine binds to tubulins in unique patterns and inhibits P-gp with a stable pharmacokinetic profile. CONCLUSION: Methylanimo replacement of the tropolonic methoxyl group of colchicine increases antitumor activity with improved therapeutic index. Methylaminocolchicine represents a new type of mitotic inhibitor with the ability of overcoming paclitaxel and vincristine resistance.

Xanthatin and 8-epi-xanthatin as new potential colchicine binding site inhibitors: a computational study.

CONTEXT: Phytocompounds xanthatin and 8-epi-xanthatin, obtained from Xanthium chinese Mill, showed antitumoral activity in vitro related to the microtubules destabilizing properties of these phytocompounds. Five binding sites for microtubule destabilizing agents have been characterized on tubulin by high-resolution X-ray crystallography: vinca domain, colchicine, pironetin, maytansine site, and more recently, the seventh site. This work aims to develop a comprehensive computational strategy to understand and eventually predict the interaction between xanthatin and 8-epi-xanthatin with the destabilizing-antimitotic binding domain of the tubulin heterodimer. In addition, we propose a putative binding site for these phytocompounds into the microtubule destabilizing binding sites on the tubulin heterodimer. Xanthanolides showed higher stability in the colchicine and pironetin binding sites, whit a greater affinity for the former. In addition, we found that xanthanolides and non-classical colchicine binding site inhibitors share a high structural similarity. METHODS: The 3D structures for xanthatin and 8-epi-xanthatin were obtained using DFT with the hybrid functional B3LYP and the base 6-31G (d,p), implemented in Gaussian 09. The 3D coordinates for tubulin proteins were downloaded from PDB. The complexes tubulin-xanthanolides were predicted using a Monte-Carlo iterated search combined with the BFGS gradient-based optimizer implemented in the AutoDock Vina. The xanthanolides-tubulin complexes were energy minimized by molecular dynamics simulations at vacuum, and their stabilities were evaluated by solvated molecular dynamics simulations during 100 ns. All molecular dynamics simulations were performed using the conjugate gradient method implemented in NAMD2 and CHARMM36 forcefield.

Discovery of potent tubulin inhibitors targeting the colchicine binding site via structure-based lead optimization and antitumor evaluation.

The colchicine binding site of tubulin is a promising target for discovering novel antitumour agents. Previously, we identified 2-aryl-4-amide-quinoline derivatives displayed moderate tubulin polymerisation inhibitory activity and broad-spectrum in vitro antitumour activity. In this study, structure based rational design and systematic structural optimisation were performed to obtain analogues C1∼J2 bearing diverse substituents and scaffolds. Among them, analogue G13 bearing a hydroxymethyl group displayed good tubulin polymerisation inhibitory activity (IC50 = 13.5 µM) and potent antiproliferative activity (IC50 values: 0.65 µM∼0.90 µM). G13 potently inhibited the migration and invasion of MDA-MB-231 cells, and displayed potent antiangiogenic activity. It efficiently increased intracellular ROS level and decreased MMP in cancer cells, and obviously induced the fragmentation and disassembly of the microtubules network. More importantly, G13 exhibited good in vivo antitumour efficacy in MDA-MB-231 xenograft model (TGI = 38.2%; i.p., 30 mg/kg).

Structural Perspective of Benzophenones Targeting Tubulin as Anticancer Agents.

Cancer is the leading cause of death and the most significant determinant of life expectancy in almost every country in this twenty-first century. According to the World Health Organization (WHO), cancer is responsible for the leading cause of death globally. Benzophenone derivatives are found in a variety of naturally occurring compounds which are known to be pharmacologically efficacious against a variety of diseases, including cancer. Microtubules are thought to be a good target for cancer chemotherapies. Microtubule polymerization and depolymerization are induced by a variety of natural, synthetic, and semisynthetic chemicals having a benzophenone nucleus, affecting tubulin dynamics. Several medications that affect microtubule dynamics are in various stages of clinical trials, including Combretastatins (phase II), Vincristine (clinically approved), Paclitaxel (in clinical usage), and epothilone (phase III), and only a few have been patented. Benzophenone derivatives target the colchicine binding site of microtubules, damage them and cause cell cycle arrest in the G2-M phase. Belonging to this class of molecules, phenstatin, a potent inhibitor of tubulin polymerization, has shown strongly inhibit cancer cell growth and arrest the G2/M phase of the cell cycle by targeting the colchicine binding site of microtubules. In the present manuscript, we described the benzophenone as tubulin polymerization inhibitors, their Structure-Activity Relationships (SARs) and molecular docking studies that reveal its binding affinity with the colchicine binding site.

Novel amino analogs of the trimethoxyphenyl ring in potent colchicine site ligands improve solubility by the masked polar group incorporation (MPGI) strategy.

The low aqueous solubility of colchicine site antimitotic agents, of which the trimethoxyphenyl (A ring) is a heavy contributor, is a serious drawback in their clinical development. We have designed new A ring analogs with chameleonic masked polar amino groups able to increase aqueous solubility and also behave as non-polar through intramolecular hydrogen bonds when bound to tubulin. We have incorporated these new A rings in several scaffolds (sulfonamides, combretastatins, phenstatins, isocombretastatins), synthesized, and assayed 43 representatives. The amino analogs show improved aqueous solubility and some of them (8, 60Z, and 67) nanomolar anti-proliferative potencies against human cancer cell lines, with the most favorable substituent being a 3-methylamino group. The antiproliferative effect relates to tubulin inhibition as shown by in vitro tubulin polymerization inhibition, immunofluorescence microscopy, and cell cycle and apoptosis analysis by flow cytometry. The compounds arrest the cell cycle of treated cells in G2/M and later develop an apoptotic response. Docking studies suggested binding at the colchicine site of tubulin with good agreement with the DFT models of the new structural variations made. The 3-methylamino-4,5­dimethoxyphenyl moiety is an example of the masked polar group incorporation (MPGI) strategy for soluble ligands binding to hydrophobic sites and a good trimethoxyphenyl ring replacement for the development of new colchicine site ligands.

ß-III Tubulin Levels Determine the Neurotoxicity Induced by Colchicine-Site Binding Agent Indibulin.

Indibulin, a microtubule-depolymerizing agent, produces minimal neurotoxicity in animals. It is also less cytotoxic toward differentiated neuronal cells than undifferentiated cells. We found that the levels of ß-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express ß-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different ß-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to ß-III tubulin with lower affinity than to other ß-tubulin isotypes. We therefore studied the implications of different ß-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of ß-III tubulin in the differentiated cells, the toxicity of indibulin and colchicine significantly increased, while sensitivity to vinblastine was unaffected. Using biochemical, bioinformatics, and fluorescence spectroscopic techniques, we have identified the binding site of indibulin on tubulin, which had not previously been established. Indibulin inhibited the binding of colchicine and C12 (a colchicine-site binder) to tubulin and also increased the dissociation constant of the interaction between tubulin and colchicine. Indibulin did not inhibit the binding of vinblastine or taxol to tubulin. Interestingly, indibulin antagonized colchicine treatment but synergized with vinblastine treatment in a combination study performed in MDA-MB-231 cells. The results indicate that indibulin is a colchicine-site binder and that the efficacy of colchicine-site binders is affected by the ß-III tubulin levels in the cells.

Discovery of potent microtubule-destabilizing agents targeting for colchicine site by virtual screening, biological evaluation, and molecular dynamics simulation.

Microtubule has been considered as attractive therapeutic target for various cancers. Although numerous of chemically diverse compounds targeting to colchicine site have been reported, none of them was approved by Food and Drug Administration. In this investigation, the virtual screening methods, including pharmacophore model, molecular docking, and interaction molecular fingerprints similarity, were applied to discover novel microtubule-destabilizing agents from database with 324,474 compounds. 22 compounds with novel scaffolds were identified as microtubule-destabilizing agents, and then submitted to the biological evaluation. Among these 22 hits, hit4 with novel scaffold represents the best anti-proliferative activity with IC50 ranging from 4.51 to 14.81 µM on four cancer cell lines. The in vitro assays reveal that hit4 can effectively inhibit tubulin assembly, and disrupt the microtubule network in MCF-7 cell at a concentration-dependent manner. Finally, the molecular dynamics simulation analysis exhibits that hit4 can stably bind to colchicine site, interact with key residues, and induce αT5 and ßT7 regions changes. The values of ΔGbind for the tubulin-colchicine and tubulin-hit4 are -172.9±10.5 and -166.0±12.6 kJ·mol-1, respectively. The above results indicate that the hit4 is a novel microtubule destabilizing agent targeting to colchicine-binding site, which could be developed as a promising tubulin polymerization inhibitor with higher activity for cancer therapy.