Structure-based virtual screening of novel tubulin inhibitors and their characterization as anti-mitotic agents

Microtubule cytoskeletons are involved in many essential functions throughout the life cycle of cells, including transport of materials into cells, cell movement, and proper progression of cell division. Small compounds that can bind at the colchicine site of tubulin have drawn great attention because these agents can suppress or inhibit microtubule dynamics and tubulin polymerization. To find novel tubulin polymerization inhibitors as anti-mitotic agents, we performed a virtual screening study of the colchicine binding site on tubulin. Novel tubulin inhibitors were identified and characterized by their inhibitory activities on tubulin polymerization in vitro. The structural basis for the interaction of novel inhibitors with tubulin was investigated by molecular modeling, and we have proposed binding models for these hit compounds with tubulin. The proposed docking models were very similar to the binding pattern of colchicine or podophyllotoxin with tubulin. These new hit compound derivatives exerted growth inhibitory effects on the HL60 cell lines tested and exhibited strong cell cycle arrest at G2/M phase. Furthermore, these compounds induced apoptosis after cell cycle arrest. In this study, we show that the validated derivatives of compound 11 could serve as potent lead compounds for designing novel anti-cancer agents that target microtubules.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Design,synthesis and molecular docking of novel diarylcyclohexenone and diarylindazole derivatives as tubulin polymerization inhibitors

New target compounds were designed as inhibitors of tubulin polymerization relying on using two types of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional groups (R¹) were attached to manipulate their physicochemical properties and/or their biological activity. The designed compounds were assessed for their antitumor activity on HCT-116 and MCF-7 cancer cell lines. Compounds 4b, 5e and 5f exhibited comparable or higher potency than colchicine against colon HCT-116 and MCF-7 tumor cells. The mechanism of the antitumor activity was investigated through evaluating the tubulin inhibition potential of the active compounds. Compounds 4b, 5e and 5f showed percentage inhibition of tubulin in both cell line homogenates ranging from 79.72% to 89.31%. Cell cycle analysis of compounds 4b, 5e and 5f revealed cell cycle arrest at G₂/M phase. Molecular docking revealed the binding mode of these new compounds into the colchicine binding site of tubulin.     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

Role of B-ring of colchicine in its binding to Zn(II)-induced tubulin-sheets

Colchicine-tubulin dimer comPlex, a Potent inhibitor of normal microtubule assembly undergoes extensive self-assembly in the Presence of 1 X 10^-4 M zinc sulPhate. Polymers assembled from colchicine-tubulin dimer comPlexes are sensitive to cold. Although colchicine can be accomodated within the Polymeric structure, the drug cannot bind to tubulin subunits in the intact Polymers. This is evidenced by the fact that (a) the colchicine binding activity of tubulin is lost when allowed to Polymerize with zinc sulPhate, (b) the loss in colchicine binding could be Prevented by Preincubation of tubulin with 1 X 10^-3 M CaCl₂ or 1 X 10^-5 M vinblastine sulPhate and finally (c) no loss in colchicine binding activity is found when tubulin is kePt at a concentration far below the critical concentration for Polymerization. Unlike colchicine, its B-ring analogues desacetamido colchicine (devoid of the B-ring subtituent) and 2-methoxy-5-(2′, 3′, 4′-trimethoxyPhenyl) troPone (devoid of the B-ring) can bind to tubulin subunits in the intact Polymers. Thus we conclude that the colchicine binding domain on the tubulin molecule is mostly (if not comPletely) exPosed in the Zn(II) -induced Polymers and the B-ring substituent Plays a major role in determining the binding ability of a colchicine analogue to tubulin in the intact Zn(II) -induced sheets.     

Binding conformations and QSAR of CA-4 analogs as tubulin inhibitors

A theoretical study on the binding conformations and the quantitative structure–activity relationship (QSAR) of combretastatin A4 (CA-4) analogs as inhibitors toward tubulin has been carried out using docking analysis and comparative molecular field analysis (CoMFA). The appropriate binding orientations and conformations of these compounds interacting with tubulin were revealed by the docking study; and a 3D-QSAR model showing significant statistical quality and satisfactory predictive ability was established, in which the correlation coefficient (R²) and cross-validation coefficient (q²) were 0.955 and 0.66, respectively. The same model was further applied to predict the pIC₅₀ values for 16 congeneric compounds as external test set, and the predictive correlation coefficient R²_pred reached 0.883. Other tests on additional validations further confirmed the satisfactory predictive power of the model. In this work, it was very interesting to find that the 3D topology structure of the active site of tubulin from the docking analysis was in good agreement with the 3D-QSAR model from CoMFA for this series of compounds. Some key structural factors of the compounds responsible for cytotoxicity were reasonably presented. These theoretical results can offer useful references for understanding the action mechanism and directing the molecular design of this kind of inhibitor with improved activity.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Optimization of substituted cinnamic acyl sulfonamide derivatives as tubulin polymerization inhibitors with anticancer activity

A new series of novel cinnamic acyl sulfonamide derivatives were designed and synthesized and evaluated their anti-tubulin polymerization activities and anticancer activities. One of these compounds, compound 5a with a benzdioxan group, was observed to be an excellent tubulin inhibitor (IC₅₀?=?0.88?µM) and display the best antiproliferative activity against MCF-7 with an IC₅₀ value of 0.17?μg/mL. Docking simulation was performed to insert compound 5a into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. 3D-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent anti-tubulin polymerization activity.     

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.     

Structure of a benzylidene derivative of 9(10H)-anthracenone in complex with tubulin provides a rationale for drug design

Microtubules are composed of αβ-tubulin heterodimers and have been treated as highly attractive targets for antitumor drugs. A broad range of agents bind to tubulin and interfere with microtubule assembly, including colchicine binding site inhibitors (CBSIs). Tubulin Polymerization Inhibitor I (TPI1), a benzylidene derivative of 9(10H)-anthracenone, is a CBSI that inhibits the assembly of microtubules. However, for a long time, the design and development of anthracenone family drugs have been hindered by the lack of structural information of the tubulin-agent complex. Here we report a 2.3 Å crystal structure of tubulin complexed with TPI1, the first structure of anthracenone family agents. This complex structure reveals the interactions between TPI1 and tubulin, and thus provides insights into the development of new anthracenone derivatives targeting the colchicine binding site.     

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.     

Hybrid Pharmacophore Design,Molecular Docking,Synthesis, and Biological Evaluation of Novel Aldimine‐Type Schiff Base Derivatives as Tubulin Polymerization Inhibitor

A series of hybrid aldimine‐type Schiff base derivatives including trimethoxyphenyl ring and 1,2,4‐triazole‐3‐thiol/thione were designed as tubulin inhibitors. The molecular docking simulations on tubulin complex (PDB: 1SA0) revealed that derivatives with nitro and/or chloro or dimethylamino substitutes (4‐nitro, 2‐nitro, 3‐nitro, 4‐Cl‐3‐nitro, and 4‐Me₂N) on the aldehyde ring were the best compounds with remarkable binding energies (?9.09, ?9.07, ?8.63, ?8.11, and ?8.07 kcal mol^?1, respectively) compared to colchicine (?8.12 kcal mol^?1). These compounds were also showed remarkable binding energies from ?10.66 to ?9.79 and ?10.12 to ?8.95 kcal mol^?1 on human (PDB: 1PD8) and Candida albicans (PDB: 3QLS) DHFR, respectively. The obtained results of cytotoxic activities against HT1080, HepG2, HT29, MCF‐7, and A549 cancer cell lines indicated that 4‐nitro and 2‐nitro substituted compounds were the most effective agents by mean IC₅₀ values of 11.84 ± 1.01 and 19.92 ± 1.36 μm , respectively. 4‐Nitro substituted compound (5 μm ) and 2‐nitro substituted compound (30 μm ) were able to strongly inhibit the tubulin polymerization compared to colchicine (5 μm ) and 4‐nitro substituted compound displayed IC₅₀ values of 0.16 ± 0.01 μm compared to that of colchicine (0.19 ± 0.01 μm ). This compound also showed the lowest MIC values on all tested microbial strains including three Gram‐positive, four Gram‐negative, and three yeast pathogens.     

A computational study of binding between 3-(4-fluorophenyl)-N-((4-fluorophenyl)sulphonyl)acrylamide and tubulin

3-(4-Fluorophenyl)-N-((4-fluorophenyl)sulphonyl)acrylamide (FFSA) is a potential tubulin polymerisation inhibitor. In this article, a theoretical study of the binding between FFSA and tubulin in colchicine site was carried out by molecular docking, molecular dynamics (MD) simulation and binding free energy calculations. The docking calculations preliminarily indicate that there are three possible binding modes 1, 2 and 3; MD simulations and binding free energy calculations identify that binding mode 2 is the most favourable, with the lowest binding free energy of ? 29.54 kcal/mol. Moreover, our valuable results for the binding are as follows: the inhibitor FFSA is suitably located at the colchicine site of tubulin, where it not only interacts with residues Leu248β, Lys254β, Leu255β, Lys352β, Met259β and Val181a by hydrophilic interaction, but also interacts with Val181α and Thr179α by hydrogen bond interaction. These two factors are both essential for FFSA strongly binding to tubulin. These theoretical results help understanding the action mechanism and designing new compounds with higher affinity to tubulin.     

(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides as tubulin polymerization inhibitors: Structure-based bioisosterism design,synthesis, biological evaluation,molecular docking and in silico ADME prediction

A series of (E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides have been synthesized and evaluated for their anticancer activity in human hepatocellular liver carcinoma HepG2 and breast adenocarcinoma MCF-7?cell lines. Among all the tested compounds, compound 3a, 3e and 3n displayed more activity than lead compound with IC₅₀ value of 0.26–0.61?μM. Meanwhile, these compounds (3a, 3e and 3n) showed potent antiproliferative activity against a panel of cancer cells and the HCT-8/T multidrug resistant cell line with IC₅₀ values in the range of 0.077– 7.44?μM. Flow cytometric analyses revealed that compound 3n induced cell cycle arrest in G2/M phases in a dose dependent manner. The compound 3n also displayed potent tubulin polymerization inhibition with an IC₅₀ value of 0.9?µM, with ten folds more active than colchicine (IC₅₀?=?9?μM). Molecular docking studies revealed that compound 3n efficiently interacted with the colchicine binding site of tubulin through hydrophobic, cation-π and hydrogen bond interaction. Furthermore, in silico pharmacokinetic prediction shown that these compounds have a good ADME-related physicochemical parameters. These results demonstrate that 3n exhibits potent cytotoxicity in cancer cells by targeting the colchicine binding site of tubulin and potentially acts as a therapeutic lead compound for the development of anticancer drugs.     

Design,synthesis and biological evaluation of (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives as a new class of tubulin polymerization inhibitors

A series of novel (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives had been synthesized and evaluated their biological activities as potential tubulin polymerization inhibitors. Among these compounds, compound 3q exhibited potent antiproliferative activities against three cancer cell lines in vitro, and antitubulin polymerization activity with IC₅₀ of 0.92 μM, which was superior to that of colchicine (IC₅₀ = 1.34 μM). Docking simulation was performed to insert compound 3q into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. These results suggested that compound 3q may be a promising antitubulin agent for the potential treatment of cancer.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

Design,synthesis and structure–activity relationship of 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]furan derivatives as a novel class of inhibitors of tubulin polymerization

The biological importance of microtubules in mitosis and cell division makes them an interesting target for the development of anticancer agents. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. Thus, a new class of inhibitors of tubulin polymerization based on the 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]furan molecular skeleton, with electron-donating (Me, OMe or OH) or electron-withdrawing (F, Cl and Br) substituents on the benzene ring, was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. Adding a methyl group at the C-3 position resulted in increased activity. The most promising compound in this series was 2-(3′,4′,5′-trimethoxybenzoyl)-3-methyl-6-ethoxy-benzo[b]furan, which inhibits cancer cell growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.     

Synthesis,cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.     

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors

Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB_VIN, and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI₅₀ values of 0.19–0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC₅₀ 1.4–1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.