Synthesis,molecular properties prediction and biological evaluation of indole-vinyl sulfone derivatives as novel tubulin polymerization inhibitors targeting the colchicine binding site

Twenty-two novel indole-vinyl sulfone derivatives were designed, synthesized and evaluated as tubulin polymerization inhibitors. The physicochemical and drug-likeness properties of all target compounds were predicted by Osiris calculations. All compounds were evaluated for their antiproliferative activities, among them, compound 7f exhibited the most potent activity against a panel of cancer cell lines, which was 2–7 folds more potent than our previously reported compound 4. Especially, 7f displayed about 8-fold improvement of selective index as compared with compound 4, indicating that 7f might have lower toxicity. Besides, 7f inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Further investigations showed that compound 7f effectively disrupted microtubule network, caused cell cycle arrest at G2/M phase and induced cell apoptosis in K562 cells. Moreover, 7f reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, the in vivo anti-tumor activity of 7f was validated in H22 liver cancer xenograft mouse model without apparent toxicity, suggesting that 7f is a promising anti-tubulin agent for cancer therapy.     

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.     

Docking study of ligands into the colchicine binding site of tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of alpha and beta-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.     

Involvement of colchicine binding site of tubulin in the polymerisation process

Role of lysine residues in the colchicine binding site and in the assembly-disassembly process was examined. It was observed that at 4 degrees C (pH 7.5-8, 8 +/- 1) lysine residues and the N-terminal methionine residue of tubulin were all buried within the molecule. Evidence indicates that epsilon-amino groups of lysine residues of tubulin are shared by both the colchicine binding site and the polymerisation process.     

Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding

Sulfhydryls and disulfides of platelet tubulin have been quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by two cycles of temperature-dependent polymerization--depolymerization was found to contain 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, one intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by one which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding, on the other hand, was only minimally influenced by blocking of sulfhydryls.     

The binding of vincristine, vinblastine and colchicine to tubulin

Preparations of tubulin were examined for their ability to bind vincristine, vinblastine, and colchicine, as measured by adsorption on DEAE impregnated filter paper. Vincristine and vinblastine were found to bind very rapidly with tubulin (<5 min), while colchicine took considerably longer (>4 hr). When varying concentrations of the alkaloids were employed, and the data examined on a Scatchard plot, it was found that colchicine had an association constant of 1.8 × 10⁶ liters/mole, while vinblastine and vincristine had constants of 6.0 × 10⁶ liters/mole and 8.0 × 10⁶ liters/mole respectively. In addition, it was found that the ratio of molar binding of colchicine was always twice that of vinblastine or vincristine.     

Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro.

The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal 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     

Biosynthesis of radiolabeled curacin A and its rapid and apparently irreversible binding to the colchicine site of tubulin.

Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [(14)C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0 degrees C, with a minimum k(f) of 4.4 x 10(3) M(-1) s(-1). We were unable to demonstrate any dissociation of the [(14)C]curacin A from tubulin. Consistent with these observations, the K(a) value was so high that an accurate determination by Scatchard analysis was not possible. The [(14)C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [(14)C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a K(a) of 3.2 x 10(-5) M(-1).     

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.     

B-ring of colchicine and its role in taxol-induced tubulin polymerization

Taxol-induced assembly of purified tubulin is not inhibited by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. Colchicine analogues having intact A, C and B-rings (without NH-CO-CH3) such as desacetamidocolchicine have also been found to be inactive. It has been observed that these two colchicine analogues are incorporated into polymers when incubated in the presence of taxol. Furthermore, preformed taxol-induced polymers of tubulin have been found to bind these two colchicine analogues. These results suggest that colchicine-binding domains on the tubulin molecule are mostly (if not completely) exposed in the taxol-induced polymers.     

Synthesis,biological evaluation and molecular docking studies of aminochalcone derivatives as potential anticancer agents by targeting tubulin colchicine binding site

A series of aminochalcone derivatives have been synthesized, characterized by HRMS, ¹H NMR and ¹³C NMR and evaluated for their antiproliferative activity against HepG2 and HCT116 human cancer cell lines. The most of new synthesized compounds displayed moderate to potent antiproliferative activity against test cancer cell lines. Among the derivatives, compound 4 displayed potent inhibitory activity with IC₅₀ values ranged from 0.018 to 5.33 μM against all tested cancer cell lines including drug resistant HCT-8/T. Furthermore, this compound showed low cytotoxicity on normal human cell lines (LO2). The in vitro tubulin polymerization assay showed that compound 4 inhibited tubulin assembly in a concentration-dependent manner with IC₅₀ value of 7.1 μM, when compared to standard colchicine (IC₅₀ = 9.0 μM). Further biological evaluations revealed that compound 4 was able to arrest the cell cycle in G2/M phase. Molecular docking study demonstrated the interaction of compound 4 at the colchicine binding site of tubulin. All the results indicated that compound 4 is a promising inhibitor of tubulin polymerization for the treatment of cancer.     

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.     

Rhizoxin binding to tubulin at the maytansine-binding site

The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for rhizoxin per molecule with a dissociation constant (Kd) of 1.7.10(-7) M. Ansamitocin P-3, a homologue of maytansine, was a competitive inhibitor of rhizoxin binding, with an inhibition constant of 1.3.10(-7) M. Vinblastine also inhibited rhizoxin binding, but was not fully competitive, and the inhibition constant was 2.9.10(-6) M. In contrast, both rhizoxin and ansamitocin P-3 were potent inhibitors of vinblastine binding. Rhizoxin inhibited tau-promoted tubulin assembly, but it, differing from vinblastine, did not induce tubulin aggregation into spirals, even at a concentration as high as 2.10(-5) M. In addition, rhizoxin strongly inhibited vinblastine-induced tau-dependent tubulin aggregation. Rhizoxin binding to tubulin was completely independent from colchicine binding. These effects resemble those of maytansine. The results suggested that rhizoxin binds to the maytansine-binding site and that the binding sites of rhizoxin and vinblastine are not the same.     

Roles of ring C oxygens in the binding of colchicine to tubulin

The roles of the oxygens in ring C of colchicine in its binding to tubulin were probed by a study of the interactions of two allocolchicine biphenyl analogues, 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB), the first one containing a methoxy group in position 4', the second a keto group. Both analogues were found to bind specifically to the colchicine-binding site on tubulin in a rapidly reversible equilibrium. The standard free energies of binding at 25 degrees C were delta G zero (TKB) = 7.19 +/- 0.11 kcal mol-1 and delta G zero (TMB) = -6.76 +/- 0.22 kcal mol-1. The binding of TKB induced the same perturbation in protein circular dichroism at 220 nm as colchicine and allocolchicine, as well as quenching of protein tryptophan fluorescence. Binding of TMB did not affect the protein CD spectrum within experimental error and induced only a marginal quenching of protein fluorescence. Comparison with the binding properties of allocolchicine and its des(ring B) analogue 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) [Medrano et al. (1989) Biochemistry 28, 5589-5599] has shown that the binding properties of the 4'-keto analogue (TKB) were closer to those of allocolchicine, even though the substituent in the 4'-position of TCB is identical with that of allocolchicine. It has been proposed that binding in the ring C subsite on tubulin, which is stabilized thermodynamically by stacking interactions, can be modulated in a nonidentical fashion by the carbonyl and the ether oxygens in the para position of ring C.     

Roles of colchicine rings B and C in the binding process to tubulin

The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.     

Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the antimitotic peptide and depsipeptide binding site on beta-tubulin

Several naturally occurring peptides and depsipeptides which include the cryptophycins, dolastatin 10, hemiasterlin, and phomopsin A have been found to be potent antimitotic agents, causing cell death at picomolar or low nanomolar concentrations. These compounds inhibit microtubule growth, modulate the dynamics of microtubules, and induce the self-association of tubulin dimers into single-walled rings and spirals. These peptides exhibit mutual competitive inhibition in binding to beta-tubulin, while noncompetitively inhibiting the binding of vinblastine and vincristine to beta-tubulin. Despite the abundance of biochemical information, the details of their molecular interactions with tubulin are not known. In this study, using a combination of molecular dynamics simulations and molecular docking studies, a common binding site for cryptophycin 1, cryptophycin 52, dolastatin 10, hemiasterlin, and phomopsin A on beta-tubulin has been identified. Application of these same methods to alpha-tubulin indicated no interaction between alpha-tubulin and any of the peptides. On the basis of the docking results, a model for the mechanism of microtubule disruption and formation of aberrant nonmicrotubule structures is proposed. Both the active site and mechanism of microtubule depolymerization predictions are in good agreement with experimental findings.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.