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.     

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.     

The effect of TN-16 on the alkylation of tubulin

The synthetic anti-tumor drug 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) is known to block microtubule assembly and colchicine binding to tubulin, although its structure does not resemble those of either colchicine, podophyllotoxin, or nocodazole (Arai, FEBS Lett. 155:273-276 (1983]. We have found that TN-16 affects the intra-chain cross-linking of beta-tubulin by N,N'-ethylene-bis(iodoacetamide) in a manner identical to that of colchicine, podophyllotoxin, and nocodazole, but different from that of vinblastine or maytansine. TN-16 also inhibits alkylation of tubulin by iodo[14C]acetamide, as do colchicine and its congeners. TN-16 appears to bind to tubulin at the colchicine binding site and one of its phenyl groups is likely to bind at the site on tubulin where colchicine's A ring binds.     

The effects of colchicine analogues on the reaction of tubulin with iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide)

We have previously found (Ludue?a, R. F., and Roach, M. C. (1981b) Biochemistry 20, 4444-4450) that colchicine and podophyllotoxin inhibit the alkylation of tubulin by iodo[14C]acetamide and the formation of an intrachain cross-link in the beta-tubulin subunit by N,N'-ethylenebis(iodoacetamide) (EBI). It was not clear whether these effects were due to conformational changes in tubulin induced by drugs or to direct steric blockage of the sulfhydryl groups involved. In an effort to characterize further these phenomena, we have examined the effects of single-ring and bicyclic analogues of colchicine on the reaction of tubulin with iodo[14C]acetamide and EBI. We have found that neither the A-ring analogues, 3,4,5-trimethoxybenzyl alcohol, 3,4,5-trimethoxybenzaldehyde, 2,3,4-trimethoxybenzaldehyde, and benzaldehyde, nor the C-ring analogues, tropolone and tropolone methyl ether, inhibited alkylation. In contrast, colchicine, podophyllotoxin, and nocodazole and the bicyclic analogues, 5-(2',3',4'-trimethoxyphenyl)-2-methoxytropone and combretastatin, inhibited tubulin alkylation. Since the presence of a bond joining the A and C rings seems to be the determining factor in the suppression of alkylation, it is likely that inhibition by colchicine of the reaction with iodo[14C] acetamide is due largely to a conformational change induced by colchicine. A different pattern was obtained when the effects on cross-link formation by EBI were examined. Here, all the A-ring analogues, the bicyclic analogues, and colchicine, podophyllotoxin, and nocodazole all inhibited formation of the cross-link, whereas the C-ring analogue tropolone methyl ether did not inhibit cross-link formation. Since compounds whose effect on alkylation is markedly different have the same effect on cross-link formation, it is possible that this effect is a steric one and that perhaps the A-ring of colchicine binds to tubulin very close to one of the sulfhydryls involved in the intrachain cross-link formed by EBI in beta-tubulin.     

Effects of inhibitors of tubulin polymerization on GTP hydrolysis

The effects of a number of antimitotic drugs on the GTPase activity of tubulin were examined. The previously reported stimulation with colchicine and inhibition with podophyllotoxin and vinblastine wee confirmed. Maytansine, which competes with vinblastine in binding to tubulin, was comparable to the latter in inhibiting GTP hydrolysis. Nocodazole, which competes with colchicine in binding to tubulin, was significantly superior to colchicine in enhancing GTP hydrolysis. This superiority arose from the more rapid bindng of nocodazole to tubulin, as the two drugs had comparable activity when drug and tubulin were preincubated prior to the addition of GTP. Both colchicine and podophyllotoxin contain a trimethoxybenzene ring, while the closest structural analogy of nocodazole to colchicine includes the trimethoxybenzene ring. To explore this apparent paradox, we examined a number of simpler colchicine analogs for their effects on tubulin-dependent GTP hydrolysis. While tropolone was without effect, 3,4,5-trimethoxybenzaldehyde and 2,3,4-trimethoxybenzaldehyde stimulated the reaction. We therefore conclude that the trimethoxybenzene ring of colchicine is primarily responsible for the drug's stimulation of the GTPase activity of tubulin and that the inhibitory effect of podophyllotoxin must derive from the latter's tetrahydronaphthol moiety.     

Tubulin Stability and Decay: Mediation by Two Distinct Classes of IKP104-Binding Sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both and subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the and subunits of tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (_ss). Moreover, IKP104 lowers the sulfhydryl titer of _ss tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of _ss tubulin. The cleavage at the C-terminal ends of both and modulates the effect of IKP104 on the subunit, but not on the subunit. Fluorometric binding data analysis suggests that IKP104 binds to the _ss tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for and and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of and and the effect of the high-affinity site is restricted largely to the subunit, while the low-affinity-site binding is modulated by the C-terminal domain of . It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.     

Immunological detection of microtubule poison-induced conformational changes in tubulin

The interaction of tubulin-microtubule poison complexes with anti-tubulin antisera has been investigated using radioimmunoassay. The binding of the major antiserum used in this study to tubulin does not interfere with the binding of colchicine to the tubulin or affect the decay of the colchicine-binding activity of the tubulin. Conversely, if colchicine is incubated with the tubulin, forming tubulin-colchicine complexes, the tubulin-colchicine complexes are less efficient competitors for antibody-binding sites than tubulin alone. This is the result of the formation of specific colchicine-tubulin complexes, since tubulin, incubated with lumicolchicine or isocolchicine, behaves as if the tubulin were incubated alone in the radioimmunoassay. When tubulin is incubated with other microtubule poisons, podophyllotoxin or vinblastine, the tubulin-drug complexes have diminished ability to compete with tubulin as did the tubulin-colchicine complexes. These changes observed in the binding of tubulin-microtubule poison complexes to anti-tubulin antisera in a tubulin radioimmunoassay suggest that the binding of colchicine, podophyllotoxin, or vinblastine to tubulin induces subtle conformational changes on the surface of the tubulin dimer involving antigenic determinant sites.     

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.     

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)     

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.     

Effect of antimitotic drugs on tubulin GTPase activity and self-assembly.

Microtubule inhibitors can be classified into two categories: 1) those which inhibit the polymerization-dependent GTPase activity of phosphocellulose-purified tubulin, but induce a significant polymerization-independent GTPase activity (e.g. colchicine, griseofulvine, daunorubicine); 2) those which inhibit the GTPase activity associated with tubulin polymerization and that induced by inhibitors of the first class (e.g. the vincaalkaloids and podophyllotoxin). The colchicine-stimulated GTPase activity of tubulin appears to be due to the tubulin.colchicine complex. This suggests that colchicine inhibits tubulin assembly by binding to a tubulin-tubulin interaction site required for the polymerization-dependent GTPase activity and induces by itself a tubulin conformational change that leads to polymerization-independent GTPase activity. Stoichiometry of inhibition by vinblastine of the colchicine-stimulated GTPase activity is 1:2. On the other hand, the inhibition by vinblastine of the tubulin self-assembly and of the polymerization-dependent GTPase activity is strongly substoichiometric at the beginning of the polymerization reaction, 1 vinblastine molecule inhibiting the ability of 10 tubulin dimers to polymerize and to hydrolyze the GTP. However, at the polymerization plateau, the inhibition effect by vinblastine appears to be lower, suggesting a selective action of vinblastine on the early stages of the polymerization reaction.     

Bis(8-anilinonaphthalene-1-sulfonate) as a probe for tubulin decay

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) has been used to detect structural correlates of the well-known but poorly understood decay of tubulin function, by which tubulin loses its ability to polymerize and bind drugs in a complex, time-dependent way. The present results indicate that the decay of tubulin is accompanied by the appearance of hydrophobic areas, which bind a total of six Bis-ANS molecules with a dissociation constant of 19 microM. This binding seems to be a result of localized structural changes that are taking place in the tubulin molecule and can be used as a probe for these changes. In particular, circular dichroism measurements revealed no significant changes in the average secondary structure of the protein during the time required for complete binding of the Bis-ANS molecules. Preincubation of tubulin with the antimitotic drugs colchicine, podophyllotoxin, and vinblastine slows the rate of appearance of the hydrophobic region. Vinblastine has the maximal effect followed by colchicine and podophyllotoxin. In contrast, preincubation with maytansine has no effect. In addition, lowering the temperature decreases the rate of appearance of this region. These results correlate with the effect of drugs on the alkylation of tubulin sulfhydryl groups by iodoacetamide [Luduena, R.F., & Roach, M.C. (1981) Biochemistry 20, 4444-4450] and with the ability of inhibitors of microtubule assembly to permit the polymerization of tubulin into nonmicrotubule structures.     

Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin

Thermodynamics of podophyllotoxin binding to tubulin and its multiple points of attachment with tubulin has been studied in detail using isothermal titration calorimetry. The calorimetric enthalpy of the association of podophyllotoxin with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -2.47 kJ mol(-)(1) K(-)(1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen-bonding forces with unfavorable negative entropic contribution at higher temperature, favored with an enthalpy-entropy compensation. Interestingly, the binding of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (AC, a colchicine analogue without the B ring) with tubulin is enthalpy-favored. However, the podophyllotoxin-tubulin association depending upon the temperature of the reaction has a favorable entropic and enthalpic component, which resembles both B- and C-ring properties of colchicine. On the basis of the crystal structure of the podophyllotoxin-tubulin complex, distance calculations have indicated a possible interaction between threonine 179 of alpha-tubulin and the hydroxy group on the D ring of podophyllotoxin. To confirm the involvement of the oxalone moiety as well as the lactone ring of podophyllotoxin in tubulin binding, analogues of podophyllotoxin are synthesized with methoxy substitution at the 4' position of ring D along with its isomer and another analogue epimerized at ring E. From these results, involvement of oxalone as well as the lactone ring of the drug in a specific orientation inclusive of ring A is indicated for podophyllotoxin-tubulin binding. Therefore, podophyllotoxin, like colchicine, behaves as a bifunctional ligand having properties of both the B and C rings of colchicine by making more than one point of attachment with the protein tubulin.     

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.     

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.     

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.     

Microtubule Assembly and Function in Chlamydomonas: Inhibition of Growth and Flagellar Regeneration by Antitubulins and Other Drugs and Isolation of Resistant Mutants

The distribution of microtubules in Chlamydomonas reinhardtii suggests that they are involved in mitosis, cell and nuclear cleavage, and generation of flagella. Vinblastine, colchicine, and podophyllotoxin bind to the protein building block of microtubules (tubulin) and prevent normal assembly. Mutants resistant to these "antitubulin" drugs are candidates to have alterations in tubulin primary structure. We report the ability to inhibit growth, and flagellar regeneration after amputation, of: vinblastine, several colchicine derivatives, two water-soluble derivatives of podophyllotoxin (succinylpodophyllotoxin and epipodophyllotoxin thiuronium bromide), and other substances which may interfere with flagellar assembly or motility (isopropyl N-phenyl carbamate, 2-methoxy-5-nitrotropone, chloral hydrate, caffeine, and nickel acetate). The ability of each drug to inhibit binding of labeled colchicine or podophyllotoxin to mammalian brain tubulin was also determined. The results suggest that only in the cases of colchicine, colcemide, and epipodophyllotoxin thiruonium bromide was the toxicity to Chlamydomonas mediated by inhibition of tubulin assembly. The requirement for high concentrations of colchicine may be due to permeability barriers, since colchicine toxicity was potentiated by deoxycholate. Mutants resistant to antitubulins were isolated after treatment with methyl methanesulfonate. The results with vinblastine were equivocal. Of three mutants resistant to inhibition of growth and flagellar regeneration by colchicine, one was also cross-resistant to epipodophyllotoxin thiuronium bromide.     

Combretastatin-like chalcones as inhibitors of microtubule polymerisation. Part 2: Structure-based discovery of alpha-aryl chalcones

Tubulin is an important molecular target in cancer chemotherapy. Antimitotic agents able to bind to the protein are currently under study, commonly used in the clinic to treat a variety of cancers and/or exploited as probes to investigate the protein’s structure and function. Here we report the binding modes for a series of colchicinoids, combretastatin A4 and chalcones established from docking studies carried out on the structure of tubulin in complex with colchicine. The proposed models, in agreement with published biochemical data, show that combretastatin A4 binds to the colchicine site of β-tubulin and that chalcones assume an orientation similar to that of podophyllotoxin. The models can be used to design a new class of podophyllotoxin mimics, the α-aryl chalcones, capable of binding to the colchicine-binding site of β-tubulin with higher affinity.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.