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.     

Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study

The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.     

Association of thiocolchicine with tubulin

Thiocolchicine, a colchicine analog in which the C-10 methoxy is replaced with a thiomethyl moiety, was shown to bind with high affinity to the colchicine site on tubulin (Ka = 1.07 +/- 0.14 x 10(6) M-1 at 23 degrees C). Like colchicine, the association kinetics were biphasic, and the rate constants of both phases were temperature dependent. The rate constant of the fast phase of the association was 4 times greater than the rate constant for colchicine binding, and the activation energy was lower (19.1 +/- 1.8 kcal/mol). X-ray crystallographic analysis shows that thiocolchicine displays greater puckering of the tropone C ring than colchicine (Koerntgen, C. and Margulis, T. N. (1977) J. Pharm. Sci. 66, 1127-1131.). These results indicate that the conformation of the C ring may have little effect on the energetics of colchicinoids binding to tubulin.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.     

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.     

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.     

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)     

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.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

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.     

Spectroscopic and kinetic features of allocolchicine binding to tubulin

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.     

Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans

The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.     

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.     

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.     

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     

Comparative molecular field analysis of colchicine inhibition and tubulin polymerization for combretastatins binding to the colchicine binding site on beta-tubulin

A molecular modeling study using Comparative Molecular Field Analysis (CoMFA) was undertaken to develop a predictive model for combretastatin binding to the colchicine binding site of tubulin. Furthermore, we examined the potential contribution of lipophilicity (log P) and molecular dipole moment and were unable to correlate these properties to the observed biological data. In this study we first confirmed that tubulin polymerization inhibition (IC50) correlated (R2 = 0.92) with [3H]colchicine displacement. Although these data correlated quite well, we developed two independent models for each set of data to quantify structural features that may contribute to each biological property independently. To develop our predictive model we first examined a series of molecular alignments for the training set and ultimately found that overlaying the respective trimethoxyphenyl rings (A ring) of the analogues generated the best correlated model. The CoMFA yielded a cross-validated R2 = 0.41 (optimum number of components equal to 5) for the tubulin polymerization model and an R2 = 0.38 (optimum number of components equal to 5) for [3H]colchicine inhibition. Final non-cross-validation generated models for tubulin polymerization (R2 of 0.93) and colchicine inhibition (R2 of 0.91). These models were validated by predicting both biological properties for compounds not used in the training set. These models accurately predicted the IC50 for tubulin polymerization with an R2 of 0.88 (n = 6) and those of [3H]colchicine displacement with an R2 of 0.80 (n = 7). This study represents the first predictive model for the colchicine binding site over a wide range of combretastatin analogues.