Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV) (original) (raw)
Related papers
European Journal of Biochemistry, 1997
2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-l-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxypheny1)-2,4,6-cycIoheptatrien-l-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K , and k, values for MDC at 25°C were 540% 65 M-' and 7 0 % 6 s-I, respectively. From the temperature dependence, a reaction enthalpy change (AH?) of the initial binding of 4 9 5 11 kJ/mol-' and an activation energy for the second step of 2 8 t 9 kJ/mol ' were calculated.
Role of B-ring of colchicine in its binding to tubulin
The Journal of biological chemistry, 1981
The chemical specificity of the colchicine-binding site of tubulin is less stringent for the presence of the B-ring than the A- and C-rings of colchicine, Colchicine analogues with modifications in the B-ring bind to tubulin at the same site as colchicine. Analogues with smaller or no substituents in the B-ring bind tubulin remarkably faster than colchicine. Thus, a compound without the B-ring [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] binds tubulin even at 4 degrees C and the binding is almost instantaneous at 37 degrees C. Colcemid and 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone bind reversibly to tubulin, whereas colchicine and desacetamidocolchicine bind almost irreversibly, suggesting that the size of the B-ring moiety of colchicine is not related to the reversibility of binding. We conclude that although the presence of the B-ring of colchicine does not appear to be an essential prerequisite for the drug-tubulin interaction, the B-ring substitu...
Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues
1996
The mechanism of the stoichiometric and substoichiometric inhibitions of tubulin self-assembly by several structural analogues of colchicine (COL) was investigated. The inhibition data were analyzed in terms of a simple model that takes into consideration K g , the normal microtubule growth constant, equal to Cr-1 (Cr is the critical concentration for microtubule formation), and K b , the binding constant of the drug to tubulin. In this manner, the value of the microtubule inhibition constant (K i), which is the binding constant of the tubulin-drug complex to the end of a growing microtubule (which stops the microtubule growth), was determined. The results of the analysis of microtubule inhibition by the various colchicine analogues show that all the inhibitions can be expressed reasonably by this model. The strongest inhibitors found were colchicine (COL), allocolchicine (ALLO), and the biphenyl keto analogue 2,3,4trimethoxy-4′-acetyl-1,1′-biphenyl (TKB), which had essentially identical values of K i) (2.1 (0.3) × 10 6 M-1. MTC, the two-ring analogue of colchicine, was weaker (K i) 5.6 × 10 5 M-1). A most striking result was that tropolone methyl ether (TME), which is ring C of COL, and which binds very weakly to tubulin (K b) 3.5 × 10 2 M-1), is a substoichiometric inhibitor. Its K i value of 8.7 × 10 5 M-1 makes it identical in strength to MTC, suggesting that ring A makes little or no contribution to the induction of assembly inhibition. The three biphenyls, which bind to tubulin with similar affinity, spanned the spectrum from strong substoichiometric inhibition (TKB) to stoichiometric inhibition for 2,3,4-trimethoxy-4′carbomethoxy-1,1′-biphenyl (TCB) and an intermediate mode for the methoxy derivative 2,3,4,4′tetramethoxy-1,1′-biphenyl (TMB). The extent of tubulin bound to drugs at 50% inhibition (r) was ca. 2% for TKB, ALLO, and COL, i.e. one liganded tubulin for every 40-50 molecules of free protein (substoichiometric). This ratio was 1:1.5 for TCB (stoichiometric) and 1:6 for TMB (intermediate). For TME, which is a single ring compound, it was 1:25. The progression of the stoichiometries varied directly with K i and was totally unrelated to the values of K b , which indicated the control of the stoichiometry by K i and the close thermodynamic linkage between r and K i. Comparison of the inhibitory capabilities of the various drugs identified the need for strong substoichiometric inhibition of a carbonyl group on ring C or C′. Furthermore, this group must be properly oriented by interaction with the protein or by the structural rigidity imparted by ring B, as in ALLO. The simple linked equilibrium model developed in this paper permits the alignment of drugs along a continuum that ranges from stoichiometric to strong substoichiometric modes of microtubule inhibition. Furthermore, it shows that the previously identified two classes are the two ends of a monotonously progressing spectrum described by a single mechanism of action.
Different Kinetic Pathways of the Binding of Two Biphenyl Analogues of Colchicine to Tubulin †
Biochemistry, 1996
The kinetics of the interaction of tubulin with two biphenyl analogues of colchicine were measured by fluorescence stopped flow. The ligands were 2,3,4-trimethoxy-4′-carbomethoxy-1,1′-biphenyl (TCB) and 2,3,4-trimethoxy-4′-acetyl-1,1′-biphenyl (TKB). The binding of both analogues is accompanied by a fluorescence increase with monophasic kinetics, which indicates that these drugs, unlike colchicine, do not discriminate between the isoforms of tubulin. The observed pseudo-first-order rate constant increases in a nonlinear way with the drug concentration, indicating that the binding of the biphenyl analogues to tubulin occurs, like colchicine, in two steps: a fast reversible equilibrium followed by an isomerization of the initial complex. Kinetic analysis shows that TCB and TKB exhibit differences in their K 1 values. At 25°C, these are 114 000 (15 000 M-1 for TCB and 8300 (900 M-1 for TKB. Both molecules show a much higher affinity than colchicine for the initial binding site. Also at 25°C, the k 2 value is 0.66 (0.04 s-1 for TCB and 3.0 (0.2 s-1 for TKB. From the temperature dependence, a reaction enthalpy change for the initial binding (∆H°1) of 44 (9 kJ‚mol-1 (TCB) and-40 (14 kJ‚mol-1 (TKB) and an activation energy for the second forward step of 64 (2 kJ‚mol-1 (TCB) and 101 (10 kJ‚mol-1 (TKB) were calculated. The dissociation kinetics were studied by displacement experiments, in which podophyllotoxin was used as a displacing ligand. The rate constant for the second step in the off direction (k-2) is 0.25 (0.05 s-1 for TCB and 0.093 (0.009 s-1 for TKB at 25°C. The activation energies for the backward isomerization of the complexes were found to be 86 (20 kJ‚mol-1 (TCB) and 79 (5 kJ‚mol-1 (TKB). Combination of these results with the kinetic parameters for association gives a full characterization of the enthalpy pathway for the binding of TCB and TKB. The pathway of TCB binding is shown to differ considerably from that of TKB binding. Since their structural difference is located in ring C′, this result points to their use of the ring C′ in the first binding step. The competitiveness of the binding of TCB and TKB with those of podophyllotoxin, MTC, and MDL 27048 indicates that the two biphenyls interact as well with the trimethoxyphenyl-specific subsite.
Biochemistry, 2005
Isotypes of vertebrate tubulin have variable amino acid sequences which are clustered at their Cterminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first order conditions. Experiments with individual isotypes established that the presence of β III in the purified tubulin is responsible for the biphasic kinetics. Since the isotypes mainly differ at the C-termini, the colchicine binding kinetics of unfractionated tubulin and the β III isotype, cleaved at the C-termini, have been tested under pseudo-first order conditions. Removal of the C-termini made no difference to the nature of the kinetics. Sequence alignment of different β isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. In order to establish whether these differences lie at the colchicine binding site or not, homology modeling of all β tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A-ring of colchicine at the colchicine-binding site on β-tubulin. While the β III isotype has two hydrophilic residues (Serine 242 and Threonine 317) both β II and β IV have two hydrophobic residues (Leucine 242 and Alanine 317). β II has Isoleucine at position 318, while β III and β IV have Valine at that position. Thus these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine binding kinetics of the different isotypes of tubulin.
2018
Theoretical investigation of the interaction between the modifications at the C-5, C-6, and C-7 positions of the B-ring of colchicine and tubulin heterodimer has been investigated by using the molecular docking simulation. The docking results provide the energetic and structural information in terms of the binding energy, binding affinity, hydrogen bonding, and conformations of docked ligand poses with residues within colchicine binding site. Overall results show that the modified C-5 (in Model A ) and C-7 (in Model C ) of B-ring analogues give the highest binding affinities to tubulin, whereas all lowest-affinity isomers belong to the C-6 substituents (in Model B ). As expected, the docked ligands of the C-5, C-6, and C-7 of B-ring analogues which are located at the a/b intradimer interface of tubulin were shifting toward the a-subunit binding space to form drug-tubulin complexes. Keywords : colchicine, B-ring analogues, tubulin, binding affinity, molecular docking
Antimitotic activity of colchicine and the structural basis for its interaction with tubulin
Medicinal Research Reviews, 2008
In this review, an attempt has been made to throw light on the mechanism of action of colchicine and its different analogs as anti-cancer agents. Colchicine interacts with tubulin and perturbs the assembly dynamics of microtubules. Though its use has been limited because of its toxicity, colchicine can still be used as a lead compound for the generation of potent anti-cancer drugs. Colchicine binds to tubulin in a poorly reversible manner with high activation energy. The binding interaction is favored entropically. In contrast, binding of its simple analogs AC or DAAC is enthalpically favored and commences with comparatively low activation energy. Colchicine–tubulin interaction, which is normally pH dependent, has been found to be independent of pH in the presence of microtubule-associated proteins, salts or upon cleavage of carboxy termini of tubulin. Biphasic kinetics of colchicines–tubulin interaction has been explained in light of the variation in the residues around the drug-binding site on β-tubulin. Using the crystal structure of the tubulin–DAMAcolchicine complex, a detailed discussion on the pharmacophore concept that explains the variation of affinity for different colchicine site inhibitors (CSI) has been discussed. © 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 1, 155–183, 2008
Interactions of long-chain homologues of colchicine with tubulin
European journal of medicinal chemistry, 2016
Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at the nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the cytotoxicity with a maximum in the range of 10-12 carbon atoms, followed by a decrease in activity with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In addition, at IC50 concentrations the analogues block the cell cycle of A549 cells in the G2/M ph...