N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin (original) (raw)
Related papers
A fluorescent stopped flow study of colchicine binding to tubulin
Journal of Biological Chemistry
The kinetics of colchicine binding to tubulin has been studied, using a fluorescence stopped flow. The measurements of Garland (Garland, D. L. (1978) Biochemistry 17, 4266-4272) have been extended to high colchicine concentrations and different temperatures. The appearance of fluorescence is biphasic. Both phases depend in a nonlinear way on colchicine concentration. The presence of colchicine dimers at these concentrations has been taken into account. The fast phase is analyzed as a two-step mechanism. The thermodynamic parameters of the fast initial binding, and the activation energy of the slow conformational change, have been determined. The relative magnitude of the slow phase depends on temperature. It is interpreted as a slow preequilibrium between two tubulin conformers. The effect of the microtubule-associated proteins on the different processes is studied. The binding of colchicine to tubulin ring-like oligomers is discussed. Colchicine is a well known microtubule inhibitor. Its mechanism of action has been studied intensively, and, depending
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...
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.
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.
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
General features of the recognition by tubulin of colchicine and related compounds
European Biophysics Journal, 1998
The kinetic mechanisms of the binding to tubulin of colchicine and eight different analogues have been studied to elucidate details of the recognition mechanism. All of the analogues follow a two step binding mechanism i.e. binding occurs via an initial step with low affinity, followed by an isomerisation of the initial complex leading to the final high affinity state. For several analogues the kinetic and thermodynamic data of both processes are compared here. For all the analogues the ∆G°1 of initial binding at 25°C varies between-13.3 and-28.8 kJ • mol-1. For the second step ∆G°2 varies between-2.4 and-27 kJ • mol-1. These limited ranges of free energy change are, however, obtained by a great variety of enthalpy changes and compensatory entropy changes. Comparison of the data for the first and second steps indicates that structural alterations of the drugs always change the thermodynamic parameters of the two steps, and the changes in the first and the second steps are in opposite directions. The fact that this range of experimental behaviour can be incorporated into a general mechanism encourages the extension of these investigations to other colchicine analogues and related compounds with potential pharmaceutical applications.
Identification of cysteine 354 of-tubulin as part of the binding site for the A ring of colchicine
1996
The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with -tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified -tubulin that had reacted with 3-(chloromethyl-[ 14 C]carbonyl)-3-demethylthiocolchicine ([ 14 C]3CTC). This -tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [ 14 C]3CTC and -tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Å of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Å distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal -tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulinbound colchicine.
Identification of Cysteine 354 of β-Tubulin as Part of the Binding Site for the A Ring of Colchicine
Journal of Biological Chemistry, 1996
The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with -tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified -tubulin that had reacted with 3-(chloromethyl-[ 14 C]carbonyl)-3-demethylthiocolchicine ([ 14 C]3CTC). This -tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [ 14 C]3CTC and -tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Å of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Å distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal -tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulinbound colchicine.