(Received for publication, July 12, 1995; and in revised form, November 20, 1995)
From the
The quenching of tryptophan fluorescence has been used to
determine the kinetic and thermodynamic parameters of binding of B-ring
analogs of colchicine to tubulin. The on rate, activation energy,
off-rate, and thermodynamics of binding reaction have been found to be
controlled at different points of analog structure. The on-rate and
off-rate of deacetamidocolchicine (DAAC) binding with tubulin is 17
times slower than that of
2-methoxy-5-(2`,3`,4`-trimethoxyphenyl)tropone-tubulin (AC-tubulin)
interaction, although both reactions have very similar activation
energies. The presence of B-ring alone does not significantly affect
the thermodynamics of the binding reactions either, since both
AC-tubulin and DAAC-tubulin interactions are enthalpy driven.
Introduction of a NH group at C-7 position of the B-ring,
as in deacetylcolchicine (NH
-DAAC) lowers the on-rate
further with a significant rise in the value of the activation energy.
However, bulkier substitutions at the same position, as in demecolcine
(NHMe-DAAC) and N-methyldemecolcine (NMe
-DAAC)
have no significant additional effect either on the on-rate or on the
value of activation energy. Introduction of NH
group in the
C-7 position of B-ring also increases the positive entropy of the
binding reaction to a significant extent, and it is maximum when
NMe
is substituted instead of NH
group. Thus,
interaction of NH
-DAAC, NHMe-DAAC, and NMe
-DAAC
with tubulin are entropy driven. Our results suggest that the B-ring
side chain of aminocolchicinoids makes contact(s) with dimeric tubulin
molecules.
Colchicine, the major alkaloid in Colchicum autumnale, is medically used for the treatment of gout (1) and Familial Mediterranean fever(2) . Due to its immense therapeutic importance, a large number of colchicine and thiocolchicine analogs have been synthesized and tested for their biological activities(3, 4, 5) . Colchicine exerts its antimitotic property upon binding to a high affinity site on the tubulin heterodimer(6, 7, 8) . It is composed of a trimethoxybenzene ring (A-ring), a methoxytropone ring (C-ring), and a seven-membered ring (B-ring), which anchors the A- and C-rings (Fig. 1). Structure activity studies indicate that the A- and C-rings of colchicine comprise the minimum structural features of the molecule necessary for its high affinity binding to tubulin. Insertion of a bulky group in the A-ring of colchicine, as in colchicoside, causes complete loss of binding, indicating that the requirement of the A-ring is stringent(4) . On the other hand, several changes in the C-ring such as different substitutions at the C-10 position or a replacement of the seven-membered ring with a six-membered ring are tolerated(9, 10, 11, 12, 13, 14) . Colchicine analogs modified at, or depleted of, the B-ring are known to retain potent antimitotic activity, self-assembly inhibitory activity, and the binding activity to tubulin at the colchicine site(15, 16, 17) . Nevertheless, the presence of B-ring alone, or substituents at C-7 position, influences the on-rate, activation energy, off-rate, reversibility, and the quantum yield of the complexes of the drug with tubulin(9, 16, 17, 18, 19, 20, 21, 22) . The thermodynamic contributions of A- and C-rings of colchicine in its binding to tubulin have also been reported. Binding of tropolone methyl ether (a C-ring analog) is characterized by negative apparent enthalpy and entropy changes, whereas N-acetylmescaline (an A-ring analog) interaction with tubulin has positive enthalpy and entropy changes(23) . Binding of AC, a simple bifunctional ligand containing A- and C-rings with tubulin has been found to be enthalpy driven(17) . Studies on the binding thermodynamics of colchicine-tubulin interaction have provided conflicting results. While early equilibrium studies on colchicine-tubulin interaction reported high entropy value for the binding reaction, calorimetric and kinetic studies reported the negative enthalpy value for the same interaction(8, 20, 24, 25) .
Figure 1: Structure of colchicine, AC, and B-ring analogs of colchicine.
In
the present study, we have determined the thermodynamic parameters for
the binding reactions of four B-ring analogs of colchicine with
tubulin: deacetamidocolchicine (DAAC), ()deacetylcolchicine
(NH
-DAAC), demecolcine (NHMeDAAC), and N-methyldemecolcine (NMe
-DAAC). Our study
indicates that the presence of B-ring per se does not affect
the entropic contribution significantly, as binding of both AC and DAAC
are enthalpy-driven reactions. It is the amino substituent at the C-7
position in the B-ring that converts an enthalpy-driven reaction into
an entropy-driven reaction. Our thermodynamic data of
colchicinoid-tubulin interactions suggest that the C-7 substituent on
the B-ring of the colchicinoids studied here make additional contact(s)
with the dimeric tubulin molecule.
Pipes, GTP, EGTA, colchicine, and demecolcine were purchased from Sigma. Deacetylcolchicine and colchicine fluorescein were obtained from Molecular Probes, Inc. All other reagents used were of analytical grade. Other colchicine analogs were gifts from T. J. Fitzgerald (Florida A & M University) and Susan Bane Hastie (SUNY, Binghamton).
Goat brain tubulin, free of microtubule-associated
proteins, was prepared by two cycles of assembly-disassembly in PEM
buffer (0.05 M Pipes, 1 mM EGTA, 0.5 mM MgCl, pH 6.9, at 25 °C) in presence of 1 mM GTP followed by two more cycles in 1 M glutamate buffer (26) and stored at -70 °C. The concentration of
protein was determined by the method of Lowry et
al.(27) .
Stock solutions of colchicine and its analogs
were prepared either in water or in dimethyl sulfoxide
(MeSO). The maximum concentration of Me
SO in
the reaction mixture was 5% for DAAC and was less than 1% for other
colchicinoids. The concentrations of the ligands were determined from
the respective extinction coefficients(22) .
where F is the fluorescence of the ligand-tubulin
complex at time t, A and B are the
amplitudes for the fast and slow phases, k and k
are the pseudo-first-order rate constants for
these two phases, respectively, and C is an integration
constant. As, for all the tubulin-colchicinoid complexes, the amplitude
of the slow phase, B, was small relative to that of the fast
phase, A, the slow phase was not analyzed
further(28) . The apparent second-order rate constant (k
), was obtained by dividing the observed rate
constant for the fast phase (k
) by the ligand
concentration. Association rate constants were determined at different
temperatures ranging from 17 to 37 °C.
where F and F are the maximum
intrinsic protein fluorescence intensity at infinite time and at time t, respectively, and k
is the
first-order dissociation rate constant. Dissociation rate constants
were determined at different temperatures ranging from 17 to 37 °C.
where F is the fluorescence at time zero, F
is the fluorescence when saturation was
reached, and r = A/B. The unknown
parameters are k
, k
, F
, F
, and r.
Each of these was systematically varied within a given range, and
statistical estimates of the quality of fit for obtained F with the experimentally determined curve were performed for each
point of iteration. The parameters obtained for the best-fit curve
giving minimum
value were thus calculated using a
BASIC program written for this purpose. For dissociation kinetics, the
two unknown parameters k
and F
were varied and the best-fit values giving
minimum
were obtained by similar iteration on .
Figure 2:
Effect of temperature on the apparent
second-order rate constant of colchicine B-ring analogs binding to
tubulin. Binding studies were carried out with DAAC (- -
-), NH-DAAC (- - - - -), NHMe-DAAC
(- - -), NMe
-DAAC (-). Inset, quenching of intrinsic protein fluorescence upon
NHMe-DAAC binding to tubulin. NHMe-DAAC (final concentration, 20
µM) was added to tubulin (concentration, 1
µM). Kinetics was followed for 1 h at 25 °C by
measuring the intensity of intrinsic protein fluorescence. Excitation
and emission wavelengths were 295 and 336 nm,
respectively.
where n is the change in the number of molecules
when the complex is formed and (
S)
is the
activation entropy. Transition state free energy, enthalpy, and entropy
of AC and DAAC binding to tubulin have recently been
measured(22) . The free energy and enthalpy values in the
transition states are very close for both AC and DAAC, whereas entropy
values differ significantly(22) . This difference in the
activation entropies of AC-tubulin and DAAC-tubulin interaction
probably arises from the restriction imposed upon DAAC by the presence
of the B-ring and thus accounts for the difference in pre-exponential
factors (Table 1).
The effect of substitutions on the B-ring
on the rate constants, activation energies, and A values of
the binding of DAAC and aminocolchicinoids are compared in Table 1. A significant drop in the association rates and a
significant enhancement in the activation energies is apparent when
NH group is present at the C-7 position of the B-ring.
However, substitution by further bulky group(s) (e.g. NHMe and
NMe
groups) did not affect either the association rate or
the activation energy. What really happens to the binding process when
-NH
is substituted at the C-7 position in the B-ring
is difficult to understand from the present state of knowledge of
B-ring analogs binding to tubulin. It is to be noted that whereas
colchicine is highly soluble in water, other aminocolchicinoids studied
here are weakly soluble, indicating that the solvation property of
those molecules are very much influenced by the side chain at C-7
position. The presence of lone pair of electrons of nitrogen will
significantly influence the carbonyl oxygen of colchicine side chain as
follows:
Thus, the oxygen of the colchicine side chain and the electron rich nitrogen atom in aminocolchicinoids can serve as potential electron donor in making hydrogen bond with surroundings. It is possible that the amino and carbonyl groups are involved in making important contacts with the protein in the complex form. Recently, it was proposed that the substituents in the B-ring of aminocolchicinoids point away from the colchicine binding site toward the exterior of the protein during their interaction with tubulin(22) . Our thermodynamic data also support this proposition (see below). We observe a dramatic change in the enthalpy and the entropy of the tubulin binding reaction of DAAC when compared with that of other aminocolchicinoids (Table 2).
Dissociation rate constants were
determined by measuring the enhancement of intrinsic protein
fluorescence due to release of colchicinoids from tubulin-colchicinoid
complexes upon a 300-fold dilution of the complex by PEM buffer. This
method has been successfully used previously to determine the
dissociation kinetics of AC-tubulin complexes(17) . The
dissociation rate constants were determined using (see
``Experimental Procedures''). The temperature dependence of
dissociation rate constants for DAAC, NH-DAAC, NHMe-DAAC,
and NMe
-DAAC were also determined by the same procedure. A
comparison of dissociation rate constants (see Table 1) indicates
that the presence of B-ring significantly lowers the dissociation rate
of drug-tubulin complexes. The dissociation rates for DAAC-tubulin
complex and aminocolchicinoid-tubulin complexes are 18-30-fold
less than that of AC-tubulin complex (Table 1). However, the
first-order dissociation rates for DAAC-tubulin and
aminocolchicinoid-tubulin complexes are comparable and are about
100-150-fold higher than that of the colchicine-tubulin complex.
It was suggested previously that the >C=O in the side chain
is responsible for the poor reversibility of the colchicine-tubulin
interaction(19) . Dissociation rate of another colchicine
analog wherein >C=O was substituted by >C=S
(colchicine fluorescein) (see Fig. 1) was determined under
identical conditions. The rate constant (1.6
10
s
at 27 °C) was similar to those obtained
for DAAC or other aminocolchicinoids and much greater than that for
colchicine, supporting the earlier hypothesis(19) .
where k and k
are
the apparent second-order association rate constant and first-order
dissociation rate constant, respectively. After calculating K
values at different temperatures, van't
Hoff plots for all four colchicinoids were done as shown in Fig. 3. Thermodynamic parameters were calculated and are
presented in Table 2. Our data (Table 2) clearly indicate
that like AC-tubulin interaction, DAAC-tubulin interaction has negative
enthalpy of binding and has small positive
S (15.8 cal
K
mol
). Both kinetic and
equilibrium studies for the DAAC-tubulin interaction provide similar
thermodynamic parameters. (
)However, when an amino group is
substituted at C-7 in the B-ring as in NH
-DAAC, the
interaction with tubulin becomes entropy driven, and the positive
S increases to 46.5 cal K
mol
. The positive
S remains
unaltered when a methyl group is substituted in NH
-DAAC, as
in demecolcine-tubulin interaction. We have reported very similar
thermodynamic parameters for demecolcine-tubulin interaction using
equilibrium method(31) . Addition of another methyl group as in
NMe
-DAAC causes a further increase in entropy to 54.9 cal
K
mol
. These data clearly
establish that the bare B-ring itself has no significant effect in the
thermodynamics of drug binding with tubulin. Rather, it is the B-ring
substituent(s) that convert an enthalpy-driven reaction into an
entropy-driven one. Early equilibrium studies on colchicine-tubulin
interaction reported high positive entropy value for the binding
reaction(8, 24) . Later, these data were questioned
for two reasons: first, these were possibly obtained in conditions
where true equilibrium has not been reached; second, proper corrections
were not made for the decay of colchicine binding site(20) . In
the study of Diaz and Andreu(25) , where corrections were made
for the decay of colchicine binding site, the colchine-tubulin
interaction was found to be accompanied by negative enthalpy change. A
negative enthalpy value for the colchicine-tubulin interaction was also
obtained from calorimetric study(20) . It should be noted that
in one of the earlier equilibrium studies, tubulin used for the binding
was in the form of vinblastine paracrystals, where tubulin is stable
for several days at room temperature(24) . Moreover,
vinblastine paracrystals and colchicine were incubated together for 24
h at room temperature for the binding study(24, 32) .
Thus, it is difficult to conceive that the decay of the colchicine
binding site and non-attainment of equilibrium is responsible for the
above reported result(20, 25) .
Figure 3:
Effect of temperature on the equilibrium
constants of B-ring analog-tubulin interactions. van't Hoff plots
of the reaction of tubulin with DAAC (A), NH-DAAC (B), NMe
-DAAC (C), and NHMe-DAAC (D) are shown.
It is interesting
to note that the thermodynamic data of the binding reaction presented
in Table 2show that the changes in enthalpy and entropy upon
binding are compensatory. This ``compensatory'' effect is
shown in Fig. 4, where H is plotted as a function
of T
S at 310 K. In this plot, the slope, i.e. d(
H)/d(T
S),
is close to 1. It is interesting to note that values of
H and T
S for the interaction of tubulin with
colchicine and AC were taken from the literature (17, 24) and plotted with that of aminocolchicinoids
studied here. Similar enthalpy-entropy compensation with slope close to
1 has been observed in many protein-ligand interactions where the
experimental conditions are fixed and only the ligand structure is
varied (Congener series)(33, 34) . Arguments have been
made that the perturbation, release, or shift in the state of water
upon the binding of ligand to a protein is the primary source of
compensating enthalpy and entropy changes. Another explanation for this
compensation arises from the assumption that the protein is in
equilibrium between two different states and that the ligand binds to
either state with different affinities(35) . Although the
ligands used in this study do not induce aggregation of tubulin dimers,
the effects of ligands on dimer-monomer equilibrium of tubulin may be
questioned. Colchicine binding to tubulin favors dimer &rlhar2; monomer
equilibrium toward dimer(36, 37, 38) . We
observed that all of these colchicinoids affect dimer &rlhar2; monomer
equilibrium and favors dimer formation very similar to that of
colchicine. Furthermore, it has now been established that colchicine
and its analogs can bind tubulin in its dimer and monomer forms equally
well(39, 40) . Since all of these ligands affects
dimer &rlhar2; monomer equilibrium similarly and in the same direction,
and as both dimer and monomer of tubulin can bind ligands equally well,
the differential affects of ligands on the state of association of
tubulin do not arise. It was recently proposed by Pyles and Bane Hastie (22) that the B-ring substituent of aminocolchicinoids resides
outside the colchicine binding site and makes contact(s) with tubulin.
Results presented in this report support their hypothesis. This contact
of the substituent with the protein would cause a reorganization of the
water structure around the protein and the ligand species toward a
greater disorder of the water molecules compared to the isolated
individually hydrated species(41) . This probably is the simple
explanation for the observed high values of entropy change accompanying
the binding reaction.
Figure 4:
Plot of H versus T
S for binding of tubulin with colchicine analogs at 37 °C. Data
for colchicine-tubulin (
) and AC-tubulin (
) complexes were
obtained from Bryan (24) and Bane et al.(17) ,
respectively. Data for aminocolchicinoid-tubulin complexes and
DAAC-tubulin complex were obtained from Table 2.