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Anti-Mitotic Activity of Colchicine

and the Structural Basis for Its


Interaction withTubulin

Bhabatarak Bhattacharyya,1 Dulal Panda,2 Suvroma Gupta,1 Mithu Banerjee1


1
Department of Biochemistry, Bose Institute, Centenary Campus P1/12, CIT Scheme VIIM,
Kolkata 700054, India
2
School of Biosciences and Bioengineering, Indian Institute of Technology,
Powai, Mumbai 400076, India

Published online 26 April 2007 in Wiley InterScience (www.interscience.wiley.com).


DOI 10.1002/med.20097
!

Abstract: 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 colchicinestubulin interaction has been explained in light of the variation in
the residues around the drug-binding site on b-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, 155183, 2008

Key words: tubulinDAMAcolchicine crystal structure; microtubule dynamics; tubulin isotypes;


C-termini of tubulin; colchicine analogs; phamacophore

1. INTRODUCTION

Microtubules, the key components of cytoskeleton are made up of ab-tubulin heterodimers. In


eukaryotic cells, they organize to form stable interphase microtubule network and highly dynamic
mitotic spindle. Microtubules are involved in a variety of cellular processes such as cell division,

Correspondence to: Bhabatarak Bhattacharyya, Department of Biochemistry, Bose Institute, Centenary Campus P1/12, CIT
Scheme VIIM, Kolkata 700054, India. E-mail: bablu@boseinst.ernet.in

Medicinal Research Reviews, Vol. 28, No. 1, 155^183, 2008


2007 Wiley Periodicals, Inc.
156 * BHATTACHARYYA ET AL.

maintenance of cell shape, cell signaling, cell migration, and cellular transport. The functional
diversity of microtubules depends on their intrinsic dynamic behaviors.19 Microtubules exhibit two
types of dynamic behaviors; one such dynamic behavior is called dynamic instability where
individual microtubule ends switch between phases of growth and shortening.1,38 Usually
microtubules display slow growth phases and rapid shortening phases. They also undergo a pause
state when there is no detectable growth or shortening at the microtubule ends. The transition from a
growth phase or a pause state to a shortening phase is called a catastrophe and the transition from a
shortening phase to a growth or a pause state is called a rescue. The transition frequencies are
thought to be important for regulating microtubule dynamics for diverse cellular tasks.39 The other
type of dynamic behavior is called treadmilling which involves a net growth at the plus end and a
net shortening at the minus end of the microtubule.2,6 A microtubule population may exhibit one or
both of these dynamic behaviors. The polymerization dynamics of microtubules mechanistically
depends on the loss or gain of a stabilizing cap composed of either tubulinGTP or tubulinGDP-Pi
at their ends.3,9 The assembly dynamics is finely regulated by several proteins including stabilizing
microtubule-associated proteins (MAPs) such as tau, MAP1, MAP2, MAP4, and destabilizing MAPs
such as stathmin.3,6,7,10,11 Microtubule dynamics is specifically important for the proper attachment
and movement of chromosomes during various stages of the mitotic phase.35 Suppression of
microtubule dynamics in cells by small molecule inhibitors blocks the cell division machinery at
mitosis leading to cell death. Therefore, the assembly dynamics of microtubule represents a potential
target for finding anti-cancer drugs. The small molecule inhibitors may imitate the action of the
natural regulators of microtubule assembly and disassembly kinetics making these agents a valuable
tool for probing the roles of microtubule dynamics in different cellular processes.
Microtubule-targeted agents can be broadly divided into two groups, namely polymerization
inhibitors and polymerization promoters. Several natural and synthetic compounds of varied
structures such as vinca alkaloids, colchicine, estramustine, and combretastatins inhibit microtubule
polymerization whereas compounds such as taxanes, laulimalides, and discodermolides promote
microtubule assembly. However, present evidence strongly suggests that both promoters and
inhibitors of microtubule assembly can suppress dynamic instability at lower concentrations without
affecting the polymer mass significantly.5,8,12 The binding sites of taxol, colchicines, and vinblastine
in tubulin are well characterized.5 Taxol and vinblastine bind to the b-subunit whereas colchicine
binds at the interphase of a and b subunits of the tubulin heterodimer. Most of the microtubule
depolymerizing agents either bind to the colchicine or vinblastine binding site on tubulin. This review
is primarily focused on colchicine and its analogs and their interactions with tubulin.
Colchicine (Fig. 1(I)), obtained from Colchicum autmnale and Gloriosa superba, is used in the
treatment of autoinflammatory diseases and gout.13 Colchicine has anti-inflammatory, anti-mitotic,
and anti-fibrotic activity.14 Colchicine also finds applications in various other diseases like

Figure 1. Structure of (I) Colchicine, (II) AC, and (III) Podophyllotoxin.


MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 157

pseudogout, familial Mediterrenian fever, cirrhosis of the liver and bile, and amyloidosis.13,15
Colchicine and colchicine-site binding agents have been widely used as probes to understand the
properties and functions of microtubules in cells. Initially tubulin was purified based on its high
affinity to colchicine. In plants, colchicine is widely used to separate chromosomes at the metaphase
and to induce polyploidy.16 Recently, colchicine is used as a selective neurotoxin in animal models to
study Alzheimers dementia.17,18

2. MECHANISM OF INHIBITION OF CANCER CELL PROLIFERATION

Colchicine blocks cell division by disrupting microtubules and the spindle microtubules are more
sensitive to colchicine than the interphase microtubules. Colchicine penetrates the cells and
equilibrates with the external colchicine rapidly, however a longer period is required to attain
saturation.19 The binding of colchicine to microtubules dissociates them into tubulin dimers. Cells in
different stages of mitosis exhibit differential sensitivity to colchicine. At higher concentrations, cells
at metaphase were blocked immediately after the addition of colchicine. At lower concentrations,
the prophase cells were more sensitive and were blocked while those in metaphase and anaphase
completed mitosis. Colchicine at a concentration of 50 nM blocks almost all the cells at mitosis.19 The
cells blocked at mitosis undergo abnormal mitotic cycle, designated as c-mitosis or colchicine-
mitosis. C-mitosis is characterized by partial or complete absence of spindle apparatus following the
breakdown of nuclear envelope, condensed chromosomes, and undivided centromeres.20 Several
studies have shown that colchicine can inhibit the function of several ion channels and the
depolymerizing effect of the drug is hypothesized to be involved in this effect.21 Colchicine was
shown to alter the membrane potential of the mitochondria resulting in the release of proapoptotic
factors like caspases, cytochrome-c, and apoptosis-inducing factors, leading to apoptotic cell death.22

3. STRUCTURE OF TUBULIN: THE RECEPTOR

Tubulin, the receptor, needs a detailed description to analyze the mechanistic aspect of drugtubulin
interaction. Electron crystallography of zinc-induced two-dimensional crystals of the protein
presented a three-dimensional model of tubulin at 6. 5 A resolution.23 The atomic model of ab tubulin
dimer was further obtained at 3.7 A resolutions by using electron crystallography of zinc-induced
tubulin sheet.24 In this structure, a and b tubulin possess identical principal structure: each monomer
being composed of a core of two beta sheets surrounded by a helices. The monomer has compact
structure, which can be divided into three functional domains: the amino-terminal domain possessing
the nucleotide-binding region, an intermediate domain where lies the Taxol-binding site, and the
carboxy-terminal domain comprising the binding site for motor proteins. This model was further
refined using standard X-ray crystallography methodology (Fig. 2).25 This model reported that each
monomer was composed of an N-terminal, nucleotide-binding domain, having six parallel b-strands
(S1S6) alternating with helices (H1H6). There is a direct involvement of the loops (T1T6),
which connects each strand with the start of the next helix in binding the nucleotide. This structure
provides a detailed description of the lateral contacts in zinc-sheets and the nucleotide and taxol
binding sites.

4. MECHANISM OF INHIBITION OF MICROTUBULE ASSEMBLY


DYNAMICS BY COLCHICINE: END POISONING MECHANISM AND
COPOLYMERIZATION MECHANISM

Colchicine has high affinity for soluble tubulin; however, it does not bind to microtubules unless
it first forms a tubulincolchicine complex, which adds to the microtubule ends. Microtubule
158 * BHATTACHARYYA ET AL.

Figure 2. The beta subunit of ab tubulin crystal structure (1JFF) showing the N-terminal region, C-terminal region, M-loop,Taxol,
GDP.The beta strands have been shown in red color, the alpha-helices in cyan and the turns are gray in color.

polymerization is inhibited by substoichiometric concentrations of colchicine implying that it


inhibits tubulin polymerization by binding to the ends of microtubules rather than to the soluble
tubulin. Colchicine first forms a reversible pre-equilibrium complex with tubulin dimer, which
induces many conformational changes in tubulin. Finally, a poorly reversible tubulincolchicine
complex is formed.26 Conformational changes that occur in tubulin upon binding to colchicine
are likely to be different from the conformational changes that normally occur during tubulin
polymerization. Careful kinetic analysis of the inhibition interaction suggests that tubulin
colchicine complex (TC-complex) binds to the microtubule ends and prevents the microtubule
growth by sterically blocking further addition of the tubulin dimers at the ends.14 It has been
suggested that the colchicine acts at the microtubule ends by an end conserving mechanism
wherein the TC-complex did not completely prevent the tubulin addition but only reduce the rate
of tubulin addition that occurs along with the addition of the TC-complex at the microtubule
ends.14,27
Microtubules in a protofilament are stabilized through both lateral and longitudinal
interactions.28 The S7-H9 loop is the central element of the interactions. This loop is defined
as the M loop (microtubule loop) (Fig. 2). It protrudes out from one side of the protofilament
and makes intimate contact with the H3 and several other loops. So any effect on the
adjoining loops gets transmitted to the M loop and consequently the microtubule becomes
destabilized. At low TC-complex concentration, the complex incorporates into microtubule
disturbing formation of lateral contacts at the newly formed end of protofilaments because
of displacement of the M loop resulting from entry of colchicine. The tubulin can no longer
remain in straight conformation since it would lead to steric clash between colchicine and the
residues a101, a181, and GTP. At low complex concentration, the proportion of lost lateral
contacts being small, the microtubules still remains intact as the propensity of lost lateral contact
remains small. On increasing the concentration of colchicines, a greater loss of lateral contacts
leads to disassembly of microtubules. Thus, the crystal structure of colchicine complexed with
alpha and beta tubulin can be used for the development of several potent anti-mitotic drugs
binding at the colchicine-binding site and their mode of interaction with tubulin can be
extensively studied.

5. TUBULIN-DAMA COLCHICINE CRYSTAL STRUCTURE AND


THE LOCATION OF THE BOUND COLCHICINE

Colchicine being the classic paradigm of anti-mitotic drugs is one of the most extensively studied
drugs. Several analogs of colchicine bind to tubulin and display unique chemical characteristics. One
of such unique property of colchicinestubulin complex is the promotion of colchicine fluorescence
upon binding to tubulin dimer. For this reason the mechanism of colchicinestubulin interaction has
attracted a great deal of attention. The exact binding site of a drug on a receptor is the key to unlock
the mechanism of drug protein interaction. Many attempts have been made to locate the precise
colchicine-binding site. The attempts mainly dealt with three types of experiments.

Figure 5. A: Model structure of the ^NH-dansyl isocolchicine^tubulin complex Showing the a-tubulin (surface representation in
salmon) and b-tubulin (cartoon representation in slate). The drug molecule viewed in sticks can be observed to have its dansyl
moiety buried deep inside the a-tubulin. The model has been generated from the tubulin^colchicine complex crystal structure
(PDB ID1SA0) using the DISCOVER program (Biosym/MSI,1995). B: Interaction of the C-ring carbonyl group of the colchicine mole-
cule with tubulin.The probable hydrogen bonds via the >CO moiety (with peptide-NH of Valine 181and g-NH2 of Lysine 352 from
a and b tubulin respectively) have been indicated using a dotted line along with the bond distance.This figure has been generated
using the software PYMOL from the tubulin^colchicine crystal structure obtained from the protein data bank (PDB ID1SA0).
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 159

Figure 2.

Figure 5.
160 * BHATTACHARYYA ET AL.

1. Direct photoaffinity labeling of tubulin with radiolabeled colchicine. In this method, a drug
protein complex is exposed to light of suitable wavelength, and ligand protein cross-link formation
is studied in details.29
2. Analog photo affinity labeling. In this method, an active drug analog having a photo reactive
moiety is synthesized, attached to tubulin, and cross link formation is induced by exposing the
complex to light of suitable wavelength.30
3. Cross-link formation with chemically reactive analogs that retain biological activity. An active
analog having a reactive chemical moiety is prepared and attached to tubulin, and cross-link
formation either occurs spontaneously or is induced by a fast change in interaction conditions.31
Two major problems are thought to be associated with all these methods. The first is the
probability for non-specific protein alkylation by the ligand, which is generally solved by taking
excess of non-reactive ligand, which inhibits the covalent interaction. The second is that enough
radiolabel must take part in cross-link formation to allow identification of the tubulin subunit (a or b),
the peptide region of the subunit, and the definite amino acid residues participating in the interaction.
Still several attempts have been made to locate the colchicines-binding site. It has been found that the
photoaffinity analogs of colchicine reacted mostly with a-tubulin or with both subunits.32,33
Whereas, direct photoaffinity labeling, using irradiation at 350 nm (the absorbance maximum of the
tropolone C-ring of colchicinoids) lead to preferential labeling of b tubulin.26 A cross-link formation
occurred between radiolabeled colchicine and amino acid (s) present in either peptide sequence 136
or peptide sequence 214241, but not with both peptides.
A unique approach was devised by placing the small chloroacetyl group (approximately 3 A in
length) at various positions in derivatives of colchicine and thiocolchicine. Upon placing at the side
chain or the C-ring, there was no specific covalent interaction with tubulin. On the other hand,
derivatized A-ring analogs 2CTC (where the methyl group of the 2-methoxy group of the A-ring has
been replaced by the chloroacetyl group) and 3CTC (where the methyl group of the 3-methoxy group
of the A-ring has been replaced by the chloroacetyl group) reacted covalently with b tubulin. 2CTC
mostly reacted with Cys b239, but there was some interaction with Cys b354 as well. It has been
shown that 3CTC shows major interaction with 354 of b subunit though there is a minor interaction
with Cys b239 as well. Thus, experiments were extensively done to define the exact colchicine-
binding site. Recently, the tubulincolchicine crystal structure has been reported.34 The structure
represents tubulin in complex with DAMAcolchicine and with the stathmin-like domain (SLD) of
RB3 at 3.5A resolution (Fig. 3). This complex throws light on the mechanism of tubulincolchicine
interaction. Colchicine binds at such a site arresting curved tubulin from assuming a straight

Figure 3. The crystal structure of animal tubulin^colchicine complex.


MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 161

structure. This structure shows the exact binding site of colchicine on tubulin and provides an insight
into the mechanism of colchicinestubulin interaction and the way it perturbs microtubule assembly
of tubulin.
Thus, the X-ray structure of tubulin has been well defined (Fig. 2). In the tubulin
DAMAcolchicine crystal structure the A and C rings interacts with the b subunit and the B ring
side chain interacts with the a subunit (Fig. 3).34
The colchicine site in the tubulin DAMAcolchicine crystal structure34 lies within the
intermediate domain of the b subunit, surrounded by strands S8 and S9, loopT7 and helices H7
and H8 (Fig. 3). Besides b-subunit, colchicine also interacts with the loop T5 of the adjacent
a-subunit. This structure supports the observation that tubulin heterodimer is stabilized upon
colchicine binding.35 Formation of the complex was done in the absence of any SLD, so the location
of colchicine in the tubulinDAMAcolchicine complex was predicted to be very similar to that in
tubulin. This structure also validates some previous data such as: tubulin having variation at the b 318
exhibits reduced sensitivity to colchicine,36 and colchicine derivatives substituted at the methoxy
positions of ring A can be cross-linked with Cys b241.37
For the entry of the colchicine molecule into the tubulin heterodimer, there is a movement of the
T7 loop and the H8 helix in the complex compared to protofilament tubulin. It is experimentally
established that binding of colchicine induces conformational change of tubulin.3840 As a
consequence of this movement, the observed colchicine site differs from the predicted site
determined based on the protofilaments structure of tubulin.

6. EFFECTS OF COLCHICINE ON MICROTUBULE DYNAMIC INSTABILITY

The effects of TC-complex on microtubule instability parameters of individual microtubules were


determined using differential interference contrast video microscopy.27 Low concentrations of TC-
complex strongly suppressed dynamic instability without reducing microtubule polymer mass
significantly. TC-complex reduced both the rate and extent of growing and shortening phases. In
addition, TC-complex also decreased the catastrophe frequency and increased the rescue frequency.
TC-complex also increases the time the microtubules spent in the pause state, neither growing nor
shortening state, and reduced the overall subunit exchange rate from the ends of microtubules.
Colchicine binding alters the lateral contacts within the microtubule and disrupts the microtubule
lattice. The number of TC-complex incorporated into the microtubules determines the stability of the
microtubule ends. Suppression of microtubule dynamics by colchicine occurs primarily by altering
the tubulinGTP or tubulinGDP-Pi cap. Colchicine binding induces conformational changes in the
tubulin so that incorporation of tubulin into the microtubule ends became energetically unfavorable.
Microtubules can resume growth once the tubulincolchicine complex dissociates from the ends.
Colchicine depolymerizes microtubules at high concentrations whereas at low concentration of
complex, it arrests microtubule growth26. The crystal structure provides the reason behind this
difference in the mode of action of colchicine at different concentrations.

7. DIFFERENT COLCHICINE ANALOGS

The colchicine molecule (Fig. 1(I)) is composed of three rings, a trimethoxy benzene ring, (ring A), a
methoxy tropone ring (ring C), and a seven-membered ring (ring B) carrying an acetamido group at its
C7 position which anchors the A and C ring. The structure of colchicine and its different analogs are
presented in Figures 1 and 4 respectively. Single ring analogs of the methoxy tropone and trimethoxy
phenyl moieties of colchicine have been tested and found to bind brain tubulin with low affinity
constant in the millimolar range.41 According to the model proposed by Andreu and Timasheff,40 the
162 * BHATTACHARYYA ET AL.

relatively weak interactions of both the two separate rings (ring A and C) of colchicine accounts
qualitatively for the much tighter binding of the complete drug to tubulin in the micromolar range.
This model takes into account the entropic advantage of colchicine as a bifunctional ligand.
Structureactivity study reveals that the A and C ring of colchicine comprises the minimal structural
feature of the molecule needed for its high affinity binding to tubulin. AC (2-methoxy-5-(2 0 ,3 0 ,4 0 -
trimethoxyphenyl)tropone) (Fig. 1(II)) is a simple bifunctional ligand that binds specifically to the
colchicine-binding site with high affinity.4246 Studies with large numbers of colchicine analogs,
established clearly that colchicine analogs modified at or even depleted of the B ring retain anti-
mitotic activity and bind tubulin at the colchicine site. However, the presence of the B ring or its side
chain at C7 position can significantly influence the binding kinetics, association rates, activation
energy, and the thermodynamics of the binding interaction.46

A. Role of A Ring
The role of the A ring of colchicine (Fig. 1(I)) towards tubulin binding has been studied in great
details. In conjunction with the ring C, the ring A constitutes an essential pharmacophore for its high
affinity binding to tubulin. Podophyllotoxin (Fig. 1(III)), another colchicine site binding ligand acts
as competitive inhibitor of colchicine because of the presence of trimethoxy phenyl ring (A ring of
colchicine).47 Insertion of a bulky group in the A ring of colchicine as in colchicoside (Fig. 4a) causes
loss of activity of the molecule.48 Thus A ring of colchicine is crucial for tubulin binding and has
attracted attention for further study on tubulin-microtubule system. For tubulin binding, the size of
methyl group of methoxy substituent of the A ring plays an important role. Substitution of methyl
group from any of the three-methoxy groups with bulky groups results in many fold reduction of the
potency of colchicine for tubulin.49 Two colchicine analogs with benzodioxole A rings have been
studied. They share structural analogies with podophyllotoxin. Cornigerine (Fig. 4b) bears equal
potential and mimics colchicine in most of the biological evaluations.50 Cornigerine was found to
arrest L1210 murine leukemia cells in mitosis and was more toxic than colchicine in these cells. But
the second one, namely the 1, 2-methylenedioxy isomer of cornigerine has been reported as an
inactive one in vivo (Fig. 4c).51 Colchicine A ring derivatives with substitutions at C4 position are
limited in numbers. Sharma et al.52 had studied one spin-labeled analog of colchicine with spin label
attached through the C-4 position. Decreased affinity of this drug for tubulin molecule had put some
questions regarding structural constraints on substitutions at the C-4 position.

B. Role of the C Ring


The tropone ring of colchicine is found to be crucial for colchicinestubulin interaction.5356 The C
ring part of colchicine has been shown to undergo photochemical decomposition giving rise to a
number of compounds (known as lumicolchicines) (Fig. 4d) with reduced binding ability
(640 M1).53 In these compounds, the tropolone ring gets transformed into a fused four and five
member ring having carbonyl and methoxy substituents.
Isocolchicine shares remarkable similarity in structure with colchicine (Fig. 4e). It is a C ring
colchicine analog differing in the relative position of the methoxy and carbonyl group. It is inactive
and is unable to inhibit tubulin assembly.5456 Isocolchicine resembles tropolone methyl ether
(a single ring analog of colchicine C-ring) with respect to its association property.40 It was assumed
that this analog binds tubulin with its altered C ring placing carbonyl and methoxy group in the same
orientation as in colchicine leaving A ring in the exterior of the binding site. Another school of
thought exists which states that not only the ring C but also the intact A-ring is a major contributor
of isocolchicinetubulin binding.57 However, both the rings contribute to the low affinity binding of
the drug. Isocolchicine has two low-affinity sites on tubulin. The molecule binds rapidly to the first
site, competing with 2-methoxy-5-(2 0 ,3 0 ,4 0 -trimethoxyphenyl)tropone (AC), and it resembles that
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 163

of A-ring analogs.11 The second site however, is not well characterized, but it does not overlap the
[2-methoxy-5-(2 0 ,3 0 ,4 0 -trimethoxyphenyl)tropone] binding site.26
From the structure activity studies, it is clear that there is a little apparent dispute among tubulin
binding activity and the tolerance of substituent on ring C of colchicine and its different analogs. As
long as the conjugated C ring of colchicine remains intact, the biological activity of this drug is
preserved. Colchicide is a colchicine analog in which the C-10 methoxy group is replaced by a
hydrogen atom (Fig. 4f). It competitively inhibits colchicine binding to tubulin and is also an inhibitor
of tubulin assembly.57 Its binding to tubulin is accompanied with quenching of tubulin fluorescence.

Figure 4. Structure of different A, B, and C ring analogs of colchicines.


164 * BHATTACHARYYA ET AL.

Figure 4. (Continued )

Colchiceine (a tropolone derivative; Fig. 4h) exists in two tautomeric forms manifesting
colchicine and isocolchicine configuration of ring C with predominance of iso form of tautomer.58,59
Since it binds to the colchicinetubulin complex, it is suggested that colchicine binds at a site
different from the colchicine site on tubulin.60
Precolchicine (a colchicine-like compound with rearranged bond system; Fig. 4i) is not
active.49 Presence of a seven (C ring as present in colchicine itself) or six membered aromatic ring in
place of ring C of colchicine is a prerequisite condition for its biological activity. Allocolchicine
(Fig. 4l) with a six membered aromatic ring in place of ring C is a member of this family with
noteworthy biological activity.61 Conformational changes on C-ring of colchicine on the other hand
are unable to generate much difference on the energetics of the binding interaction. The above
conclusion can be made from the results of the binding of thiocolchicine and allocolchicine to tubulin
that occurs with high energy of activation (Table I).
Thiocolchicine binds tubulin faster in contrary to the prediction of its slow binding. Again
allocolchicine binds to tubulin in a rapid and reversible manner in the fast step of the binding
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 165

Table I. Association Rate Constants and Activation Energies of Binding of Colchicine and
Its C Ring Analogs to Tubulin

interaction. But its activation energy was lowered only by 2 kcal/mole from that of colchicines
tubulin binding62 as observed from the Table I.

C. Role of B-Ring and Its Side Chain at C7 Position


A and C ring of colchicine comprise the main building scaffold for tubulin binding. The B ring is also
important, though it is not essential for tubulin binding activity. As mentioned earlier, AC binds
tubulin almost to the same extent as colchicine and inhibits tubulin polymerization.41 B ring analogs
mostly modulate the kinetic property of colchicine tubulin binding namely, on-rate, off-rate,
activation energy reversibility, and quantum yield of the drugtubulin complex.46 B ring analogs
with substitutions at C-7 position is tolerated by tubulin and resulted in a number of active
compounds. But N-dimethyl colcemid, a quarternary ammonium salt of iodide, and the dia-
stereoisomers of N-[2,2,5,5,-tetra methyl-1-oxy- 3-pyrrolidinyl) carbonyl] deacetyl colchicine are
exceptions in having reduced binding affinity.26,52
Another colchicine derivative 5,6-dihydro-6-hydroxymethyl-1,2,3 trimethoxy-9-methyl thio-
8Hcyclohepta [a] napthalen-8-one, possess a six membered in place of seven membered B ring with
additional minor differences. This molecule binds rapidly and reversibly to tubulin.62 Chakraborty
et al.46 studied a number of B ring analogs with C-7 substituent with tubulin. As these compounds are
non-fluorescent so their association with tubulin have been measured through quenching of tubulin
tryptophan fluorescence.46 These analogs behave similar to colchicine with high energy of activation
and low association rate for tubulin binding, probably arising because of the bulkiness of the
substituent at C7 position on ring B.46 A detailed analysis of their binding parameters has been
discussed in next section.

8. KINETICS AND THERMODYNAMICS OF


COLCHICINE TUBULIN INTERACTION

The kinetics of binding of AC to tubulin has comparatively lower activation energy with respect to
colchicinestubulin binding. The interaction of AC with tubulin is monitored from the enhancement
of AC fluorescence upon binding tubulin. The association process can be resolved into a fast and slow
phase similar to that of colchicine. The apparent second order rate constant for the fast phase is
5.2  104 M1s1 at 37 C and the activation energy is 13 kcal/mol (Table I).41 An explanation to
justify the observed low activation energy for AC-tubulin binding is provided with the idea that the
binding happens through a low energy pathway because of uninterrupted free rotation about the biaryl
bond.41 So it is tempting to speculate that the B ring portion of colchicine is responsible for its
high activation energy. It imparts some rigidity to the colchicine structure. To unravel the mystery,
the association rate constants of tubulin binding to DAAC along with some derivatives of DAAC,
(NMe2-DAAC, NHMe-DAAC, NH2-DAAC) were determined. Values of the second order rate
constants for the fast phase along with the activation energies are shown in Table II. Comparison of
the association rates of AC and DAAC clearly delineates the fact that the B-ring itself has a dramatic
166 * BHATTACHARYYA ET AL.

Table II. Association Rate Constants and Activation Energies of Binding of Colchicine and Its
B Ring Analogs to Tubulin

a
From Pyles and Bane Hastie (1993).
b
From data previously publishedby Bane etal. (1984).
c
From data previouslypublishedby Chabin & Hastie (1989).
d
From data previously publishedby Hastie (1989).
e
From data previouslypublishedby Chakraborti etal. (1996).
f
From Sengupta etal. (1993, 2000).
g
From Das etal. (2005).

effect on the association rates although they have identical activation energies of binding. While the
activation energies of AC and DAAC binding to tubulin are almost identical (13 and 12.3 kcal/mol for
AC and DAAC respectively), AC binds tubulin 17 times faster than DAAC.63 According to the
Arrhenious equation, the rate constant of an interaction is a product of its activation energy term (Ea)
and the pre-exponential factor A, that is, kon A.eEa/RT. Two interactions with the same activation
energy might thus possess different rate constants because of differences in the A values, and
vice versa. The pre-exponential factor (A) is related to the activation entropy (DS*) by the following
equation:
A en  kT=h  eS=R
where n is the change in the number of molecules when the complex is formed and DS* is the
activation entropy. The A value for AC was calculated to be 70 times higher than that of DAAC. The
kinetics of a number of colchicine analogs binding to tubulin with altered B ring (i.e., containing
different C7 substituents) has been investigated.46 However, the activation energies of tubulin
colchicinoid interaction undergo a hike when NH2 group is present at the C-7 position of the B ring
(Table II). The presence of the B ring itself retards the interaction rate compared to AC. No
extra effect is added either on the on rate or on the value of activation energy from introduction of
bulkier groups at the same position, for example, as in demecolcine (NHMeDAAC) and in
N-methyldemecolcine (NMe2DAAC) as evident from Table I B. What really is responsible for the
change when NH2 is substituted at the C-7 position in the B-ring is difficult to understand from the
current knowledge of B ring analogs binding to tubulin.

9. EFFECT OF pH ON COLCHICINE TUBULIN INTERACTIONS: ROLE OF


B RING SIDE CHAIN OF COLCHICINE AND C-TERMINI OF TUBULIN

The interaction of colchicine with tubulin is strongly influenced by the pH of the binding interaction.
Both colchicine as well as tubulin structure play crucial role in determining pH sensitivity of
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 167

colchicinetubulin interaction. The colchicinetubulin interaction has pH optimum near 7 and the
extent of interaction decreases on both side of it that is at low as well as at high pH. When tested for
other analogs, a somewhat variable picture was observed. The binding of a colchicine analog lacking
B ring (such as AC) or without side chain at C7 (such as DAAC) with tubulin is influenced scarcely
by the pH of the binding interaction. The pH sensitivity of other B-ring analogs of colchicine
such as deacetylcolchicine (NH2  DAAC), colcemid (NHCH3  DAAC), and N-methylcolcemid
(N(CH3)2  DAAC) was tested using quenching of the tryptophan fluorescence of tubulin since
it is known that these B-ring analogs fluoresce poorly upon binding to tubulin.64 Like colchicine,
pH-sensitive binding (optimum near pH 7.0) of deacetylcolchicine (NH2  DAAC), colcemid
(NHCH3  DAAC), and N-methylcolcemid (N(CH3)2  DAAC) to tubulin was observed.64 There-
fore, on the basis of the above results, it appeared that the negatively charged C-terminus of a-tubulin
is involved in a long distance conformational change when a colchicine analog with B-ring side chain
at C-7 position binds tubulin.
Colchicine structure remains unaffected in this pH range and it is the conformation of tubulin that
gets altered during changes in pH and is responsible for these findings. Mukhopadhyay et al.65 has
shown earlier that the affinity constant of colchicine binding to ab-tubulin and abs-tubulin are pH
dependent and decreased on either side of the pH 6.8. However, asbs-tubulin binds colchicine less
tightly in a pH-independent manner compared to ab-tubulin.65 With peptide (P2) having the
sequence NLRKLRGGRLKRLN, the role of the a-C-terminus in colchicine binding has been
elucidated.66 P2 protects alpha tail of tubulin from subtilisin digestion compared to beta tail. It is
suggested that it might be because of the tighter binding of the basic peptide P2 to the negatively
charged C-terminus of a-tubulin than to the C-terminus of b-tubulin. Microtubule proteins (MTP)
consisting of several basic microtubule-associated proteins (MAPs) bound to its negatively charged
C-termini bind colchicine independent of pH similar to asbs-tubulin binding to colchicine.
Colchicine binding to ab-tubulin at different pHs in the presence of 0.1 M NaCl is also pH
independent like MTP or P2-bound tubulin similar to asbs-tubulin. The on-rate and the activation
energy of colchicinetubulin interactions under the above conditions were determined under
pseudo-first order conditions (analysis was done according to Lambier and Engelborghs).67 The on-
rate and activation energy values for the colchicinetubulin interaction are presented in Table III for
the fast phase. While the removal of the C-terminus of both subunits (asbs) lowers the activation
energy significantly to 10.68 kcal/mole, hybrid tubulin (abs) (where the C-terminus of only b-tubulin
is cleaved) has a high activation energy level of 19.58 kcal /mole.64 The dissociation of subunits of
tubulin (a b) also lowers the activation energy to 13.09 kcal /mole.68 The activation energies of
colchicine binding with P2 peptide-bound tubulin and MTP are 13.89 kcal/mole and 13.58 kcal/mole

Table III. Association Rate Constants and Activation Energies of Binding of Colchicine to
Modified Tubulin

h
From Chakraborty etal. (2004).
168 * BHATTACHARYYA ET AL.

respectively (Table III).64 The presence of NaCl lowers the activation energy to 12.68 kcal /mole
(Table III).64

10. DESIGN OF ACTIVE ISOCOLCHICINE ANALOGS

Previous studies involving different colchicine site analogs have established beyond reasonable
doubt that the drug makes at least two-points of attachment with tubulin through its A and C-rings.
For example, the trimethoxyphenyl ring (ring A) has been shown to be involved in tubulin binding as
colchicine analogs with bulky substituent in the A-ring (colchicoside) are unable to inhibit
podophyllotoxin-tubulin binding.47 Similarly, analogs having an identical A-ring but a modified
C-ring such as isocolchicine (4e) and lumicolchicine (4d) are biologically inactive and bind tubulin
with lower affinity.56 This has been substantiated by the fact that individual ring compounds such as
mescaline (an A-ring analog) and methoxy tropone (C-ring analog), both bind tubulin with lower
affinity as compared to colchicine.40 Recently, however, studies from our laboratory have shown that
isocolchicine becomes biologically active, binds tubulin with higher affinity, inhibits tubulin self
assembly at low drug concentrations, and competes with [3H]-colchicine for binding to tubulin upon
introducing suitable hydrophobic groups like NBD or a dansyl moiety at the C-7 position of
isocolchicine.69,70 It has been found that the congeners of isocolchicine family [with hydrophobic
substitution on B ring namely NBD-isocolcemid (4j) or NH-dansyl isocolchicine (4k)] have rate
constants varying linearly with drug concentrations. The parent molecule, isocolchicine (4e) binds to
tubulin in a single step because of its inability to fit itself into an altered second more planar
conformation. Since the high activation energy of colchicinetubulin interaction depends upon this
second conformational change step, so it can be predicted that NBD-isocolcemid binding to tubulin is
also confined to a one-step process.69 The second step leading to conformational change of both drug
and protein is missing. This observation is supported from the absence of GTPase activity and from
the identical cleavage pattern of native protein for NBD-isocolcemid (4j)tubulin complex.69 On
the contrary, the binding of NBD-colcemid (4q) to tubulin is a two-step process like colchicine.71
NH-dansylcolchicine (4r), another B-ring analog of colchicine acts like NBD-colcemid (4q) and
binds tubulin in a two-step process whereas binding of its iso-analog to tubulin occurs in one step.70
For this drug, the kinetics is manifested with the conventional linear dependence of the observed rate
constant on drug concentration with its parent compound, isocolchicine.70 Enhancement of GTPase
activity of tubulin is not observed with this analog indicating the absence of second slow step of the
tubulin binding. The affinity constant of this iso analogtubulin interaction is 0.7  105 M1, which
is approximately three times lower than that of NH-dansyl colchicine (4r)tubulin interaction. On
the other hand, the affinity constant of isocolchicinetubulin interaction is approximately 500 times
lower than that of colchicinetubulin interaction.56 There is justification to assume that the altered
C-ring of NH-dansyl isocolchicine (4k) does not contribute towards the binding affinity, and the
increase in the affinity must be a consequence of the dansyl substitution on the B ring at the C-7
position. This substitution would promote -NH-dansyl isocolchicine (4k) to bind as a bifunctional
ligand, by making two points of attachment to tubulin through its A and B-ring side chain. This also
provides a way to increase the affinity of a drug for a target protein by proper substitution on the drug
moiety. Molecular modeling based on the recently determined crystal structure of the tubulin
DAMAcolchicine complex34 further adds momentum to the above proposition concerning the dansyl
grouptubulin interaction. Replacement of colchicine with NH dansyl colchicines (4r) or NH
dansyl isocolchicine (4k) in the complex shows that the dansyl group gets buried deep inside the
a-subunit of tubulin,70 leading to a large change in the accessible surface area of the drug upon
binding (Fig. 5A). In accordance with the earlier biochemical observations, the model shows that the
A-ring of the drug interacts with b-tubulin, while the B-ring side chain interacts with the a-chain. On
the other hand, the C-ring seems to interact with both chains through the formation of hydrogen bonds
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 169

via the >CO moiety (with peptide-NH of Valine 181 and g-NH2 of Lysine 352 from a and b-tubulin
respectively) (Fig. 5B).70 Thus, it seems quite natural that an interchange of the >CO and OCH3
groups as in isocolchicine would affect these hydrogen bond formation in addition to steric
constraints.71 These two drugs as seen from models have different orientation of their colchicine
moiety, while the dansyl moieties occupy almost the same position. Moreover, the conformations of
a-tubulin are not significantly altered in these complexes (rmsd < 0.1A), but large deviations in the
respective b subunits are noticeable (rmsd 0.9 A). These conformational differences in the protein
and the drug may shed light on the difference in the biochemical properties of the NH dansyl
colchicine (4r) and NH dansyl isocolchicine (4k). These studies further demonstrate that two-
points of attachment of the drug with tubulin are essential for higher binding affinity and have
confirmed the previous hypothesis that the B-ring side chain of colchicine also makes contact with
tubulin and contributes toward drug binding affinity.72

11. STRUCTURAL BASIS OF THE DIFFERENTIAL INTERACTION OF


COLCHICINE WITH DIFFERENT ISOTYPES OF TUBULIN

Carboxy terminal tails of tubulin are heterogeneous in nature, flexible, containing several glutamic
acid residues, solvent exposed, and sensitive to proteolysis.73,74 Carboxy termini of tubulin have
attracted immense attention for their role in tubulin polymerization. Microtubular proteins as well as
divalent calcium ions bind at the C-termini and regulate the assembly of tubulin.73,74 Carboxy termini
of tubulin are also responsible for its chaperone-like activity.75 Though colchicine binds at the alpha
beta interface of tubulin far from its carboxy terminal end, several properties of colchicinestubulin
interactions such as pH-sensitivity, off-rate, on-rate, and the stability of the colchicines-binding site
are greatly influenced by the a C-terminal of tubulin as discussed in the previous section.67 Earlier Pal
et al.76 suggested the presence of tailbody interaction between the negatively charged a-C-
terminus of tubulin with the positively charged residues of the main body of tubulin. Vertebrate
tubulin contains four b-tubulin classes designated as bI, bII, bIII, and bIV in relative amounts of 3, 58,
25, and 13% respectively.77 The kinetics of colchicine binding to tubulin indicates biphasic pattern.
b-tubulin isotypes differ from each other apparently at the C-termini (b430444). The origin of the
two phases in the binding kinetics was not clear before the findings that the bovine kidney tubulin
lacking bIII tubulin isotype (which accounts for 25% of the total brain tubulin) binds colchicine in a
monophasic manner.78 So naturally the question crops up whether C-termini is responsible for all
these findings?
Experiments with C-termini cleaved tubulin showed biphasic kinetics similar to native tubulin
(uncleaved) so it became clear that the C-termini, which lies far away from the colchicine binding site
plays no role in modulating colchicine tubulin binding kinetics. Crystal structure of tubulin
DAMAcolchicinestathmindomain complex, has been solved at 3.5 A resolution and the amino
acid residues defining the binding site of colchicine on tubulin have been identified.34 From
the crystal structure, it has been observed that the colchicine binding site is mostly embedded in
the intermediate domain of the b-subunit while the B-ring side chain of colchicine interacts with
the a-subunit. Sequence alignment of the b-subunit of the crystal structure and the b-isotypes
of tubulin indicates that the isotypes differ in many other regions of their sequences besides the
C-terminal ends.

12. DISTINGUISHING ISOTYPES ON THE BASIS OF RESIDUES


SURROUNDING THE BOUND COLCHICINE MOLECULE IN TUBULIN

Table IV presents the amino acid residues lying within 5 A and 8 A from the colchicine molecule, in
the crystal structure. They are identical for bI and bIV. Isotypes bII and bIV differ at only position 318:
170 * BHATTACHARYYA ET AL.
Table IV. Residues of Different b-Tubulin Isotypes Lying in Close Proximity to Colchicine Molecule

Isoleucine (318) for bII while Valine (318) for bIV. However, isotype bIII has three changes: Serine
(242), Threonine (317), Valine (353) in place of Leucine (242), Alanine (317), Threonine (353) for
bIV. Inspite of a single difference in amino acid residue, a mixture of (abII abIV) tubulin exhibits
monophasic kinetics for colchicinetubulin interaction. Addition of abIII isotype to either abII or
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 171

Table V. Affinity Constant and On-rate Constants of Different Isotypes of Tubulin

a
From Banerjee etal.

abIV to form (abIII abII) or (abIII abIV) changes the binding kinetics from monophasic to
biphasic.78 Thus, it seems reasonable that abIII is responsible for the biphasic kinetic pattern. These
differences in nature of amino acids may explain the difference in the association rates as well as the
affinity constant values for the abIII-colchicine interaction.
Previously, an analysis was done to canvass a part of the colchicine-binding site with the help of
experimentally determined association constants with some available primary beta tubulin
sequences.36 It was suggested that the relative affinities of different tubulins for colchicine depend
upon residues in the immediate vicinity of b316. Comparison of residues with the experimental
association constants revealed that tubulin with Iso b316 in bI must bind colchicine significantly more
weakly than with Val b316 in bII, but more strongly than Metb316 in Caenorhabditis elegans or
Phenylalanine b316 in Saccharomyces cerevisiae system.36 As observed from Table V, the
colchicines-binding affinity constant for abIV is approximately 14-fold greater than that of abII
whereas the corresponding rate constant for abIV is twice that of abII. Similarly, the affinity constant
for colchicine binding for abIV is approximately 28-fold higher than that of abIII and the rate constant
is about 8 times greater. bII has an Isoleucine residue at position 318 whereas bIV has a Valine residue
at the same position. Indeed the side chains of Isob318 and Val b318 differ only in the presence
or absence of an ethyl group. It is clear from Figure 6A that the distance between the carbon of
3-methoxy group of ring A and the side chain methyl carbon of Val b318 in bIV is 4.21 A. On the other
hand, the distance between the hydrogen of 3-methoxy group of ring A and the side chain ethyl carbon
of Isob318 in bII is 3.62 A (Fig. 6B). The ethyl group, being a bulkier group than a methyl group,
suffers from van der Waals repulsive interaction with the 3-methoxy group of ring A of colchicine
which results in the lowering of affinity constant and rate constant of bII relative to bIV. When bIII is
compared to bIV, there are two changes within 5 A of the colchicine molecule. Leu b242 and Ala b317
of abIV have been replaced by Ser b242 and Thr b317 respectively in abIII (Fig. 6C). A shift from a
hydrophobic to a hydrophilic environment for bIII can be correlated with earlier evidences that
colchicine binding involves hydrophobic interaction. These two alterations lower the Ka (affinity
constant) of colchicine binding as well as the on-rate constant of abIII relative to abIV. In case of
kidney b tubulin, lacking the bIII isotype79 the apparent on-rate constant of binding is very close to
that of the faster binding component (abIV) of brain tubulin.
For abII and abIII, the rate constant of the former is about four times that of abIII, while the
corresponding affinity constant for colchicine binding is about two times greater. Iso b318, Leu b242,
Ala b317 in abII have been replaced by Val b318, Ser b242, Thr b317 in abIII. Hydrophobic residues
(Leucine and Alanine) in the case of isotype abII have been substituted by hydrophilic residues
(Serine and Threonine) in case of abIII. This difference in the nature of residues might be responsible
for the differential binding kinetics as well as the difference in affinity constant between bII and bIII.
So, the variation in the nature of residues encompassing the colchicine molecule influences the
kinetics and affinity constant of the colchicinetubulin interaction. Interestingly, we have found that
the variation in nature of residues (in a proximal region of 5 A) lies around the A-ring of colchicine
only, as discussed later.
172 * BHATTACHARYYA ET AL.

Figure 6. Residues of different b-tubulin isotypes lying in close proximity of colchicine molecule: (A) shows residues of bIV denoted
by B353, B317, B318 and B242, (B) shows the residues of bII denoted by B353, B317, B318, and B242, and (C) Shows the residues of
bIII denoted by B353, B317, B318, and B242. [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 173

13. EFFECT OF COLCHICINE STRUCTURE IN REGULATING DIFFERENT


ISOTYPE COLCHICINE INTERACTION

The B-ring and the C-7 side chain of colchicine mainly control the kinetics of colchicinetubulin
interaction. Thus, AC (Fig. 1(II)); having only the A and C-ring of colchicine) binding to tubulin is
instantaneous and reversible whereas colchicine binding is slow and poorly reversible.41,80 The
kinetics of colchicine binding to unfractionated tubulin indicates biphasic pattern. The association
rates and affinity constants values are tabulated in Table V. The binding of desacetamidocolchicine
(DAAC) (Fig. 4m), to tubulin follows biphasic kinetics similar to that of colchicine. Moreover like
colchicine, the affinity constant of DAAC for abIII is much less than that for abII and abIV.81
Therefore, it is obvious that the B-ring substituent does not distinguish different b isotypes. Again
there is a report that AC [2-methoxy-5-(2 0 , 3 0 , 4 0 -trimethoxyphenyl) tropone] (Fig. 1(II)), which is
devoid of the B-ring, also exhibits biphasic kinetics in its binding to tubulin.82 In an effort to study the
role of the B-ring substituents, interaction of two compounds of thiocolchicine series, (THC 5 and
THC 18) has been tested with tubulin isoforms from bovine brain.83 It was observed that THC 18,
having a side chain with a pi-bonded SP2 conformation, binds differently to the tubulin isoforms.
THC 5 with a slightly different side chain does not. THC 18 was found to be 1,600-fold more potent
than THC 5 in inhibiting the growth of RPMI 7951 melanoma cells.83 The results indicate that the
conformation of the B-ring side chain plays a major role in the differential interaction of a colchicine
derivative with different tubulin isoforms. There are reports that colchicine analogs with modified
C-ring such as colchicide (Fig. 4f) and MD [2-methoxy-5-(2 0 ,4 0 -dimethoxy phenyl)-2,4,6-
cycloheptatriene-1-one] (Fig. 4s) with a modified A-ring do not recognize tubulin isotypes.84
Colchicide has a modified C-ring, where the C-10 methoxy group of the ring C has been replaced by a
hydrogen atom. This analog does not distinguish kinetically among different tubulin isotypes.85 MD
is fast binding colchicine analog like AC (without B-ring) having a modified A-ring, where 3 0
methoxy group in A-ring has been replaced by a hydrogen.84 Like colchicide, MD cannot
differentiate among tubulin isotypes. For both analogs, the replacement of a bulky methoxy group by
a hydrogen atom abolished completely their ability to recognize different tubulin isotypes. So, may
be the presence of a methoxy group is the crucial determinant for this type of recognition. Since a
larger methoxy group is replaced by a smaller hydrogen atom, methoxy group of those analogs
may play some role in generating biphasic kinetics of the colchicinestubulin interaction. Perhaps
the 3-methoxy group of ring A of colchicine suffers steric repulsion with residue Iso b318 in bII.
Such interaction is absent in the case of bIV (Fig. 6A) as well as in the case of bIII (Fig. 6C) since both
of them have a Valine residue at the same position and Isoleucine is much bulkier than Valine.

14. PREDICTION OF A PHARMACOPHORE FOR COLCHICINE


SITE INHIBITORS

Microtubule is the target of several anti-cancer drugs. Drugs binding at the vinca and taxol sites of
tubulin play important roles in the treatment of human cancers. Even though colchicine is widely
used to elucidate the structure and properties of microtubules, its use in cancer treatment is limited
because of its toxicity. The adverse effects of colchicine include vomiting, nausea, fatigue, and
nephrotoxicity.86 However, several colchicine analogs and drugs designed to bind to the colchicine-
binding site have recently gained interest as potent anti-cancer agents because of their ability to
inhibit multidrug-resistant (MDR) tumors. Many of these agents are in various phases of clinical
trials. These drugs have been grouped as colchicine site inhibitors (CSI).87 Some of these drugs show
structural resemblance to colchicine whereas others have entirely diverse structure. This gives rise to
the question what are the essential structural features required for activity of a drug and where does
174 * BHATTACHARYYA ET AL.

these drugs show similarity. The identification of a common pharmacophore among these structures
provides an answer to these questions.
A pharmacophore is a three-dimensional substructure of a molecule that carries the essential
features responsible for a drugs biological activity.87 Molecular docking can be used to find what
kinds of groups are required to bind to the available amino acids and where they should be
positioned.88,89
Molecular dynamics simulations and docking studies have been deployed to construct binding
models for some structurally different CSIs, using the tubulincolchicine crystal structure as a
template.87 The compounds were selected on the basis of occupation of same chemical space,
consistent topology, and binding modes. Since the crystal structure of colchicine and podophyllotoxin
with dimeric tubulin were available, they were the first choice for mapping the binding mode of other
CSI. Relative to colchicine and podophyllotoxin, some characteristics were identified such as number
of hydrogen bonding groups, number of rotatable bonds, number of aromatic rings etc since the
conformational flexibility of a molecule can be predicted from all such parameters. This comparison
showed that some drugs were rigid whereas others were flexible. Because of the lack of structural
information of the binding modes of the CSIs detailed molecular docking procedure was employed.
Models of each CSI were analyzed using the steric and electrostatic parameters of the colchicine-
binding site and the characteristics of each binding mode were analyzed.
The drugs could be divided into two groups. The first group included drugs having structural
resemblance to colchicine and three important features: A diaryl system, a trimethoxyphenyl (TMP)
moiety, and a constrained conformation. The second group did not possess at least one of the
characteristics stated above and hence were structurally more diversified than the first group. On the
basis of structural similarity the binding model of the first group of drugs was determined using
docking experiments taking into account the TMP moiety as the template. The superimpositions in
case of the other drugs were difficult but similar method was adapted, keeping in mind that all of them
bind to the colchicine-binding site. In spite of being structurally dissimilar, all of them occupied
similar Cartesian space in the colchicine site.
An analysis of the binding modes of these compounds showed that they could be connected
through a seven-point pharmacophoric points. They comprised of three hydrogen bond acceptors
(A1, A2, A3), one hydrogen bond donor (D), two hydrophobic center (H1 and H2), and one planar
group (R1) on the drug scaffold. One acceptor, two hydrophobic centres and the planar group were the
minimum features for the drug to be active.
On the tubulin molecule (receptor), the following residues participated in bond formation and in
promoting hydrophobic stabilization. The hydrogen bonds were to the corresponding amino acid
residues on the receptors:A1 to amide Nitrogen of Val (a179), A2 to sulfur atom of Cys b241, A3 to
the amide nitrogen atoms of Ala b248, Asp b249, and Leu b250, D1 to the carbonyl oxygen atom of
Thr a177. H1 got hydrophobically stabilized by remaining wedged between the side chains of Val
a179 and Met b257.
The greater the number of pharmacophoric points of a drug with a receptor, the better is the
binding affinity of the drug for that particular receptor. All drugs did not have the same number of
pharmacophoric points. The drugs could be grouped on the basis of number of pharmacophoric points
(Table VI).
Table VI shows the pharmacophoric points for colchicine. The drugs lying within Group I
domain contain five pharmacophoric points. We are mainly interested in the drugs binding at the
colchicine binding site and which lie within Group I. There are five pharmacophoric points which
includes, Hydrogen bonds: A1, between amide nitrogen of Val (A181) and the carbonyl oxygen atom
of the C ring of colchicine and A2, between sulphur atom of Cys b239 and the methoxy oxygen of the
3-methoxy group of the A ring of colchicine. Hydrophobic stabilization: H1 is wedged between the
side chains of A181 (VAL) and B259 (MET), H2 characterized by trimethoxyphenyl moiety,
Hydrophobic plane: R1.
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 175

15. COLCHICINE SITE AGENTS AS ANTI-CANCER DRUGS

As discussed in the previous section, there are several therapeutically important drugs that bind to the
colchicine-binding site of tubulin. The pharmacophoric points for some of the drugs are enlisted in
Table VI. Structures of several agents that bind to the colchicine site have been displayed in Figure 7.
A brief mode of action of these drugs from the point of view of the pharmacophoric points along with
their mechanism of action has been outlined below.
Combretastatin (Fig. 7A), a strong inhibitor of tubulin polymerization, also belongs to this
group. Combretastatin A-4-Phosphate (CA4P), a disodium phosphate prodrug of Combretastatin, is
active against different cancer cells, including multidrug resistant cells.90 CA4P, is currently
undergoing phase 2 clinical trials for the treatment of solid tumors.91,92 However, early clinical trials
of CA4P show that it has significant toxicities.93 CA4P, which inhibits microtubule assembly also

Table VI. Table Enlisting the Drugs with Their Pharmacophoric Points

(Continued )
176 * BHATTACHARYYA ET AL.

Table VI. (Continued )

Thefollowingarethepharmacophoricpointsonthe drugs:Hydrogen Bond Acceptors: A1, A2, and A3.Hydrogen Bond Donor:D1.Hydrophobicgroups:
H1and H2.Hydrophobicplane:R1.

exhibits selective toxicity to tumor vasculature.94,95 A functional tumor vasculature is necessary for
the growth and survival of tumors and tumor vasculature is a major target of many of the colchicine
site agents.94 Tubulin and microtubules are important to maintain the elongated shape of the vascular
endothelial cells. When the cellular microtubule network is disrupted by the drugs, the elongated
endothelial cells round up and block the blood flow through the blood vessels.
Phenstatin (Fig. 7B), a benzophenone type of CA4 analog, has been derived by replacing the
olefinic bridge of CA4 with a carbonyl group.96 Though phenstatin shows structural resemblance to
combretastatin, it is a better inhibitor than the former. It has six pharmacophoric points (A1-A2-A3-
H1-H2-R1), and possesses an extra pharmacophoric point A3 enabling the CO group of the drug to
hydrogen bond with the backbone NH groups of residues b248,-b249-b250. Such a hydrogen bond is
absent in Combretastatin thereby making it a less potent inhibitor. It has cytotoxic activity and
inhibits tubulin polymerization. Replacing hydroxy group at the C2 position with an amino group
yields 2-aminobenzophenone (Fig. 7C). This also exhibits significant extent of cytotoxicity against
a number of cancer cell lines. 2-aminothiophene (Fig. 7D) in turn has been synthesized from
2-aminobenzophenone by replacing an ethylene group with a sulfur atom. The existence of
bioisosteric relationship between benzene and thiophene paved the way for further generation of anti-
cancer compounds. Consequently a number of p-fluore, p-methyl, and p-methoxy phenyl substituted
analogs of the above have been developed and among them p-fluoro derivative (Fig. 7D) seemed to be
a very promising candidate. Tubulin polymerization inhibition study together with inhibition of
colchicine binding to tubulin showed that these groups of compounds interfere with microtubule
assembly originating from interaction with the colchicine-binding site of tubulin. Microscopic
evaluation along with flow cytometric study of cells treated with this group of compounds showed
lengthening of the G2/M phase of the cell cycle. This p-fluoro derivative of thiophene was also
docked at the colchicine-binding site applying AutoDock and using the recently reported crystal
structure of tubulinDAMA colchicine complex. It showed similar orientation and the binding
was stabilized through favorable hydrogen bonding. This result was in line with the information
obtained relating to possible binding mode of various strong colchicine site inhibitors of tubulin
polymerization.97,98
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 177

Figure 7. Structure of some important drugs inhibiting tubulin polymerization and binding at the colchicine-binding site.

2-Methoxyestradiol (2-ME) (Fig. 7E), an estradiol metabolite, inhibits the growth of a variety of
cancer cells but does not harm normal cells. 2-ME is a promising chemotherapeutic agent for
advanced prostate cancer. 2-ME inhibits the cancer cell growth by inducing cell cycle arrest,
disrupting microtubules, inducing apoptosis, inhibiting angiogenesis, and increasing oxidative
damage.99 2-ethoxyestradiol and its congeners (Fig. 7F and G) possess five pharmacophoric points
and their occupancy of the colchicine-binding site showed that an ethyl group (Fig. 7F) could be
placed without encountering any steric hindrance. Similarly, the activity of 2-ME showed that
substitution on opposite ends of ligands could also be accommodated. Curacin A (Fig. 7H) having an
entirely different structure from colchicine also belongs to this group. It was found to be a better
inhibitor of tubulin polymerization than its analog (Fig. 7I). Both Curacin A (Fig. 7H) and its analog
(Fig. 7I) differ in having a single methyl group. The methyl group gains hydrophobic stabilization
being surrounded by the side chains of Leu B240, Leu (B250), and Leu (B253). The analog (Fig. 7I)
fails to achieve such hydrophobic stabilization and thus is a weaker inhibitor of tubulin
polymerization than Curacin A (Fig. 7H). Modeling data showed that the activity of 2-aroyl indole
(Fig. 7J) and Steganacin (Table VI) depends on the substitution at the 3-position instead of the TMP
moiety. Both of them are members of group III (Table VI). The drug indanocine (Fig. 7K) is a member
of group IV. Indanocine exhibits toxicity towards multidrug-resistant cell lines.100 Modeling data
provides an explanation to the resistance imparted on indanosinetubulin interaction upon making
178 * BHATTACHARYYA ET AL.

point mutation on the receptor. The mutation caused a Lys350 to arginine substitution in the
b-tubulin.101 Lys350 represents an important interacting site for indanocine on b-tubulin. The
pharmacophoric points of nocodazole have been shown in Table V. Nocodazole is a potent inhibitor
of tubulin polymerization. Its congener mebendazole (Fig. 7L) also belongs to this group. Modeling
studies showed that the pi electron clouds of the thiophene and the phenyl rings can act as hydrogen
bond acceptors by forming a weak hydrogen bond coupled with hydrogen bond donor such as the
thiol group of Cys b239.
The pharmacophoric points of podophyllotoxin are shown in Table VI, it contains six
pharmacophoric points. Podophyllotoxin binds to tubulin faster than colchicine and the binding is
reversible. Podophyllotoxin disassembles microtubules at high concentrations and the end-
dependent disassembly occurs through the formation of a podophyllotoxintubulinGTP ternary
complex, which is inactive in assembly.102 Podophyllotoxin affects the dynamics of tubulin at
substiochiometric concentrations, by forming a tubulinGTPpodophyllotoxin complex so that
elongation of the microtubule ends is inhibited. Removal of the tubulinGTP from the microtubule
ends causes the shrinking of the ends, resulting in microtubule disassembly. Therefore,
the suppression of microtubule dynamics by podophyllotoxin occurs by the inactivation of
the microtubule ends, which prevents further growth. Even though podophyllotoxin is used
in the treatment of genital warts, it is not used as a chemotherapy agent because of its toxicity.
However, etoposide, a podophyllotoxin analog is an anti-cancer agent, and blocks the cell cycle at
G1/S phase by acting as a topoisomerase II inhibitor. Steganacin (Table VI) in spite of having
structural resemblance to podophyllotoxin belongs to Group III and has five pharmacophoric points
since it lacks D1 of podophyllotoxin.
Colchicine is toxic but the drug E7010 occupying the colchicine-binding site has been designed
as a successful anti-cancer drug progressing from Phase I to Phase II clinical trial (Table VI). E7010

Figure 8. Proposed mechanism ofaction of indole sulfonamides. Indole sulfonamides suppressed the spindle assembly dynamics
and blocked the cells at mitosis. The mitotically blocked cells had either multipolar spindles or abnormal bipolar spindles with
condenseddisorganized chromosomes.Theblocked cells were eliminatedbyapoptosis mediatedbyhyperphosphorylationof bcl2.
MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 179

blocks cells at mitosis by inhibiting tubulin polymerization. E7010 binds reversibly to the colchicine-
binding site of b-tubulin and it displays anti-tumor activity against various types of drug resistant
tumor cell lines. This drug has six pharmacophoric points, which are shown in Table VI. Recently, a
new class of indole sulfonamides has been prepared where the amino substituted pyridine ring of
E7010 was replaced by an indole group. Docking studies by Nguyen et al.87 indicates that E7010 and
the indole derivative have distinct binding modes at the colchicine site of tubulin. The indole
sulfonamides inhibited HeLa cell proliferation and blocked cell cycle progression at mitosis by
depolymerizing cellular microtubules and disorganizing chromosomes.103 They also inhibited the
microtubule formation in vitro and at low concentrations, suppressed the dynamic instability
behavior at plus ends of individual steady state microtubules in vitro. The indole sulfonamides
perturbed the assembly dynamics of spindle microtubules that arrested the cell proliferation at
mitosis. The mitotically arrested cells eventually underwent apoptotic cell death mediated by the bcl-
2 pathway.103 The proposed mechanism of action of the indole sulfonamides is given in Figure 8.
A minor modification of E7010 can lead to the generation of an ideal drug having all seven-
pharmacophoric points. Here lies the beauty as well as utility of the discovery of a common
pharmacophore for all colchicine site inhibitors whereby a more potent anti-cancer drug can be
generated by proper chemical modification of a known drug. The pharmacophoric concept has been
and will be instrumental in the synthesis of several therapeutically useful drugs in the near future.

ACKNOWLEDGMENTS

Authors thank Renu Mohan and Rathinasamy K for critical reading and helpful suggestions. The
review is supported by a CSIR grant to BB and a Swarnajayanti fellowship to DP.

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Bhabatarak Bhattacharyya: Born at Calcutta in 1944 and graduated from the University of Calcutta in 1965.
He obtained the M.Sc. and Ph.D. degrees from the same university in 1967 and 1974 respectively. He carried out
postdoctoral research at the NIH, Bethesda, Maryland from 1972 to 1976 in the laboratory of Dr. Jan Wolff.
Returning to India, he joined the faculty of the Bose Institute, Calcutta after being a Pool Officer (C.S.I.R) for
1 year. He became Professor in the same institute in 1989 and the Chairman of the Biochemistry Department,
since 1998. He is a recipient of the Shanti Swaroop Bhatnagar Award, P.S. Sharma Memorial Award, Bose
Institute Foundation Day Award, and Biresh Chandra Guha Memorial Lecture award, Recipient of Fogarty
International Fellowship and Recipient of International Cell Biology Fellowship for Young Scientist. Professor
Bhattacharyya has been elected as a fellow of: Third World Academy of Science, Indian National Science
Academy, New Delhi, Indian Academy of Sciences, Bangalore, and National Academy of Sciences, Allahabad.
His area of research includes biophysical chemistry, structural biology, biochemistry, and molecular
spectroscopy. He has supervised 19 Ph.D. students and has published more than one hundred papers in
reputed journals.
Dulal Panda: Born at Kui, a small village in West Bengal, India. He has a Ph.D. degree in Biochemistry from the
Bose Institute Kolkata, India under the supervision of Dr. Bhattacharyya. He did postdoctoral research with
Dr. Leslie Wilson at the University of California, Santa Barbara. At present, he is a faculty member at the Indian
Institute of Technology, Bombay. His research interest includes biochemistry of eukaryotic and prokaryotic cell
division, and microtubule targeted anticancer and antifungal drugs.
Suvroma Gupta: Born at Calcutta, Suvroma Gupta graduated with Chemistry and has done her M.Sc. in
Biochemistry from University of Calcutta. She worked in the Department of Biotechnology, East India
Pharmaceuticals Limited for 5 years. Mrs. Suvroma Gupta has done her Ph.D. from Bose Institute under the
supervision of Prof. Bhattacharyya and at present is a Research Associate in the same lab. Her research work is
focused on anticancer drugtubulin interaction.
Mithu Banerjee: Born at Calcutta, Mithu Banerjee graduated in Chemistry and has done her M.Sc. in
Chemistry from University of Calcutta. She is a final year Ph.D. student under the supervision of Prof.
Bhattacharyya. Her research work revolves around studying anti-cancer drug protein interaction using
calorimetric, spectroscopic, and computational tools.

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