anti-mitoticactivityofcolchicine andthe structural basis ... · anti-mitoticactivityofcolchicine...

Anti-Mitotic Activity of Colchicineand the Structural Basis for Its

Interaction withTubulin

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

1Department of Biochemistry, Bose Institute, Centenary Campus P1/12, CIT Scheme VIIM,

Kolkata 700054, India2School 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

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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 colchicines–tubulin 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, 155–183, 2008

Key words: tubulin–DAMAcolchicine crystal structure; microtubule dynamics; tubulin isotypes;

C-termini of tubulin; colchicine analogs; phamacophore

1 . I N T R O D U C T I O N

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

SchemeVIIM,Kolkata 700054, India.E-mail: [email protected]

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

� 2007 Wiley Periodicals, Inc.

maintenance of cell shape, cell signaling, cell migration, and cellular transport. The functional

diversity of microtubules depends on their intrinsic dynamic behaviors.1–9 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.1,3–8 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.3–9 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 tubulin–GTP or tubulin–GDP-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.3–5 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.

156 * BHATTACHARYYA ET AL.

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 Alzheimer’s dementia.17,18

2 . M E C H A N I S M O F I N H I B I T I O N O F C A N C E R C E L L P R O L I F E R A T I O N

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 . S T R U C T U R E O F T U B U L I N : T H E R E C E P T O R

Tubulin, the receptor, needs a detailed description to analyze the mechanistic aspect of drug–tubulin

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 ofab 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

(S1–S6) alternating with helices (H1–H6). There is a direct involvement of the loops (T1–T6),

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 . M E C H A N I S M O F I N H I B I T I O N O F M I C R O T U B U L E A S S E M B L YD Y N A M I C S B Y C O L C H I C I N E : E N D P O I S O N I N G M E C H A N I S M A N DC O P O L Y M E R I Z A T I O N M E C H A N I S M

Colchicine has high affinity for soluble tubulin; however, it does not bind to microtubules unless

it first forms a tubulin–colchicine complex, which adds to the microtubule ends. Microtubule

MECHANISM OF COLCHICINE AND TUBULIN INTERACTION * 157

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 tubulin–colchicine

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 . T U B U L I N - D A M A C O L C H I C I N E C R Y S T A L S T R U C T U R E A N DT H E L O C A T I O N O F T H E B O U N D C O L C H I C I N E

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 colchicines–tubulin complex is the promotion of colchicine fluorescence

upon binding to tubulin dimer. For this reason the mechanism of colchicines–tubulin 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 drugmolecule 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 theDISCOVERprogram (Biosym/MSI,1995).B: Interactionof theC-ringcarbonyl groupof the colchicinemole-cule with tubulin.The probable hydrogen bonds via the>COmoiety (with peptide-NHof Valine181and g-NH2 of Lysine 352 from

a andb tubulin respectively) havebeen indicatedusingadotted linealongwith thebonddistance.This figurehasbeengenerated

using the software PYMOL fromthe tubulin^colchicine crystal structure obtained from theproteindatabank (PDB ID1SA0).

Figure 2. The beta subunit of ab tubulin crystal structure (1JFF) showing theN-terminal region,C-terminal region,M-loop,Taxol,

GDP.Thebetastrandshavebeenshown in red color, thealpha-helices in cyanand the turnsare gray incolor.


Figure 2.

Figure 5.


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 orb),

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 1–36

or peptide sequence 214–241, 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 tubulin–colchicine crystal structure has been reported.34 The structure

represents tubulin in complex with DAMA–colchicine and with the stathmin-like domain (SLD) of

RB3 at 3.5A resolution (Fig. 3). This complex throws light on the mechanism of tubulin–colchicine

interaction. Colchicine binds at such a site arresting curved tubulin from assuming a straight

Figure 3. The crystal structure ofanimal tubulin^colchicine complex.


structure. This structure shows the exact binding site of colchicine on tubulin and provides an insight

into the mechanism of colchicines–tubulin 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 tubulin–DAMAcolchicine 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.38–40 As a

consequence of this movement, the observed colchicine site differs from the predicted site

determined based on the protofilaments structure of tubulin.

6 . E F F E C T S O F C O L C H I C I N E O N M I C R O T U B U L E D Y N A M I C I N S T A B I L I T Y

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 tubulin–GTP or tubulin–GDP-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 tubulin–colchicine 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 . D I F F E R E N T C O L C H I C I N E A N A L O G S

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


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.

Structure–activity 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.42–46 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 colchicines–tubulin interaction.53–56 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 M�1).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.54–56 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 isocolchicine–tubulin 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


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 ofdifferent A,B, andCringanalogsofcolchicines.


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 colchicine–tubulin 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

Figure 4. (Continued )


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 drug–tubulin 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-

8H–cyclohepta [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 . K I N E T I C S A N D T H E R M O D Y N A M I C S O FC O L C H I C I N E – T U B U L I N I N T E R A C T I O N

The kinetics of binding of AC to tubulin has comparatively lower activation energy with respect to

colchicines–tubulin 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 M�1s�1 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

Table I. Association Rate Constants and Activation Energies of Binding of Colchicine and

Its C Ring Analogs to Tubulin


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 ¼ A.e�Ea/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 ¼ e��n � kT=h � eð�SÞ�=R

where n is the change in the number of molecules when the complex is formed and DS* is the

activation entropy. The Avalue 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 . E F F E C T O F p H O N C O L C H I C I N E – T U B U L I N I N T E R A C T I O N S : R O L E O FB R I N G S I D E C H A I N O F C O L C H I C I N E A N D C - T E R M I N I O F T U B U L I N

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

Table II. Association Rate Constants and Activation Energies of Binding of Colchicine and Its

B Ring Analogs to Tubulin

aFromPylesandBaneHastie (1993).

bFromdatapreviouslypublishedbyBaneetal. (1984).

cFromdatapreviouslypublishedbyChabin&Hastie (1989).dFromdatapreviouslypublishedbyHastie (1989).

eFromdatapreviouslypublishedbyChakrabortietal. (1996).fFromSenguptaetal. (1993,2000).gFromDasetal. (2005).


colchicine–tubulin interaction. The colchicine–tubulin 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 chargedC-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 colchicine–tubulin 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 colchicine–tubulin 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 theC-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 (aþ 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

hFromChakrabortyetal. (2004).


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

(Table III).64

1 0 . D E S I G N O F A C T I V E I S O C O L C H I C I N E A N A L O G S

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 colchicine–tubulin 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 analog–tubulin interaction is 0.7� 105 M�1, which

is approximately three times lower than that of NH-dansyl colchicine (4r)–tubulin interaction. On

the other hand, the affinity constant of isocolchicine–tubulin interaction is approximately 500 times

lower than that of colchicine–tubulin 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

group–tubulin 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


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

1 1 . S T R U C T U R A L B A S I S O F T H E D I F F E R E N T I A L I N T E R A C T I O N O FC O L C H I C I N E W I T H D I F F E R E N T I S O T Y P E S O F T U B U L I N

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 theC-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 colchicines–tubulin

interactions such as pH-sensitivity, off-rate, on-rate, and the stability of the colchicines-binding site

are greatly influenced by the aC-terminal of tubulin as discussed in the previous section.67 Earlier Pal

et al.76 suggested the presence of ‘‘tail–body 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 (b430–444). 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 theC-termini, which lies far away from the colchicine binding site

plays no role in modulating colchicine tubulin binding kinetics. Crystal structure of tubulin–

DAMAcolchicine–stathmin–domain 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.

1 2 . D I S T I N G U I S H I N G I S O T Y P E S O N T H E B A S I S O F R E S I D U E SS U R R O U N D I N G T H E B O U N D C O L C H I C I N E M O L E C U L E I N T U B U L I N

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:


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 þ abIV) tubulin exhibits

monophasic kinetics for colchicine–tubulin interaction. Addition of abIII isotype to either abII or

Table IV. Residues of Different b-Tubulin Isotypes Lying in Close Proximity to Colchicine Molecule


abIV to form (abIIIþ abII) or (abIIIþ 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 Isob316 inbI 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 inbIV 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 tobIV, there are two changes within 5 A of the colchicine molecule. Leub242 and Alab317

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. Isob318, Leub242,

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 colchicine–tubulin 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.

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

aFromBanerjee etal.


Figure 6. Residues ofdifferent b-tubulin isotypes lying in closeproximityofcolchicinemolecule: (A) shows residues of bIVdenotedby B353,B317,B318 andB242, (B) shows the residuesof bII denotedby B353,B317,B318, andB242, and (C) Shows the residues of

bIII denotedby B353, B317, B318, and B242. [Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]


1 3 . E F F E C T O F C O L C H I C I N E S T R U C T U R E I N R E G U L A T I N G D I F F E R E N TI S O T Y P E – C O L C H I C I N E I N T E R A C T I O N

The B-ring and the C-7 side chain of colchicine mainly control the kinetics of colchicine–tubulin

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 colchicines–tubulin 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.

1 4 . P R E D I C T I O N O F A P H A R M A C O P H O R E F O R C O L C H I C I N ES I T E I N H I B I T O R S

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


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 drug’s 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 tubulin–colchicine 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.


1 5 . C O L C H I C I N E S I T E A G E N T S A S A N T I - C A N C E R D R U G S

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 )


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 tubulin–DAMA 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

Table VI. (Continued )

Thefollowingarethepharmacophoricpointsonthedrugs:HydrogenBondAcceptors:A1,A2,andA3.HydrogenBondDonor:D1.Hydrophobicgroups:

H1andH2.Hydrophobicplane:R1.


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 indanosine–tubulin interaction upon making

Figure 7. Structure of some importantdrugs inhibiting tubulinpolymerizationandbindingat the colchicine-bindingsite.


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 podophyllotoxin–tubulin–GTP ternary

complex, which is inactive in assembly.102 Podophyllotoxin affects the dynamics of tubulin at

substiochiometric concentrations, by forming a tubulin–GTP–podophyllotoxin complex so that

elongation of the microtubule ends is inhibited. Removal of the tubulin–GTP 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. Proposedmechanismofactionof indole sulfonamides. Indole sulfonamides suppressed thespindleassemblydynamics

and blocked the cells at mitosis. The mitotically blocked cells had either multipolar spindles or abnormal bipolar spindles with

condenseddisorganizedchromosomes.Theblockedcellswereeliminatedbyapoptosismediatedbyhyperphosphorylationofbcl2.


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.

A C K N O W L E D G M E N T S

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 theM.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 inWest 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 drug–tubulin 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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