Interactions of long-chain homologues of colchicine with tubulin

Article abstract of DOI:10.1016/j.ejmech.2016.11.049

Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at the nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the cytotoxicity with a maximum in the range of 10–12 carbon atoms, followed by a decrease in activity with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In addition, at IC₅₀concentrations the analogues block the cell cycle of A549 cells in the G2/M phase. Molecular docking studies suggest that, while interactions of the colchicine analogues with the colchicine binding site at β-tubulin are still present, the increase in the acyl chain length leads to the progressive development of new interactions, not present in colchicine itself, with the neighboring α-tubulin subunit. Indeed, sufficiently long acyl chains span the intradimer interface and contact with a hydrophobic groove in α-tubulin. It is worth noting that some of the compounds show cytotoxicity at concentrations three orders of magnitude lower than colchicine. Their pharmacological use in cancer therapy could possibly be performed with lower dosages and be thus endowed with less acute toxicity problems than in the case of colchicine.

Full text of DOI:10.1016/j.ejmech.2016.11.049

Accepted Manuscript
Interactions of long-chain homologues of colchicine with tubulin
Ana Marzo-Mas, Pascale Barbier, Gilles Breuzard, Diane Allegro, Eva Falomir, Juan
Murga, Miguel Carda, Vincent Peyrot, J. Alberto Marco
PII:
S0223-5234(16)30987-4
10.1016/j.ejmech.2016.11.049
EJMECH 9082
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2016
Revised Date: 21 November 2016
Accepted Date: 22 November 2016
Please cite this article as: A. Marzo-Mas, P. Barbier, G. Breuzard, D. Allegro, E. Falomir, J. Murga, M.
Carda, V. Peyrot, J.A. Marco, Interactions of long-chain homologues of colchicine with tubulin, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.049.
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ACCEPTED MANUSCRIPT
Interactions of long-chain homologues of colchicine with tubulin
Ana Marzo-Mas, Pascale Barbier, Gilles Breuzard, Diane Allegro, Eva Falomir, Juan Murga,* Miguel
Carda, Vincent Peyrot,* and J. Alberto Marco
Graphical Abstract
Several N-acyl colchicine analogues have been synthesized and their cytotoxicities and ability to inhibit
microtubule assembly have been measured.
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European Journal of Medicinal Chemistry
Manuscript draft
Title: “Interactions of long-chain homologues of colchicine with tubulin”
Article type: Original paper
Keywords: tubulin, colchicine, microtubules, abnormal polymerization, cytotoxicity, docking studies
Abstract: Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic,
straight-chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at
the nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit
activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude
more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related
to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the
cytotoxicity with a maximum in the range of 10-12 carbon atoms, followed by a decrease in activity
with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation
of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In
addition, at IC50 concentrations the analogues block the cell cycle of A549 cells in the G2/M phase.
Molecular docking studies suggest that, while interactions of the colchicine analogues with the
colchicine binding site at β-tubulin are still present, the increase in the acyl chain length leads to the
progressive development of new interactions, not present in colchicine itself, with the neighboring α-
tubulin subunit. Indeed, sufficiently long acyl chains span the intradimer interface and contact with a
hydrophobic groove in α-tubulin. It is worth noting that some of the compounds show cytotoxicity at
concentrations three orders of magnitude lower than colchicine. Their pharmacological use in cancer
therapy could possibly be performed with lower dosages and be thus endowed with less acute toxicity
problems than in the case of colchicine.
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Interactions of long-chain homologues of colchicine with
tubulin
Ana Marzo-Mas,a Pascale Barbier,b Gilles Breuzard, b Diane Allegro, b Eva Falomir, a Juan Murga, a*
Miguel Carda a Vincent Peyrot, b* and J. Alberto Marcoc
aDepart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
bAix-Marseille Université, Inserm, CRO2 UMR_S 911, Faculté de Pharmacie, 13385, Marseille,
France
cDepart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
*Authors to whom correspondence should be addressed. E-Mail addresses: jmurga@uji.es,
vincent.peyrot@univ-amu.fr.
ABSTRACT
Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-
chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at the
nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit
activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude
more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related
to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the
cytotoxicity with a maximum in the range of 10-12 carbon atoms, followed by a decrease in activity
with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation
of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In
addition, at IC50 concentrations the analogues block the cell cycle of A549 cells in the G2/M phase.
Molecular docking studies suggest that, while interactions of the colchicine analogues with the
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colchicine binding site at β-tubulin are still present, the increase in the acyl chain length leads to the
progressive development of new interactions, not present in colchicine itself, with the neighboring α-
tubulin subunit. Indeed, sufficiently long acyl chains span the intradimer interface and contact with a
hydrophobic groove in α-tubulin. It is worth noting that some of the compounds show cytotoxicity at
concentrations three orders of magnitude lower than colchicine. Their pharmacological use in cancer
therapy could possibly be performed with lower dosages and be thus endowed with less acute toxicity
problems than in the case of colchicine.
KEYWORDS
tubulin, colchicine, microtubules, abnormal polymerization, cytotoxicity, docking studies.
1. Introduction
Since life began on Earth, Nature has been producing small-molecule products which can interact
inside living organisms with macromolecular targets that contain structural domains similar to human
proteins. The result of this natural selection process has been a plethora of structures that developed
optimal interactions with many biological targets [1]. Natural product research therefore is a growing
domain at the interface of chemistry and biology that continuously generates new chemical entities
with high pharmacological interest [2].
During the past few years we have been investigating a range of analogues of natural products [3]
for their potential value in anticancer therapy [4]. Research has shown that the onset of cancer may be
triggered by a number of both internal and external factors. This has motivated the development of
various types of therapeutic treatments [5]. Among these is the use of cytotoxic drugs which often lead
to cell apoptosis [6].
Microtubules are dynamic polymers that play a very important role in many cellular processes,
particularly in cell division, as they are the key constituents of the mitotic spindle. They are hollow
tubes having an external diameter of approximately 25 nm. Microtubules are composed of a
heterodimeric protein formed through the non-covalent binding of two monomeric constituents, α- and
β-tubulin [7]. For cell division to occur in the normal way, microtubules must be in a constant state of
formation and disruption, a process called microtubule dynamic instability in which GTP hydrolysis
into GDP plays an essential role [8].
It is clear that any molecule which interacts with microtubules can influence the cell division
process of both normal and tumoral cells. This influence may be exerted by molecules that interact with
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tubulin, which would explain why tubulin-binding molecules (TBM) constitute a very important
category of anticancer agents [9]. TBM interfere with microtubule assembly and functions, either by
causing disruption of the microtubules or else through their stabilization. Most of the active drugs
which have been describe earlier are either natural products or their derivatives [10]. Major drugs can
already be found on the market, while many more promising compounds are now in clinical trials [9,
10].
Obviously, discovery and subsequent investigation of new drugs constitute an important activity in
chemistry and pharmacology. Our interest in natural product analogues has led us to prepare several
structural classes and to investigate their action on the aforementioned types of biological activity.
Among the natural products included in our research, colchicine (Fig. 1) is now become of particular
interest to us. Colchicine has been successfully employed to treat gout and is also being used for the
treatment of familial Mediterranean fever. Unfortunately, the marked toxicity of colchicine has
prevented its use in cancer therapy [11]. Very recently, we have published two reports on the
cytotoxicity of several colchicine analogues [4, 12].
Colchicine inhibits microtubule assembly in vitro through a substoichiometric poisoning
mechanism. The compound binds to tubulin at a specific binding site at the β-subunit proximal to the
intradimer interface that separates the latter from the neighbor α-subunit. Colchicine forms complexes
which then add to the microtubule ends capping them and making further free tubulin dimer addition
impossible [13].
2. Research purpose
In continuation of our research on novel natural product analogues with potential utility in cancer
therapy [4, 12] and on the basis of the aforementioned aspects, we wanted to ascertain whether
modifications in the N-acyl residue of the colchicine molecule might give rise to noticeable changes in
the biological activity. Thus, we have prepared amides 1-10 where the N-acetyl residue of colchicine
has been replaced by straight-chain, aliphatic N-acyl moieties bearing up to eighteen carbon atoms in
the carbon chain.
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Figure 1. Structure of colchicine derivatives investigated in this study.
3. Synthetic work
N-acyl colchicine derivatives 1-10 were prepared from the trifluoroacetate salt of N-deacetyl
colchicine 11 [14] as depicted in Scheme 1. Treatment of 11 with the corresponding carboxylic acid in
the presence of DCC and DMAP gave rise to N-acyl derivatives 1-10 with fair to good yields. Details
about the precise reaction conditions and yields are indicated in the Experimental Part (graphical
spectra and physical, IR, NMR and Mass spectrometry data are provided in the Supporting
Information) [15].
RCOOH (2 eq),
DCC (6 eq),
DMAP (2 eq)
O
MeO
MeO
MeO
MeO
NH3
N
H
R
CF3COO
MeO
MeO
CH2Cl2, RT,
3 h (44-82%)
O
O
OMe
OMe
11
1-10
Scheme 1. Synthesis of colchicine derivatives 1-10.
4. Biological work
4.1. Cytotoxic effects
The cytotoxic ability of compounds 1-10 was established by means of their IC50 values towards the
tumoral cell lines HT-29 (human colon adenocarcinoma), MCF-7 (breast adenocarcinoma) as well as
towards the non-tumoral cell line HEK-293 (human embryonic kidney cells). Results are presented in
Table 1 along with the calculated selectivity indexes SIA (for HT-29 cell line) and SIB (for MCF-7 cell
line), obtained by dividing the IC50 values of the non-tumoral cell line (HEK-293) by those of the
corresponding tumoral cell line. The higher the SI index, the higher the therapeutic safety margin.
Table 1. Cytotoxicity of colchicine analogues 1-10 toward tumoral cell lines HT-29 and MCF-7 and
one non-tumoral cell line HEK-293.a
b
c
Drug
HT-29
MCF-7
12 ± 7
HEK-293
5 ± 1
SIA
0.1
1.4
2.1
SIB
Colchicine
50 ± 3
0.42
1.0
1
2
10 ± 2
13 ± 1
13.5 ± 0.5
7.1 ± 0.3
3.37 ± 0.15
7.02 ± 0.05
1.0
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3
4
1.69 ± 0.09
23.0 ± 0.5
18.9 ± 1.1
0.242 ± 0.005
0.17 ± 0.04
0.23 ± 0.06
1.5 ± 0.3
11.2 0.8
0.143 ± 0.011 29 ± 4
1.7
4.2
0.9
0.2
1.3
1.3
0.8
< 0.1
5
0.041 ± 0.004 1.77 ± 0.13
0.1
0.4
0.2
2.3
0.3
6.4
6
0.27 ± 0.03
7.0 ± 0.7
36.0 ± 1.0
184 ± 6
0.53 ± 0.23
6.8 ± 1.7
20 ± 7
7
8
46 ± 3
9
720 ± 60
120 ± 50
244 ± 7
10
1010 ± 80
766 ± 10
aIC50 values are expressed as the compound concentration (nM) that inhibits the cell growth by 50%. Data are the
average (±SD) of three experiments. bSIA = IC50(HEK-293)/IC50(HT-29). cSIB = IC50(HEK-293)/IC50(MCF-7).
The observed IC50 values are in most cases in the medium to low nanomolar range. The results for
the HT-29 cell line deserve particular mention, as the lowest IC50 values are observed with this cell
line. Thus, all compounds except 9 and 10 showed IC50 values in the low nanomolar range, with 4, 5
and 6 exhibiting very high cytotoxicities situated in the picomolar range, therefore two to three orders
of magnitude more cytotoxic than colchicine itself (values highlighted in italics). A clear correlation is
observed between the length of the acyl chain and the potency of the compound: the IC50 values
decrease with increasing length of the aliphatic chain up to the 11-carbon acyl chain, and then increase
again. Indeed, the highest cytotoxicity (lowest IC50 value) is observed in compound 5, with an aliphatic
acyl chain of 11 carbon atoms. In addition, compound 5 also shows a high selectivity index in the HT-
29 cell line. The decrease of cytotoxicity for the compounds having the longest acyl chains (8-10, ≥14
carbon atoms) could be related to an increase in hydrophobicity, the diffusion through the plasmatic
membrane of cells becoming more difficult for these compounds. Five compounds (2-6) were thus
selected for further biological evaluations. The selection was made on the basis of products showing
particularly low IC50 values (high cytotoxicity) in the HT-29 cell line.
4.2. Tubulin assembly
Effects on tubulin self-assembly was determined in 20 mM sodium phosphate (NaPi), 10 mM
MgCl2, 1 mM EGTA, 3.4 M glycerol and 0.1 mM GTP at pH = 6.5 in the presence of 10 µM of
colchicine (as a positive control inhibitor) and 10 µM of compounds 2-6. Figure 2 shows the effects on
the microtubule formation studied by turbidity time-course measurements. A solution of 15 µM of
tubulin was incubated at room temperature with 10 µM colchicine (CLC hereafter in figures) and
compounds 2-6 for 10 min, which is the time necessary to reach the equilibrium state of the binding
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reaction between drugs and tubulin. Subsequently, the microtubule formation was started by increasing
the temperature to 37ºC and absorbance at 400 nm was followed over time.
Figure 2. Effects of colchicine and compounds 2-6 on the in vitro tubulin assembly. The lines in the
figure show the turbidity time course of polymerization of tubulin alone (black) at 15 µM, colchicine
(blue) at 10 µM, 2 (red) at 10 µM, 3 (gray) at 10 µM, 4 (orange) at 10 µM, 5 (green) at 10 µM and 6
(purple) at 10 µM.
As structural analogues of colchicine, the compounds under study were expected to inhibit the
formation of microtubules. Indeed, and as in the case of colchicine (blue line, overlapped by the red
line), compound 2 at 10 µM concentration showed a full inhibition of microtubule formation (red line).
Compound 3 inhibits around 80% of the microtubule formation (grey line). Interestingly, an increase in
turbidity was observed with compounds 4, 5 and 6 that could be due to the formation of abnormal
polymers. Indeed, formation of the tubulin-colchicine complex is known to induce tubulin
conformational changes which result in GTPase activity [16] and polymerization into abnormal
polymers [17].
In view of the above results, it was thus necessary to examine the effects of the drugs on tubulin
self-assembly at lower concentrations in order to avoid the formation of abnormal polymers of tubulin.
Figure 3 displays, as an example, the effect of compound 4 on tubulin self-assembly in vitro whereby a
clear inhibition was noticed as a function of the concentrations of drug added. It turns out that the rate
of assembly and the final amount of microtubules are lower in the presence of substoichiometric
concentrations of the compound 4. Indeed, the electron micrographs (see Supplementary Information,
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Figure SI-1) show much less microtubules in the presence of compound 4 at 0.1 µM concentration than
in the absence of it.
Figure 3. Effect of compound 4 on the turbidity time-course of in vitro microtubule formation. The left
panel shows tubulin polymerization at 15 µM without 4 (control) and in the presence of 0.1, 0.25, 0.5,
0.75 and 1 µM of compound 4. The right panel presents the correspondent percentage of inhibition by
4.
The inhibitory effects on microtubule formation of these colchicine analogues (2-6) by means of
measuring the IC50 values are reported in Table 2.
Table 2. Inhibitory effects on microtubule assembly at 37ºC and 15 µM of tubulin.a
Compound
Tubulin inhibition IC50 (µM)
1.8 ± 0.3
Colchicine
2
3
4
5
6
2.06 ± 0.20
1.08 ± 0.05
0.70 ± 0.07
0.79 ± 0.06
1.21 ± 0.14
a Data are the average (±SD) of three experiments
.
Compound 2 showed an inhibitory effect on microtubule assembly similar to colchicine. With
compounds 3-6, however, a 50% of microtubule assembly inhibition was measured at concentrations
lower than colchicine, with compound 4 being the most active one.
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Then, it was of interest to examine the ability of the compounds under study to induce the formation
of abnormal polymers [18]. This is a characteristic feature of the interaction of colchicine and its
analogues with tubulin [19]. Indeed, the 1:1 tubulin-colchicine complex polymerizes into anomalous
structures in the absence of glycerol (tubulin alone does not polymerize in these conditions) [18,20]. In
order to check the formation of such abnormal polymers, a new buffer was used consisting in 20 mM
NaPi, 16 mM MgCl2 and 0.1 mM GTP at pH 7 [19]. A solution of 15 µM of tubulin and 50 µM
solutions of colchicine and compounds 2-6 were incubated for 10 min a room temperature. The
polymerization reaction was then started by increasing the temperature to 37ºC. The results of these
experiments are shown in Figure 4: large increases in turbidity were observed for all compounds.
However, compounds 4-6 were more active than compounds 2 and 3. The formation of abnormal
polymers with compounds 2-6 was also followed by electron microscopy (see Supplementary
Information, Figure SI-2). As expected, we observed the absence of microtubules and the presence of
poorly defined filamentous sheet structures for colchicine and for the two studied compounds.
Figure 4. Effect of colchicine and compounds 2-6 on in vitro abnormal polymer formation. The lines in
the figure show the effect of tubulin alone (black) at 15 µM, colchicine (blue) at 50 µM, 2 (red) at 50
µM, 3 (grey) at 50 µM, 4 (orange) at 50 µM, 5 (green) at 50 µM and 6 (purple) at 50 µM.
4.3. Specificity and localization of the interaction
We decided to determine whether the structural analogues of colchicine were able to bind to tubulin
liganded to colchicine. Figure 5 (panel A) shows the time course of fluorescence change induced by the
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association of 10 µM of colchicine and 10 µM of tubulin at 25°C (red trace). The equilibrium of the
binding process is reached after 50 minutes. Addition of 20 µM of compound 4 to tubulin (black trace)
produced a large increase of the fluorescence; furthermore, the equilibrium is reached faster than with
colchicine alone (red trace). Colchicine has a very low kinetic rate constant for the dissociation process,
so that we used this property to perform the competition experiments. The tubulin-colchicine complex
was first formed and then 20 µM of 4 was added (as it was done with tubulin alone). In Figure 5 (panel
B), no large increase of fluorescence signal is observed after the addition of 4. This indicates that it
cannot bind to tubulin. This also indicates that compound 4 binds to or near the colchicine site.
Figure 5. (A) Time course of the fluorescence change upon binding 20 µM of compound 4 (black
arrow, and black trace) and 10 µM of colchicine (red arrow and red trace) to 10 µM of tubulin at 25°C.
Arrows indicate the addition of compound 4 and colchicine. (B) Time course of the fluorescence
change when 20 µM of compound 4 is added to 10 µM tubulin-colchicine complex. The black arrow
indicates the addition of compound 4. The observed increase in fluorescence was not the same as that
observed in the absence of colchicine. This indicates that 4 competes with the natural product for the
same binding site.
The poor amplitude of the residual signal obtained is probably the consequence of a weak
displacement of colchicine by the compound and association of the latter with free tubulin. This
competition experiment indicates that compounds bind to tubulin at a site involving the colchicine site.
4.4. Interaction with tubulin: binding parameters
The binding of colchicine analogues implicated tryptophan residues and quenched the intrinsic
tubulin fluorescence. The intrinsic fluorescence emission spectra of tubulin was obtained by exciting
the tryptophan residues at 295 nm. Then, examination of the fluorescence of tubulin in presence of 4
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revealed a decrease in the intrinsic protein fluorescence signal. The measurements were performed after
15 minutes, which is the time required to ensure that the equilibrium state of the binding reaction
between tubulin and the ligands has been reached. As an example, Figure 6 shows the results observed
for the specific case of 4. A decrease in the fluorescence emission intensity at 330 nm was observed
with an increase of the concentrations of 4. This quenching of fluorescence was used to carry out
binding titration experiments. The results are indicated in the graph inserted in Figure 6, which shows
the titration curve for the association of 4 to tubulin. Similar results are obtained for the other
compounds and for colchicine [18]. Quenching of tubulin fluorescence suggests that compounds 2-6
bind to tubulin close to a fluorophoric residue, i.e., tryptophan.
Figure 6. Tryptophan fluorescence modifications of tubulin upon binding of compound 4 at 25ºC in
PG buffer, pH 7. The excitation wavelength was 295 nm. Fluorescence emission of 2 µM tubulin alone
(black line), with 2 µM (···), 10 µM (-·-) and 20 µM (-··-) of 4. The inset shows the quenching
fluorescence titration curve inverted produced by 4 binding to 2 µM of tubulin; the solid line is the
fitting curve obtained as described in the Experimental Part
.
The apparent affinity stoichiometric constants (Ka) were then calculated for colchicine and for
compounds 2-6 (Table 3) [21].
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Table 3. Binding Parameters of Colchicine and Compounds 2-6 to tubulin in PG buffer at 25ºCa.
Equilibrium binding constant
Compound
to tubulin Ka x 104 [M-1]
Colchicine
9 ± 3
2
3
4
5
6
6.4 ± 2.0
5.2 ± 1.1
17.9 ± 2.3
18.4 ± 1.6
19 ± 2.3
a Data are the average (±SD) of three experiments
.
Colchicine analogues 2 and 3 have apparent affinity constants slightly lower than colchicine. In
contrast, compounds 4, 5 and 6 present higher apparent affinity binding constants than the natural
alkaloid. These molecules were able to induce the formation of abnormal polymers at lower
concentrations values than colchicine. These apparent binding constant values were close to that of the
colchicine analogue MTC [22].
4.5. Mitotic arrest and inhibition of interphase microtubules of cultured cells
Further in vitro and in-cell studies have been done in order to establish a relationship between the
lengths of the acyl chain and the biological activity of compounds 2-6, which showed the highest
cytotoxicities towards the HT-29 cell line. Thus, IC50 values were also measured on the A549 tumoral
cell line (adenocarcinome human alveolar basal epithelial cells), after 72 h of incubation (see Table 4,
which also includes the calculated selectivity indexes SIC).
Table 4. Cytotoxicity of colchicine analogues 2-6 towards the tumoral cell line A549a.
b
Compound
A-549
SIC
Colchicine
14 ± 6
0.36
0.39
0.14
< 0.1
< 0.1
< 0.1
2
3
4
5
6
18 ± 2
133 ± 23
25 ± 4
62 ± 4
71.9 ± 0.8
aIC50 values are expressed as the drug concentration (nM) that inhibits the cell growth by 50%. Data are
the average (±SD) of three experiments
bSIC = IC50(HEK-293)/IC50(A549).
.
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Most of the IC50 values are in the low nanomolar range although the selectivity indexes SIC are in all
but one compound lower than colchicine. These SIC values slightly diverge from the SIA values of the
HT-29 line, in which SI values are higher than those of colchicine. Since our colchicine derivatives
bind to tubulin and inhibit in vitro microtubule assembly, it was convenient to establish whether they
also could inhibit microtubule assembly in living cells and to characterize their effects on microtubules,
mitoses and DNA content. Accordingly, subsequent biological studies were carried out using flow
cytometry to investigate whether the IC50 concentrations of the compounds could induce a G2/M block
after a 24 h treatment. Thus, an indirect immunofluorescent assay on the A549 cells line was realized to
observe the effect on microtubules during the interphase.
In order to establish the cell cycle distribution, A549 cells were treated for 24 hours with colchicine
and compounds 2-6 at IC50 values. The observed results show that all compounds are able to arrest the
cell cycle at the G2/M phase, as expected for colchicine derivatives [4,23]. Among all compounds, 5
showed the highest capacity to arrest the cell cycle at its IC50 concentration value (see Figure SI-3 in
the Supplementary Information).
Since compounds 2 and 4 displayed cytotoxicity values similar to that of colchicine in the A549
line, they were selected to perform the immunofluorescence assays. The results are displayed in Figure
7. Colchicine (panel B), compound 2 (panel C) and compound 4 (panel D) are able to completely
depolymerize cellular microtubules at 100 nM after 4 hours of treatment.
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Figure 7. Effects of compounds 2 and 4 as compared to colchicine on the microtubule network. A549
cells were treated for 4 hours and processed for immunofluorescence microscopy: (A) microtubules of
cells without inhibitor, (B) 100 nM colchicine, (C) 100 nM compound 2 and (D) 100 nM compound 4.
Microtubules were stained with α-tubulin antibodies. The scale bar represents 50 µm.
4.6. Molecular docking
After having demonstrated that synthetic compounds bind to tubulin at/or near the colchicine
binding site, and with the aim at gaining insight about the binding site of these colchicine derivatives
on tubulin dimers, molecular docking was performed using Autodock 4.2. The crystal structure of αβ
tubulin (PDB ID 1SA0) was used as a template [24]. Figure 8 (A and B panels) shows a
superimposition of colchicine (two-carbon acyl chain), 4 (ten-carbon acyl chain), 5 (eleven-carbon acyl
chain), and 6 (twelve-carbon acyl chain) over the structure of DAMA-colchicine [N-deacetyl-N-(2-
mercaptoacetyl)colchicine], obtained from the PDB ID 1SA0 [25].
These docking experiments suggest that these derivatives exhibit conformations very close to that of
DAMA-colchicine. Furthermore, the calculations suggest that, while the binding site of the colchicine
fragment is situated, as expected, at the β-tubulin domain, the aliphatic chain spans the intradimer
interface zone that separates the α and β subunits and inserts itself in a hydrophobic groove located in
the α-tubulin domain. This is more clearly shown in Figure 8, which contains docking structures of
colchicine, 4, 5 and 6 complexed with tubulin (C-F panels). As commented above, the colchicine
moiety of these derivatives is located in the colchicine binding site at β-tubulin, represented in green
color in the images. However, the N-acyl residue points to a groove in the α-tubulin domain (yellow).
Sufficiently long aliphatic chains (e.g. 4, 5 or 6) fit well into a hydrophobic groove in α-tubulin, and
develop more hydrogen bonding interactions than DAMA-colchicine itself (see 2D interactions in
Figure SI-4 in Supplementary Information). However, shorter aliphatic chains, as in colchicine itself,
are unable to reach this α-tubulin groove. Among these three ligands, compound 6 is able to insert its
aliphatic chain into the aforementioned groove more deeply than the others. This is underscored by an
increased number of hydrogen bonding interactions (see Figure SI-4 in Supplementary Information).
We have also calculated for colchicine and compounds 2-6 the Gibbs free energies from the docking
binding parameters and from the apparent affinity constants, experimentally obtained. These values
show the same trend (see Graphic SI-1 in the Supplementary Information). Thus, it can be concluded
that the greater in vitro activity of compounds 4-6 on tubulin could be due to the new interactions that
they develop with α-tubulin.
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Figure 8. Superimposition of the structures of colchicine (black), 4 (orange), 5 (red) and 6 (purple) on
the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Images A and B correspond
to the same superimpositions observed from two different angles. Structures of colchicine (C), 4 (D), 5
(E) and 6 (F) at the colchicine binding site. The α- and β-tubulin subunits are coloured in yellow and
green, respectively.
5. Summary and conclusions
Colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-chain
acyl moieties show cytotoxic activities in the medium to low nanomolar range. Of the tested
compounds, those with N-acyl residues of 10-12 carbon atom length (compounds 4, 5 and 6) show
particularly high cytotoxicities (picomolar range) towards the HT-29 cell line and form abnormal
tubulin polymers. This contrasts with the behaviour observed with colchicine, which gives rise to
inhibition of tubulin polymerization. By means of competition experiments we have shown that all
the studied derivatives bind to tubulin at/or near the colchicine binding site. Compounds 2 and 3,
having the shorter acyl chains, bind to tubulin with similar affinity constants than colchicine,
whereas compounds 4, 5 and 6 having longer acyl chains bind to tubulin with a higher affinity than
colchicine. By means of immunofluorescence experiments on A549 cells, we have shown that the
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compounds inhibit the formation of microtubules at concentrations and incubation times identical to
those of colchicine. Moreover, all the studied derivatives cause an extensive arrest of the cell cycle
in the G2/M phase at concentrations corresponding to the IC50 values. For the sake of comparison,
compound 5 and colchicine are able to arrest 41% and 8%, respectively, of the cells in the G2/M
phase at such a concentration. It is worth noting that docking calculations have shown that while the
binding site of the colchicine fragment is situated, as expected, at the β-tubulin domain, near the
intradimer interface zone that separates the α and β subunits, the aliphatic chain of sufficiently long
acyl residues (compounds 4-6) spans this interface zone and inserts in a hydrophobic groove located
in the α-tubulin domain. In addition, 2D representations of compounds 4-6 show more interactions
than DAMA-colchicine with the α-subunit of tubulin. This may explain the differences in behavior
between colchicine and its long-chain homologues. A most interesting aspect of this research is the
fact that compounds such as 4-6 show cytotoxicity at concentrations three orders of magnitude
lower than colchicine. Their pharmacological use in cancer therapy could possibly be performed
with lower dosages and be thus endowed with less acute toxicity problems than in the case of
colchicine. Nevertheless, widely in vivo studies should be carried out in order to assess the toxicity
of these compounds.
6. Experimental
6.1. Chemistry
6.1.1. General procedures
NMR spectra were measured at 25°C. The signals of the deuterated solvent (CDCl3) was taken as
the reference. Multiplicity assignments of 13C signals were made by means of the DEPT pulse
sequence. Complete signal assignments in 1H and 13C NMR spectra were made with the aid of 2D
homo- and heteronuclear pulse sequences (COSY, HSQC, HMBC). High resolution mass spectra were
run by the electrospray mode (ESMS). IR data were measured with oily films on NaCl plates (oils) and
are given only for relevant functional groups (C=O, NH). Optical rotations were measured at 25 °C.
Experiments which required an inert atmosphere were carried out under dry N2 in flame-dried
glassware. THF was freshly distilled from sodium/benzophenone ketyl and transferred via syringe.
Commercially available reagents were used as received. Where solutions were filtered through a Celite
pad, the pad was additionally washed with the same solvent used, and the washings incorporated to the
main organic layer.
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6.1.2. General procedure to prepare colchicine analogues 1-10.
Compound 11 (283 mg, 0.6 mmol) and the appropriate acid (1.2 mmol) were dissolved under N2 in
dry CH2Cl2 (60 mL) and treated with N,N'-dicyclohexylcarbodiimide (743 mg, 3.6 mmol) and 4-(N,N-
dimethylamino)pyridine (147 mg, 1.2 mmol). The mixture was stirred for 3 h at room temperature and
then filtered through Celite. The filtrate was evaporated under reduced pressure, and the residue was
subjected to column chromatography on silica gel (EtOAc-acetone, 8:1) to afford the desired
compound 1-10. The physical and spectral data are indicated in the Supplementary Information.
Yields: 1, 44%; 2, 51%; 3, 82%; 4, 44%; 5, 44%; 6, 61%; 7, 44%; 8, 72%; 9, 46%; 10, 77%.
6.2. Biological studies. Materials and methods
6.2.1. Cell culture
Cell culture media were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was a
product of Harlan-Seralab (Belton, U.K.). Supplements and other chemicals not listed in this section
were obtained from Sigma Chemical Co. (St. Louis, MO). Plastics for cell culture were supplied by
Thermo Scientific BioLite. All tested compounds were dissolved in DMSO at a concentration of 10
µg/mL and stored at −20°C until use.
HT-29, MCF-7 and HEK-293 cell lines were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing glucose (1g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50
µg/mL), and amphotericin B (1.25 µg/mL), supplemented with 10% FBS.
Cells from human non-small lung carcinoma (clone A549; ATCC n°: CCL2, MD, USA) were
routinely grown at 37°C in a humidified atmosphere of 5% CO2. Cells were maintained in a standard
medium composed of RPMI 1640 (Lonza, France) supplemented with 10% fetal bovine serum (FBS), 2
mM L-glutamine and 1% penicillin and streptomycin (Invitrogen). Cells were free from mycoplasma as
determined by mycoalert tests (Lonza).
6.2.2. Cytotoxicity assays
For cell viability assay on the cell line A549, cells harvested from sub-confluent monolayers were
seeded at 25,000/mL in 96-well microtiter plate (Cambridge Technology, France) and cultured 24
hours under standard conditions. Standard medium was then replaced by fresh medium containing no
drugs (control or “0”) or compounds at different concentrations. The surviving cells were quantified
after 72 h by the MTT assay, according to the manufacturer’s instructions. Briefly, 20 µL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution at 5 mg.mL-1 were added to
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cells for 2 h at 37°C. The supernatant was discarded and replaced by 200 µL of DMSO to dissolve
formazan crystals. The absorbance was then read at 540 nm by spectrophotometry. For all
concentrations of compound, cell viability was expressed as the percentage of the ratio between the
mean absorbance of treated cells and the mean absorbance of untreated cells. Three independent
experiments were performed, and the IC50 values (i.e., concentration half inhibiting cell proliferation)
were graphically determined.
A total of 5 × 103 HT-29, MCF-7, or HEK-293 cells in a total volume of 100 µL of their respective
growth media were incubated with serial dilutions of the tested compounds. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) dye reduction
assay in 96-well microplates was used, as previously described [26]. After 3 days of incubation (37 °C,
5% CO2 in a humid atmosphere), 10 µL of MTT (5 mg/mL in phosphate-buffered saline, PBS) was
added to each well, and the plate was incubated for a further 4 h (37 °C). The resulting formazan was
dissolved in 150 µL of 0.04 N HCl/2-propanol and read at 550 nm. All determinations were carried out
in triplicate.
6.2.3. Fluorescence microscopy
For immunofluorescence microscopy of the microtubule network, 106 cells were plated on
coverglass and incubated with the different drugs. Cells were then fixed in 3.7% formaldehyde (in PBS
pH 7.4) for 20 min at room temperature, permeabilized with PBS-Triton X-100 0.5% for 10 min at
room temperature. Direct immunostaining was carried out for 2 h at room temperature with a primary
FITC-conjugated anti-α-tubulin antibody (dilution 1:400 in PBS-BSA 1% from a 1 mg/mL solution;
monoclonal antibody, clone DM1A, Sigma-Aldrich, France). Next, cells were incubated for 45 min at
room temperature in darkness with secondary Alexa fluor 488-conjugated antibodies (dilution 1:200
each from 1.5 mg/mL; Molecular Probe, France). Then, cells were washed in PBS and coverglasses
were mounted with a drop of ProLong® anti-fade solution (Invitrogen). The cytoskeleton was imaged
by a confocal laser scanning microscope (CLSM) Leica SP5 with a Leica inverted microscope,
equipped with a Plan-Apochromat 63× oil immersion objective (NA=1.4). Each image was recorded
with the CLSM’s spectral mode selecting specific domains of the emission spectrum. The FITC
fluorophore was excited at 488 nm with an argon laser and its fluorescence emission was collected
between 496 nm and 535 nm. The public-domain ImageJ software was used for image analysis [27].
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6.2.4. Cell cycle analysis and mitosis index
For flow cytometric analysis of DNA content, 4 x 105 cells in exponential growth were treated with
the IC50 value and ten times this value of each compound, so 14 nM and 140 nM for CLC, 18 nM and
180 nM compound 2, 133 nM and 1.33 µM compound 3, 25 nM and 250 nM compound 4, 62 nM and
620 nM compound 5 and 72 nM and 720 nM compound 6 for 24 h for 24 h. The supernatant and
trypsinated cells were harvested then centrifuged (1200 rpm, 5 min, 4°C). The cell pellet was re-
suspended in 1 mL of cold 70% ethanol for 30 min at -20°C. Cells were centrifuged (2000 rpm, 5 min,
4°C) to remove ethanol then the cell pellet was re-suspended in the staining mix containing 50 µg/mL
propidium iodide (Molecular Probes, France) and 100 µg/mL RNAse A (Sigma, France) in PBS for 20
min at room-temperature in darkness. Samples were analyzed on a Becton Dickinson FACScan flow
cytometer using the CellQuest software, which was also used to determine the percentage of cells in the
G2/M phases of the cell cycle. PI was excited at 488 nm and fluorescence analyzed at 620 nm on
channel Fl-2.
6.2.5. Tubulin purification, microtubule assembly and formation of abnormal polymers
Lamb brain tubulin was purified from brain soluble extracts by a modified Weisenberg procedure
consisting in ammonium sulfate fractionation and ion-exchange chromatography [22, 28]. Pure protein
was stored in liquid nitrogen and prepared as described for used [29]. Protein concentrations were
determined spectrophotometrically at 275 nm with a Perkin-Elmer spectrophotometer Lambda 800
with an extinction coefficient of 1.07 L.g-1.cm-1 in 0.5% SDS in neutral aqueous buffer or with an
extinction coefficient of 1.09 L.g-1.cm-1 in 6 M guanidine hydrochloride.
Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM
MgCl2, 3.4 M glycerol and 0.1 mM GTP (pH 6.5 adjusted). The reaction was started by warming the
samples at 37°C and the mass of polymer was monitored by turbidimetry with a spectrophotometer
Beckman DU 7400 at 400 nm in order to minimize light absorption by compounds. Samples containing
compounds and their controls had less than 2% residual DMSO. For the IC50 determation turbidimetry
was measured at 400 nm with a POLARstar BMG Labtech spectrophotomether using a 96-well plate.
Samples containing the compounds and control had less than 1% residual DMSO. Three independent
experiments were done in each case.
Formation of abnormal polymers induced by CLC and compounds 1-10 in 20 mM sodium
phosphate buffer, 16 mM MgCl2, 0.1 mM GTP (pH 7) at 37°C was monitored by turbidimetry at 400
nm in order to minimize light absorption by the compounds. Tubulin was mixed with saturating
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concentration of compounds 1-10 in thermostated cuvettes. The residual quantity of DMSO was less
than 2%.
6.2.6. Transmission electron microscopy
For standard negative staining, aliquots of 3 µL of the protein samples were absorbed onto 200 mesh
Formvar carbon-coated copper grid (Canemco Imc. & Marivac Inc., Lakefield, QC, Canada) negatively
stained with 2% (w/v) uranyl acetate. Grids were observed onto a JEOL (Tokyo, Japan) JEM-1400
transmission electron microscope operated at 80 kV.
6.2.7. Binding measurement by fluorimetric titration
Fluorescence emission was reported at 330 nm with the fluorescence spectrophotometer Horiba
Jobin Yvon Fluoromax-3. Fluorescence measurements were performed with an excitation wavelength
of 295 nm in order to excitate the tubulin tryptophan residues. PG buffer (pH 7) was used to obtain the
uncorrected fluorescence spectra using 0.2 cm (excitation direction) x 1 cm cells (Hellma) thermostated
at 25 ºC by an external water bath. Tubulin (1-3 µM) was titrated with the corresponding compound.
The inner filter effect was presented in all compounds and was corrected according to the following
equation [30].
Fcorr and Fobs are the corrected and observed fluorescence values at the emission wavelengths. Aexc
and Aem are the absorptions at the excitation and emission wavelengths, calculated with the formula
Ax=εxlxC, where x is the excitation or emission direction, ε is the extinction coefficient, l is the path
length of the cell in the excitation and emission direction, and C is the ligand concentration. The
corrected quenching fluorescence titration curves were inverted and fitted to the saturation curve
equation by means of nonlinear least-squares regression analysis.
Fmax is the plateau fluorescence value. Concentrations (bound [Lb] and free [Lf] ligand) and Kd the
stoichiometric dissociation binding constants were calculated using the following equation:
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in which [L0] and [P0] are the total ligand and protein concentrations. The algorithm starts with an
arbitrary opening Kd value. With this value, [Lb] and [Lf] are calculated and then the nonlinear least-
squares regression analysis is executed. The initial set is corrected in the next step by a Newton-Gauss
procedure. This iterative procedure is continued until the minimum sum of squared deviations between
experimental and calculated values of Fcorr is reached.
Fluorescence time course of binding of AMM4 and colchicine are monitored in a fluorescence
spectrophotometer Horiba Jobin Yvon Fluoromax-3 at 25°C using 0.2 cm (excitation direction) x 1 cm
cells (Hellma) thermostated at 25 ºC. Emission fluorescence intensities were collected at 443 nm using
an excitation wavelength of 350 nm and with emission and excitation slit widths of 5 nm.
The concentrations of compounds were detected spectrophotometrically with a Beckman DU 7400.
The extinction coefficients (ε) were determined by means of dissolving the compounds in DMSO and
diluting this solution in 20 mM NaPi buffer at pH 7, followed by measurement of the UV visible
spectra. Three independent determinations allowed us to determine the value of the extinction
coefficient at 354 nm (Table 5). The other values were calculated graphically.
Table 5
compound
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
ε
2
3
4
5
6
354nm = 14100 ± 2500
354nm = 12800 ± 1700
354nm = 9100 ± 2500
354nm = 10300 ± 1400
354nm = 9300 ± 1500
295nm = 3760
295nm = 2844
295nm = 2395
295nm = 3679
295nm = 3100
330nm = 9870
330nm = 8533
330nm = 6705
330nm = 6621
330nm = 5580
6.2.8. Molecular docking
Molecular docking was performed using Autodock 4.2 [24]. The crystal structure of αβ tubulin
(PDB ID 1SA0) was used as a template. Nevertheless, the C and D subunits, stathmin, water molecules
and bound ligands were previously removed from the protein structure in order to perform docking
simulation. The GaussView 5.0 program [31] was used to build the structures of the colchicine
derivatives 2-6. Local docking was made in such a way that the grid box covered the entire αβ-tubulin
interface. The grid map was used with 126 points equally in each x, y and z direction and with grid
spacing 0.247 Å. The cluster was compared on the basis of the binding energy. The Lamarckian genetic
algorithm was chosen to search for the best conformers. Molecular graphics were done with the
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program Visual Molecular Dynamics (VMD) [32]. LigPlot+ [33] has been used to show the interaction
in 2-D between the compounds and the tubulin.
Acknowledgments
This research has been funded by the Ministerio de Economía y Competitividad (project CTQ2014-
52949-P), by the Universitat Jaume I (project PI-1B2015-75) and by the Conselleria d’Educaciò,
Investigaciò, Cultura i Sport de la Generalitat Valenciana (project PROMETEO 2013/027). A. M.-M.
thanks the Conselleria d’Educaciò, Investigaciò, Cultura i Esport de la Generalitat Valenciana for
financial support to a research stay in the laboratories in Marseille. The biological part has been
supported by Inserm and the Université of Aix-Marseille.
Supplementary Information
Additional figures, graphics, experimental data, physical, IR, NMR and Mass spectrometry data of
all new synthetic compounds are provided in the Supplementary Information.
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List of captions
Scheme 1. Synthesis of colchicine derivatives 1-10.
Figure 1. Structure of colchicine derivatives investigated in this study.
Figure 2. Effects of colchicine and compounds 2-6 on the in vitro tubulin assembly. The lines in the
figure show the turbidity time course of polymerization of tubulin alone (black) at 15 µM, colchicine
(blue) at 10 µM, 2 (red) at 10 µM, 3 (gray) at 10 µM, 4 (orange) at 10 µM, 5 (green) at 10 µM and 6
(purple) at 10 µM.
Figure 3. Effect of compound 4 on the turbidity time-course of in vitro microtubule formation. The left
panel shows tubulin polymerization at 15 µM without 4 (control) and in the presence of 0.1, 0.25, 0.5,
0.75 and 1 µM of compound 4. The right panel presents the correspondent percentage of inhibition by
4.
Figure 4. Effect of colchicine and compounds 2-6 on in vitro abnormal polymer formation. The lines in
the figure show the effect of tubulin alone (black) at 15 µM, colchicine (blue) at 50 µM, 2 (red) at 50
µM, 3 (grey) at 50 µM, 4 (orange) at 50 µM, 5 (green) at 50 µM and 6 (purple) at 50 µM.
Figure 5. (A) Time course of the fluorescence change upon binding 20 µM of compound 4 (black
arrow, and black trace) and 10 µM of colchicine (red arrow and red trace) to 10 µM of tubulin at 25°C.
Arrows indicate the addition of compound 4 and colchicine. (B) Time course of the fluorescence
change when 20 µM of compound 4 is added to 10 µM tubulin-colchicine complex. The black arrow
indicates the addition of compound 4.
Figure 6. Tryptophan fluorescence modifications of tubulin upon binding of compound 4 at 25 ºC in
PG buffer, pH 7. The excitation wavelength was 295 nm. Fluorescence emission of 2 µM tubulin alone
(black line), with 2 µM (···), 10 µM (-·-) and 20 µM (-··-) of 4. The inset shows the quenching
fluorescence titration curve inverted produced by 4 binding to 2 µM tubulin; the solid line is the fitting
curve obtained as described below
.
Figure 7. Effects of compounds 2 and 4 as compared to colchicine on the microtubule network. A549
cells were treated for 4 hours and processed for immunofluorescence microscopy: (A) microtubules of
cells without inhibitor, (B) 100 nM colchicine, (C) 100 nM compound 2 and (D) 100 nM compound 4.
Microtubules were stained with α-tubulin antibodies. The scale bar represents 50 µm.
Figure 8. Superimposition of the structures of colchicine (black), 4 (orange), 5 (red) and 6 (purple) on
the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Images A and B correspond
to the same superimpositions observed from two different angles. Structures of colchicine (C), 4 (D), 5
(E) and 6 (F) at the colchicine binding site. The α- and β-tubulin subunits are coloured in yellow and
green, respectively.
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Table 1. Cytotoxicity of colchicine analogues 1-10 toward tumoral cell lines HT-29 and MCF-7 and
one non-tumoral cell line HEK-293.
Table 2. Inhibitory effects on microtubule assembly at 37ºC and 15 µM of tubulin.a
Table 3. Binding parameters of colchicine and compounds 2-6 to tubulin in PG buffer at 25ºCa.
Table 4. Cytotoxicity of colchicine analogues 2-6 towards the tumoral cell line A549a.
Table 5. Extinction coefficients (M-1cm-1) of colchicine derivatives 2-6.
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