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Experimental Therapeutics |
Biotechnology Unit, Biomaterial Institute of Quebec, Centre Hospitalier Universitaire de Quebec, Pavillon St-François d’Assise, Laval University, Quebec City, Quebec, Canada, G1L 3L5
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ABSTRACT |
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To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the ßIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that ßIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with ß-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of ß-tubulin.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cytotoxicity of CEU has been evaluated in human breast cancer (MDA-MB-231), human colon adenocarcinoma (LoVo), and mouse lymphocytic leukemia (P388D1) cell lines (3) . Several CEU, including 4-tBCEU, 4-iPCEU, and 4-sBCEU, were shown to be significantly more cytotoxic than chlorambucil and carmustine themselves (2 , 3) . Interestingly, CEU were not mutagenic in the Ames test (2) . We also found that 4-tBCEU did not show any differential cytotoxicity in a panel of cell lines that have acquired resistance to several chemotherapeutic agents (4) . CEU cytotoxicity was unaffected by several mechanisms of chemotherapeutic resistance, including increased P-glycoprotein expression, increased DNA repair, increased intracellular glutathione concentration, and glutathione-S-transferase activity, as well as alteration of topoisomerase II activity (4) . The lack of resistance of tumor cells to CEU suggests that CEU could be potentially useful to treat various types of tumors, including those that are resistant to conventional chemotherapy.
4-tBCEU was shown to have significant antineoplastic activity in the L1210 cell graft model (5) , with a median survival time enhanced 1.77 times relative to the untreated control at 10 mg/kg/day, which is higher than chlorambucil (2) . Moreover, in the B16 melanoma model (5) , CEU significantly decreased tumor weight by almost 50% after 14 days of transdermic administration.4 The mechanism of CEU cytotoxicity is still unknown. However, evidence was provided that vimentin and ß-tubulin synthesis was altered by CEU (6) , suggesting that CEU might behave as antimicrotubule agents, such as colchicine, vinblastine, or podophyllotoxin. In the present study, we provide evidence that CEU are antimicrotubule agents that covalently bind to ß-tubulin and consequently prevent microtubule assembly.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drugs.
Colchicine, vinblastine, paclitaxel, and iodoacetamide were
purchased from Sigma (St. Louis, MO). CEU derivatives and EBI were
prepared as already described (3
, 4
, 10)
. Synthesis of
[urea-14C]-4-tBCEU was carried out
as described previously (11)
. All drugs were dissolved in
DMSO, and the final concentration of DMSO in the culture medium was
maintained at 0.5% (v/v).
Cytotoxicity Assay.
Cytotoxicity was assessed using the MTT assay
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as
described by Carmichael et al. (12)
. Cytotoxic
activity of these compounds was expressed as the concentration of CEU
inhibiting MDA-MB-231 cell growth by 50% (IC50).
Kinetics of Alkylation of 4-(4-nitrobenzyl)pyridine by CEU
Derivatives.
The rate constant of alkylation (K') of CEU
derivatives and chlorambucil was evaluated by a colorimetric assay as
described by Bardos et al. (13)
. Briefly, 1 ml
of a 10% (v/v) solution of 4-(4-nitrobenzyl)pyridine in ethanol and 1
ml of 50 mM sodium acetate (pH 4.3) were added to
an ethanol solution (95%) containing 400 nmol/ml of either
chlorambucil or CEU and heated to 80°C in a shaking water bath for
60, 90, 120, or 150 min. The reaction was stopped by cooling the
mixtures on ice for 5 min. Then, 1.5 ml of 0.1 M
KOH:ethanol (1:2; v/v) were added to the reaction mixture. Samples were
vortexed for 12 s and set aside for 2.5 min before reading the
absorbance at 570 nm. The values were compared with those obtained
using a blank sample containing all reagents except the alkylating
agent.
Cell Cycle Analysis.
After incubation of MDA-MB-231 cells with 4-tBCEU,
4-iPCEU, or 4-sBCEU, the cells were harvested,
resuspended in 1 ml of PBS, and fixed by the addition of 2.4 ml of
ice-cold anhydrous ethanol. Then, 5 x 105
cells from each sample were centrifuged for 3
min at 1000 x g. Cell pellets were
resuspended in PBS containing 50 µg/ml of propidium iodide and 40
units/ml of RNase A (Boehringer Mannheim, Laval, Canada). Mixtures were
incubated at room temperature for 30 min, and cell cycle distribution
was analyzed using an Epics Elite ESP flow cytometer (Coulter
Corporation, Miami, FL).
Separation of Soluble and Polymerized Tubulins.
Separation of soluble and polymerized tubulins from MDA-MB-231 cells
was carried out as described by Minotti et al.
(14)
with minor modifications. Briefly, after drug
exposure, about 5 x 106 cells in
100-mm Petri dishes were washed with PBS at 37°C and harvested in 3
ml of PBS containing 0.4 µg/ml of paclitaxel using a rubber
policeman.
Cells were centrifuged and lysed using 250 µl of microtubule-stabilizing buffer [20 mM Tris-HCl (pH 6.8), 140 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 0.5% NP40, and 0.4 µg/ml paclitaxel] and then transferred to 1.5-ml microcentrifuge tubes. Samples were centrifuged at 12,000 x g for 10 min at 4°C, and the supernatants containing soluble tubulin were placed in separate microcentrifuge tubes containing 250 µl of 2x Laemmli sample buffer (15) . Pellets containing the polymerized tubulin were resuspended in 250 µl of water, followed by two freeze/thawing cycles and the addition of 250 µl of 2x Laemmli sample buffer. Samples were analyzed by SDS-PAGE, and immunoassay was performed as described below.
Subcellular Fractionation of MDA-MB-231 Cells.
Cells (
5 x 106) were incubated
with 30 or 100 µM
[14C]-tBCEU for 12 or 24 h and
then washed with PBS and harvested by scraping in lysis buffer [5
mM HEPES (pH 7.4), 1 mM
MgCl2, 10 mM KCl, 1
mM CaCl2, 1
mM phenylmethylsulfonyl fluoride, 1
mM benzamidine, and 1 mM
aprotinin]. Cell lysates were homogenized using a tissue grinder, and
the final sucrose concentration of each sample was adjusted to 250
mM. Samples were centrifuged at 600 x g for 10 min at 4°C to isolate the postnuclear
supernatant. The pellets, containing nuclei and intact cells, were
discarded, and the postnuclear supernatant was recentrifuged at 90,000
rpm using a Rotor TLA-100.1 in a Beckman TL-100 ultracentrifuge for 30
min to separate the cytosolic fraction (C) from the insoluble fraction
(M) containing the membrane components and mitochondria. One volume of
2x Laemmli sample buffer was then added to the supernatant (C), and
the pellet (M) was resuspended in 200 µl of Laemmli sample buffer.
Samples were boiled for 5 min and kept at -20°C until analysis.
SDS-PAGE Analysis and Immunoblotting of ß-tubulin.
Samples (1 x 105
cells)
were analyzed by 10% SDS-PAGE using the Laemmli system
(15)
. Membranes were then incubated with PBSMT [PBS (pH
7.4), 5% fat-free dry milk, and 0.1% Tween-20] for 1 h at room
temperature and then with 1:500 monoclonal anti-ß-tubulin (clone TUB
2.1, Sigma) or 1:400 anti-ß-tubulin (clone no. SDL.3D10, Sigma) for
1 h. This monoclonal antibody is specific to ßIII-tubulin and
does not cross-react with other ß-tubulin isoforms. Membranes were
washed with PBSMT and incubated with 1:2500 peroxidase-conjugated
antimouse immunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT
for 30 min. Detection of the immunoblot was carried out with the ECL
Western blotting detection reagent kit (Amersham Canada, Oakville,
Canada).
Preparation of Protein Extract and Two-dimensional SDS-PAGE.
MDA-MB-231 cells (5 x 106)
incubated with 30 µM
[urea-14C]-4-tBCEU (11)
for 48 h were harvested by scraping and transferred to a 1.5-ml
microcentrifuge tube. Cell pellets were lysed by the addition of 1 ml
of lysis buffer containing 4% (w/v)
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
4.6% Ampholines [comprised of 3.6% Ampholine (pH range 5–7) and 1%
Ampholine (pH 3–10; Sigma, St. Louis, MO], 50
mM DTT, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM
benzamidine. Samples were homogenized by 10 passages through a 26-G
needle and incubated with 1 mg/ml DNase and 0.25 mg/ml RNase A for 5
min on ice. Then, EDTA/EGTA (1:1) and urea were added to final
concentrations of 1 mM and 8.5
M, respectively. Samples were centrifuged at
14,000 rpm for 2 min at 4°C, and 0.03% (w/v) bromphenol blue was
added to the supernatant. Samples were kept at -80°C until
processed. Protein extracts were separated by isoelectric focusing
according to the procedure described by O’Farrell (16)
with minor modifications. Briefly, samples were applied to a 4.5%
polyacrylamide gel containing 8.5 M urea, 2%
(w/v) CHAPS, and 2% (v/v) Ampholines. Samples were prefocused at 200,
300, and 400 V for 15, 30, and 30 min, respectively (17)
.
Isoelectric focusing was performed at 200, 400, 800, and 600 V during
0.5, 15, 1, and 1.5 h, respectively. Gels were equilibrated twice
for 15 min in buffer A containing 50 mM Tris-HCl
(pH 6.8), 6 M urea, 30% glycerol, 2% (v/v) SDS,
and 2% (w/v) DTT and for 10 min in buffer B containing 50
mM Tris-HCl (pH 6.8), 6 M
urea, 30% glycerol, 2% (v/v) SDS, and 0.5% (w/v) iodoacetamide. In
the second dimension, proteins were separated according to their
molecular weight by using a 10% polyacrylamide SDS gel. The gels were
then transferred onto a nitrocellulose membrane.
Detection of Proteins Alkylated by
[urea-14C]-4-tBCEU.
Nitrocellulose membranes were dried for 3 days at room temperature and
fixed under a FBTIV-816 UV transilluminator at 312 nm (Fisher
Scientific, Ottawa, Canada) for 3 min. Membranes were incubated for
1 h in Entensify Aqueous Fluor Solution B (DuPont, Boston, MA),
dried, and exposed to X-ray film (Kodak, Biomax MR Film) for a week.
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RESULTS |
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shows that CEU are soft alkylators of 4-(4-nitrobenzyl)pyridine as
compared to chlorambucil. The K' of CEU are almost 13 times
lower than for chlorambucil, a known alkylating agent derived from
aromatic nitrogen mustards (18)
. In addition, the
cytotoxicity of different CEU did not correlate with their alkylation
potency. Indeed, active CEU, such as 4-tBCEU,
4-iPCEU, or 4-sBCEU having
IC50s of 4, 2, and 2 µM
respectively, and inactive CEU, such as CEU, 2-ECEU, 4-sBEU,
and 4-tBCPU, have almost the same K' values
(2.5–3.5 µM). Furthermore, CEU did not show
detectable alkylation of either DNA, glutathione, or glutathione
reductase (data not shown). However, substitution of the 2-chloroethyl
moiety of active CEU analogues with methyl, ethyl, or 3-chloropropyl
groups abrogated their cytotoxicity. This suggests that, albeit very
weak, the alkylation potency of CEU is nevertheless involved in the
mechanisms of their cytotoxicity.
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, the cytotoxicity of 4-tBCEU, 4-iPCEU, and
4-sBCEU was higher in CHO-TAX 5–6 cells (e.g.,
IC50 = 4.6 ± 0.3
µM for 4-tBCEU) and lower in CHO-VV
3–2 cells (e.g., IC50 = 21.3 ± 1.1 µM for
4-tBCEU). The cytotoxicity of 4-tBCEU in
CHO-10001 cells was 11.6 ± 0.7
µM. These results strongly suggest that the
cytotoxicity of CEU is due to microtubule depolymerization.
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,24-h incubation with CEU derivatives caused a significant accumulation
of cells in the G2 + M, suggesting that
CEU derivatives, like colchicine and vinblastine, might induce
microtubule disruption and consequently prevent mitosis. Induction of
mitosis blockage in prophase by CEU derivatives was confirmed by
mitotic index analysis as described by Drouin et al. (Ref.
22
; data not shown). Moreover, the cell cycle arrest in
G2 + M was induced more rapidly with
4-iPCEU and 4-sBCEU than with 4-tBCEU.
As expected, antimicrotubule agents such as colchicine or vinblastine
also led to a G2 + M block in the
MDA-MB-231 cell cycle (data not shown).
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shows that complete microtubule depolymerization was
observed after incubation with 30 µM
4-tBCEU, 4-iPCEU, or 4-sBCEU for 24
and 48 h. Furthermore, a second immunoreactive band with an
apparent lower molecular weight (Mr)
than native ß-tubulin was detected. Interestingly, this second
immunoreactive band, herein called modified ß-tubulin, was only
observed in the free tubulin fraction. Formation of the modified
ß-tubulin was induced in a dose-dependent manner by
4-tBCEU (data not shown). Furthermore, its presence was
specific to active CEU because no such band was observed with
colchicine, vinblastine, or paclitaxel (data not shown). Finally, the
effect of 4-tBCEU seemed to be specific to ß-tubulin
because no electrophoretic evidence for 
-tubulin modification could
be found (data not shown). To assess the structure-activity
relationships between the molecular structure of other CEU derivatives,
we compared their relative rates of microtubule depolymerization and
formation of the modified ß-tubulin. To this end, CEU derivatives
were grouped into three subclasses of activity: (a) highly
active CEU (IC50 between 2 and 5
µM; Fig. 2A
), (b) weakly
active CEU (IC50 between 10 to 50
µM; Fig. 2B
), and (c)
essentially inactive CEU (IC50 > 50
µM; Fig. 2C
). Fig. 2A
shows that the effects of the highly active 4-sBCEU and
4-iPCEU on the formation of modified ß-tubulin are
slightly faster than 4-tBCEU.
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. Moreover, no depolymerization and no effect on
ß-tubulin were observed with inactive 2-ECEU (Fig. 2B)
and
also with CEU with modified 2-chloroethyl moiety, such as
4-tBEU, 4-iPEU, and 4-sBEU (Fig. 2C)
, suggesting that alkylation is required for ß-tubulin
inactivation and microtubule disruption. Finally, 4-tBCPU,
as well as unsubstituted CEU and CEU-bearing polar groups such as
4-methoxy were also completely inactive (Fig. 2C)
. Taken
together, these results suggest that the alteration of ß-tubulin,
possibly by alkylation and microtubule disassembly, might at least in
part be responsible for the cytotoxic activity of CEU.
Alkylation of Cellular Proteins by [urea-14C]-4-tBCEU
in MDA-MB-231 Cells.
Because the appearance of the modified ß-tubulin was the hallmark of
active CEU, we assessed the possibility that CEU could specifically
alkylate ß-tubulin. MDA-MB-231 cells were incubated with
[urea-14C]-4-tBCEU, and cellular
proteins were analyzed by SDS-PAGE. In Fig. 3A
, a small number of radiolabeled proteins were detected with
main radioactive species with Mr
>200,000, 50,000, 34,000, and 29,000, respectively. The
appearance of these bands was time- and dose-dependent, suggesting that
they are specific targets of the radiolabeled 4-tBCEU.
Subcellular localization of these proteins indicated that alkylated
proteins with Mr >200,000 and an
Mr 34,000 species were found in the
insoluble fraction (M) containing the membrane proteins, whereas the
Mr 50,000 and
Mr 29,000 proteins were found in the
cytosolic fraction (C). The protein of
Mr 50,000 is more specifically labeled
at low concentrations of
[urea-14C]-4-tBCEU, and the
molecular weight of this protein band coincided with that of the
modified ß-tubulin (data not shown), suggesting that
4-tBCEU could alkylate ß-tubulin.
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, whereas the native and modified
ß-tubulin were revealed by immunoblotting (Fig. 4A)
. As
depicted in Fig. 4B
, the 14C-labeled
Mr 50,000 protein and modified
ß-tubulin colocalized on the gel, indicating that ß-tubulin
modification likely resulted from direct alkylation by
4-tBCEU. This was confirmed by isolation by two-dimensional
gel electrophoresis of the Mr 50,000
14C-labeled protein or modified ß-tubulin and
mass spectral analysis using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (Ref.
23
; data not shown). To determine whether alkylated or
modified ß-tubulin induce microtubule disassembly, MDA-MB-231 cells
were incubated with
[urea-14C]-4-tBCEU for various
time intervals followed by separation of soluble and polymerized
tubulins and SDS-PAGE analysis. Native and modified ß-tubulin were
detected by a monoclonal ß-tubulin antibody (Fig. 5A)
, and 14C-labeled modified
ß-tubulin was detected by autoradiography (Fig. 5B)
. Fig. 5A
shows that complete microtubule disruption occurs after
8–12 h of incubation, whereas modified ß-tubulin appears only at
12 h. Interestingly, Fig. 5B
illustrates that
14C-labeled modified ß-tubulin appears after
only 1–4 h of incubation with the drug. These results confirm that the
apparent alkylation of ß-tubulin occurs before microtubule
disruption, suggesting that ß-tubulin alkylation by
[urea-14C]-4-tBCEU induces
microtubule disassembly. Interestingly, alkylated ß-tubulin was found
solely in the soluble protein fraction, suggesting that the alkylation
of ß-tubulin is possible only when the protein is in its
unpolymerized state. Thus, the site of ß-tubulin alkylation by CEU
might be involved in the microtubule assembly process through as yet
unknown mechanisms.
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shows that treatment of MDA-MB-231 cells with
4-tBCEU, 4-iPCEU, or 4-sBCEU in the
presence of paclitaxel, a microtubule stabilizing agent (29
, 30)
, abrogated the formation of modified ß-tubulin.
Furthermore, immunoblotting experiments (Fig. 6B)
showed
that colchicine, but not vinblastine, prevented the formation of
modified ß-tubulin induced by 4-tBCEU or
4-sBCEU. These data suggest that the binding site of CEU on
ß-tubulin is in the vicinity of the colchicine-binding site. This was
confirmed by the treatment of cells with
[urea-14C]-4-tBCEU in the presence
of colchicine or vinblastine, which showed the inhibition of the
formation of the Mr 50,000
radiolabeled band by the former, but not the latter drug (data not
shown). Two potential sites of alkylation have been identified in the
vicinity of the colchicine-binding site in ß-tubulin
(25)
. These putative sites of alkylation are two
nucleophilic sulfhydryl groups of cysteine residues at position 239 and
354, respectively (25)
. To assess the possible alkylation
of these sulfhydryl residues by CEU, MDA-MB-231 cells were treated with
4-tBCEU in the presence of iodoacetamide, a specific thiol
alkylating agent (31
, 32)
. Fig. 6B
shows that
iodoacetamide completely prevented ß-tubulin alkylation by
4-tBCEU, indicating that 4-tBCEU alkylates
cysteine residues. Similar experiments were carried out using EBI, a
bifunctional alkylating agent that specifically induces cross-linking
between Cys239 and Cys354 of ß-tubulin (32)
.
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. These results strongly
suggest that EBI and CEU share the same alkylation site on ß-tubulin,
which is either Cys239, Cys354, or both.
Localization of the Cysteine Residue(s) Alkylated by CEU.
The results presented above suggest that alkylation of cysteine
residues of ß-tubulin could occur either on Cys239, on Cys354, or on
both residues. Alkylation of both residues is most unlikely because CEU
are monoalkylating agents. To discriminate between alkylation of Cys239
and Cys354, we compared the relative alkylation induced by
[urea-14C]-4-tBCEU on a
ßIII-tubulin isoform (Fig. 7A)
using an antibody that specifically recognizes
ßIII-tubulin without cross-reactivity with other tubulin isotypes.
ßIII-tubulin is characterized by the substitution of the Cys239 by a
serine residue. In this case, serine residues are not nucleophilic
enough to be alkylated by a soft alkylating agent such as
[urea-14C]-4-tBCEU. SK-N-SH cells,
which express significant amounts of neuronal-specific ßIII-tubulin
(33)
as well as several others isoforms, such as ßI-,
ßII- and ßIV-tubulin, were treated with
[urea-14C]-4-tBCEU. In contrast with
other ß-tubulin isoforms containing Cys239 (cf. Fig. 5
), Fig. 7A
shows that
[urea-14C]-4-tBCEU does not alter
migration of the ßIII-tubulin on SDS-PAGE and does not decrease the
cytosolic level of ßIII-tubulin. Moreover, the autoradiogram (Fig. 7B)
shows that ßIII-tubulin is not alkylated by
[urea-14C]-4-tBCEU because
ßIII-tubulin and the 14C-labeled band did not
colocalize on the gel. The 14C-labeled band
observed in Fig. 7B
corresponds to other ß-tubulin
isoforms, such as ßI-, ßII-, and ßIV-tubulin. Taken together,
these results suggest that the residue alkylated by CEU is most likely
Cys239.
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DISCUSSION |
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We determined that the most likely reactive site of 4-tBCEU
was either Cys239 or Cys354 in the vicinity of the colchicine-binding
site because colchicine inhibits ß-tubulin alkylation by CEU.
Moreover, we demonstrated that ß-tubulin with a Cys
Ser
substitution at position 239 is not alkylated by the drug, suggesting
that Cys239 might be the residue alkylated by CEU, such as
4-tBCEU. Previous evidence has established that Cys239, but
not Cys354, is specifically alkylated by synthetic compounds such as
2,4-dichlorobenzyl thiocyanate (36)
and
2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene
(37)
inducing microtubule disassembly. These results
suggest that Cys239 is more sensitive and more accessible to alkylation
than Cys354. Thus, the integrity of Cys239 is most likely essential in
the microtubule assembly process. However, we cannot discard the
possibility that 4-tBCEU alkylates ß-tubulin at other
residues of the protein and that Cys239 is essential to maintain the
proper conformation of ß-tubulin that is reactive with CEU.
Alkylation of ß-tubulin by CEU induces the formation of a modified
ß-tubulin, which migrates ahead of native ß-tubulin on SDS-PAGE.
The electrophoretic behavior of the modified ß-tubulin obtained by
alkylation of ß-tubulin by CEU is similar to the modified ß-tubulin
observed after the formation of a cross-link between Cys239 and Cys354
by EBI (32)
. It is important to mention that CEU are
monoalkylating agents and are therefore unlikely to induce such
cross-links in ß-tubulin.
The probability that CEU could carbamoylate proteins through reaction of the carbonyl group of the urea moiety with lysine or cysteine residues in the vicinity of Cys239 or Cys354 is most unlikely. The chemical stability of aromatic ureas is very high and does not allow nucleophilic reactions, even with highly nucleophilic entities, such as glutathione and glutathione reductase (data not shown). Moreover, there are no other nucleophilic entities present in the hydrophobic pocket or in the vicinity of the hydrophobic pocket that are available for such a reaction (25) .
A plausible explanation to the formation of the modified
ß-tubulin by CEU is illustrated in Fig. 8
. That putative mechanism of alkylation of ß-tubulin by CEU is based
on the analysis of the three-dimensional structure of ß-tubulin
recently published by Nogales et al. (25)
and
on the results of the present study. Briefly, the nucleophilic Cys239
is present in the drug-binding domain of colchicine, which is delimited
by residues 206–381 that contain four mixed ß-sheets and five
surrounding -helices. Two of the ß-sheets, ß8 and ß9, are in
close proximity, leading to the formation of a hydrophobic pocket (8
Å across between Cys239 and Cys354; Ref. 25
). The
existence of such a hydrophobic pocket in ß-tubulin has previously
been suggested to be in the vicinity of the colchicine-binding site
(38)
. This hydrophobic cavity could accommodate the
aromatic ring substituted by lower alkyl groups at the 4 position. We
speculate that the hydrophobic moiety of CEU readily forms hydrophobic
bonds with several amino acids, such as valine (316, 342, 349, 353),
alanine (314, 315, 352), and leucine (240) residues present in the
cavity. In addition, a strong hydrogen bond might be formed between the
aromatic amino group of CEU and the glutamic acid residue at position
343. That hydrogen bond could be stabilized by electron resonance using
the arginine residue at position 241.
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In conclusion, CEU are soft monoalkylating agents that are unreactive toward most cellular nucleophiles, such as DNA, glutathione, and glutathione reductase. On the other hand, CEU might alkylate specific proteins bearing strong nucleophilic centers that present a spatial environment favoring close and prolonged contacts between the drug and the nucleophilic moiety. These elements describe the concept of "soft alkylation," which introduces new perspectives about the rational design of drugs that might be able to inactivate specific cellular proteins with resulting cytotoxic effects toward tumor cells. Our results also suggest that the "soft alkylation" properties of CEU could be coupled with specific ligands for a more selective targeting of proteins to be alkylated and inhibited. The preparation of such molecules might lead to drugs able to specifically modulate metabolic processes and transduction pathways. Preliminary proofs of concept have been obtained using CEU derivatives that were shown to be cytotoxic in the micromolar range and to block selectively the cell cycle in S or in G1/G0. Most interestingly, these new cytotoxic CEU derivatives did not alkylate ß-tubulin and did not disrupt the microtubule network.5 Studies of the pharmacological properties of these new CEU derivatives are in progress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Cancer
Institute of Canada, the Fonds de la Recherche en Santé du
Québec (to P. P.) and Fonds de La Recherche en Santé
du Québec/Hydro-Québec. 
2 To whom requests for reprints should be
addressed, at Biomaterial Institute of Quebec, CHUQ, Pavillon
St-François d’Assise, 10, rue de L’Espinay, Quebec City,
Quebec, Canada, G1L 3L5. Phone: (418) 525-4485; Fax: (418) 525-4372;
E-mail: Rene.C-Gaudreault{at}crsfa.ulaval.ca
3 The abbreviations used are: CEU,
1-aryl-3-(2-chloroethyl)urea; 4-tBCEU,
4-tert-butyl [3-(2-chloroethyl)ureido] benzene;
4-iPCEU, 4-iso-propyl
[3-(2-chloroethyl)ureido] benzene; 4-sBCEU,
4-sec-butyl [3-(2-chloroethyl)ureido] benzene; 2-ECEU,
2-ethyl [3-(2-chloroethyl)ureido] benzene; 3-ECEU, 3-ethyl
[3-(2-chloroethyl)ureido] benzene; 4-ECEU, 4-ethyl
[3-(2-chloroethyl)ureido] benzene; 4-tBEU,
4-tert-butyl [3-(ethyl)ureido] benzene;
4-iPEU, 4-iso-propyl [3-(ethyl)ureido] benzene;
4-sBEU, 4-sec-butyl [3-(ethyl)ureido]
benzene; 4-tBCPU, 4-tert-butyl
[3-(2-chloropropyl)ureido] benzene; EBI,
N,N'-ethylenebis (iodoacetamide).
4 J. Legault, L. Lacroix, and R. C.
Gaudreault, unpublished results.
5 J. Legault et al.,
unpublished results.
Received 10/30/98. Accepted 12/16/99.
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REFERENCES |
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