ABSTRACT
Alkylating distamycin derivative FCE 24517 (I) is the prototype of a novel class
of alkylating agents. In the present study we have investigated the effect of
further chemical modifications introduced in the alkylating distamycin-derived molecule with the aim of improving their ability to bind DNA. The
new compound, MEN 10710 (II), has a four pyrrolecarboxamide backbone linked at
its N-terminus and through a butanamido residue to a 4-[bis(2-chloroethyl)amino]phenyl moiety. We have demonstrated that the
presence of the flexible trimethylene chain confers to the novel distamycin
derivative a peculiar mode of interaction with DNA as compared with I or
melphalan. In fact, interstrand cross-links are detected in DNA samples treated even with low concentrations of
II (being 200-fold more efficient than melphalan) but not with I. Similar results were
obtained with a related compound of II containing a three pyrrole ring
backbone. Compound II induces a conformational change in the DNA structure as
deduced from the inhibition of T4 DNA ligase activity. In alkylation
experiments, unlike melphalan, both I and II induce DNA breaks at bases closely
located to AT-rich tracts, however II was more potent than I in producing greater amount
of covalent adducts. These data suggest that the new compound shows a different
and peculiar mechanism of interaction with DNA.
Nitrogen mustard derivatives, such as melphalan and chlorambucil, still have a
place in the chemotherapy of several types of leukemias and solid tumours (
1
). The biological activity of these typical alkylating agents is mediated
through alkylation at N-7 of guanine and subsequent DNA interstrand cross-linking (
2
-
4
). In 1989, a novel class of distamycin analogues bearing a benzoyl mustard
group have been described as new potential anticancer agents (
5
). The prototype of this novel class of alkylating agents, namely compound FCE
24517 (tallimustine, structure
I
of Fig.
1
), exerted relevant growth inhibitory activity in several murine and human
experimental tumours (
5
-
7
) and is currently under clinical investigation. Modalities of DNA interaction
of
I
are different when compared with typical alkylating agents: the compound
exhibits a greater affinity for AT-rich sequences in the minor groove of DNA (
7
), coupled to a weak, albeit characteristic, alkylating activity. In particular,
a preferential alkylation at N-3 of adenine, but not at N-7 of guanine, together with the absence of DNA cross-links has been reported (
8
). Further studies have indicated that compound
I
induces breaks at any bases in the proximity of AT regions (
9
). Compound
I
has also been described as an inhibitor of DNA ligase activity (
10
). Recently, a series of new compounds have been synthetized in our laboratories
with the aim at evaluating the effect of the insertion of a flexibile carbon
chain between the oligo-pyrrolecarboxamido backbone and the alkylating moiety. Among them, MEN
10710 (
II
, Fig.
1
) emerged as an interesting compound possessing, as compared with
I
and melphalan, (i) a greater cytotoxic activity against different tumour cell
lines
in vitro
, (ii) a higher potency in the antitumour tests
in vivo
and (iii) a reduced myelotoxicity in an
in vitro
system (
11
). Compound
II
differs from
I
for two structural features: an additional residue of 4-amino-1-methylpyrrole-2-carboxylic acid in the backbone and the presence
of a butanamido residue linking the alkylating moiety to the oligo-pyrrole carboxamido backbone (Fig.
1
). It appeared worthwhile investigating whether the mode of DNA interaction of
II
could be different as compared to typical (melphalan) and atypical alkylating
agents such as
I
. With this aim, these compounds have been compared for their ability to induce
cross-links and to exhibit specific alkylation sites as well as to interfere
with DNA joining and relaxation (T4 ligase). Present findings indicate that
II
possesses a peculiar mode of DNA interaction, different from either that shown
by
I
or by melphalan.
pBR322 DNA, T4 polynucleotide kinase, T4 ligase,
Hin
dIII,
Eco
RI and bacterial alkaline phosphatase were purchased from Boeheringer. AMP, ATP,
chloroquine phosphate, l-phenylalanine mustard (melphalan) were obtained from Sigma. A 226 bp
Eco
RI-
Ava
I fragment, used in alkylation assay, was obtained by restriction enzyme
digestion of a commercial plasmid (SureTrack footprinting kit, Pharmacia,
Sweden). FCE 24517, MEN 10710 and MEN 10569 (
III
, Fig.
1
) were synthetised in the Department of Chemistry of Menarini Ricerche Sud
S.p.A., by F. Animati, G. Giannini and C. Rossi. Their characterisation and
purity were assessed by comparing spectral data for FCE 24157 with those
reported in the literature (
5
) and by NMR for new compounds [
II
: [delta] 1.82 (2H, m), 2.25 (2H, t), 2.45 (2H, t), 2.64 (2H, t), 3.50 (2H, m),
3.70 (8H, s), 3.82 (3H, s) 3.85 (3H, s), 3.86 (3H, s), 3.87 (3H, s), 6.69 (2H,
d), 6.90 (1H, d), 6.97 (1H, d), 7.04 (2H, d), 7.08 (2H, bs), 7.15 (1H, d), 7.18
(1H, d), 7.22 (2H, bs), 8.19 (1H, t), 8.57-8.95 (4H, bd), 9.76 (1H, bs), 9.90 (2H, bs), 9.95 (1H, bs);
III
: [delta] 1.80 (2H, m), 2.22 (2H, t), 2.48 (2H, t), 2.60 (2H, t), 3.50 (2H, m),
3.70 (8H, s), 3.78-3.80 (9H, s), 6.65 (2H, d), 6.89 (1H, s), 6.92 (1H, s), 7.02 (1H, s),
7.03 (1H, s), 7.12 (1H, s), 7.15 (1H, s), 7.19 (1H, s), 8.19 (1H, t), 8.60-8.97 (4H, bd), 9.75 (1H, s), 9.85 (2H, s)].
Cross-link assay was carried out according to Hartley
et al
. (
12
). Briefly, pBR322 plasmid DNA was linearized by digestion with
Hin
dIII and dephosphorylated by treatment with bacterial alkaline phosphatase. The
DNA was 5'-end-labeled using T4 polynucleotide kinase and [[gamma]-
32
P]ATP (5000 Ci/mmol, Amersham). Following precipitation and removal of
unincorporated ATP, the DNA was resuspended in sterile double-distilled water at 1 mg/ml. Labeled DNA (~10 ng) was used for each experimental point. Reactions with drug were
performed in 25 mM triethanolamine, 1 mM EDTA (pH 7.2) at 37oC for 2 h and terminated by the addition of an equal volume of stop
solution (0.6 mM sodium acetate, 20 mM EDTA, 100 [mu]g/ml tRNA). DNA was precipitated by the addition of 3 vol of 95% ethanol.
Following centrifugation and supernatant removal, the DNA pellet was obtained
by lyophilization. Samples were dissolved in 10 ml strand separation buffer
(30% dimethyl sulfoxide, 1 mM EDTA, 0.04% bromophenol blue, 0.04% xylene
cyanol), heated at 90oC for 2 min and chilled immediately in a ice-water bath prior to loading. Control undenaturated samples were
dissolved in 10 ml 6% sucrose, 0.04% bromophenol blue and loaded directly.
Electrophoresis was performed on 20 cm long 0.8% submerged horizontal agarose
gels at 40 V for 16 h. The gel and running buffer was 40 mM Tris, 20 mM acetic
acid, 2 mM EDTA (pH 8.1). Gels were dried at 80oC onto one layer of Whatman 3MM paper on a Bio-Rad Model 583 gel drier connected to a vacuum. Autoradiography was
performed with X-OMAT Kodak films for 4 h at -70oC using DuPont-Cronex Lightening-plus intensifying screen. Sharper images were
obtained by overnight exposure without the intensifying screen. Quantitation
was achieved by autoradiograph microdensitometry.
DNA joining activity was assayed by the method of plasmid DNA circularization (
13
). Linear pBR322 DNA was incubated with T4 DNA ligase in the following reaction
buffer (20 ml): 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl
2
, 1 mM DTT, 0.7 mM ATP plus the drug at the desired concentration. After
incubation at 15oC for 30 min, reactions were stopped by addition of EDTA (20 mM final
concentration). Ligated samples were analysed on 1% agarose gels made with
Tris-acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 2 mM EDTA, 18 mM NaCl,
final pH 8). The gel was then stained with ethidium bromide (1 mg/ml) for 30
min and destained with H
2
O for 15 min. DNA was visualized and the gel photographed on Polaroid 55 films,
with the aid of an ultraviolet illuminator.
AMP-dependent DNA relaxation catalyzed by T4 ligase was assayed as previously
described (
14
): the reaction mixture (20 ml) contained 20 mM Tris-HCl pH 8.0, 3 mM MgCl
2
, 100 mg/ml BSA, 1 mM EDTA and 200 ng of naturally supercoiled pBR322 DNA, 1 mM
AMP and 20 mU of T4 ligase. After 30 min at 30oC, reactions were stopped by adding 2 ml of a solution containing 1 mg/ml
Bromophenol Blue, 50% (v/v) glycerol and 5 mg/ml sodium dodecyl sulfate and
analysed on a 1.2% agarose gel.
Enzyme adenylation was evaluated by the reported technique (
15
) in which DNA ligase is incubated at 37oC in the presence of [
35
S]adenosine-5'-(
O
-1-thiotriphosphate) (400 Ci/mmol) in the same reaction mixture (10 ml)
utilized for DNA ligation. The reaction was stopped by the addition of a
solution (20 ml) containing 10 mM EDTA and 150 mg of BSA/ml; then 25 ml of the
reaction mixture was spotted on to a Whatman GF/C filter; the filter was batch-washed with trichloroacetic acid, dried and counted for radioactivity.
A 226 bp
Eco
RI-
Ava
I (
16
) fragment was labeled at the
Ava
I site and incubated with the compound for 5 h at room temperature in 0.1* standard saline citrate (1* standard saline-citrate is 15 mM NaCl-1.5 mM sodium citrate). After two sequential ethanol
precipitations, samples were resuspended in 50 ml of 0.1* standard saline citrate, heated at 90oC for 30 min and ethanol precipitated. DNA fragments were resolved
on 6% denaturing gels.
The presence of a cross-link between the two DNA strands prevents complete separation of the
strands upon denaturation so that the cross-linked DNA reanneals in a neutral agarose gel to run as double stranded.
Quantitation of the amount of double-stranded versus single-stranded therefore gives a measure of the extent of drug-induced cross-linking in a given DNA fragment (
12
). A gel autoradiograph is shown in Figure
2
, illustrating the cross-link adducts induced by
II
but not by
I
. In fact no cross-linking was induced by
I
in the concentration range 0.5-200 [mu]M (Figs
2
and
3
). On the other hand,
II
was very efficient in inducing DNA cross-links starting at concentrations as low as 0.5 nM and reaching a maximal
effect at 5-50 nM (Figs
2
and
3
). As expected, also melphalan induced cross-linking although at higher concentrations (50 nM-1 [mu]M). In order to better understand the reason for the different
ability of the two drugs to form cross-links, we have analysed the activity of
III
, that differs from
I
only for the presence of the trimethylene chain (Fig.
1
). Interstrand cross-links are clearly formed by
III
at doses that are similar to those of
II
(Fig.
3
). Compounds
I
,
II
and
III
showed a similar pattern of sequence preference for their alkylating action. In fact, alkylation occured at tracts of DNA with sequences: 5'-AAGAATTGGAT-3', 5'-ATATTGGCTT-3', 5'-AAAATGGAT-3',
5'-TTTGACA-3'. The sites susceptible to attack by the active
distamycin derivative were the residues preceded by (A/T)
n
rich sequences, that were important for binding of the distamycin (
15
). In fact, the same DNA sequences were recognized by no alkylating distamycin
derivatives in footprinting studies (
16
). The new derivatives
II
and
III
alkylated more efficiently than
I
(Fig.
4
). Melphalan as most of the available alkylating agents which are used in cancer
therapy (
17
-
19
), produced only breaks corresponding to guanine sites, indicating N-7 guanine alkylation (
8
) (data not shown).
Montecucco
et al
. (
10
,
13
,
20
) have indicated that DNA intercalating agents or minor groove binders interfere
with DNA ligase. In the present study,
Eco
RI-digested pBR322 DNA was the substrate and different, increasing amounts of
the drugs were used. Inhibition of the ligation was deduced from a progressive
reduction of the amount of ligated linear product. Quantitation was achieved by
microdensitometry of the photograph and the percentage of T4 ligase activity
was calculated. Both distamycin analogues
I
and
II
inhibited T4 ligase joining activity in a concentration-dependent manner (2-20 [mu]M) (IC
50
12 and 17 [mu]M for
II
and
I
, respectively) (Fig.
5
). Compound
III
showed an inhibitory effect that was similar to that exhibited by
I
(IC
50
12 [mu]M). By contrast, melphalan in the concentration range 0.5-400 [mu]M was devoid of any inhibitory effect. Interestingly, both
I
and
II
(up to 200 [mu]M) were ineffective against T4 DNA ligase-induced DNA adenylation (data not shown), suggesting that for
inhibition of DNA joining the DNA and not the enzyme was their target.
We have also investigated whether these drugs interfered with DNA
topoisomerization, the so-called `reverse reaction' of DNA ligation (
21
). In this experimental setting T4 DNA ligase can generate fully relaxed DNA
molecules starting from naturally supercoiled plasmid DNA in an AMP-and Mg
2+
dependent reaction (
13
,
22
). Unlike melphalan (1-400 [mu]M), either
I
or
II
inhibited the reaction in the range 1-5 [mu]M in a concentration-dependent manner. Reference
I
, however, showed slightly lower activity than
II
(Fig.
6
).
The novel distamycin analogue MEN 10710 (
II
) shows strong cytotoxic activity against tumour cell lines and a remarkable
antitumour activity in a variety of human tumour xenografts in immunodepressed
mice (
11
). In this study, we have investigated the DNA interaction properties of
II
in an attempt to relate the marked biological effects with a specific molecular
mechanism of action.
The results of the cross-linking assay suggest that the flexible trimethylene chain, present as a
linker between the peptidic backbone and the bis(2-chloroethyl)aminophenyl moiety, gives rise to a novel class of distamycin
related alkylating agents with improved ability to strongly bind DNA sequences
as compared with FCE 24517 (
I
) type derivatives. The trimethylene chain interrupts the conjugation through
the molecule resulting in a different charge distribution. The alkylating group
becomes more electron-rich, providing most likely
II
with stronger affinity for double stranded DNA and, in essence, a higher
alkylation rate as compared with
I
. In addition, the length and flexibility of the chain allows the formation of
double strand cross-links making the new compound, from this standpoint, more similar to the
classic agents like, for example, melphalan.
Recently, we have shown that modifications in the N-terminal residue of distamycin analogues have a consequence in their
properties of competing with nuclear factors for the binding to their target
DNA sequences (
16
,
23
). Most likely, this portion of the molecule is strongly implicated in the DNA
recognition and therefore in the corresponding interference with the specific
DNA-protein interaction. In this study we have demonstrated the ability of
the new compounds to form molecular complexes with DNA and to induce
conformational changes in the DNA structure, through the inhibition of T4
ligase. In fact the inhibition of the DNA joining or topoisomerizing activity
of the enzyme reflects the complexation of the molecules with the DNA substrate
causing interference with the DNA-protein interaction as observed by Montecucco
et al
. for
I
(
13
,
20
).
The present work shows that structural variations of molecules of the distamycin
type, as obtained by introducing selected reactive groups together with
modifications of the backbone, can vary the DNA binding properties of these
drugs and improve antitumour potency. The presence of the trimethylene spacer
in
II
and
III
determines a higher affinity for double stranded DNA of these compounds in
respect to
I
and is responsible for the formation of interstrand cross-links. This is particularly evident when the behaviour of
III
is compared with that of
I
, as the two compounds differ only in the presence of the said spacer in the
former and not in the latter. On the other hand, the presence of an additional
residue of 4-amino-1-methylpyrrole-2-carboxylic acid in
II
might be responsible for a greater sequence specificity. The latter point has
already been proved in a physico-chemical study concerning the complexation of DNA oligomers with the
corresponding derivatives with three and four of the said residues but not
possessing the alkylating N-terminal groups (
24
).
In conclusion, compound
II
has peculiar mode of DNA interaction that differs either from FCE 24517
(induction of cross-links and either potency in DNA alkylation and T4 ligase inhibition) or
from melphalan (different pattern of sequence preference and alkylation). This
mode of DNA interaction could be of revelance to the interesting cytotoxic and
antitumor properties exhibited by this compound.
This work was carried out in the frame of a joint project of A. Menarini,
Industrie Farmaceutiche Riunite, Florence and of Bristol-Meyers Squibb Italia, Rome. It was supported by a grant of the Istituto
Mobiliare Italiano (contract No 53658).
REFERENCES
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