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DNA groove. Taking into consideration that DBP(1) is a dimer of
MB (Ht derivative) and, according to the X-ray data, Ht is known
to bind to DNA in the minor groove,3,4 we supposed that upon
the complex formation DBP(1) was also located in the DNA minor
groove. Similar variations in the CD spectra were observed for
DBP(2–4) (spectra are not shown), and this allowed us to conclude
that DBP(2–4) are also DNA-specific minor groove ligands.
For the evaluation of dissociation constants (Kd) of DBP(n)–DNA
complexes we studied the fluorescence spectra of the DBP(n) bind-
ing to a 30-bp DNA duplex A containing the CpG recognition site of
prokaryotic MTase M.SssI (target Cyt residues are underlined):
100
80
60
40
20
0
B
0
50 100 150 200 250 300
[DBP(3)], μM
Figure 2. Inhibition of the methylation activity of M.SssI by DBP(3). (A) Cleavage of
fA duplex with R.Hin6I endonuclease after its methylation with M.SssI in the
presence of 0–200
lM DBP(3). [fA] 300 nM, [S-adenosyl-L-methionine] 25 lM,
[M.SssI] 2 M. 20% PAG under denatured conditions (7 M urea) followed by gel
imaging on a FUJIFILM FLA-3000 device. (B) The relative methylation degree R of
duplex fA plotted against the DBP(3) inhibitor concentration.
l
The fluorescence of compounds DBP(n) enhanced upon increasing
the DNA duplex concentrations (data not shown). The saturation
binding curves, that is, the DNA concentration dependence of the
fluorescence intensity at 460 nm, were obtained (Fig. S3). Using
the one site hyperbolic binding equation we calculated Kd values
(Table 1).
Table 2
Inhibition of methylation of fA duplex by M.SssI in the presence of DBP(n)
The efficacy of DBP(1) binding to duplex A was nearly the same
as for compound DB(11), for which Kd was 0.39 lM (Cherepanova
Compound
IC50 SEM*
DBP(1)
DBP(2)
DBP(3)
DBP(4)
63
et al., unpublished data). DBP(2), DBP(3) and DBP(4) bound to
duplex A even better than DB(11). Thus we showed the capacity
of DBP(n) to bind to duplex A and used these substrates for the
studies of the methylation reaction inhibition.
(lM)
74 11
76 10
64 6.5
5
*
Mean values of three experiments.
Previously we found and studied the inhibitory properties of
DB(n) toward murine Dnmt3a MTase13 and received the first data
on the demethylation activity of these compounds in normal and
tumor cells.18 However, due the low solubility of DB(n) in aqueous
solutions the stock solutions could be only prepared in dimethyl-
sulfoxide, which was toxic for the cells. An increased solubility
due to the introduction of a 1,4-piperazine residue in the oligo-
methylene linker made it possible DBP(n) aqueous solutions. The
DNA methylation is one of the most intensely studied epigenetic
modifications playing an important role for the control of gene
expression in eukaryotic cells.19 Local hypermethylation of pro-
moter gene regions was found in human tumor cells, particularly,
in the promotor regions of tumor suppressor genes resulting in
their inactivation.20–22 The DNA methylation status depends on
the MTases functioning. This is the reason for an increased interest
in the inhibition of the activity of these enzymes.23
120
100
80
60
40
20
0
− − DBP(4), IC 63 5 μM
50
⎯ DB(11), IC 18 0.5 μM
50
0
50
100
150
200
[inhibitor], µM
Figure 3. The relative methylation degree R of duplex fA by M.SssI plotted against
the dDB(11) and j DBP(4) inhibitor concentrations.
Inhibitory effects of the DBP(n) synthesized toward DNA
methylation were studied using prokaryotic M.SssI MTase, which,
similarly to mammalian MTases, can recognize and methylate
DNA CpG sites in positions 5 of the cytosine residues. Duplex A
labeled at the 50-end of the upper strand with 6(5)-carboxyfluores-
ceine (fA) was used as a substrate. For the complex formation,
and 30-membered uncleaved oligonucleotide (Fig. 2A). Relative
methylation degrees R were calculated using the samples lacking
the inhibitor24. The IC50 values were determined using the function
of the R value versus DBP(n) concentrations (Fig. 2B and Table 2).
As is seen in Table 2, all the four DBP(n) inhibited the M.SssI-
mediated methylation of duplex fA at micromolar concentrations.
No essential differences in the inhibition activities were observed
within the series.
We compared inhibitory activities of DBP(4) and dimeric bis-
benzimidazole DB(11) lacking a piperazine residue (Fig. 1). This
pair of compounds was chosen due to approximately the same lin-
ker lengths between the bisbenzimidazole blocks (Fig. 1).
Compound DBP(4) was a less effective inhibitor of fA methylation
than DB(11), although its IC50 remained within the micromolar
range (Fig. 3). Despite the lower inhibitory activity of DBP(4) if
compared with the DB(n) counterpart, the study of DBP(n) as
MTase inhibitors in normal and tumor cells is of a considerable
interest.
duplex fA was incubated in the presence of 0–200 lM DBP(n) for
3 days. The fA methylation was visualized by digestion with the
methylation-sensitive restriction enzyme R.Hin6I (GCGC recogni-
tion site). Nonmethylated molecules were cleaved at the GCGC site
to give 14- and 16-membered fragments, whereas methylated
molecules were not affected. After the electrophoretic separation
in denaturing PAG the methylation degree of duplex fA was deter-
mined on the basis of fluorescence of the 14-membered product
Table 1
Binding of DBP(n) to duplex A
Compound
Kd M)
DBP(1)
DBP(2)
DBP(3)
DBP(4)
(
l
0.37
0.12
0.07
0.07