298 N. A. Cherepanova et al.
1
(2H, d, J= 8.7, H5); 8.75 (2H, s, H7); 9.19 (2H, t, J= 5.3,
CONH); 11.23 (2H, brs, NH(Bim)). MALDI TOF-MS
(monoisotop.): calcd for C43H16N14O2: 790.92; found: m/z
(M + H)+ 793.4, (M + Na + K) + 855.7.
bis-HT(NMe)·7 HCl: H-NMR (400MHz, 23°C): δ 2.25
(4H, quint, J=6.5, OСН2СН2); 2.85 (9H, brs, CH3); 3.12 (8H,
m, H(3′″, 5′″)); 3.35 (4H, m, СН2N); 3.36 (8H, m, H(2″′,
6″′)); 4.23 (4H, m, OСН2); 7.18 (6H, m, H(2, 6,’ 7″)); 7.28 (2H,
dd, J=1.9, J 9.3, H5″); 7.67 (2H, d, J=9.3, H4″); 7.86 (2H, d,
J=8.7, H4’); 8.24 (2H, d, J=8.7, H5’); 8.30 (4H, d, J=8.7, H(3,
5)); 8.62 (2H, s, H7’). MALDI TOF-MS (monoisotop.): calcd
for C57H61N13O2: 960.09; found: m/z (M + H)+ 961.0.
1
DB(2)·6 HCl: H-NMR (400MHz, 32°C): δ 2.63 (4H, s,
CH2CO); 2.84 (6H, brs, CH3); 3.23 (8H, m, H(3″, 5″)); 3.54
(4H, m, H(2″, 6″)); 3.87 (4H, м, (2″, 6″)); 4.74 (4H, d, J=5.6,
CH2NH); 7.17 (2H, brs, H7’); 7.32 (2H, d, J=8.7, H5’); 7.68
(2H, d, J=9.3, H4’); 7.87 (2H, d, J=8.7, H4); 8.32 (2H, d,
J=8.7, H5); 8.72 (2H, s, H7); 8.99 (2H, t, J=5.6, CONH);
11.24 (1H, brs, NH(Bim)). MALDI TOF-MS (monoisotop.):
calcd for C44H48N14O2: 804.94; found: m/z (M + H)+ 806.3.
Enzyme inhibition assay
Duplexes A or B (300nM) were incubated in the presence
of increasing concentrations of a DB(n) inhibitor (0.1–200
µM) in buffer B (for Dnmt3a-CD) or C (for M.SssI) for 3 days
at 4°C or for 10min at 25°C. Dnmt3a-CD (2 µM of mono-
mers) or M.SssI (2 µM) and AdoMet (25 µM) were added to
the reaction mixtures and the mixtures were incubated for
40min at 37°C. Reaction mixtures lacking either the enzyme
or the inhibitor were used as controls. e DNA was precipi-
tated with ethanol (EtOH) in the presence of 0.4M sodium
acetate. e precipitate was washed with 80% EtOH and
evaporated on a SpeedVac to remove remained EtOH. e
methylation was studied by protection of the methylated
DNA from cleavage by 2U of R.HhaI in the 20 µL Tango
buffer for an hour at 37°C. e cleavage products were
suspended in 10 µL 80% formamide and separated in 20%
polyacrylamide gel under denatured conditions (7M urea)
followed by gel imaging on a FUJIFILM FLA-3000 device.
e methylation % (M) was determined as the ratio of
methylated to total DNA based on the intensities of the
corresponding bands in gel using the Image Quant 5.0
program. To determine inhibitor concentration which
reduced enzyme activity by 50% (IC50) the dependence of
relative methylation percentage (R, %) versus inhibitor con-
centration were plotted. R values were calculated from the
formula:
1
DB(3)·6 HCl: H-NMR (400 MHz, 23°C): δ 1.86 (2Н,
quintet, J= 7.5, СН2СН2СН2), 2.33 (4Н, t, J= 7.5, СН2СО),
2.84 (6Н, d, J= 3.7, CH3), [3.38 (8H, m, H(3″,5″)), 3.58 (8H,
m, H(2″,6″)—at 97°С)], 4.72 (4Н, d, J= 5.0, СН2NH), 7.18
(2H, brs, H7’), 7.34 (2Н, dd, J= 1.9, J= 9.3, H5’), 7.70 (2Н,
d, J= 9.3, H4’), 7.94 (2Н, d, J= 8.7, H4), 8.34 (2Н, d, J= 8.7,
H5), 8.73 (2Н, s, H7), 8.97 (2Н, t, J= 5.3, CONН), 11.13 (2H,
brs, NH(Bim)). MALDI TOF-MS (monoisotop.): calcd for
C45H50N14O2: 818.88; found: m/z (M + H)+ 820.2.
1
DB(4) 6 HCl: H-NMR (400MHz, 32°C): δ 1.60 (4H, м,
CН2CH2CO), 2.32 (4H, м, CH2CO), 2.83 (6H, brs, CH3),
3.24 (8H, m, H(3″,5″)), 3.53 (4H, m, H(2″,6″)), 3.86 (4H, m,
H(2″,6″)); 4.77 (4H, d, J=5.0, СН2NH), 7.18 (2H, brs, H7’),
7.33(2H, d, J=9.3, H5’), 7.69(2Н, d, J=8.7, H4’), 7.96(2Н, d,
J=8.7,H4),8.47(2Н,d,J=9.3,H5);8.84(2Н,s,H7),8.95(2Н,
t,J=5.3,CONН),11.46(1H,brs,NH(Bim)).MALDITOF-MS
(monoisotop.): calcd for C46H52N14O2: 832.91; found: m/z
(M + H)+ 833.7.
1
DB(5) 6 HCl: H-NMR (400 MHz, 32°C): δ 1.32 (2Н,
quintet, J= 7.5, CO(CH2)2CН2), 1.58 (4Н, quintet, J= 7.5,
COCH2CH2), 2.26 (4H, t, J= 7.5, COCH2,), 2.85 (6H, s, CH3),
[3.39 (8H, m, H(3″,5″)); 3.78 (8H, m, H(2″,6″)—at 84°C],
4.63 (4H, d, J= 5.6, CH2NH,), 7.20 (2H, d, J= 1.9, H7’), 7.31
(2H, dd, J= 1.9, J= 8.7, H5’), 7.68 (2H, d, J= 8.7, H4’), 7.89
(2H, d, J= 8.7, H4), 8.26 (2H, d, J= 8.7, H5), 8.66 (2H, s,
H7), 8.68 (2H, t, J= 5.3, CONH), 11.03 (1H, brs, NH(Bim)).
MALDI TOF-MS (monoisotop.): calcd for C47H54N14O2:
847.02; found: m/z (M + H)+ 848.3.
M − Menzyme free
(
)
×100
Minhibitor free − Menzyme free
e curves were subjected to regression analysis in Origin
6.0 program using simple exponential function:
1
DB(7) 6 HCl: H-NMR (400MHz, 32°C): δ 1.28 (6Н, m,
CO(CH2)2(CН2)3), 1.55 (4Н, m, COCH2CH2), 2.23 (4H, t,
J=7.5, COCH2), 2.83 (6H, s, CH3), [3.38 (8H, m, H(3″,5″));
3.55(8H,m,H(2″,6″))—at84°C],4.58(4H,d,J=5.0,CH2NH),
7.19 (2H, s, H7’), 7.26 (2H, d, J=8.7, H5’), 7.66 (2H, d, J=8.7,
H4’),7.80(2H,d,J=8.1,H4),8.20(2H,d,J=8.1,H5),8.60(4H,
m, CONH, H7), 11.19 (1H, brs, NH(Bim)). MALDI TOF-MS
(monoisotop.): calcd for C49H58N14O2: 875.08; found: m/z
(M + H)+ 876.5.
1 /t
R = R0 + A⋅e[ ]
,
where input parameters were R and inhibitor concentra-
tion, [I] (µM); R0, A, and t were dependent parameters for
fitting. All data were fitted with correlation coefficients
more than 0.95. e IC50 values were then determined
by interpolation. For DB(4,5) when IC50 values exceed
the inhibitor concentration range, they were estimated
by extrapolation of the above mentioned function to 50%
inhibition. e values of standard errors were calculated
using 3–6 independent experiments.
1
DB(11)·6 HCl: H-NMR (400MHz, 23°C): δ 1.22 (14Н,
m, СО(СН2)2(СН2)7), 1.52 (4Н, m, СОСН2СН2), 2.23 (4Н, t,
J=7.2, СОСН2), 2.83 (6Н, brs, СH3), 3.21 (8H, m, H(3″, 5″)),
3.53 (4H, m, H(2″,6″)), 3.88 (4H, m, H(2″,6″)), 4.66 (4Н, d,
J=5.0, СН2NH), 7.19 (2H, s, H7’), 7.33 (2Н, dd, J=1.2, J=8.7,
H5’), 7.70 (2Н, d, J=8.7, H4’), 7.91 (2Н, d, J=8.7, H4), 8.34
(2Н, d, J=8.7, H5), 8.73 (2Н, s, H7), 8.78 (2Н, t, J=5.3, CONН),
11.19 (2H, brs, NH(Bim)). MALDI TOF-MS (monoisotop.):
calcd for C53H66N14O2: 931.18; found: m/z (M + H)+ 931.8.
results and discussion
e inhibitory activities of DB(n) and bis-HТ(NMe)
were studied in the reaction of Dnmt3a-CD cata-
lyzed methylation of a 30-mer DNA duplex: 5’-FAM-
Journal of Enzyme Inhibition and Medicinal Chemistry