The in vitro methylation of DNA by a minor groove binding methyl sulfonate ester

Article abstract of DOI:10.1021/tx9501849

The preparation of sequence and groove specific DNA methylating agents based on N-methylpyrrolecarboxamide subunits appended with an O-methyl sulfonate ester functionality (MeOSO₂(CH₂)₂-Lex) has previously been described [Zhang, Y., Chen, F.-X., Mehta, P., and Gold, B. (1993) Biochemistry 32, 7954-7965]. In contrast to simple methyl sulfonate esters, e.g., methyl methanesulfonate (MMS), which predominantly methylate at 7- guanine, MeOSO₂(CH₂)₂-Lex affords N3-methyladenine (3-MeAde) as its major adduct. Using competitive ELISA determinations, the methylation at major and minor groove sites in calf thymus DNA by MeOSO₂(CH₂)₂-Lex has been precisely quantitated. The yields of N7-methylguanine (7-MeGua), 3-MeAde, and O⁶-methyldeoxyguanosine (6-Me-dGuo) are 0.424, 3.195, and 0.0027 mmol of adduct/mol of DNA, respectively, using 10 μM MeOSO₂(CH₂)₂-Lex and 100 μM DNA. This compares to 0.773, 0.072, and 0.0033 mmol of adduct/mol of DNA for 7-MeGua, 3-MeAde, and 6-Me-dGuo, respectively, using MMS. The increase in the yield of 3-MeAde due to the minor groove equilibrium binding properties of MeOSO₂(CH₂)₂-Lex is ~40-fold relative to MMS.

Full text of DOI:10.1021/tx9501849

APRIL/MAY 1996
VOLUME 9, NUMBER 3
© Copyright 1996 by the American Chemical Society
Articles
Th e in Vitr o Meth yla tion of DNA by a Min or Gr oove
Bin d in g Meth yl Su lfon a te Ester
Lance Encell,†,‡ David E. G. Shuker,§ Peter G. Foiles,| and Barry Gold*,†,‡
Eppley Institute for Research in Cancer and Allied Diseases and Department of Pharmaceutical
Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, MRC Toxicology
Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN United Kingdom,
and American Health Foundation, 1 Dana Road, Valhalla, New York 10595
Received November 2, 1995X
The preparation of sequence and groove specific DNA methylating agents based on
N-methylpyrrolecarboxamide subunits appended with an O-methyl sulfonate ester function-
ality (MeOSO2(CH2)2-Lex) has previously been described [Zhang, Y., Chen, F.-X., Mehta, P.,
and Gold, B. (1993) Biochemistry 32, 7954-7965]. In contrast to simple methyl sulfonate esters,
e.g., methyl methanesulfonate (MMS), which predominantly methylate at 7-guanine, MeOSO2-
(CH2)2-Lex affords N3-methyladenine (3-MeAde) as its major adduct. Using competitive ELISA
determinations, the methylation at major and minor groove sites in calf thymus DNA
by MeOSO2(CH2)2-Lex has been precisely quantitated. The yields of N7-methylguanine (7-
MeGua), 3-MeAde, and O6-methyldeoxyguanosine (6-Me-dGuo) are 0.424, 3.195, and 0.0027
mmol of adduct/mol of DNA, respectively, using 10 µM MeOSO2(CH2)2-Lex and 100 µM
DNA. This compares to 0.773, 0.072, and 0.0033 mmol of adduct/mol of DNA for 7-MeGua,
3-MeAde, and 6-Me-dGuo, respectively, using MMS. The increase in the yield of 3-MeAde
due to the minor groove equilibrium binding properties of MeOSO2(CH2)2-Lex is ∼40-fold
relative to MMS.
cancer (1). In addition, the mechanism of action for many
antineoplastic agents also involves DNA alkylation. Most
simple alkylating agents react to some extent with
DNA at all four nucleobases, and at multiple nucleo-
philic sites on each base (1, 2). As a result of the
complexity of the adduct profile, it is difficult to elucidate
the relationship between the physical structure of an
individual adduct and its biological activity, i.e., mu-
tagenesis and cytotoxicity. In addition, an appreciation
of the roles of different DNA repair enzymes in the
removal of specific lesions is complicated by the presence
of numerous types of DNA modifications. Understanding
In tr od u ction
DNA alkylation by xenobiotics and endogenous com-
pounds plays an important role in the etiology of human
* To whom all inquiries should be addressed: Voice, 402-559-5148;
FAX, 402-559-4651; Email, bgold@unmc.edu.
† Eppley Institute for Research in Cancer and Allied Diseases,
University of Nebraska Medical Center.
‡ Department of Pharmaceutical Sciences, University of Nebraska
Medical Center.
§ University of Leicester.
| American Health Foundation. Present address: Alteon Inc., 170
Williams Dr., Ramsey, NJ 07446.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
0893-228x/96/2709-0563$12.00/0 © 1996 American Chemical Society
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564 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Encell et al.
Ma ter ia ls a n d Meth od s
Rea gen ts. MeOSO2(CH2)2-Lex was prepared as previously
described (7). O6-Methylguanosine was synthesized according
to the method of Balsiger and Montgomery (8) and coupled to
bovine serum albumin (fraction V, >95%; Gibco) according to
the procedure of Erlanger and Beiser (9). The N3-methyl-N6-
[(5-carboxypentyl)-ovalbumin]Ade (10) and N7-methyl-N2-[(2-
carboxyethyl)-bovine serum albumin]guanine were prepared as
previously described (11). These conjugates were used as
coating antigens in the competitive ELISA studies described
below. The other chemical and biological reagents were pur-
chased from commercial sources and used without any further
purification. Ca u tion : MeOSO2(CH2)2-Lex has been shown to
be cytotoxic in bacteria and rodent cells and should be handled
accordingly.
In Vitr o In cu bation s. The methyl methanesulfonate (MMS)
or MeOSO2(CH2)2-Lex was incubated with calf thymus DNA
(100 µM) in 10 mM Tris-HCl buffer (pH 8.0) for the time
specified at room temperature. At the end of the incubation,
the reactions were heated at 90 °C for 30 min to release 3-MeAde
and 7-MeGua. After cooling in an ice bath, the partially
apurinic DNA was precipitated with ice cold 0.1 N HCl (2). The
supernatant was used for 3-MeAde and 7-MeGua quantitation,
and the precipitate for 6-Me-dGuo analysis.
7-MeGu a a n d 3-MeAd e An a lyses. The supernatant (see
above) was concentrated in vacuo and the residue dissolved in
10 mM Tris-HCl buffer (pH 7.4) containing 0.9% NaCl, 2.5 mM
MgCl2, and 0.1% of either chicken egg albumin (7-MeGua
analysis) or bovine serum albumin (3-MeAde analysis). Samples
were assayed for 3-MeAde and 7-MeGua using specific antibod-
ies and competitive ELISA. Standard ELISA inhibition curves
using authentic adducts were generated simultaneously with
the unknown determinations for each 96-well plate. The
standard curve was calculated using SigmaPlot 4.01 by least
squares fitting of the data to the equation:
F igu r e 1. Structures of compounds.
the mutational and cytotoxic contribution of different
DNA lesions is of paramount importance for both mecha-
nistic and pragmatic reasons. It has long been appreci-
ated that effective treatment of leukemia, as well as other
cancers, with combination radiotherapy/chemotherapy
(including alkylating agents) can eventually cause sec-
ondary leukemias in a significant number of patients (3).
Clearly, the design of effective cytotoxic antineoplastic
agents that do not generate mutations would be an
important step in the improvement of cancer chemo-
therapy (4).
P ) YMIN + (YMIN - YMAX)(In/Kn)/(1 + In/Kn)
where P is the % inhibition, I is the amount of inhibitor (7-
MeGua or 3-MeAde), YMIN and YMAX are the y-axis values where
the curve plateaus, n is the Hill coefficient, and K is the
dissociation constant (12).
6-Me-d Gu o An a lysis. The acid-precipitated DNA from the
incubation (see above) was washed with cold EtOH and then
resuspended in 10 mM Tris-HCl buffer (pH 7.0) containing 5
mM MgCl2. The DNA was then digested to the deoxynucleoside
level with DNase, snake venom phosphodiesterase, and alkaline
phosphatase as previously described (13). The 6-Me-dGuo was
separated from the unmodified nucleosides, and any
other potential adducts, by reverse phase HPLC (semipre-
parative column, 10 × 250 mm YMC-Pack ODS A-323-10
S-10 120 Å; solvent system, 40 min gradient of H2O to 40%
aqueous MeOH; flow rate, 3 mL/min). The fraction where
6-Me-dGuo elutes was collected and concentrated in vacuo.
The residue was dissolved in 10 mM Tris-HCl (pH 7.4) contain-
ing 0.9% NaCl, 2.5 mM MgCl2, and 0.1% dried milk and
analyzed for 6-Me-dGuo by competitive ELISA (13-15)
with a monoclonal antibody (15). The 6-Me-dGuo was quanti-
tated as described above using a standard ELISA inhibition
curve generated with authentic 6-Me-dGuo in the same 96-well
plate.
Because it is generally thought that N3-methyladenine
(3-MeAde)1 is toxic, but not particularly promutagenic
(5, 6), it was of interest to prepare compounds that would
be able to selectively introduce 3-MeAde modifications
into DNA under in vivo conditions. Accordingly, we have
described the synthesis of methyl sulfonate esters teth-
ered to a minor groove equilibrium binding N-methylpyr-
rolecarboxamide dipeptide (MeOSO2(CH2)2-Lex, Figure
1 for structure) (7). In contrast to other methyl sulfonate
esters that predominantly methylate 7-Gua (2), these
compounds were shown in sequencing gels to selectively
react at 3-Ade due to their minor groove binding proper-
ties (7). HPLC analysis coupled with UV detection also
indicated that little 7-MeG was formed. We now detail
the quantitation of DNA methylation at 3-Ade, 7-Gua,
and 6-dGuo by MeOSO2(CH2)2-Lex using more sensitive
competitive ELISA.
Resu lts
The 7-MeGua, 6-Me-dGuo, and 3-MeAde adducts were
quantitated using competitive ELISA (10, 11, 14, 15). In
all experiments, a control reaction was run in which no
drug was added, and the background values for these
incubations were subtracted from those in which methy-
lating agent was added. These background levels were
1
Abbreviations: Ade, adenine; Gua, guanine; dGuo, deoxygua-
nosine; Lex, lexitropsin (N-methylpyrrolecarboxamide-based informa-
tion reading peptide); 3-MeAde, N3-methyladenine; 3-MeGua, N3-
methylguanine; 7-MeGua, N7-methylguanine; 6-Me-dGuo, O6-methyl-
deoxyguanosine; MMS, methyl methanesulfonate; MNNG, N-methyl-
N′-nitro-N-nitrosoguanidine.
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MeOSO2(CH2)2-Lex Methylation Adducts
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 565
Ta ble 1. Yield s of 3-MeAd e, 7-MeGu a , a n d 6-Me-d Gu o fr om a 24 h In cu ba tion of MeOSO2(CH2)2-Lex or MMS w ith 100 µM
Ca lf Th ym u s DNA in 10 m M Tr is-HCl (p H 7.8) Bu ffer a t Room Tem p er a tu r e
adduct ratios
adduct yields (mmol of adduct/mol of DNA)
3-MeAde/
7-MeGua
3-MeAde/
6-Me-dGuo
6-Me-dGuo/
7-MeGua
[compound] (µM)
3-MeAde
7-MeGua
6-Me-dGuo
CH3OSO2(CH2)2-Lex
1
5
10
25
0.603 ( 0.099
1.408 ( 0.317
3.195 ( 0.250
5.065 ( 0.357
0.046 ( 0.013
0.234 ( 0.020
0.424 ( 0.055
1.093 ( 0.086
nda
nd
13.1
6.0
0.0027 ( 0.0002
0.0046 ( 0.0004
7.5
4.6
1183
1101
0.006
0.004
MMS
10
0.072 ( 0.020
0.773 ( 0.155
0.0033 ( 0.0004
0.093
22
0.004
a
nd, not determined.
F igu r e 2. Time course for the in vitro methylation of 100 µM
calf thymus DNA by 10 µM MeOSO2(CH2)2-Lex in 10 mM Tris-
HCl (pH 7.8) buffer at room temperature: 3-MeAde (b);
7-MeGua (3); and 6-Me-dGuo (1).
F igu r e 3. Time course for the in vitro formation of 3-MeAde
and 7-MeGua from the reaction of 100 µM calf thymus DNA
with 10 µM MeOSO2(CH2)2-Lex in 10 mM Tris-HCl (pH 7.8)
buffer at room temperature: 3-MeAde (b); 7-MeGua (3).
quite low, despite the fact that a small percentage of the
unmodified bases are hydrolyzed from the DNA during
the neutral thermal hydrolysis step (90 °C for 30 min at
pH 7.0) that is used to release 3-MeAde and 7-MeGua
from the DNA. This low background is consistent with
the low cross-reactivity of the antibodies for unmodified
Ade and Gua (10, 11). It was also established that the
high levels of 3-MeAde formed from MeOSO2(CH2)2-Lex
did not cross react with the 7-MeGua or 6-Me-dGuo
antibodies (data not shown). The background noise in
the 6-Me-dGuo assay is virtually zero due to the HPLC
purification of the adduct prior to the competitive ELISA.
The HPLC separation is necessary because the large
amount of unmodified dGuo formed in the enzymatic
digestion binds, albeit with low affinity, with the anti-
body. In control studies in which 27.4 pmol of authentic
6-Me-dGuo was injected onto the HPLC column and
quantitated using the antibody, the recovery of the
adduct was >90%. The adduct data in Table 1 are not
corrected for recovery.
linear dose response, but 3-MeAde does not. The adduct
levels for 10 µM MMS are also included in Table 1. The
ratio of 3-MeAde/7-MeGua for MMS is 0.09, which is in
the range of literature values (2). The 6-Me-dGuo/7-
MeGua ratio for MMS is 0.004, and this is also very close
to values previously reported using radiolabeled MMS
(2). The yield of 3-MeAde from 10 µM MeOSO2(CH2)2-
Lex is approximately 44-fold higher than that observed
with the same dose of MMS. In contrast, the levels of
7-MeGua and 6-Me-dGuo from MeOSO2(CH2)2-Lex are
45% and 20% lower, respectively, than that seen with
MMS (Table 1). The 3-MeAde/7-MeGua ratio is ∼140-
fold higher (13.1 vs 0.093) with MeOSO2(CH2)2-Lex
relative to MMS. The 6-Me-dGuo/7-MeGua ratios are
similar for both MMS and MeOSO2(CH2)2-Lex.
Discu ssion
Based on previous sequencing gel studies, MeOSO2-
(CH2)2-Lex selectively methylates Ade in, or immediately
adjacent to, the dipeptide’s minor groove equilibrium
binding sites (7). In sequencing gels, both 7-MeGua and
3-MeAde (but not 6-Me-dGuo) can be detected since they
are converted into strand breaks by sequential heating
and treatment with alkali. The methylation process
for MeOSO2(CH2)2-Lex involves initial association of the
Lex moiety with its DNA binding sites followed by
transfer of the methyl group from the sulfonate ester to
accessible nucleophilic atoms in the minor groove. In
the sequencing gel studies, there was little evidence
The time course for the in vitro formation of the three
adducts from the reaction of MeOSO2(CH2)2-Lex with calf
thymus DNA is plotted on a log scale in Figure 2. By
plotting the 3-MeAde and 7-MeGua data with a linear
ordinate axis, it is clear that the reaction at 3-Ade is still
increasing at 48 h (Figures 2 and 3); however, the yields
of 7-MeGua and 6-Me-dGuo have plateaued by 6 h
(Figure 2).
The dose-response for the reaction of MeOSO2(CH2)2-
Lex with calf thymus DNA is shown in Table 1. The
yields of the 7-MeGua and 6-Me-dGuo adducts show a
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566 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Encell et al.
that MeOSO2(CH2)2-Lex reacted at 7-Gua, which is
located in the major groove. If MeOSO2(CH2)2-Lex was
quantitatively associated with the minor groove, little
methylation at major groove sites would be expected.
However, this was not anticipated because the binding
affinity of the dipeptide moiety used in these studies is
not as strong as that of the naturally occurring Lex
molecules, e.g., distamycin and netropsin (16). This is
because MeOSO2(CH2)2-Lex was designed to be a neutral
peptide: distamycin and netropsin are mono- and dica-
tionic species, respectively, at physiological pH (Figure
1). The lack of an electrostatic interaction between Lex
and DNA results in a more transient binding of the
neutral dipeptide, which cannot be footprinted (16) using
standard DNA cleaving reagents such as methidiumpro-
pyl-EDTA-Fe(II) (17) or Fe(II)-EDTA-H2O2 (18). In
these studies nonalkylating neutral and charged Lex
analogues were used (16). However, the sequence selec-
tive methylation of DNA by MeOSO2(CH2)2-Lex is un-
equivocal evidence that it selectively equilibrium binds
to classical A/T rich Lex recognition sites (7). Since it
was assumed that MeOSO2(CH2)2-Lex would generate
some major groove adducts, its reaction with DNA was
re-examined using a more sensitive approach than
sequencing gels.
The quantitative data in Table 1 show that MeOSO2-
(CH2)2-Lex does in fact methylate DNA in both grooves,
although the normal dominance of major groove alkyla-
tion, i.e., 7-Gua (2), is surmounted. It is significant that
the absolute and relative yields of 7-MeGua and 6-Me-
dGuo from MeOSO2(CH2)2-Lex are very similar to that
seen with MMS. This suggests that minor groove and
major groove adduction are not competitive events under
the conditions studied. We attribute this result to an
excess of drug. This conclusion is supported by the
observation that the coaddition of distamycin, a competi-
tive inhibitor of Lex binding, to the incubation does not
increase the yield of 7-MeGua, but significantly inhibits
3-MeAde formation (ref 7 and data not shown). If some
reasonable assumptions are made concerning the stoi-
chiometry of drug binding, i.e., two MeOSO2(CH2)2-Lex
molecules/binding site (19), and the requirement of an
A/T run of g4 (20-22), then the concentration of potential
drug binding sites is approximately 1.6 µM when the
reactions are run with 100 µM DNA. This estimation is
based on the 6.25% probability of finding an (A/T)4 run,
and the requirement of 8 nucleotides (4 bases/strand) for
each binding site. This is a conservative estimate since
it assumes (i) that any (A/T)4 run will be a good equilib-
rium binding site for Lex, which is not the case (7, 16);
and (ii) that the efficiency of methylation, i.e., the % of
bound Lex molecules that deliver a methyl group to
DNA, is 100%. This estimate, with all of the caveats,
does indicate that, at the higher doses, MeOSO2(CH2)2-
Lex affinity binding sites may well be saturated. Under
such conditions MeOSO2(CH2)2-Lex methylates DNA
in the major groove via a mechanism similar to that of
other diffusible SN2 methylating agents (2). The increase
in the 3-MeAde/7-MeGua ratio as a consequence of
decreasing MeOSO2(CH2)2-Lex concentration (Table 1)
and the lack of a dose-response for 3-MeAde formation
are suggestive that minor groove binding derived me-
thylation does increase over nonspecific methylation
when drug concentrations are not saturating (near 1.5
µM).
and significantly protracted over that observed for
7-MeGua and 6-Me-dGuo (Figures 2 and 3). The origin
of this difference is not understood, but it is possible
that MeOSO2(CH2)2-Lex is rapidly sequestered in the
minor groove and that the dominant mode of complex-
ation does not produce methylation. This could involve
nonspecific DNA equilibrium binding, which inhibits
MeOSO2(CH2)2-Lex from binding to its A/T recognition
sites, where methylation takes place, and “protects”
MeOSO2(CH2)2-Lex from solvolysis. Such an explanation
is consistent with the results obtained when a N-(2-
chloroethyl)-N-nitrosourea was attached to Lex or to a
charged Lex analogue. The equilibrium binding of the
charged Lex analogue to DNA was stronger than Lex,
but its rate of alkylation was much slower (16). Both
compounds hydrolyzed at the same rate in solution in
the absence of DNA. These data imply that the equilib-
rium binding of a compound to DNA may alter the
solvolytic chemistry of reactive groups for steric reasons.
It is also possible that the lower polarity of the minor
groove, due to displacement of water molecules by Lex,
could retard the SN2 formation of the cationic 3-MeAde
lesion.
If 3-MeAde is a highly cytotoxic lesion (5, 6, 23, 24),
MeOSO2(CH2)2-Lex should be far more cytotoxic than an
equimolar dose of MMS, and its toxicity should be
exaggerated in cells that are ineffective at repair of
3-MeAde. This has been demonstrated in mouse ES cells
and in methyladenine-DNA glycosylase homozygous null
mutants (25). In a colony forming assay, MeOSO2(CH2)2-
Lex is approximately 20-fold more toxic (10% survival)
than MMS in wild type ES cells. The enhanced cytotox-
icity of MeOSO2(CH2)2-Lex over MMS persists in the null
mutants. MeOSO2(CH2)2-Lex is also 10-20-fold more
cytotoxic than MMS in wild type Escherichia coli and in
mutants defective in constitutive 3-methyladenine-DNA
glycosylase I (Tag) and/or inducible 3-methyladenine-
DNA glycosylase II (alkA)-mediated repair.2 A complete
characterization of the toxicity and mutagenicity of
MeOSO2(CH2)2-Lex in bacterial and mammalian cells is
ongoing, as is the design of more specific DNA methy-
lating agents.
Ack n ow led gm en t. This work was supported by NIH
Grant CA29088 (B.G.), Cancer Center Support Grant
CA36727 from the National Cancer Institute, American
Cancer Society Center Grant SIG-16, UNMC Environ-
mental Toxicology and Carcinogenesis Fellowship (L.E.),
UNMC Graduate Assistantship (L.E.), and University of
Nebraska Emley Presidential Fellowship (L.E.).
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