(Received for publication, March 20, 1995; and in revised form, June 19, 1995)
From the
5`-Triphosphates of -D and
-L-enantiomers of
2`,3`-dideoxycytidine (ddC), 2`,3`-dideoxy-5-fluorocytidine (FddC),
1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine
(FOddC) were evaluated as inhibitors and substrates for human DNA
polymerases
,
,
,
, and
. L-ddCTP was
not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA
polymerases and was a less potent inhibitor of DNA polymerases
and
than its D-enantiomer by 2 orders of magnitude. In
contrast, all L-dioxolane analogs were potent inhibitors and
chain terminators for all cellular DNA polymerases studied. The K
values of their 5`-triphosphates for
DNA polymerase
were found to be in the following order: D-ddC < D-FddC
L-OddC
D-FOddC < L-FOddC L-FddC. The K
values of L-OddCTP for the
reactions catalyzed by DNA polymerases
,
,
,
, and
were 6.0, 1.9, 0.4, 3.0, and 0.014 µM, respectively,
and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06
µM, respectively. The K
values for incorporation of L-OddCTP into the
standing points of primer extension were also evaluated and determined
to be 1.3, 3.5, 1.5, 2.8, and 0.7 µM for DNA polymerases
,
,
,
, and
, respectively. The incorporation
of dioxolane analogs into DNA by replicative DNA polymerases could
explain their potent cellular toxicity.
Several 2`,3`-dideoxynucleoside analogs have been approved for
the treatment of patients with AIDS(1, 2) . Among
them, ddC ()has been shown to be one of the most potent
inhibitors of HIV replication. Recently, SddC was found to have potent
activity against HIV (3, 4) and hepatitis B virus (5) in cell culture. It was subsequently shown that the
stereoisomer L-SddC (3TC) was responsible for the antiviral
effect, while cytotoxicity was associated mainly with the D-enantiomer(6, 7, 8, 9) . L-ddC and L-FddC were also found to be active against
hepatitis B virus and HIV in culture without much
toxicity(10, 11, 12) . In contrast to L-SddC or L-FddC, L-OddC and L-FOddC were markedly more toxic than their D-enantiomers against human leukemic CEM cells (13) .
The spectrum of L-OddC toxicity against human tumor cells
differed from that of cytosine arabinoside. L-OddC was also
shown to have activity against solid human tumors in nude
mice(14) . Currently, L-OddC is being evaluated
further as an anticancer agent. The reason why L-OddC but not L-ddC or L-SddC has potent antitumor activity is not
clear, but it is probably associated with its interference in cellular
DNA synthesis. L-SddC and L-OddC were shown to be
phosphorylated by cellular kinases to the corresponding 5`-triphosphate
metabolites inside cells. The antiviral effects of nucleoside analog
are due to preferential interference with viral replication caused by
incorporation into DNA by viral DNA polymerase. The interaction of
5`-triphosphate metabolites of L-OddC or L-FddC with
cellular DNA polymerases may be one of the key factors in determining L-OddC cytotoxicity.
In the present paper, the interaction
of 5`-triphosphates of D- and L-enantiomers of
2,3-dideoxycytidine and 1,3-dioxolane-cytidine as well as their
5-fluoro-derivatives with human pol , pol
, pol
, pol
, and pol
is reported.
ssDNA cellulose was obtained from Sigma. DEAE
cellulose (DE-52), phosphocellulose (P11), and S-Sepharose fast flow
were purchased from Whatman. [-
P]dCTP (3000
Ci/mmol), [
-
P]ATP (6000 Ci/mmol) and T4
polynucleotide kinase were obtained from Amersham Corp. M13 mp10 and
M13 mp18 phage ssDNA were isolated as described
previously(16) . Oligonucleotides were synthesized on an
Applied Biosystems 380A DNA synthesizer at the Yale Oligonucleotide
Synthesis Facility. The primer oligonucleotides were labeled at the 5`
position with T4 polynucleotide kinase using
[
-
P]ATP, annealed to M13 phage ssDNA as
described in (16) , purified on a Sephadex G-25 column, and
used as substrates for elongation reactions. BuAdATP was a kind gift
from Professor George Wright (University of Massachussetts).
pol and pol
activities were
routinely assayed in 20 µl of 20 mM Tris-HCl buffer (pH
7.4) containing 6 mM MgCl
, 1 mM
dithiothreitol, 0.5 mM EDTA, 200 µg/ml heat-inactivated
bovine serum albumin, 150 µg/ml activated calf thymus DNA, 20
µM each of dATP, dTTP, and dGTP, and 1 µCi of
[
H]dCTP or [
-
P]dCTP
at concentrations no less than the K
values for
the appropriate enzymes. The same assay conditions were used for other
polymerases with the following modifications: pol
, pH 8.5; pol
, pH 8.0 and 80 mM KCl; and pol
, 8 mM MgCl
. Assays for DNA polymerases contained 2 µl of
enzyme (1 unit of pol
, pol
, or pol
, 2 units of pol
, 0.75 unit of pol
). One unit of enzyme activity is defined
as the amount of enzyme needed to incorporate 1 nmol of
[
H]dTMP/h into the acid-insoluble fraction at 37
°C. The reactions were allowed to proceed at 37 °C for
10-60 min, after which 15-µl aliquots were removed and
spotted onto Whatman DE-81 discs. The filters were washed with 0.3 M KCl containing 0.5 mM EDTA and then fixed with
ethanol. The incorporation of the radiolabeled substrate into the DNA
chain was measured by liquid scintillation counting.
The K values of each compound for all DNA polymerases
studied were determined using a competitive inhibition equation as
described in (25) .
Figure 6:
Autoradiograph of chain terminating
sequencing reaction with L-OddCTP using pol (lanes
2-6) and M13 mp10 phage DNA annealed with
5`-
P-15 mer primer. The letters on the leftside of the figure indicate the sequence of the
growing DNA chain after the primer. Lane1 (control)
shows the DNA sequence by Klenow fragment with 20 µM ddCTP. Lanes2-5, DNA sequence by pol
with L-OddCTP at concentrations 10, 20, 20, and 40
µM, respectively. Lane4, after the
reaction, 100 µM mixture containing all four dNTP was
added, and the reaction continued 20 min more. Track6, reaction without L-OddCTP. The mixture for
pol
contained 2.5 µM dCTP and 20 µM each of three other dNTPs.
Complexes of M13 phage DNA and primer used in this study
were as follows.M13mp18 phage DNA 3`-CATTTTGCTGCCGGTCACGG-5`
5`-GTAAAACGACGGCCAGT-3` 17-mer primer
M13mp10 phage DNA
3`-GGTCAAGTGCTGCAACATTTTGCTGCCGG-5` 5`-CCAGTTCACGACGTTGTAAAACGA-3`
14-mer primer
15-mer primer
Figure 1: Structure of L- and D-enantiomers of 2`,3`-dideoxycytidine 5`-triphosphate analogs.
Figure 2:
Incorporation of L- and D-enantiomers of ddCTP analogs into the 3`-end of a 17-mer
primer annealed with M13 mp18 phage DNA by pol (lanes
1-9) and pol
(lanes 10-17). The
reactions were performed as described under ``Experimental
Procedures.'' Lane1, incorporation of dGMP
residues into the first position after primer; lane2, 1 µMD-FddCTP; lane3, 10 µML-ddCTP; lanes4 and 5, 5 and 10 µML-FddCTP, respectively; lanes6 and 7, 1 and 6 µML-FOddCTP; lanes8 and 9, 0.2 and 2 µML-OddCTP, respectively. Lane10, 5
µMD-ddCTP; lane11, 10
µMD-FddCTP; lane12, 10
µML-ddCTP; lane13, 10
µML-FddCTP; lanes14 and 15, 1 and 6 µML-FOddCTP, respectively; lanes16 and 17, 2 and 5 µML-OddCTP, respectively.
Figure 3:
Incorporation of L- and D-enantiomers of ddCTP analogs into the 3`-end of
5`-P-15-mer primer annealed with M18 mp10 phage DNA by pol
(A) and pol
(B). A, 10
µMD-ddCTP (lane1); 10
µMD-FddCTP (lane2); 10
µML-ddCTP (lane3); 10
µML-FddCTP (lane4); 5 and 20
µMD-FOddCTP (lanes5 and 6); 0.5 and 2 µML-FOddCTP (lanes7 and 8); 0.5, 1, and 2 µML-OddCTP (lanes9, 10, and 11, respectively); 10 µML-SddCTP (lane12). B, 5 µMD-ddCTP (lane1); 10 µML-ddCTP (lane2); 1 and 5 µMD-FddCTP (lanes3 and 4,
respectively); 2 and 10 µML-FddCTP (lanes5 and 6, respectively); 2 and 10 µML-OddCTP (lanes7 and 8); 2
and 5 µML-FOddCTP (lanes9 and 10, respectively); 1 and 5 µMD-FOddCTP (lanes11 and 12,
respectively).
Figure 4:
Concentration-dependent incorporation of L-OddCTP into DNA by pol (lanes 1-7) and
pol
(lanes 8-14) into the 3`-end of
5`-
P-15-mer primer annealed with M13 mp10 phage DNA. DNA
primer extension was carried out as described under ``Experimental
Procedures'' with the exception that incubation time was 10 min,
and 0.2 unit of pol
and 0.25 unit of pol
were used to
assure that less than 30% of the primer was consumed during
experiments. A, incorporation of 2.5 uM dCTP (lanes1 and 8); 0.2, 0.5, 1, 2, 5, and 10
uML-OddCTP (lanes2-7 and lanes 9-14, respectively. B, the intensity of
each track from A was quantitated by computer densitometry and
plotted as 1/Vversus 1/[S] for the
reactions catalyzed by DNA pol
(-+-) and pol
(-
-). 1/V is presented as relative
value of density.
As previously shown(6, 20) , L-SddCTP was
a weak inhibitor of pol and pol
and was not a substrate for
either enzyme. We compared the ability of L-SddCTP and L-OddCTP to be incorporated at standing and running points of
a DNA chain by pol
under similar conditions. As one can see in Fig. 5, L-SddCTP is incorporated neither into the first
nor into the eighth position of an elongated primer. Conversely, L-OddCTP is a good substrate for pol
, and the DNA
fragments terminated by L-OddCMP are accumulated in both
cases.
Figure 5:
Incorporation of L-OddCTP (A, lane 2, and B, lanes 1 and 2) and L-SddCTP (A, lanes 3 and 4, and B, lanes 3-5) in standing (A) and running (B) points of 15-mer primer (A) or 14-mer primer (B) annealed to M13 mp10 phage
DNA by pol . PanelA, lane1,
shows the incorporation of dCTP. The reactions were performed as
described under ``Experimental
Procedures.''
Recently, nucleoside derivatives with the unnatural L-configuration were evaluated against a broad spectrum of
viruses, and some of these analogs proved to be very potent against
hepatitis B virus and
HIV(6, 7, 8, 9, 10, 11, 12) .
At the same time, differences in their cellular toxicities were
noticed. In contrast to L-SddC or L-ddC, both L- and D-enantiomers of FOddC and OddC were shown to
be very cytotoxic(13, 14) . The interaction of their
5`-triphosphate metabolites with cellular DNA polymerases may be one of
the key factors in their varying toxicities. This possibility prompted
us to evaluate the 5`-triphosphates of L- and D-enantiomers of dioxolane-cytidine as potential inhibitors or
substrates of human DNA polymerases. For comparison, nucleoside
5`-triphosphates with the natural D-configuration were
included in the study. The results presented here show that L-OddCTP and its 5-fluoro-derivative are potent inhibitors and
chain terminators of cellular DNA polymerases including pol , pol
, and pol
. The inhibition of polymerase activity is due to
the incorporation of L-OddCTP into the DNA chain at cytidine
residue sites but not to the inhibition of the rate of incorporation of
other dNTPs. If the inhibition of synthesis was due to a dual mechanism
including both incorporation and interference with the incorporation of
other nucleosides, we would expect to see DNA fragments of a size
inconsistent with DNA products terminated at cytidine residues (pauses) (Fig. 6). The interaction of L-OddCTP and L-FOddCTP with human DNA polymerases is the first example of
the lack of enantioselectivity described for human replicative DNA
polymerases. This phenomenon was described for HIV reverse
transcriptase, pol
, and pol
with respect to the L-
and D-enantiomers of SddCTP (6, 20, 32) and to carbovir
triphosphate(33) . It should be mentioned that replicative DNA
polymerases are believed to be more sensitive to changes in the
conformation of substrates. Indeed, this is true for SddCTP whose L-isomer is not a substrate for replicative DNA polymerases.
However, substitution of sulfur for oxygen at the 3`-position of the
ribose residue leads to the opposite effect. L-OddCTP is a
good terminating substrate for replicative DNA polymerases, inhibiting
them with K
values in the pharmacologically
relevant concentration range.
The amount of L-OddCMP
present at the DNA terminus depends not only on the efficiency of
incorporation of L-OddCTP by polymerases but also on the rate
of excision by 3` 5`-exonucleases(34) . The ability of
3`
5`-exonucleases associated with pol
and pol
are
currently evaluated. The anticancer activity of L-OddC may be
a result of termination of DNA synthesis after L-OddCTP
incorporation into proliferating cells coupled with inefficient
excision of incorporated L-OddCMP from DNA.
In the present
study, we also made an attempt to address the impact of the
substitution of fluorine for hydrogen at the 5-position of cytidine
5`-triphosphate analogs on their ability to serve as substrates for
cellular DNA polymerases. As shown in Table 1, both L-
and D-enantiomers of ddCTP and FddCTP were not substrates for
pol , pol
, and pol
and inhibited pol
and pol
at the same range of concentrations. Similar results were
obtained for the L-enantiomers of OddCTP and FOddCTP. No
significant differences were seen between L-OddCTP and its
5-fluoro-derivatives in terms of their interaction with DNA
polymerases. Both L-enantiomers were equally potent inhibitors
for these DNA polymerases. With respect to pol
, L-OddCTP
was 6 times more potent than its 5-fluoro-analog. At present, we cannot
explain the differences in the interaction of L-OddCTP and its
5-fluoro-derivative with pol
.
In summary, L-OddC is the first L-nucleoside shown to have potent antitumor activity. This activity could be related to the ability of L0OddCTP to be utilized as a substrate by human replicative DNA polymerases. Surprisingly, this property is not shared with L-SddC and L-ddC, which are relatively noncytotoxic. The discovery that the L-enantiomers of chain-terminating nucleotides can be incorporated by replicative DNA polymerases could lead to the development of a new class of anticancer compounds.