Competitive inhibition of uracil DNA glycosylase by a modified nucleotide whose triphosphate is a substrate for DNA polymerase

Full text of DOI:10.1021/ja807705z

Published on Web 01/09/2009
Competitive Inhibition of Uracil DNA Glycosylase by a Modified Nucleotide
Whose Triphosphate is a Substrate for DNA Polymerase
Haidong Huang,† James T. Stivers,‡ and Marc M. Greenberg*,†
Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218, and
Department of Pharmacology & Molecular Sciences, Johns Hopkins UniVersity School of Medicine, 725 North
Wolfe Street, Baltimore, Maryland 21205
Received September 29, 2008; E-mail: mgreenberg@jhu.edu
DNA base excision repair (BER) is an essential process in
prokaryotes and eukaryotes for protecting the integrity of the
genome. The first step in BER of modified nucleotides is carried
out by glycosylases, such as uracil DNA glycosylase (UDG), which
produces an abasic site (AP) via an oxocarbenium ion from 2′-
deoxyuridine that arises from deamination of 2′-deoxycytidine
(Scheme 1).1,2 UDG homologues exist in bacteria, viruses (e.g.,
HIV, HSV-1), as well as humans, and are potential antiviral and
anticancer targets.3,4 Small molecule competitive inhibitors that bind
UDG in the micromolar range have been discovered.5 In addition,
synthetic oligonucleotides containing modified nucleotides and nucle-
otide analogues that inhibit UDG or other BER glycosylases have also
been reported.6-11 However, the most potent of these inhibitors are
not incorporated into DNA by polymerases because they lack a
nucleobase. In addition, some 2′-deoxynucleotide triphosphates of
inhibitors containing 2′-fluoro substituents are substrates for thermo-
stable polymerases used in PCR and Pol R, but not Pol III, which
limits their potential as therapeutic candidates.12-14
Klenow exo- (apparent Km ) 15.6 ( 1.1 µM, Vmax ) 6.7 (
Scheme 1
1.0%·min-1, 1 nM), albeit ∼350-fold less efficiently than dT
(Figure 2B). CNdU was not incorporated opposite dG or dC, and
only weakly opposite dT in the presence of high concentrations
(0.3 mM) of 2. In addition to selective incorporation opposite dA,
complete extension was achieved when the primer-template com-
plex (50 nM) was in excess of enzyme (5 nM), indicating that
multiple molecules of CNdU can be incorporated.17
We postulated that 1′-cyano-2′-deoxyuridine (CNdU) would be
a UDG inhibitor whose respective nucleotide triphosphate (2) would
also be a substrate for DNA polymerase. The strong destabilization
of carbocations by R-cyano groups suggested that CNdU would
be a potent UDG inhibitor in DNA. Furthermore, molecular
modeling of the enzyme-DNA complex in which CNdU was
substituted for pseudouridine suggested that the cyano group would
only weakly perturb the protein’s structure (Figure 1). The
nucleotide triphosphates of various 2′-fluoro substituted nucleotides
that inhibit BER glycosylases when incorporated in synthetic
oligonucleotides are not accepted as substrates for DNA poly-
Figure 1. Molecular modeling showing the accommodation of a 1′-cyano
merases because they alter the pucker of the sugar ring. C1′-
substituted 2′-deoxyribonucleotide triphosphates (dNTPs) have not
been reported as substrates for DNA polymerases. However, the
acceptance of C4′-modified dNTPs as substrates by these enzymes
encouraged us to investigate 2 in this regard.15
Incorporation of 2 by the Klenow fragment of E. coli DNA
polymerase I that lacks proofreading capability (Klenow exo-) was
examined quantitatively under steady-state conditions using primer-
template complex 3 and dNTP 2 that was synthesized from CNdU
via standard methods.16,17 The dNTP was accepted as a substrate
by Klenow exo+ (apparent Km ) 14.8 ( 1.1 µM, Vmax ) 6.5 (
1.2% ·min-1, 0.33 nM), which contains proofreading ability (Figure
2A). CNdU was also incorporated slightly less efficiently by
substituent on pseudo-2′-deoxyuridine containing DNA cocrystallized with
human UDG (PDB ID: 1EMH). The arrow points toward the cyano
substituent.
Having established that CNdU can be incorporated into DNA
we turned our attention to its ability to inhibit UDG when present
in the biopolymer. Oligonucleotides containing CNdU were
synthesized by solid-phase synthesis from the respective phos-
phoramidite (1). DNA containing CNdU (4b) was not a substrate
for UDG. Hydrolysis of CNdU by UDG would produce 2-deox-
yribonolactone, which like AP is cleaved under mild alkaline
conditions.17,18 However, no evidence of reaction of 5′-32P-4b was
observed even upon prolonged exposure (24 h, 37 °C) to E. coli
UDG. In contrast, 4b was a potent inhibitor. Measurement of the
apparent Km (K′m) of E. coli UDG acting on 4a as a function of
concentration of an otherwise identical duplex containing CNdU
† Johns Hopkins University.
‡ Johns Hopkins University School of Medicine.
9
1344 J. AM. CHEM. SOC. 2009, 131, 1344–1345
10.1021/ja807705z CCC: $40.75
2009 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Melting Thermodynamics of DNA Containing CNdU and
(4b) yielded Ki ) 1.4 ( 0.1 nM (Figure 3A). The Ki of 4b was
lower than the Km (10.7 ( 0.2 nM) for the substrate. The strength
of the inhibition was independently verified using a method in which
the ratio of observed rate constants at various concentrations of
inhibitor (4b) relative to that in the absence of inhibitor were
measured when the substrate is present at a concentration much
lower than its Km (Figure 3B, Table 1).19 The Ki (4.6 ( 1.2 nM)
is very close to that determined from the plot of K′m versus 4b
concentration. Control experiments using duplex DNA containing
thymidine (4c) in place of CNdU showed that inhibition was not
due to nonspecific binding. For instance, addition of 20 nM 4c
(>4× the Ki of 4b) diminished the hydrolysis of the substrate by
<15%. Finally, 4b also effectively inhibits human UDG
(Table 1).
dU
duplex
TM (°C)a
∆H (kcal/mol)
∆S (cal/mol·deg)
∆G298 (kcal/mol)
5a
5b
48.9 ( 0.1
44.7 ( 0.3
92.9 ( 0.7
85.2 ( 4.8
260.8 ( 0.1
240.2 ( 0.1
15.5
13.6
a [duplex] ) 2.5 µM.
increase in entropy (Table 2). The thermodynamic differences are
consistent with a destabilized duplex, which would be expected to
make binding to UDG more favorable by decreasing the energy
required to flip the base out of the helix.
In summary, we have described the first competitive inhibitor
of UDG that is incorporated into DNA by the Klenow fragment of
DNA polymerase I, a replicative polymerase. The presence of the
molecule within the DNA scaffold contributes significantly to its
potency. Nucleosides are often useful as therapeutic agents. 1′-
Cyano-2′-deoxyuridine (CNdU) and related molecules may prove
useful as a new family of therapeutic or experimental agents that
target DNA repair by using the cells’ polymerase(s) to incorporate
them into DNA. To be useful in this way, CNdU or a pro-drug of
it will need to be a substrate for cellular kinases, which at this
time is unknown. A potential benefit of such a mechanism is that
multiple incorporations can occur for longer DNA molecules leading
to amplification of the inhibitory effect beyond that seen here with
short DNA duplexes. The in vivo effectiveness of such a strategy
has been validated for the inhibition of cytosine 5-methyl DNA
methyltransferases by the nucleoside prodrugs 5-azadeoxycytidine
and deoxyzebularine.20
Figure 2. Acceptance of 2 as a substrate by (A) Klenow exo+ (B) Klenow
exo- using 3 as a template under steady-state conditions.
Acknowledgment. We are grateful for generous support from
the National Institute of General Medical Sciences (Grant GM-
063028 to M.M.G. and Grant GM-056834 to J.T.S.).
Supporting Information Available: Experimental procedures,
autoradiogram of full-length extension using 2, Van’t Hoff plots, and
oligonucleotide characterization. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 3. Determination of Ki of E. coli UDG by 4b by (A) determining
the apparent Km (K′m) of 4a as a function of inhibitor concentration and
(B) measuring the rate constant ratio in the presence of varying [4b] (ki)
versus no inhibitor (k0) at [4a] , Km.
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