Herpes simplex virus type 1 uracil-DNA glycosylase: Isolation and selective inhibition by novel uracil derivatives

Abstract

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

Full text

Biochem.
J.
(1993)
292,
883-889
(Printed
in
Great
Britain)
Herpes
simplex
virus
type
1
uracil-DNA
glycosylase:
isolation
and
selective
inhibition
by
novel
uracil
derivatives
Federico
FOCHER,*§
Annalisa
VERRI,*
Silvio
SPADARI,*II
Roberto
MANSERVIGI,t
Joseph
GAMBINOt
and
George
E.
WRIGHTt
*Istituto
di
Genetica
Biochimica
ed
Evoluzionistica,
CNR,
via
Abbiategrasso
207,
1-27100
Pavia,
Italy,
tDiparimento
di
Microbiologia,
Universita
degli
Studi
di
Ferrara,
Ferrara,
Italy,
and
$Department
of
Pharmacology,
University
of
Massachusetts
Medical
School,
Worcester,
MA
10655,
U.S.A.
We
have
purified
Herpes
simplex
type
1
(HSV1)
uracil-DNA
glycosylase
from
the
nuclei
of
HSVI-infected
HeLa
cells
harvested
8
h
post-infection,
at
which
time
the
induction
of
the
enzyme
is
a
maximum.
The
enzyme
has
been
shown
to
be
distinct
from
the
host
enzyme,
isolated
from
HeLa
cells,
by
its
lack
of
sensitivity
to
a
monoclonal
antibody
to
human
uracil-DNA
glycosylase.
Furthermore,
several
uracil
analogues
were
synthesized
and
screened
for
their
capacity
to
discriminate
between
the
viral
and
human
uracil-DNA
glycosylases.
Both
enzymes
were
inhibited
by
6-(p-alkylanilino)uracils,
but
the
viral
enzyme
was
significantly
more
sensitive
than
the
HeLa
enzyme
to
most
analogues.
Substituents
providing
the
best
inhibitors
of
HSV1
uracil-DNA
glycosylase
were
found
to
be
in
the order:
p-
INTRODUCTION
Herpes
simplex
virus
(HSV),
after
primary
infection
in
peripheral
mucocutaneous
tissue,
can
penetrate
nerve
cells
and
establish
latent
infection
in
neuronal
ganglia
(Challberg
and
Kelly,
1989;
Roizman
and
Sears,
1990).
Re-infection
with
the
same
virus
type
has
been
described
in
humans,
but
it
is
extremely
rare;
most
recurrent
infections
represent
re-activation
of
the
same
virus
type
from
latency.
Although
the
molecular
mechanism
leading
to
viral
re-activation
is
still
unclear,
it
is
known
that
re-activation
is
the
viral
answer
to
environmental
injury
to
the
host
cell.
For
example,
ultraviolet
light,
trauma
to
neuronal
ganglia,
immuno-
suppression
or
general
stress
situations
could
damage
the
host
cell
and
seriously
compromise
virus
survival
(Roizman
and
Sears,
1990).
Starting
in
the
lytic
cycle,
HSV
suppresses
host
DNA
and
protein
synthesis
(Roizman
and
Sears,
1990)
and
induces
its
enzymic
machinery
controlling
the
efficient
bio-
synthesis
of
DNA
precursors,
the
replication
of
its
genome,
and
the
maturation
of
the
virus
particle.
Among
the
variety
of
enzymes
induced
by
HSV
during
the
re-
activation
event,
some
replace
the
suppressed
host
counterparts,
for
instance
dUTPase
(Caradonna
and
Cheng,
1981),
and
others
supply
cellular
enzymes
that
are
lacking.
The
enzymes
of
this
second
class
are
either
those
catalysing
specific
viral
reactions,
such
as
virus
assembly,
or
those
whose
cellular
counterparts
are
developmentally
absent,
such
as
DNA
polymerases
and
thymidine
kinase
(TK).
Our
laboratory
has
demonstrated
that
adult
neurons
lack
not
only
TK,
but
also
replicative
DNA
polymerases
a
(Hiibscher
et
al.,
1977,
1978)
and
d/e
(Spadari
et
al.,
1988),
and
uracil-DNA
n-butyl
<
p-n-pentyl
=
p-n-hexyl
<
p-n-heptyl
<
p-n-octyl.
The
most
potent
HSV1
enzyme
inhibitor,
6-(p-n-octylanilino)uracil
(OctAU),
with
an
IC50
of
8
,uM,
was
highly
selective
for
the
viral
enzyme.
Short-term
[3H]thymidine
incorporation
into
the
DNA
of
HeLa
cells
in
culture
was
partially
inhibited
by
OctAU,
whereas
it
was
unchanged
when
6-(p-n-hexylanilino)uracil
was
present
at
concentrations
that
completely
inhibited
HSV1
uracil-
DNA
glycosylase
activity.
These
compounds
represent
the
first
class
of
inhibitors
that
inhibit
HSV1
uracil-DNA
glycosylase
at
concentrations
in
the
micromolar
range.
The
results
suggest
their
possible
use
to
evaluate
the
functional
role
of
HSV1
uracil-DNA
glycosylase
in
viral
infections
and
re-activation
in
nerve
cells.
glycosylase
(Focher
et
al.,
1990).
In
neurons,
the
activity
of
these
enzymes
decreases
during
fetal
development
and
disappears
at
birth,
in
synchrony
with
the
pattern
of
neural
division
(Hiibscher
et
al.,
1977,
1978;
Spadari
et
al.,
1988;
Focher
et
al.,
1990).
This
means
that
in
neurons,
where
viral
re-activation
takes
place,
HSV
replication
strictly
depends
on
its
own
enzymes.
This
is
supported
by
the
observation
that
TK-
HSV
can
sustain
lytic
infections
in
mucosal
and
skin
surfaces
(TK+
cells),
but
fails
to
re-activate
from
adult
explanted
ganglia
(TK-
cells).
The
pivotal
role
of
HSV
TK
in
re-activation
was
clearly
demonstrated
by
the
use
of
non-substrate
inhibitors
of
viral
TK
synthesized
and
characterized
in
our
laboratories
(Focher
et
al.,
1988;
Spadari
and
Wright,
1989).
Used
in
a
murine
model,
these
compounds
significantly
reduced
the
number
of
latently
infected
trigeminal
ganglia
that
yielded
virus
upon
explant
culture
(Leib
et
al.,
1990).
Induction
of
HSV
uracil-DNA
glycosylase
activity
was
observed
during
viral
infection
(Caradonna
and
Cheng,
1981),
and
the
coding
sequence
of
the
viral
enzyme
has
been
identified
(Caradonna
et
al.,
1987;
Mullaney
et
al.,
1989;
Worrad
and
Caradonna,
1988).
Uracil-DNA
glycosylase
belongs
to
the
class
of
enzymes
involved
in
post-replicative
DNA
repair
processes.
Its
function
consists
of
the
specific
removal
of
uracil
residues
from
DNA,
deriving
either
from
cytosine
deamination
or
dUTP
incorporation,
by
cleavage
of
the
N-glycosidic
bond
linking
the
base
to
the
deoxyribose
phosphate
backbone.
In
this
work,
we
present
details
of
the
isolation
and
characterization
of
the
enzyme
from
HSVl-infected
cells,
and
report
the
first
specific
inhibitors
of
HSV1
uracil-DNA
glycosylase
acting
in
the
micromolar
range.
These
compounds
Abbreviations
used:
HSV1,
Herpes
simplex
virus
type
1;
TK,
thymidine
kinase;
PMSF,
phenylmethanesulphonyl
fluoride;
DTT,
dithiothreitol;
NP-40,
Nonidet
P-40;
DMEM,
Dulbecco's
modified
Eagle's
medium;
BuAU,
6-(p-n-butylanilino)uracil;
OctAU,
6-(p-n-octylanilino)uracil;
HexAU,
6-(p-n-
hexylanilino)uracil.
11
To
whom
correspondence
should
be
addressed.
§
To
whom
reprint
requests
should
be
addressed.
883

884
F.
Focher
and
others
might
be
valuable
in
defining
whether
this
enzyme,
like
the
viral
TK,
plays
a
key
role
in
viral
re-activation
from
normal
UDG-
nerve
cells.
MATERIALS
AND
METHODS
Chemicals
and
enzymes
Deoxyribonucleoside
triphosphates
were
from
Boehringer
or,
for
3H-labelled
nucleotides
and
nucleosides,
from
Amersham.
Heparin-Sepharose
CL-6B
was
from
Pharmacia,
pepstatin
A
and
phenylmethanesulphonyl
fluoride
(PMSF)
were
from
Sigma,
BSA
(A
grade)
was
from
Calbiochem.
Sodium
metabisulphite
and
plastic
t.l.c.
sheets
(polyethyleneimine-cellulose
F;
0.1
mm)
were
from
Merck.
Nonidet
P-40
was
from
BDH,
and
dithio-
threitol
(DTT)
was
from
Fluka.
Phosphocellulose
P11
and
DE-32
were
from
Whatman.
DNA
polymerase
I,
Klenow
fragment,
was
purchased
from
Boehringer.
HSV1
Infection
and
harvesting
of
Infected
cells
Confluent
monolayers
of
HeLa
BU
(TK-)
cells
on
225
cm2
flasks
were
infected
with
HSV1
strain
F
(Ejercito
et
al.,
1968)
at
a
multiplicity
of
infection
of
5
plaque-forming
units/cell.
After
a
1
h
adsorption
period,
the
monolayers
were
rinsed
twice
with
phosphate-buffered
saline,
overlaid
with
Dulbecco's
modified
Eagle's
medium
(DMEM)
containing
10
%
fetal
calf
serum,
and
incubated
for
8
h.
The
cells
were
then
detached
with
a
rubber
policeman,
the
suspension
was
divided
into
portions
in
plastic
tubes,
and
cells
were
kept
frozen
at
-70
°C
until
use.
Uracil-DNA
glycosylase
assay
Assays
in
a
final
volume
of
25
,1
each
contained
100
mM
Tris/HCl
(pH
8.0),
5
mM
DTT,
10
mM
EDTA,
500
ng
of
[3H]dUMP-labelled
DNA
(40
c.p.m./ng;
220
c.p.m./pmol
of
uracil;
3.6
1sM
uracil)
prepared
as
described
(Focher
et
al.,
1990),
4
,ug
of
unlabelled
activated
DNA
and
the
enzyme
(0.3
unit)
to
be
tested
(when
HSV1
enzyme
was
used,
BSA
was
added
in
the
test
tube
in
order
to
obtain
a
protein
concentration
comparable
with
that
for
the
human
enzyme).
After
incubation
at
37
°C
for
30
min,
20
,tl
portions
of
the
mixtures
were
spotted
on
to
GF/C
filters
(Whatman).
The
filters
were
washed
three
times
in
5
%
(v/v)
trichloroacetic
acid
for
5-10
min
and
twice
in
ethanol.
The
filters
were
dried
and
the
acid-insoluble
radioactivity
was
measured
by
scintillation
counting
in
1
ml
of
scintillation
fluid.
One
unit
of
uracil-DNA
glycosylase
removes
1
umol
of
uracil
from
DNA
in
1
h
at
37
'C.
PurfflcatIon
of
HSV1
uracil-DNA
glycosylase
HeLa
cells
(5
x
108)
were
infected
by
HSV1
as
described
above
and
collected
8
h
post-infection.
The
cells
were
resuspended
in
6
vol.
of
ice-cold
buffer
A
(1O
mM
NaCl,
1
mM
potassium
phosphate,
pH
6.8,
1
mM
DTT
and
1
%
dimethyl
sulphoxide,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin).
After
5
min
on
ice
the
cells
were
homogenized
in
a
Dounce
homogenizer.
The
homogenate
was
centrifuged
at
1400
g
for
15
min
in
order
to
precipitate
the
nuclei.
The
nuclei
were
suspended
in
5
vol.
of
buffer
B
(1
mM
potassium
phosphate,
pH
7.0,
0.32
M
sucrose,
1
mM
MgCl2,
0.3
%
NP-40
detergent
and
1
mM
DTT,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin).
In
this
buffer
the
nuclei
were
homogenized
and
centrifuged
at
1400
g
for
15
min.
The
nuclear
pellet
was
resuspended
in
5
vol.
of
buffer
C
(400
mM
potassium
phosphate,
pH
7.0,
1
mM
DTT,
0.1
%
NP-40
and
1
%
dimethyl
sulphoxide,
containing
1
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin).
After
5
min
on
ice
the
nuclei
were
disrupted
in
a
Dounce
homogenizer.
The
homogenate
was
centrifuged
at
48
000
g
for
1
h.
The
supernatant
was
loaded
on
a
DE-32
column
(4
ml)
previously
equilibrated
with
10
column
volumes
of
buffer
C
(to
remove
nucleic
acids).
The
flow-through
and
the
first
20
ml
of
column
wash
with
buffer
C
were
collected
and
dialysed
against
three
changes
of
buffer
D
(20
mM
potassium
phosphate,
pH
7.5,
1
mM
DTT
and
0.1
%
NP-40,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,cM
pepstatin)
in
order
to
lower
the
ionic
strength.
The
dialysed
solution
was
loaded
on
to
a
DE-32
column
(4
ml)
previously
equilibrated
with
buffer
D.
The
flow-through
and
the
first
16
ml
of
the
column
wash
with
buffer
D
containing
the
enzyme
activity
were
loaded
on
to
a
phosphocellulose
column
(2
ml)
equilibrated
with
buffer
E
(20
mM
potassium
phosphate,
pH
7.5,
20
%
glycerol,
1
mM
DTT
and
0.1
%
NP-40,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,#M
pepstatin).
The
phosphocellulose
column
was
washed
with
4
column
volumes
of
buffer
E,
and
the
enzyme was
eluted
with
20
ml
of
a
linear
gradient
from
20
to
400
mM
potassium
phosphate
(pH
7.5)
in
buffer
E.
The
pooled
fractions,
which
eluted
at
approx.
200
mM
potassium
phosphate,
were
dialysed
against
buffer
F
(20
mM
potassium
phosphate,
pH
7.0,
30
%
glycerol,
1
mM
DTT,
0.5
mM
EDTA
and
0.1
mM
EGTA,
containing
0.2
mM
PMSF
and
1
,uM
pepstatin),
and
then
loaded
on
to
a
heparin-Sepharose
column
(1
ml)
equilibrated
with
buffer
F.
The
column
was
washed
with
4
ml
of
buffer
F,
and
the
enzyme
was
eluted
with
18
ml
of
a
linear
gradient
from
0
to
600
mM
KCI
in
buffer
F;
the
enzyme
eluted
at
250
mM
KCI.
The
pooled
fractions
were
immediately
frozen
and
stored
in
liquid
nitrogen
in
small
aliquots.
The
final
preparation
had
a
specific
activity
of
11080
units/mg
and
there
was
no
contamination
by
nucleases.
The
sedimentation
coefficient
was
3.17.
The
purification
procedure
is
summarized
in
Table
1.
Purfficaton
of
human
uracil-DNA
glycosylase
Human
uracil-DNA
glycosylase
was
purified
from
the
cytoplasm
of
HeLa
cells
by
a
procedure
partially
derived
from
that
of
Krokan
and
Wittwer
(1981).
HeLa-S3
cells
(2
x
109)
were
re-
suspended
in
28
ml
of
ice-cold
buffer
G
(1O
mM
Tris/HCl,
pH
7.5,3
mM
MgCl2,
2
mM
EGTA
and
1
mM
DTT,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin).
After
5
min
on
ice
the
cells
were
homogenized
in
a
Dounce
homogenizer,
and
then
14
ml
of
buffer
H
(340
mM
Tris/HCl,
pH
8.1,
3
mM
MgCl2,
150
mM
glucose,
2
mM
EGTA,
0.15
%
Triton
X-100
and
1
mM
DTT,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
#tM
pepstatin)
was
added.
The
resulting
solution
was
centrifuged
at
680
g
for
5
min.
The
supernatant
was
saved,
and
the
pellet
containing
the
nuclei
was
washed
in
a
Dounce
homogenizer
in
8
ml
of
buffer
I
(buffer
G/buffer
H,
2:
1,
v/v)
and
then
centrifuged
at
680
g
for
5
min.
The
supernatant
was
combined
with
the
previous
one
and
centrifuged
at
100000
g
for
1
h.
The
derived
supernatant
was
dialysed
for
8
h
against
20
vol.
of
buffer
J
(20
mM
Tris/HCl,
pH
7.5,
20
%
glycerol,
0.1
0%
Triton
X-100
and
1
mM
DTT,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin)
and
then
loaded
on
to
a
DE-32
column
(10
ml)
equilibrated
with
buffer
J.
The
flow-through
and
the
column
wash
with
buffer
J
containing
the
enzyme
activity
were
loaded
on
to
a
phosphocellulose
column
(5
ml)
equilibrated
with
buffer
K
(20
mM
potassium
phosphate,
pH
7.5,
20
%
glycerol,
0.1
%
Triton
X-100
and
1
mM
DTT,
containing
0.2
mM
PMSF,
4
mM
sodium
metabisulphite
and
1
,uM
pepstatin).
The
column
was
washed
with
10
column
volumes
of
buffer
K,
and
the
enzyme
was
eluted
with
a
linear
gradient
(50
ml)
of
20-300
mM
potassium

Selective
inhibitors
of
Herpes
simplex
virus
type
1
uracil-DNA
glycosylase
Table
1
PurIffcation
of
HSV1
uracil-DNA
glycosylase
Specific
Protein
Activity
activity
Recovery
Purification
(mg)
(units)
(u/mg)
(%)
(-fold)
Nuclear
extract
20
1640
82
-
-
48
000
g
4.62
1530
331
93
4.0
centrifugation
DEAE
(I)
4.24
2328
549
142
6.7
DEAE
(II)
1.08
2268
2100
138
25.6
Phosphocellulose
0.28
950
3386
58
41.3
P11
Heparin-Sepharose
0.05
554
11
080
34
135.1
Table
2
Yields
and
properties
of
6-(p-alkylanilino)uracils
Compounds
were
synthesized
by
reaction
between
6-chlorouracil
and
the
aniline
in
refluxing
2-methoxyethanol
as
described
(Wright
and
Brown,
1980).
Abbreviations:
EtOH,
ethanol;
HOAc,
glacical
acetic
acid. C,
H
and
N
analyses
agreed
to
within
±0.4%
of
calculated
values.
The
general
formula
of
the
derivatives
is
given
below:
0
HN)
0
N
N
\
R
HH
Melting
Crystallization
R
Yield
(%)
point
(OC)
solvent
Formula
Isobutyl
78
327-333
EtOH
C14H17N302
Isopentyl
88
319-320
EtOH
C15H19N302
n-Pentyl
67
321-324
EtOH
C15H19N302
n-Hexyl
70
317-321
EtOH
C16H21N302,NEtOH
n-Heptyl
47
303-304
EtOH
C7H23N302,
lEtOH
n-Octyl
52
298-301
HOAc
C18H25N302
phosphate,
pH
7.5,
in
buffer
K.
The
pooled
fractions
(15
ml)
were
dialysed
against
500
ml
of
buffer
L
(20
mM
potassium
phosphate,
pH
7.0,
30%
glycerol,
0.5
mM
EDTA,
0.1
mM
EGTA,
0.2
mM
PMSF
and
1
,#M
pepstatin)
and
then
loaded
on
to
a
heparin-Sepharose
column
(1
ml)
equilibrated
with
buffer
L.
The
column
was
washed
with
20
ml
of
buffer
L,
and
the
enzyme
was
eluted
with
a
linear
gradient
(10
ml)
of
0-600
mM
KCI
in
buffer
L.
The
enzyme
was
further
purified
as
follows:
100
,l
portions
of
the
pooled
fractions
from
the
heparin-Sepharose
column
were
desalted
on
a
Sephadex
G-50
(fine)
column
and
then
loaded
on
a
poly(U)-Sepharose
column
(50
,ul)
equilibrated
in
50
mM
Tris/HCl,
pH
7.5,
2
mM
DTT,
10%
glycerol
and
0.5
mM
PMSF.
The
column
was
washed
with
the
same
buffer
and
then
with
increasing
concentrations
of
KC1
in
the
same
buffer.
Enzyme,
eluted
at
400
mM
KCI,
was
immediately
frozen
and
stored
in
small
aliquots
in
liquid
nitrogen.
The
final
preparation
had
a
specific
activity
of
1060
units/mg
and
there
was
no
contamination
by
nucleases.
It
had
a
sedimentation
coefficient
of
3.5.
Slot-blot
analysis
The
same
number
of
units
(2.5
units)
or
the
same
amount
of
proteins
(250
ng)
of
HSV1
and
human
uracil-DNA
glycosylases
were
loaded
on
a
nitrocellulose
filter
in
a
slot-blot
filtration
manifold
(Hoefer).
After
a
wash
with
phosphate-buffered
saline,
the
filter
was
removed
and
processed
as
described
(Towbin
et
al.,
1979).
The
blot
was
tested
with
1
,g/ml
anti-(human
uracil-
DNA
glycosylase)
monoclonal
antibody
(42.08.07).
Anti-(mouse
IgG)
conjugated
with
biotin
was
added,
and
the
blot
was
developed
with
streptavidin-alkaline
phosphatase
reagent
(Bio-
Rad).
[3H]Thymidine
Incorporation
Into
DNA
of
HeLa
cells
Cells
were
grown
in
suspension
at
37
°C
in
DMEM
containing
10%
fetal
calf
serum.
At
a
density
of
0.6
x
106/ml
cells
were
collected
by
centrifugation
and
suspended
in
fresh
medium
without
fetal
calf
serum
at
a
density
of
106/ml.
Aliquots
of
0.4
x
106
cells
were
incubated
at
37
°C
in
the
presence
of
40
,uM
[3H]thymidine
(25
#Ci/ml;
25
Ci/mmol)
in
the
absence
or
pres-
ence
of
inhibitors
at
various
concentrations.
At
0,
15,
30
and
45
min
80
,1
samples
of
culture
were
spotted
on
to
25
mm
GF/C
filters
(Whatman).
The
filters
were
washed
immediately
in
a
large
volume
of
ice-cold
5
%
trichloroacetic
acid.
The
filters
were
washed
twice
in
trichloroacetic
acid
and
twice
in
ethanol,
dried
and
counted
in
Omnifluor
scintillation
fluid.
Synthesis
of
Inhibitors
The
syntheses
of
several
of
the
compounds
tested
have
been
described
(Baker
and
Rzeszotarski,
1968;
Wright
and
Brown,
1980).
New
6-(p-alkylanilino)uracils
were
prepared
by
reaction
between
6-chlorouracil
and
thep-alkylanilines
(Aldrich
Chemical
Co.)
in
refluxing
2-methoxyethanol
as
described
(Wright
and
Brown,
1980).
Yields
and
properties
of
new
compounds
are
presented
in
Table
2.
Proton
n.m.r.
spectra
of
all
compounds
in
[2H6]dimethyl
sulphoxide
(300
MHz;
Varian
Unity
300
instru-
ment)
were
fully
consistent
with
the
proposed
structures.
RESULTS
Induction
of
HSV1
uracil-DNA
glycosylase
HSV1-infected
HeLa
cells
were
collected
at
0,
2,
4,
6,
8,
10,
12
and
18
h
post-infection,
and
their
cytoplasmic
and
nuclear
fractions
were
tested
for
uracil-DNA
glycosylase
activity.
Most
of
the
enzyme
activity
was
present
in
the
nuclear
fraction
(results
not
shown)
with
a
peak
at
8
h
after
infection
(Figure
1),
indicating
a
viral
induction
of
the
enzyme.
Purfflcation
of
HSVI
uracil-DNA
glycosylase
The
enzyme
was
purified
from
the
nuclei
of
HSVl-infected
HeLa
cells
at
the
peak
of
enzyme
induction,
as
described
in
the
Materials
and
methods
section,
and
Table
1
summarizes
the
yields
and
degree
of
purification
obtained.
Since
most
of
the
inducible
uracil-DNA
glycosylase
activity
was
observed
in
the
nuclei
we
used
only
the
nuclear
fraction
as
starting
material.
The
purification
sequence
resulted
in
an
enzyme
preparation
with
a
specific
activity
of
11080
units/mg
and
there
was
no
con-
tamination
by
nucleases.
For
comparative
studies,
the
human
uracil-DNA
glycosylase
was
purified
from
HeLa
cells
based
on
the
method
of
Krokan
and
Wittwer
(1981)
(see
the
Materials
and
methods
section).
The
latter
preparation
had
a
specific
activity
of
1060
units/mg,
with
no
contamination
by
nucleases.
The
lower
specific
activity
of
the
human
compared
with
the
viral
enzymic
preparation
may
be
due
to:
(i)
the
5-fold
higher
protein
con-
centration
of
the
human
crude
extract
derived
from
the
cellular
cytoplasm,
(ii)
the
low
levels
of
human
uracil-DNA
glycosylase
activity
in
non-infected
cells
compared
with
the
high
levels
of
the
viral-induced
activity
at
the
time
of
induction,
and
(iii)
the
885

886
F.
Focher
and
others
140
1
20
'5
100
80
D
.
o
60
<404
z
20
0
4
8
12
16
20
Time
post-infection
(h)
Figure
1
Induction
of
uracil-DNA
glycosylase
activity
In
nuclear
extracts
of
HeLa
cells
after
HSV1
Infection
At
each
time
point,
2
x
107
cells
were
collected,
sonicated
three
rtimes
at
100
W
in
3
vol.
of
10
mM
Tris/HCI,
pH
7.5,
10
mM
KCI,
1
mM
DTT
and
0.1
mM
EDTA,
containing
1
mM
PMSF
and
1
/M
pepstatin,
and
centrifuged
at
8000
g
for
10
min.
The
supernatant
is
the
cytoplasmic
fraction.
The
pellet,
containing
most
of
the
nuclei,
was
made
400
mM
in
KCI
in
3
vol.
of
the
same
buffer.
Sonication
and
centrifugation
were
repeated
as
above.
The
resulting
supernatant
is
the
nuclear
fraction.
HSV
H
A
=
B
C
Figure
3
Slot-blot
analysis
ot
HSV1
(lane
HSV)
and
human
(lane
H)
uracil-
DNA
glycosylases
A,
2.5
units
of
each
enzyme;
B,
0.22
,ug
of
total
protein
of
each
enzyme
preparation;
C,
2.5
units
of
each
enzyme
in
2.3
,ug
of
total
protein
(HSV1
enzyme
preparation
was
complemented
with
BSA).
The
filter
was
incubated
in
the
presence
of
10
,ug
of
monoclonal
anti-(human
uracil-
DNA
glycosylase)
antibody
42.08.07
(Arenaz
and
Sirover,
1983),
kindly
supplied
by
Professor
M.
Sirover,
as
described
in
the
Materials
and
methods
section.
Table
3
Effects
of
6-anilinouraclls
on
HSV1
and
human
uracil-DNA
glycosylases
The
IC5w
is
the
concentration
of
compound
that
caused
half-maximal
inhibition
of
[3H]uracil
release
from
[3H]dUMP-containing
DNA.
Assays
were
performed
as
described
in
the
Materials
and
methods
section
in
the
presence
of
various
concentrations
of
test
compounds.
Control
assays
contained
the
same
concentration
of
the
compound
solvent,
dimethyl
sulphoxide.
The
general
formula
is
given
below:
0
0HN
H
3
N
34
H
'C50
(,uM)
Substituent(s)
HSV1
Human
Figure
2
SDS/PAGE
of
human
(0.5
ug;
lane
H)
and
HSV1
(0.25
pg;
lane
HSV)
uracil-DNA
glycosylases
Electrophoresis
was
conducted
in
an
SDS/10%-polyacrylamide
gel,
and
proteins
were
stained
with
Coomassie
Blue.
Lane
M,
protein
standards:
1
u.tg
each
of
phosphorylase
b
(92.5
kDa),
BSA
(66.2
kDa),
ovalbumin
(45
kDa),
carbonic
anhydrase
(31
kDa)
and
soybean
trypsin
inhibitor
(21.5
kDa).
greater
stability
of
the
viral
uracil-DNA
glycosylase
during
the
purification.
The
electrophoretic
pattern
of
the
viral
enzyme
was
compared
with
that
of
the
human
enzyme
by
SDS/PAGE
(Figure
2).
Both
enzymes
showed
a
band
at
a
molecular
mass
of
37
kDa,
but
the
human
enzyme
had
higher
molecular
mass
bands
as
well.
This
apparent
molecular
mass
of
37
kDa
is
consistent
with
the
reported
molecular
masses
of
39
kDa
for
the
HSV1
enzyme
(Caradonna
et
al.,
1987)
and
37
kDa
for
the
human
placental
enzyme
(Seal
et
al.,
1987).
That
the
uracil-DNA
glycosylase
isolated
from
virus-infected
HeLa
cells
is
indeed
viral
in
origin
was
demonstrated
by
a
slot-
blot
experiment
with
a
monoclonal
antibody
to
the
human
placenta
enzyme
(Arenaz
and
Sirover,
1983).
Figure
3
shows
that
4-Hydroxy
3,4-Dimethoxy
3,4-Dichloro
3-Ethyl-4-methyl
4-n-Propyl
4-isopropyl
4-n-Butyl
(BuAU)
4-Isobutyl
4-n-Pentyl
4-isopentyl
4-n-Hexyl
(HexAU)
4-n-Heptyl
4-n-Octyl
(OctAU)
>
500
>
500
500
500
>500
>
500
150
400
30
140
30
20
8
the
human
HeLa
enzyme,
but
not
the
HSVI
with
the
monoclonal
antibody
42.08.07.
>
500
>
500
>
500
>
500
>
500
>500
>
500
>
500
250
200
>
300
140
>
300
enzyme,
reacted
Uracil
analogues
discriminate
between
human
and
HSV1
uracil-DNA
glycosylases
Screening
of
a
large
number
of
uracil
derivatives
and
related
compounds
against
the
purified
HSV1
and
human
HeLa
uracil-
(kDa)
92.5
-
66.2
-
45
-
31
-
21.5
-
M
H
HSV

Selective
inhibitors
of
Herpes
simplex
virus
type
1
uracil-DNA
glycosylase
100w
80
60
40
20
0
100
200
[HexAUl
(uM)
0
50
100
[OctAUI
(pM)
300
150
Figure
4
Dose-response
curves
tor
uracli-ONA
glycosylase
inhibition
by
6-(p-n-alkylanilino)uracils
The
HSV1
(0)
and
human
(0)
enzymes
were
assayed
as
described
in
the
Materials
and
methods
section
with
the
addition
of
stock
solutions
of
inhibitors
dissolved
in
dimethyl-sulphoxide.
Control
activity
corresponds
to
enzyme
activity
in
the
presence
of
an
identical
concentration
of
the
solvent.
PentAU,
6-(pn-pentylanilino)uracil;
HeptAU,
6-(p-n-heptylanilino)uracil.
DNA
glycosylases
revealed
several
6-anilinouracils
that
showed
weak
activity
against
the
viral
enzyme.
Many
substituted
derivatives
were
only
marginally
active
at
500
,uM
in
inhibiting
the
release
of
[3H]uracil
in
the
standard
uracil-DNA
glycosylase
assay
(see
the
Materials
and
methods
section),
but
6-(p-n-
butylanilino)uracil
(BuAU)
inhibited
the
viral
enzyme
by
50
%
at
150
,uM
and
was
ineffective
at
500
,uM
against
the
human
enzyme.
This
result
prompted
the
synthesis
and
testing
of
additional
p-
alkyl
derivatives
in
an
attempt
to
identify
more
potent
and
selective
inhibitors
of
the
HSV1
enzyme.
The
properties
of
these
new
compounds
are
summarized
in
Table
2.
The
results
of
testing
of
representative
6-anilinouracils
and
the
new
derivatives
are
summarized
numerically
in
Table
3,
and
the
selectivity
of
the
more
potent
inhibitors
is
displayed
graphically
in
Figure
4.
6-
Anilinouracils
with
n-alkyl
groups
in
thep
position
of
the
anilino
ring
were
progressively
more
potent
as
inhibitors
of
the
HSV1
enzyme
and
retained
a
high
degree
of
selectivity
for
the
viral
enzyme.
The
most
potent
compound,
6-(p-n-octylanilino)uracil
(OctAU)
had
an
IC50
of
8
,uM
against
the
viral
enzyme,
but
one
of
>
300
,uM,
the
highest
concentration
tested,
against
the
human
enzyme.
Interestingly,
the
n-heptyl
derivative
was
less
selective
than
the
n-hexyl
or
n-octyl
derivatives
(Figure
4).
Both
the
n-
hexyl
and
n-octyl
derivatives
were
found
to
be
inactive
against
viral
TK
and
DNA
polymerase
(results
not
shown).
Mechanism
of
Inhibition
Insight
into
the
mechanism
by
which
6-(p-alkylanilino)uracils
inhibit
HSV1
uracil-DNA
glycosylase
was
sought
by
studying
the
dependence
of
inhibition
on
the
concentration
of
the
substrate
in
the
reaction.
The
effect
of
OctAU
at
varying
concentrations
of
uracil,
as
dUMP
concentration
in
DNA,
are
displayed
in
the
double-reciprocal
plot
in
Figure
5.
The
results
suggest
that
the
-1
0
1
2
3
1/[Uracill
(M-1)
Figure
5
Double-reciprocal
plot
of
the
inhibitlon
of
HSV1
uracil-DNA
glycosylase
by
OctAU
as
a
function
of
substrate
concentraton
Assays
were
done
as
described
in
the
legend
to
Figure
4
in
the
presence
of
various
concentrations
of
[3H]dUMP-labelled
DNA.
Concentrations
of
OctAU
used:
0,
0uM;
*,
10,uM;
V.
16MuM.
inhibition
is
competitive
with
the
substrate,
perhaps
as
a
result
of
inhibitor
binding
to
the
catalytic
site
of
the
enzyme.
This
contrasts
with
the
reported
non-competitive
effect
of
uracil
itself
as
an
inhibitor
of
human
uracil-DNA
glycosylase
(Caradonna
and
Cheng,
1981).
Uracil
probably
represents
a
product
inhibitor
of
1004
80
60
40
20
c
0
0
4-
0
0
3
100
80
60
40
20
0
lPentAUI
(,M)
[HeptAUI
(uM)
0
....
I....
887

888
F.
Focher
and
others
E
100
E
0
80
0
4-
60
0
o
c
40
0.
20
2-
n
HexAU
15
30
45
lime
(min)
100
80
60
40
20
OctAU
15
30
Time
(min)
45
Figure
6
Effect
of
6-(p-n-alkylanilino)uraclls
on
short-term
incorporation
of
VH]thymIdIne
Into
DNA
of
HeLa
cells
In
culture
Cells
were
incubated
at
37
°C
in
medium
containing
inhibitors
or
inhibitor
solvent
and
[3H]thymidine.
Aliquots
were
removed
at
various
times
and
processed
as
described
in
the
Materials
and
methods
section.
Maximum
incorporation
at
45
min
corresponded
to
8.37
pmol/1
06
cells.
uracil-DNA
glycosylase,
whereas
the
anilinouracils
may
bind
to
the
enzyme
as
analogues
of
the
substrate.
Effect
of
uracil
analogues
on
short-term
V3H]thymidine
Incorporation
Into
DNA
of
HeLa
cells
Two
of
the
most
potent
inhibitors
of
HSVl
uracil-DNA
glycosylase,
6-(p-n-hexylanilino)uracil
(HexAU)
and
OctAU,
were
tested
with
cultured
HeLa
cells
in
order
to
evaluate
their
effect
on
DNA
synthesis.
Experiments
were
performed
as
de-
scribed
in
the
Materials
and
methods
section
to
evaluate
short-
term
[3H]thymidine
incorporation
into
the
DNA
of
untreated
and
inhibitor-treated
HeLa
cells.
The
results
depicted
in
Figure
6
show
that
HexAU
did
not
affect
DNA
synthesis
by
HeLa
cells
at
300
,#M,
but
OctAU
inhibited
DNA
synthesis
even
at
5
,uM.
The
apparent
IC50
for
the
effect
of
OctAU
was
about
25
,uM,
but
even
concentrations
as
high
as
300,uM
did
not
completely
suppress
thymidine
incorporation
(Figure
6).
The
effect
of
OctAU
on
cellular
DNA
synthesis
is
similar
to
that
previously
reported
for
the
analogue
BuAU,
a
selective
inhibitor
of
DNA
polymerase
a
(Wright
et
al.,
1980).
BuAU
inhibited
the
incorporation
of
[3H]thymidine
by
HeLa
cells
in
culture,
but
not
incorporation
of
labelled
uridine
or
leucine,
markers
for
RNA
and
protein
synthesis
respectively.
Thus,
although
OctAU
is
a
more
potent
inhibitor
of
HSVl
uracil-DNA
glycosylase
than
HexAU,
the
latter
compound
lacks
an
effect
on
cellular
DNA
synthesis
at
concentrations
at
which
the
viral
enzyme
is
strongly
inhibited.
The
unexpected
observation
that
OctAU
inhibited
[3H]-
thymidine
incorporation
by
HeLa
cells
in
culture,
and
the
similarity
of
its
structure
to
that
of
a
selective
inhibitor
of
DNA
polymerase
a,
BuAU
(Wright
et
al.,
1980),
prompted
us
to
ask
if
this
new
derivative
inhibited
one
or
more
of
the
eukaryotic
replicative
DNA
polymerases
a,
a
and
e.
In
standard
DNA
polymerase
reactions
with
nicked
DNA,
assayed
as
described
(Weiser
et
al.,
1991),
OctAU
had
little
effect
on
HeLa
DNA
polymerase
a
(IC50
>
500
uM),
but
inhibited
HeLa
DNA
polymerase
e
with
an
IC50
of
80
,uM.
HexAU,
on
the
other
hand,
weakly
inhibited
both
enzymes
(IC50
>
500
,M)
under
these
conditions,
thus
explaining
the
lack
of
effect
of
this
compound
on
cellular
DNA
synthesis.
Furthermore,
both
DNA
polymerases
e
and
a
[proliferating
cell
nuclear
antigen
(PCNA)-dependent]
from
calf
thymus
were
more
sensitive
to
OctAU
than
polymerase
a
(results
not
shown).
These
results
suggest
that
OctAU
may
represent
a
prototype
of
selective
inhibitors
of
DNA
polymerase
a
and/or
e,
just
as
BuAU
was
the
prototype
of
a
series
of
useful
selective
inhibitors
of
DNA
polymerase
a
(Brown
et
al.,
1986).
DISCUSSION
The
DNA
repair
enzyme
uracil-DNA
glycosylase
has
been
purified
from
HSVl-infected
HeLa
cells.
By
comparison
with
the
enzyme
isolated
from
uninfected
HeLa
cells,
this
enzyme
has
been
shown
to
be
of
viral
origin
by
virtue
of:
(1)
its
time-
dependent
induction
upon
virus
infection
of
cells
(Figure
1);
(2)
its
apparent
molecular
mass
of
37
kDa
(Figure
2),
and
(3)
its
insensitivity
to
a
monoclonal
antibody
to
the
human
enzyme
(Figure
3).
The
enzyme
further
differs
from
its
cellular
counter-
part
in
its
potent
and
selective
inhibition
by
6-(p-n-alkylanilino)
uracils
(Table
3),
compounds
that
appear
to
act
mechanistically
as
substrate analogues.
The
enzyme
isolated
from
HeLa
cells
was
consistently
less
sensitive
to
inhibition
by
all
compounds
in
this
series.
Few
inhibitors
of
uracil-DNA
glycosylases
have
been
reported.
Among
a
group
of
simple
pyrimidines,
only
uracil
was
found
to
inhibit
the
enzyme
from
leukaemic
blast
cells
(Caradonna
and
Cheng,
1980);
uracil
caused
81
%
inhibition
at
1
mM,
and
was
non-competitive
with
the
substrate
(as
[dUMP]).
Uracil,
6-
aminouracil
and
5-azauracil
were
reported
to
inhibit
uracil-DNA
glycosylase
from
HeLa
cells,
but
with
IC50
values
of
1-2
mM
(Krokan
and
Wittwer,
1981).
In
contrast,
5-fluorouracil
and
5-
bromouracil
at
1
mM
inhibited
uracil-DNA
glycosylase
from
human
placenta
only
after
preincubation
with
the
enzyme
at
4
°C
(Seal
et
al.,
1987).
Neither
the
2'-deoxyribonucleosides
nor
the
corresponding
5'-monophosphates
of
the
5-halouracils
substan-
tially
inhibited
the
placenta
enzyme
even
after
the
preincubation
period.
The
results
in
the
present
paper
reveal
the
first
potent
and
selective
inhibitors
of
the
viral
uracil-DNA
glycosylase
that
can
be
used
as
probes
of
the
mechanism
of
enzyme
action,
and
these
may
prove
to
be
useful
in
studying
the
role
of
this
enzyme
in
virus
growth
and
re-activation.
It
is
known
that
uracil
can
arise
in
DNA
by
two
mechanisms:
(i)
the
misincorporation
of
dUMP
residues
by
DNA
polymerases,
u

Selective
inhibitors
of
Herpes
simplex
virus
type
1
uracil-DNA
glycosylase
and
(ii)
the
spontaneous
deamination
of
cytosine.
In
particular
with
regard
to
the
nervous
system,
where
herpes
viruses
establish
latency,
it
is
worthwhile
to
recall
that
the
only
DNA
polymerase
present
in
adult
neurons,
DNA
polymerase
/3,
in
contrast
to
the
replicative
DNA
polymerases
a
and
e
(which
utilize
dUTP
70-80
%
less
efficiently
than
dTTP),
does
not
discriminate
between
dUTP
and
dTTP
(Focher
et
al.,
1990).
This
suggests
that
dUTP
could
be
introduced
into
DNA
during
DNA
repair
in
adult
neurons,
although
most
of
the
uracil
in
DNA
results
from
deamination
of
cytosine.
These
observations
suggest
that
dUMP
residues
may
accumulate
in
DNA
during
the
life-span
of
adult
neurons
(Mazzarello
et
al.,
1990).
The
continuous
spontaneous
deamination
of
cytosine,
the
fact
that
both
viral
DNA
polymerase
(Focher
et
al.,
1992)
and
DNA
polymerase
,3
(Focher
et
al.,
1990)
incorporate
dUTP
and
dTTP
with
comparable
efficiency,
and
the
lack
of
cellular
uracil-DNA
glycosylase
in
nerve
cells
(Focher
et
al.,
1990)
suggest
a
role
of
the
virus-encoded
uracil-DNA
glycosylase
in
the
re-activation
and
replication
of
Herpes
simplex
virus
in
nerve
cells.
This
role
is
supported
by
our
finding
that
uracil
in
DNA
affects
specific
DNA-protein
interactions
(Verri
et
al.,
1990;
Focher
et
al.,
1992).
In
conclusion,
we
hypothesize
that
HSV1
uracil-DNA
glycosylase,
which
is
non-essential
for
the
proliferation
of
HSV1
in
cell
cultures,
could
play
a
key
role
in
nerve
cells
in
'cleansing'
of
the
viral
genome
before
DNA
replication
as
well
as
in
the
removal
of
misincorporated
uracil
during
viral
DNA
replication.
We
intend
to
use
the
specific
inhibitors
described
in
this
paper
in
order
to
verify
the
role
of
viral
uracil-DNA
glycosylase
during
virus
re-activation
and
replication
in
nerve
cells
in
vivo.
One
derivative,
HexAU,
by
virtue
of
its
lack
of
effect
on
cellular
DNA
synthesis
and
on
viral
TK
and
DNA
polymerase
at
concen-
trations
that
completely
inhibit
the
viral
enzyme,
has
been
selected
for
such
studies.
This
work
was
supported
by
an
ISS-AIDS
grant
and
by
the
P.F.
CNR
RAISA,
Biotecnologie
e
Biostrumentazione
and
Chimica
Fine.
A.V.
was
supported
by
a
fellowship
from
the
P.F.
CNR
Biotecnologie
e
Biostrumentazione.
The
n.m.r.
instrument
was
purchased
with
Shared
Instrumentation
Grant
RR04659
from
the
National
Institutes
of
Health.
We
thank
Dr.
M.
A.
Sirover
for
the
gift
of
anti-(human
uracil-DNA
glycosylase)
monoclonal
antibody.
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accepted
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889