239
J. gen. Virol. (I974), 25, 239-244
Printed in Great Britain
The Effect o f Isatin fl Thiosemicarbazone (IBT)-related C o m p o u n d s
on IBT-resistant and on IBT-dependent Mutants o f Vaccinia Virus
By E. K A T Z , EVA M A R G A L I T H AND B E L A W I N E R
The Chanock Centre for Virology, The Hebrew University - Hadassah Medical School
Jerusalem, Israel
(Accepted 24 July I974)
SUMMARY
IBT-related compounds were examined for their ability to inhibit wild-type
and IBT-resistant mutants of vaccinia virus and for their capability to support the
growth of an IBT-dependent mutant. Among thirteen compounds tested, six did
not affect any one of the three virus strains and five behaved similarly to IBT, but
two inhibited the growth of all three viruses and therefore differed from IBT. It was
found that the Iast two compounds also differed from IBT in that they interfered
with synthesis of the virus DNA. This difference is surprising in view of a substantial
similarity in chemical structure.
INTRODUCTION
Vaccinia virus growth is inhibited by IBT (Sheffield, Bauer & Stephenson, 196o). The
mechanism of action of this antiviral drug is not yet clear. It was found that early events in
poxvirus replication such as virus DNA synthesis, are not interrupted by the drug (Easterbrook, I96I ; Appleyard, Hume & Westwood, I965; Woodson & Joklik, I965). Woodson &
Joklik (1965) found that polyribosomes that are formed by late mRNA are rapidly broken
down in the presence of IBT. Recently we found that several late virus polypeptides are
made in the presence of IBT, and this suggested that the block in virus replication caused by
IBT occurred at very late stages of virus maturation (Katz et al. I973 a). We also isolated two
mutants of vaccinia virus, one resistant to IBT and the other dependent on the drug for
growth (Katz et al. I973 b, c; Katz, Margalith & Winer, t973). This paper presents a study
of the effects of several IBT-related compounds on these two mutants and on the wild type
strain.
METHODS
Cells. HeLa S-3 and BSCI cells were grown in monolayer cultures in MI99 medium
supplemented with IO % calf serum.
Virus. Stocks of WR strain (wild t y p e - wt), IBT dependent (iBTD) and IBT resistant
(IBTR) mutants of vaccinia virus were prepared in HeLa cells and titrated on BSCI monolayers, as previously described (Katz et aL 1973 c).
Assay of virus. BSCI cell monolayers in 60 mm plastic Petri dishes (Nunc, Denmark)
were infected with serial end-point dilutions of vaccinia virus strains. After 45 min at 37 °C,
the monolayers were overlayed with Eagle's media containing 1% Special Agar Noble (Difco
Laboratories, Detroit, Mich.) and 5 % inactivated calf serum. Neutral red (o.0o25 %) was
added 4 days after infection and plaques were counted on the following day.
Virus DNA synthesis. At different times after infection, HeLa cells in 60 mm Petri dishes
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24o
E. K A T Z , E. M A R G A L I T H
A N D B. W I N E R
were incubated for Io rain with 20 #Ci of [3H]-thymidine (I8"9 mCi/mmol, Amersham,
England). The pulses were terminated by cooling the cultures and washing the cells with cold
saline. The cells were resuspended in I ml of IO mM-tris (hydroxymethyl) aminomethane,
I o mM-NaC1 and I "5mM-MgCI~,pH 7"4 (RSB), containing 0"5 % NP4o. Nuclei were removed
by sedimentation at iooo g for two min. Radioactivity of trichloroacetic acid precipitable
material from the cytoplasm of the cells was measured by liquid scintillation counting.
Chemicals. Isatin fl-thiosemicarbazone was obtained from Mann Research Laboratories,
New York, N.Y.; formylthiosemicarbazide and 4-formylacetanilide thiosemicarbazone
from K. and K. Laboratories Inc., Plain View, N.Y. ; thiourea from Riedel De Haen AG,
Seelze, Hanover, Germany; thiosemicarbazide from Hopkin and Williams Ltd, Essex,
England and semicarbazide from Eastman Kodak Co. Rochester, N.Y. The compounds
5-formylthiazole thiosemicarbazone, 5-bromoisatin 3-thiosemicarbazone, I-methylisatin-3semicarbazone and isatin-4'-methyl-3-thiosemicarbazone were kindly donated by D.J.
Bauer, Wellcome Research Laboratories, Kent, England. Picolinaldehyde-5-hydroxythiosemicarbazone (NSC IO7392) was donated by H. B. Wood, National Institutes of Health,
Bethesda, Md; 5-cyanothiophene-2-carboxaldehyde thiosemicarbazone (Hoe IO5) by H.
Rolly, Farbwerke Hoechst AG, Frankfurt, Germany; I-formylisoquinoline thiosemicarbazone (IQ-I) by F. A. French, Mount Zion Hospital Medical Center, Palo Alto, Calif. and
7-thiochromanone-4-thiosemicarbazone by H. Isoyama, Meiji Seika Ltd, Tokyo, Japan.
RESULTS
The ability of the three virus strains, wild type, IBT-resistant (IBT~) and IBT-dependent
(IBTD) mutants, to form plaques on BSCt cell monolayers treated with IBT-related compounds was examined (Table T, 2). it is possible to divide the compounds into three groups:
(I) compounds which behave similarly to IBT, i.e. inhibit the growth of wt strain, do not
affect the plaque formation of IBT ~ and support the growth of IBTD; (2) compounds which
do not inhibit the growth ofwt strain and do not support the growth of IBTD; (3) compounds
which inhibit the growth of all three virus strains.
The first group of compounds, which behave similarly to IBT, includes: 5-formylthiazole
thiosemicarbazone, 5-bromoisatin-3-thiosemicarbazone, 4-formylacetanilide thiosemicarbazone, 5-cyanothiophene-2-carboxaldehyde thiosemicarbazone and y-thiochromanone-4thiosemicarbazone (Table Q. All these compounds are composed of a combination of
a thiosemicarbazone chain and a closed ring. The thiosemicarbazone side chain, in all these
compounds, has the structure--CNNHCSNH2 while the structure of the attached ring is
I
quite different from one to another.
The antiviral activity against poxviruses of compounds of the first group was previously
reported: that of 5-formylthiazole thiosemicarbazone and of 5-bromoisatin 3-thiosemicarbazone by D. J. Bauer (t963, and personal communication); that of 5-cyanothiophene-2carboxaldehyde thiosemicarbazone by Rolly & Winkelman (I97I) and that of 7-thiochromanone-4-thiosemicarbazone by Tsunoda et al. (~97I).
The second group of compounds, which do not affect the growth of any strain, includes:
thiourea, thiosemicarbazide, semicarbazide, formylthiosemicarbazide, isatin-4'-methyl3-thiosemicarbazone and i-methylisatin-3-semicarbazone (Table r, 2). Among these
compounds one can find a compound which consists just of the side chain of isatin fl-thiosemicarbazone (thiosemicarbazide), a compound which is a shortened side chain (thiourea),
and a compound which is a modification of thiosemicarbazide, containing oxygen instead of
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I B T mutants o f vaccinia virus
241
Table I, Effects of IBT-related compounds on wt, IBT ~ and IBT ~ strains of vaccinia virus
Group
Compound
Cone.
(pM)
wt
Virus growth*
IBT D IBT g
S
I
Group
Compound
Cone.
Virus growth*
(pM)
wt
14
+
N.D.t
14
+
N.D.
14
+
-
N.D.
14
+
--
N.D,
14
+
--
N.D.
14
+
--
N.D.
14
-
14
-
IBT D IBT R
H
S
--NH--C--NHz
14
-
+
II
+
HzN--C--NHz
thiourea
H
isatin-3-~hiosemicarbazone
S
II
H2N--NH--C--NHz
thiosemicarbazide
CH~N--NH--
--NHz
14
-
+
+
O
II
H 2 N - - N H - - C --NHz
5-formylthiazole thiosemicarbazone
S
II
Br~C~N--NH--C
semicarbazlde
--NHz
14
-
+
O
S
It
II
H--C--NH--NH--C--NH2
+
for mylt hiosemica rbazide
H
5-bromoisatin-3-t hiosemicarbazone
S
II
C=N--NH--C--NH
~
S
It
HC = N - - N H - - C - - N H 2
112
--
+
+
=O
CH3
N
H
isatin 4' methyl 3-thlosemlcarbazone
O
II
~
NH--C--CHs
11
O
4-formylacetanilide thiosemicarbazone
\=N--NH--C--NH,
NC=O
I
CH3
N---- C-g.,, S ~ - . C H = N--N H - - C --N Hz
56
-
+
l-methylisatin~3-semicarbazone
+
5-cyanot hlophene-2-earboxaldehyde
thiosemicarbazone
/H
S
Cf
S
II
17" - C H = N - N H - C - N H *
HO'--'K'~,,#
N--NH--C--NH2
3"5
--
+
+
picolinaldehyde-5-hydroxy
thiosemicarbazone
7 tthiochromanone-4-thiosemiearbazone
CH~N--NH--C--NH2
l-formylisoquinoline ~hlosernic~rbazoae
* Virus growth (p.f.u./ml) was determined as described in Methods. IBT and the other IBT-related compounds were supplemented in the agar overlay. The/ZMvalues given for compounds included in group I are
the highest active non-toxic concentrations.
t Not done.
sulphur (semicarbazide). This group of compounds also includes formyl-thiosemicarbazide,
which has a formyl group instead of the isatin and also two compounds which, although
having an aromatic ring, have changes in the thiosemicarbazone structure: one is isatin-4'methyl-3-thiosemicarbazone, in which the hydrogen of the N terminal is exchanged with a
methyl group and another is I-methylisatin-3-semicarbazone, in which sulphur was replaced
by oxygen in the thiosemicarbazone residue of the molecule.
The two compounds that we found to inhibit the growth of all three virus strains (wt,
IBT ~ and IBT D) are picolinaldehyde-5-hydroxythiosemicarbazone and ~-formylisoquinoline
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242
E. KATZ~E. M A R G A L I T H AND B. W I N E R
Table 2. Growth of wt, I B T ~ and I B T n strains o f vaccinia virus in the
presence o f I B T and two I B T related compounds
Virus growth*
¢
Group
I
Compound
Wild type
Isatin-~-thiosemicarbazone
II
Thiosemicarbazide
III
Picolinaldehyde-5-hydroxy
thiosemicarbazone
None
Control
<
10 5
I'3X
IO 7
< lO5
1'5 X
IO 7
IBT •
I'I X I0 s
9"OX IO 7
< Io5
I'3
× I05
IBTD
I'I X I0 7
<
10 5
< lO5
<
105
* Virus growth (p.f.u./ml) was determined as described in Methods. IBT and the two IBT-related compounds were supplemented in the agar overlay at a concentration of I4/ZM.
I
I
1
2
I
I
!
3 -
4-"
I
×
j
.-~ 2 -
,2.,
I --
0
3
4
5
Time (h)
Fig. I. Effect of picolinaldehyde-5-hydroxythiosemicarbazone( 0 - - ( 3 ) and I-formylisoquinoline
thiosemicarbazone (m--m) on virus strain wt DNA synthesis as measured by Io min pulses of
[3H]-thymidine given at the indicated times. The drugs were added at a concentration of 28 #M,
following adsorption of the virus for 3o min at 37 °C. 0 - - 0 , untreated infected cells; & - - A ,
uninfected cells.
t h i o s e m i c a r b a z o n e (Table I, 2). W e l o o k e d for the step in virus replication which is inhibited
b y these c o m p o u n d s in o r d e r to find o u t w h e t h e r it is similar to the one b l o c k e d b y IBT.
Vaccinia virus D N A is synthesized in H e L a cells m a i n l y between 2 a n d 5 h after infection
(Salzman, 196o; J o k l i k & Becker, I964). Since vaccinia virus multiplies in the c y t o p l a s m o f
the cell, it is possible to follow virus D N A synthesis b y d e t e r m i n a t i o n o f the i n c o r p o r a t e d
t h y m i d i n e into c y t o p l a s m i c trichloroacetic acid-precipitable material, after s h o r t pulses
(Joklik & Becker, i964). Infected cells were pulsed with r a d i o a c t i v e thymidine, a n d cytop l a s m i c fractions were p r e p a r e d b y lysing the cells in RSB c o n t a i n i n g the non-ionic detergent
N P 4 o (o'5 %). The nuclei were then r e m o v e d b y s e d i m e n t a t i o n (Zylber & P e n m a n , I97o).
The q u a n t i t y o f radioactively labelled D N A in the c y t o p l a s m i c fraction was m e a s u r e d after
trichloroacetic acid precipitation.
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243
[ B T mutants o f vaccinia virus
Table 3. Growth o f wt, I B T n and I B T ~ vaccinia virus strains and o f attenuated
poliomyelitis virus type I in the presence of I B T or two IBT-related compounds
Virus growth*
22 h
A
r
Virus strain
Waccinia wt
Vaccinia IBT a~
Vaccinia IBT D
Polio I
End of
adsorption
period
9'0 × I 0 5
--
IBT
1"3 x i o 6
I ' 2 X IO 6
5 ' 0 X 108
6"5 × IO 8
8" 5 × IO 5
8" 5 X I 0 5
3"8 × IO 8
5 " 0 X IO 6
2"0)< IO 6
I'7×
6"5× lO 7
I08
Picolinaldehyde5-hydroxy
thiosemicarbazone
I'0 × I@
4 ' 3 × IO5
6'5 x I o 5
I'2X
IOa
I-formylisoquinoline
thiosemicarbazone
1"9× I 0 5
I ' O × IO 6
× Io 5
4'8
3'8×
IO 7
* HeLa cells in 60 m m plastic Petrie dishes were infected with 2 p.f.u./cell o f w t , IBT ~, IBT D, or poliovirus
type I. Following adsorption for 3o min at 37 °C, 5 ml o f M 199 medium, were added, supplemented with 2 %
calf serum and with IBT, picolinaldehyde-5-hydroxy thiosemicarbazone or l-formylisoquinoline thiosemicarbazone at a concentration o f 28 #M. Virus infectivity (p.f.u./culture) at the end o f the adsorption period
and 22 h after infection was assayed as described in Methods. To the cultures in which IBT D was assayed
IBT (I4 #M) was added to the agar overlay.
D N A synthesis in wild type virus-infected cells started between ~ and 2 h after infection,
and the rate of synthesis reached the highest level at 3 h and then declined (Fig. I). Picolinaldehyde-5-hydroxythiosemicarbazone and I-formylisoquinoline thiosemicarbazone virtually
completely inhibit vaccinia virus D N A synthesis (Fig. I). These findings are well correlated
with previously reported observations indicating that these compounds are inhibitors of D N A
synthesis during S phase in cell division and also of D N A viruses of the herpes family
(Brockman et al. I97O; DeConti et al. I97Z). Since these two compounds are inhibitors of
vaccinia virus, a DNA-containing virus, we examined in parallel their effect on the growth
of poliomyelitis virus, an RNA-containing virus. Table 3 indicates that neither picolinaldehyde-5-hydroxythiosemicarbazone or I-formylisoquinoline thiosemicarbazone inhibited
the replication of poliomyelitis virus. This is a proof that the effect of these compounds was
not just unselective toxicity on the host cell, since an RNA-containing virus was able to grow
while the growth of a DNA-containing virus was inhibited in the drug-treated cells.
DISCUSSION
The aim of the present study was to find out whether vaccinia strains wt, IBT ~ and IBT D
were similarly affected by IBT and several IBT-related compounds. The activities of these
compounds fell into three groups. All five compounds of group I were exactly similar to IBT
in their effect on the three strains of vaccinia virus, and accordingly are very likely to act in
the same step in virus replication. Six compounds (group II) did not inhibit the growth of
the wild type strain but also could not support the growth of the IBT-dependent mutant.
Two compounds (group III) inhibit the growth of all three strains, and therefore probably
act on a step in replication different from that of IBT. This was confirmed by showing that
the compounds of group III inhibit initial steps (i.e. D N A synthesis) in vaccinia virus replication, while IBT is an inhibitor of late stages of virus maturation. It is suggested that the
thiosemicarbazone part of these two molecules does not play a primary role in their antiviral
activity and that their involvement in nucleic acid metabolism would be caused by the other
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244
E. K A T Z , E. M A R G A L I T H
A N D B. W I N E R
p a r t o f t h e m o l e c u l e . A f u r t h e r s t u d y o f t h e a n t i v i r a l effects o f d i f f e r e n t p a r t s o f t h e s e t w o
c o m p o u n d s will c l a r i f y t h e i r i n v o l v e m e n t i n t h e i n h i b i t i o n o f v a c c i n i a v i r u s g r o w t h .
W e w o u l d like t o t h a n k D r N . G o l d b l u m f o r his a d v i c e a n d e n c o u r a g e m e n t i n t h e c o u r s e
o f this study. The w o r k was s u p p o r t e d by a g r a n t f r o m the Ministry o f Health, State o f
Israel.
REFERENCES
APPLEVARD, ~., HUME,V. B. M. & WESTWOOD,J. C. N. 0965). The effect of thiosemicarbazones on the growth
of rabbitpox virus in tissue culture. Annals of the New York Academy of Sciences x3o, 92-IO4.
BAUER, O. J. (I963). The chemotherapy of ectromelia infection with isatin `8-dialkylthiosemicarbazones. The
British Journal of Experimental Pathology 44, 233-242.
BROCKMAN, R. W., SrDWELL, R. W., ARNETT, ~. & SHADDIX, S. (I970). Heterocyclic thiosemicarbazones: correlation between structure, inhibition of ribonucleotide reductase and inhibition of D N A viruses,
Proceedings of the Society for Experimental Biology and Medicine x33, 6o9-614.
DECONTI~ R. C., TOFTNESS, B. R.~ AGRA~VAL~K. C., TOMCHICK~R.~ MEAD, J. A. R., BERTINO, J. R.~ SARTORELLI~
A. C. & CREASEY, W. A. 0972). Clinical and pharmacological studies with 5-hydroxy-2-formylpyridine
thiosemicarbazone. Cancer Research 32, I455-1462.
EASTERBROOK,K. B. 0961). Interference with the maturation of vaccinia virus by isatin ,8-thiosemicarbazone.
Virology x7, 245-25I.
SOKLnq W. K. & BECKER,Y. (1964). The replication and coating of vaccinia DNA. Journal of Molecular Biology
xo, 452-474.
KATZ, r., MAR~ALIT~, E. & WINER, B. (I973). An isatin `8-thiosemicarbazone (IBT)-dependent mutant of
vaccinia virus: the nature of the IBT-dependent step. Journal of General Virology 2x, 477-484.
KATZ, E., MAROALITH,r., WINER, B. & ~OLDBLUM, N. 0973a). Synthesis of vaccinia virus polypeptides in the
presence of isatin-fl-thiosemicarbazone. Antimicrobial Agents and Chemotherapy 4, 44-48.
KATZ, E., MARGALITH,E., WINER, B. & LAZAR,A. (I973b). Characterization and mixed infections of three strains
of vaccinia virus: wild type, IBT-resistant and IBT-dependent mutants. Journal of General Virology 2x,
469-475.
KATZ, E., WINER, B., MARGALITH, E. & GOLDBLUM, N. (I973e). Isolation and characterization of an IBTdependent mutant of vaccinia virus. Journal of General Virology x9, 16I-I64.
ROLLY,H. &WINKELMAN,E. (197I). A new thiosemicarbazone active in vivo against vaccinia virus. In Advances'
in Antimicrobial and Antineoplastic Chemotherapy 3o5-3o7. Edited by M. Hejzlar, M. Semonsky &
S. Masak. Munchen and Berlin: Urban & Schwarzenberg.
SALZMAN,Y. P. (I960). The rate of formation of vaccinia deoxyribonucleic acid and vaccinia virus. Virology
io, I5O-I52.
SHEFFIELD,R. W., BAUER,D. J. & STEPHENSON,S. (1960). The protection of tissue cultures by isatin ,8 thiosemicarbazone from the cytopathic effects of certain pox viruses. The British JournalofExperimental Pathology
4x, 638-647.
TSUNODA, A., MIYAZAKI,K., AOTA, T., MATSUMOTO,S., KUMAGAI,K. & 1SHADA,N. (I97I). Antivaccinia activity
of 3' thiochromanone-I-thiosemicarbazone in vitro and in vivo. Japanese Journal of Microbiology x6,
61-66.
WOODSO~, B. & JOKLIK, W. K. (I965). The inhibition of vaccinia virus multiplication by isatin fl thiosemicarbazone. Proceedingsof the National Academy of Sciences of the United States of America 54, 946-953.
ZYLBER, E.a. & PENMAN, S. (I970). The effect of high ionic strength on monomers, polyribosomes, and
puromycin-treated polyribosomes. Biochimica et Biophysica Acta o-o4, 221-229.
(Received 3 April I974)
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