Effects of Some Antiviral Isatinisothiosemicarbazones on

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ANTnMcIoBiAL AGENTS AND CHEMOTHERAPY, Apr. 1974, p. 393-397 Copyright i 1974 American Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Effects of Some Antiviral Isatinisothiosemicarbazones on Cellular and Viral Ribonucleic Acid Synthesis in Mengovirus-Infected FL Cells MARION TONEW MD E.

1 ANTnMcIoBiAL AGENTS AND CHEMOTHERAPY, Apr. 1974, p Copyright i 1974 American Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Effects of Some Antiviral Isatinisothiosemicarbazones on Cellular and Viral Ribonucleic Acid Synthesis in Mengovirus-Infected FL Cells MARION TONEW MD E. TONEW GDR Academy of Science, Research Center for Molecular Biology and Medicine, Central Institute for Microbiology and Experimental Therapy, Jena, German Democratic Republic Received for publication 12 September 1973 Three antiviral isatinisothiosemicarbazones strongly inhibited the incorporation of [8H ]uridine into the ribonucleic acid (RNA) of FL cells as a consequence of the inhibition of uridine transport. After prelabeling of cells at a low temperature (1 h at 16 C) with uptake of [3H]uridine into the acid-soluble nucleotide pool, the later addition of the test compounds revealed only a sniall or negligible influence on host-directed RNA synthesis. The pulse-labeled soluble nucleotide pool of FL cells was sufficient to give a gradual increase in incorporation into RNA over a period of 7 h. With the same method of prelabeling at the beginning of the experiment, it was also possible to detect virus-induced RNA synthesis in the presence of actinomycin D. In this way the specific inhibitory action of the three isatinisothiosemicarbazones on viral RNA synthesis could be demonstrated. The antiviral activity of some isatinisothiosemicarbazones (IITS) against mengovirus in FL cells has been described in detail previously (20). The present paper is concerned with the influence of these substances on ribonucleic acid (RNA) synthesis in noninfected and mengovirus-infected cells. For the measurement of the rate of RNA synthesis, [3H ]uridine is generally used. However, this labeled substance is not a direct precursor. First, it must be taken up into the cell; secondly, there are some phosphorylation steps to uridine 5'-triphosphate (UTP) which must occur before incorporation into RNA takes place. Recently, it has been shown that a number of compounds inhibit the incorporation of uridine into RNA by interfering with these steps, i.e., uridine transport into the cell or the subsequent phosphorylation, without any real inhibition of RNA synthesis itself (3-5, 7, 10-12, 14, 16, 17). By controlling the incorporation of [H Juridine in the presence of different HTS in the experiments, a remarkable reduction in the incorporation into acid-insoluble material was found (more than 80%). However, when cells were supplied in advance with uridine, which was phosphorylated for the most part, it was not possible to find such a strong inhibitory effect of the tested compounds on host-directed RNA 393 synthesis. Therefore, one cannot interpret the above fmdings as indicative of the true inhibition of cellular RNA synthesis. On the other hand, the results of the present report demonstrate the specific inhibitory action of 1ITS on viral RNA synthesis. MATERIALS AND METHODS Test compounds. Compound no. 7 is 1-ethylisatin- S-ethyl-isothiosemicarbazone, no. 9 is 1-ethylisatin- S-n-butyl-isothiosemicarbazone, and no. 5 is 1- methyl-isatin-s-ethyl-isothiosemicarbazone. Synthesis was carried out according to Heinisch et al. (1, 2). Chemicals. Actinomycin D was obtained from the Zentralinstitut fur Mikrobiologie und experimentelle Therapie, Jena, GDR; sodium deoxycholate was from VEB Chemisches Werk, Berlin-GrUnau, GDR; filter disks of chromatography paper FN 3, 90 to 100/30 min, were from VEB Niederschlag/thiUr., GDR; [5-'Hluridine was from Radiochemical Centre, Amersham, England (specific activity of 5.83 Ci/mmol); and PPO and dimethyl-popop were from Packard Instruments International S. A., Zurich, Switzerland (for toluene scintillator). FL cells and mengovirus. Cultivation methods and media were used as described previously (18, 19). Incorporation of [5-'Hluridine. For method A, FL cells were labeled from 0 to 6 h in the presence of the test compounds. The radioactivity of acid-insoluble material was determined at different times and compared with untreated controls (18). For method B, FL cells were prelabeled with 1 ACi of uridine per tube (ca. 400,000 cells) at 4 or 16 C for 30 to 90 min. Residues of the labeled precursor were carefully washed out three times with cold, phosphate-buffered

2 394 TONEW AND TONEW saline. The test compounds were dissolved in fresh, cold Eagle minimal essential medium and added at 0 h. Their influence on cellular RNA synthesis was studied at different times by analysis of three tubes from each group. Virus infection was carried out with a multiplicity of infection of approximately 40 mean tissue culture infective doses per cell at 16 C in the presence of [3HJuridine and also actinomycin D (0.2 ILg/ml) if indicated. Further treatment was as described under method B. The inhibitory effect of the compounds on virus multiplication was controlled in parallel experiments by estimation of the virus yield in the usual manner (19). Determination of radioactivity. Radioactivity was estimated as described by Tonew and Bleecken (18) when the values are given in disintegrations per minute or as follows. The monolayer was shaken off from the glass wall with 0.15% sodium deoxycholate in distilled water for nearly 5 min, precipitated with 6% trichloroacetic acid, collected on filter disks, and washed three times with 6% trichloroacetic acid and then with ethanol and ethyl ether. The paper disks were placed into vials containing 5 ml of toluene scintillator and subsequently analyzed for radioactivity (counts per minute) by using a Packard Tri-Carb liquid scintillation counter. V Q, ZS ANTIMICROB. AG. CHEMOTHER. RESULTS Incorporation of [3H]uridine into cellular RNA of FL cells in the presence of several IITS. Figure 1 demonstrates the effect of IITS on the incorporation rate of [3H ]uridine when isotope and test compounds were added simultaneously at 0 h. In this case, a remarkably reduced incorporation of the radioactive precursor into cellular RNA was found when compared with the untreated control. At the highest concentrations used, only 6 to 18% of the radioactivity was detected in the acid-insoluble material at the sixth hour. The following experiments will show that this cannot be considered to be a true inhibition of cellular RNA synthesis. Incorporation of [3H]uridine into cellular RNA of FL cells prelabeled at low temperature. [3H]uridine is not a direct precursor of RNA synthesis as mentioned above. However, at low temperature it has been shown to be taken up into the cells and also phosphorylated to uridine 5'-monophosphate, uridine 5'-diphosphate, and UTP, without significant incorporation into RNA (6, 8, 13). Therefore, a prelabeling of cells at low temperatures accompanied with uptake of the radioactive nucleoside into the soluble cellular nucleoside pool, followed by addition of the inhibitory compounds, allows a direct test of the effects of substances on RNA synthesis (7, 11, 14). In a preliminary test, time and temperature for prelabeling of FL cells were investigated to find out the optimal conditions TIME (HOURS) FIG. 1. Incorporation of [3H]uridine into acidinsoluble material of FL cejls untreated and in the presence of IITS. Symbols: control, 0; no. 7, 0 (70 iamol) and 0 (30 gmol); no. 9, x (30 umol) and x (10 umol); no. 5, + (200 jamol). [3H]uridine and the test compounds were added together at 0 h. Radioactivity was determined at various times. Points represent averages of analyses of three tubes. for the subsequent incorporation of [3H ]uridine into RNA. The results are listed in Table 1. The optimal increased incorporation from the prelabeled soluble nucleotide pool into RNA was found to take place in the group that was pulse-labeled at 16 C for 1 h. Therefore, the experiments which follow were carried out under such conditions. In this case, the incorporation of uridine into RNA after washing (O h) reached nearly 3% of the control value at 7 h. This value at 0 h was subtracted from all others in each experiment. Kinetics of uridine incorporation into cellular and viral RNA after prelabeling and the action of inhibitory compounds. The prelabeled nucleotide pool in FL cells suffices to measure an increase of cellular RNA synthesis from 0 to 7 h (Fig. 2). When the substances were added after the surplus of uridine was washed out, they induced (at 2 h) no or only a small

3 VOL. 5, 1974 TABLE 1. Prelabeling procedure MENGOVIRUS-INFECTED FL CELLS 395 Kinetics of uridine incorporation into cellular RNA after different prelabeling procedures of the FL cellsa Radioactivity in acid-insoluble material after Time 2 h 4 h 6 h Temp (min) dpmb dpm dpm 4 C C x 10' x 10' x 10' a Expressed in percentage of the group prelabeled at 16 C for 1 h. bdpm, Disintegrations per minute. diminution of cellular RNA synthesis at 4 and 7 15 / h in comparison with the untreated control (Fig. 2). The method of prelabeling at low temperatures was also useful for obtaining evidence of virus-specific RNA synthesis in infected cells //x treated with actinomycin D (Fig. 3). In accordance with previous results (18), viral RNA // // synthesis started 4 h after infection and reached 10 <, ////+ a maximum of more than 300% at 8 h (calculated by comparison with the value at the fourth hour). The curve of virus control without addi- Z.) SQ /- dtion of actinomycin D demonstrates a first peak at the second hour, followed by a further in- crease after 4 h which nearly parallels that with a 2u W e /f/the addition of actinomycin D. This course in the first hours illustrates the 5- E i ///inhibition of cellular RNA synthesis. Conse- I 5 quently, the second increase can be considered ^ t 11/ to be caused by viral RNA synthesis. All three test compounds (no. 7, 9, and 5) give a complete l//_- - inhibition of virus-specific RNA synthesis in the ;/t-.._ ~_-+_ >.~ ~ ~~ presence of actinomycin D (Fig. 3). Up to hours,,'_4~-the curves show a decrease similar to uninfected,o ~~~~~~-X cells under the influence of the substances and actinomycin D (Fig. 2). -1 O l Table 2 shows the inhibitory capacity of the TIME (HOURS) three tested compounds on virus yield after one replication cycle studied in parallel under the FIG. 2. Incorporation of [3Hluridine into cellular samtes conditions in paboe. RNA after prelabeling of FL cells (1 h at 16 C). After a same test conditions mentioned above careful washing out of the nucleoside not taken up, test substances were added (0 h). Their effects on DISCUSSION RNA synthesis were studied at different times by the It has been shown previously that the rateanalysis of radioactivity in comparison with the limiting step in the incorporation of uridine into untreated control. Symbols: control, 0; no. 7, O (70 the in ton ofuriin into,gmol); no. 9, x (30 ;mol); no. 5, + (200 Amol). Dotted the nucleotde pool and intorna of cells is the lines give the results of the same groups under permeation of the nucleoside into the cell (3-5, simultaneous influence of actinomycin D (0.2 gg/ml). 7, 8, 10, 11, 14, 16, 17). Immediately after entry All points represent averages of analyses of three into the cell, uridine becomes phosphorylated. tubes. It has been reported (6, 13) that after a short

396 TONEW AND TONEW x E lu-q :C:3 :ti TIME (HOURS) FIG. 3. Incorporation of [3H]uridine into mengovirus-infected FL cells without (0) and with (A) actinomycin D (0.

4 396 TONEW AND TONEW x E lu-q :C:3 :ti TIME (HOURS) FIG. 3. Incorporation of [3H]uridine into mengovirus-infected FL cells without (0) and with (A) actinomycin D (0.2 pg/ml) after prelabeling (1 h at 16 C) and virus adsorption. Effects of test substances, added at 0 h with actinomycin D (0.2 gg/ml) on virus-specific RNA synthesis were studied at different times by the analysis of radioactivity. Symbols: no. 7, 0 (70 gmol); no. 9, x (30 Amon); and no. 5, + (200 gmol). All points represent averages of analyses of three tubes. TABLE 2. Inhibition of virus yield after one replication cycle of mengovirus Infectious virus titer (TCID a of Inhibition Test compound 0.2 ml) in log of control units at (% Virus control Virus control with actinomycin D (0.2 &g/ml) No. 7 (70 umol) and actinomycin D No. 9 (30 umol) and actinomycin D No. 5 (200,mol) and actinomycin D Oh 8h atcid.o, Mean tissue culture infective dose. ANTIBUCROB. AG. CHEMOTHER. pulse most of the uridine taken up is located in UTP (60 to 80%o depending on the temperatures) and that synthesis of RNA is extensively depressed at low temperatures (4 and 16 C). To measure RNA synthesis under various circumstances and to test the action of substances on cellular or viral RNA synthesis, Plagemann and Shia (11) have advised labeling the nucleotide pool by incubating the cells with uridine at low temperatures and then chasing out the isotope under specific test conditions. In this way, he could reveal the competitive inhibition of uridine transport by phenethylalcohol, persantin, and adenosine (7, 11) without affecting viral RNA synthesis per se. Our results above indicate that the three ]ITS tested also inhibit the uridine transport and, to the same extent, reduce incorporation of uridine into cellular RNA by about 80% of the control. It is likely that the phosphorylation steps are unaffected by the compounds studied because one of these compounds (no. 7) has failed to inhibit uridine kinase activity when tested in cell-free extracts (unpublished data). On the other hand, 70 to 80%o of the nucleoside can be assumed to be rapidly phosphorylated to UTP (6, 13) before addition of the substances, which, therefore, could not influence phosphorylation. FL cells were pulse-labeled for 1 h at 16 C at the beginning of the experiment. Both actinomycin D and virus infection have no effect on uridine transport (11, 12). For that reason, actinomycin D and virus were added together with the labeled nucleoside. After washing out the residual uridine, incorporation into RNA was studied. The uninfected controls showed increases in radioactivity in the acid-insoluble material over a period of 7 h. Contrary to this, after prelabeling at 4 C, incorporation had ceased after 10 to 15 min in Novikoff rat hepatoma cells (11). In young chicken embryo fibroblast cells, incorporation had taken place for at least 2 h (15). To confirm that FL cells incorporate [3H juridine really from the labeled nucleotide pool and not from uridine left behind in the medium, persantin, which is a specific inhibitor of uridine transport (5, 10, 14), was also used. In the presence of persantin, a high and long incorporation rate into RNA was recovered in prelabeled and chased cells (Tonew and Dseguse, manuscript in preparation). Therefore, prelabeled FL cells are very useful for obtaining information about the influence of substances on RNA synthesis itself. The results with the tested IITS demonstrate only low inhibitory effects on host-directed RNA synthesis of less than 25% after 4 and 7 h of incubation. The question of whether FL cells also have two independent nucleotide pools for cellular and viral RNA synthesis, as suggested for Novikoff rat hepatoma cells (9), was not studied.

VOL. 5, 1974 However, with the same method of prelabeling as described here, it was also possible to demonstrate virus-specific RNA synthesis under the influence of actinomycin D, beginning at the

5 VOL. 5, 1974 However, with the same method of prelabeling as described here, it was also possible to demonstrate virus-specific RNA synthesis under the influence of actinomycin D, beginning at the fourth hour after infection and increasing to 8 h in accordance with previous findings (18). Hence, it is not necessary to interrupt the replication cycle for labeling (11), and the test compounds can be added at 0 h. In this case, all substances, no. 7, 9, and 5, showed a highly specific inhibition of virus-induced RNA synthesis in the concentrations used. ACKNOWLEDGMENT The excellent technical assistance of E. Lippmann and E. Klimke is gratefully acknowledged. LITERATURE CITED 1. Heinisch, L., and K. Kramarcyk Antivirale Thiosemicarbazone und verwandte Verbindungen. I. Zur Synthese und Struktur substituierter Isatinthiosemicarbazone und -isothiosemicarbazone. J. Prakt. Chem. 214: Heinisch, L., M. Tonew, and E. Tonew Verfahren zur Herstellung virostatisch wirksamer substituierter Isatinisothiosemicarbazone. Patentschrift WP 12p/ kl. 12p,2. Int. Cl. C 07 d, DDR. 3. Korbecki, M., and P. G. W. Plagemann Competitive inhibition of uridine incorporation by 6-azauridine in uninfected and mengovirus-infected Novikoff hepatoma cells. Proc. Soc. Exp. Biol. Med. 132: Nakata, Y., and J. P. Bader The uptake of nucleosides by cells in culture. II. Inhibition by 2-mercapto-1-,-4-pyridethyl-benzimidazole. Biochim. Biophys. Acta 190: Peters, J. H., and P. Hausen Effect of phytohemagglutinin on lymphocyte membrane transport. 1. Stimulation of uridine uptake. Eur. J. Biochem. 19: Plagemann, P. G. W Effect of temperature on the transport of nucleosides into Novikoff rat hepatoma cells growing in suspension culture. Arch. Biochem. Biophys. 140: Plagemann, P. G. W Effects of phenethyl alcohol on transport reactions, nucleoside pools and macromolecular synthesis in Novikoff rat hepatoma cells growing in suspension culture. J. Cell. Physiol. 75: Plagemann, P. G. W Nucleotide pools of Novikoff MENGOVIRUS-INFECTED FL CELLS 397 rat hepatoma cells growing in suspension culture. 1. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle. J. Cell. Physiol. 77: Plagemann, P. G. W Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. 2. Independent nucleotide pools for nucleic acid synthesis. J. Cell. Physiol. 77: Plagemann, P. G. W., and M. F. Roth Permeation as the rate-limiting step in the phosphorylation of uridine and choline and their incorporation into macromolecules by Novikoff hepatoma cells. Competitive inhibition by phenethyl alcohol, persantin, and adenosine. Biochemistry 8: Plagemann, P. G. W., and M. A. Shia Transport as the rate-limiting step in the incorporation of uridine into mengovirus ribonucleic acid in Novikoff rat hepatoma cells. J. Virol. 7: Plagemann, P. G. W., G. A. Ward, B. W. J. Mahy, and M. Korbecki Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells. J. Cell. Physiol. 73: Scholtissek, C Nucleotide metabolism in tissue culture cells at low temperatures. 1. Phosphorylation of nucleosides and deoxynucleosides in vivo. Biochim. Biophys. Acta 145: Scholtissek, C Studies on the uptake of nucleic acid precursors into cells in tissue culture. Biochim. Biophys. Acta 158: Scholtissek, C Detection of an unstable RNA in chick fibroblasts after reduction of the UTP pool by glycosamine. Eur. J. Biochem. 24: Scholtissek, C., and H. Becht Action of acridines on RNA and protein synthesis and on active transport in chick embryo cells. Biochem. Biophys. Acta 123: Steck, T. L., Y. Nakata, and J. P. Bader The uptake of nucleosides by cells in culture. 1. Inhibition by heterologous nucleosides. Biochim. Biophys. Acta 190: Tonew, M., and S. Bleecken Antivirale Wirkung von Imidazolderivaten. 2. Untersuchungen zur RNSund Proteinsynthese. Arch. Gesamte Virusforsch. 33: Tonew, M., and E. M. Tonew Antivirale Wirkung von Imidazolderivaten. 1. Die Hemmung der Vermehrung des Mengovirus in FL-Zellen. Arch. Gesamte Virusforsch. 33: Tonew, M., E. Tonew, and L. Heinisch Antiviral thiosemicarbazones and related compounds. II. The antiviral activity of substituted isatinisothiosemicarbazones. Acta Virol. 18:17-24.

Inhibition of the Multiplication of Vesicular Stomatitis and

JOURNAL OF VIROLOGY, June 1974, p. 1186-1193 Copyright 1974 American Society for Microbiology Vol. 13, No. 6 Printed in U.S.A. Inhibition of the Multiplication of Vesicular Stomatitis and Newcastle Disease