Antipoxvirus Antibiotics

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1972, p Vol. 1, No. 6 Copyright 1972 American Society for Microbiology Printed in U.S.A. Congocidine and Distamycin A, Antipoxvirus Antibiotics YECHIEL BECKER, YAEL ASHER, AND ZICHRIYA ZAKAY-RONES Department of Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 11 April 1972 The antibiotics congocidine and distamycin A inhibited the replication of Shope fibroma and vaccinia viruses in BSC, cells. Treatment of infected cultures with congocidine for a 12-hr period resulted in an irreversible inhibitory effect on virus development. A longer period of treatment with the antibiotic also resulted in inhibition of the residual infectious virions present in the treated cells. Distamycin A inhibited the replication of poxviruses, but its inhibitory effect was reversed when the antibiotic was removed from the infected cells even as long as 24 hr after treatment. The inhibitory effect of distamycin A and its reversibility resembled the antipoxvirus activity of rifampin. Distamycin A (1) and netropsin (4) are oligopeptide antibiotics isolated as fermentation products of Streptomyces distallicus and S. netropsis, respectively. The chemical structure of distamycin A has been determined by a complete synthesis (1). Congocidine, which is closely related to netropsin (Fig. 1), also belongs to this group of antibiotics. These antibiotics were reported to possess antiviral properties and to inhibit the replication of deoxyribonucleic acid (DNA)-containing viruses in cell cultures in vitro (2, 13, 14) and in animals in vivo (11, 13, 14). Studies on the mechanism of action of distamycin A and netropsin demonstrated that the antibiotic molecules interact with adenine-thymine (A-T) rich regions in DNA molecules. Because of this interaction, the replication and transcription of the DNA molecules is prevented (3, 8, 9, 16). The binding to A-T rich regions of the DNA template is stronger with netropsin than with distamycin A because the former contains two guanidine side chains, whereas distamycin A has only one (1; M. A. Verini, A. M. Casazza, and A. Fioretti, unpublished data). As vaccinia virus DNA is rich in A + T (about 64%; 5), it seems that the ability of the antibiotics to interact with A-T rich regions in the viral DNA might explain the antipoxvirus activity of the antibiotics (2, 11). It was therefore of interest to determine the antipoxvirus activity of congocidine, as well as of derivatives of distamycin A (Fig. 1), and to compare it with the antipoxvirus activity of both distamycin A and rifampin (12, 15). The present study demonstrates that treatment of poxvirus-infected cells with congocidine inhibits the replication of the virus similarly to distamycin A. However, after 24 hr of treatment with congocidine, the inhibitory effect is irreversible. Not only virus replication, but residual infectious virus as well, is inhibited by congocidine. The inhibitory effect of distamycin A is reversible whenever the antibiotic is removed. MATERIALS AND METHODS Cells. BSC, cells (15) were serially propagated in Eagle's medium supplemented with 1% calf serum. Monolayer cultures in milk bottles were used. For titrations of virus progeny, monolayer cultures of BSC, cells in 6-mm plastic dishes (NUNC, Denmark) were used. Viruses. Shope fibroma virus (SFV; 15) was obtained from Leo Sachs, The Weizmann Institute, Rehovot, Israel. The virus was propagated in primary rabbit kidney monolayers and later in BSC, cells. Virus from the latter cultures retains its ability to produce local tumors in rabbit skin. Vaccinia virus, Noguchi strain, was obtained from F. Fenner, the Australian National U., Canberra. Virus infection. Infection of BSC1 monolayers, to determine the virus growth cycle, was carried out at a multiplicity of infection ranging from.2 to 1. plaque-forming unit/cell. The virus was adsorbed to the cells for a 2-hr period; then medium was added and the cultures were reincubated at 37 C. At different time intervals, cultures were removed, and the cells were scraped into the medium and disintegrated by four cycles of freezing and thawing. The virus yield was determined by titration in BSC1 monolayers. To determine the effect of the antibiotics on the development of viral plaques, monolayer cultures in 6-mm plastic plates were infected with a virus preparation which yielded 2 to 3 plaques per plate. After a 2-hr adsorption period, fresh medium was added and the cultures were reincubated at 37 C. 483

2 484 H2 N-C-NH CH2-CNH III2_1 CNH N N BECKER, ASHER, AND ZAKAY-RONES IULCONH-(CH2)-C N NH2 CH 3 CON GOCID INE TABLE 1. ANTIMICROB. AG. CHEMOTHER. Effect of congocidine on the formation of Shope fibroma virus plaques Antibiotic concn (ug/ml) Plaque-forming units/plate Expt 1 Expt 2 Expt 3 H2N-C-NHCH2- CNH --1 NH N H vo H v CONH-(CH2)2- N NH2 NET ROPSIN HCNH-Ti CN-CON m NvlCONH an tzconh m NH N2 LCONH-(CH) C\ DISTAMYCIN A. NLCONH --3 CONH m NH C H3 N econh -(CH2)2 -C N NH2 COMPOUND 11 CHO- NH- L N NH Nt3 -NHN HH L c COMPOUND XII 2 c FIG. 1. Chemical composition of congocidine, distamycin A, and its derivatives, compounds II and XIIl. After incubation for 48 hr, the medium was removed and the cell monolayers were stained with a solution of.1% crystal violet prepared in.1 M citric acid. Antibiotics. Congocidine (Rhone-Poulenc Laboratories, France), distamycin A, and its derivatives (Verini et al., unpublished data), compounds II and XIII (Fig. 1), were obtained from Farmaceutici Italia, Milan, Italy. Rifampin was obtained from Gruppo Lepetit, Milan, Italy. Congocidine was dissolved in water, whereas distamycin A was dissolved in a.4% aqueous solution of dimethylformamide. Various concentrations of the antibiotics were added to infected monolayers. RESULTS Effect of congocidine on poxvirus replication. Congocidine (75 Ag/ml) inhibited the development of SFV (Table 1) and vaccinia virus (Table 2) in monolayers of BSC, cells. In this respect, 2 N TABLE 2. Effect of congocidine, distamycin A, and its derivative compoiinds II and XIII on the development of vacciniia virus (Noguchi strain) plaques Plaques/plate Antibiotic concn (pg/ml) Congoci- Distamy- Compound Compound dine cin A II XIII congocidine resembled distamycin A (Table 2). Regardless of the stage in the virus replication cycle at which it was added, congocidine inhibited development of SFV (Fig. 2). These results were taken to indicate that the antibiotic congocidine interferes with the synthesis of poxvirions in the infected cells whenever added to the infected cultures. Irreversible inhibitory effect of congocidine. Treatment of SFV-infected BSC1 cells with congocidine (25,g/ml) was found to inhibit virus replication (Fig. 3) in accordance with the results in Fig. 2. In addition, congocidine decreased the amount of residual infectious virus present in the infected cells. It is possible to assume that, after exposure for 24 hr to congocidine, the virions were inhibited by the antibiotic. It was also found that, when congocidine was removed from the infected cultures after treatment for 24 hr, the virus did not replicate. Further (Fig. 4), the extent of irreversibility of the inhibitory effect of congocidine depended on the duration of contact of the antibiotic with the infected culture. If the antibiotic was removed early (3-hr contact), little effect upon virus replication was observed. Longer periods of treatment produced progressively greater degrees

3 6 UNTREATED E LL' (D Qj -E IL - 5 I / 41 3 CONGOCIDINE TREATED 2 ANTIBIOTIlC REMOVED REVERSED,,A~~ FIG. 2. Effect of congocidine on the replication of Shope fibroma virus (SFV). Cell cultures were infected with SFV. Part of the cultures were left untreated (solid lines); the others were treated with congocidine, 2,ug/ml (dashed lines) added at different times after infection (indicated by arrows). The cultures were harvested at 48 hr after infection, and the virus yield in the treated and untreated cultures was determined FIG. 3. Irreversibility of the effect of congocidine on the replication of Shope fibroma virus (SFV). Monolayer cultures of BSC1 cells were infected with SFV and treated with congocidine (2,ug/ml) after infection. Untreated infected cultures served as a control. At various time intervals, samples of infected congocidine-treated (a) and untreated () cultures were removed, and the virus progeny was determined. Part of the infected, congocidine-treated cells were carefully washed at 24 hr after infection to remove the antibiotic. Fresh medium was added to the cultures, which were reincubated at 37 C. Samples were obtained at different time intervals (-, reversed), and the virus progeny was determined. E :D Li ID FIG. 4. Reversibility of congocidine inhibition of poxvirus replication. BSC1 monolayers were infected with Shopefibroma virus and treated with congocidine (), 2,ug/ml. Untreated cells served as a control for the virus growth cycle (). Samples were obtained at different time intervals, and the virus progeny was determined by the plaque assay. At the times indicated by arrows, congocidine was washed out from the infected untreated cells, which were reincubated at 37 C (A; the number in the circle corresponds to the number above the arrow, indicating the time of congocidine removal). 485

4 486 BECKER, ASHER, AND ZAKAY-RONES ANTIMICROB. AG. CHEMOTHER. TABLE 3. Effect of distamycin A and its derivative compounds II and XIII on the formation of Shope fibroma virus plaques Plaques/platea Antibiotic concn (ug/ml) Distamycin Compound Compound A II XIII a Average of three experiments. of irreversibility, with complete loss of virus occurring after exposure of infected cells to congocidine for 24 hr or longer. These results indicated that congocidine is capable of inhibiting SFV replication in an irreversible manner. Effect of distamycin A and its derivatives on poxvirus replication. Treatment of BSC1 monolayers with distamycin A at a concentration of 5 &g/ml inhibited the development of vaccinia virus plaques (Table 2). Inhibition of SFV plaques was attained at a concentration of 2 jig/ml (Table 3). The effects of distamycin A (Fig. 5A) and rifampin (Fig. SB) upon replication of SFV resembled that already shown for congocidine (Fig. 2). With all three antibiotics, regardless of when in the viral replication cycle treatment was initiated, inhibition of virus activity was observed. To obtain information regarding the molecular site of the antipoxvirus activity in the distamycin A molecules, the effect of two derivatives of the antibiotic (Fig. 1) were studied. Compound II had no inhibitory effect on the replication of SFV and vaccinia virus. Compound XIII had no effect on SFV and a slight effect on vaccinia virus plaque formation. These results indicated that modifications in the distamycin A molecules resulted in the loss of the antiviral activity of the antibiotic. Further studies on additional derivatives of distamycin A are needed before the active site in the antibiotic will be determined. Reversibility of distamycin A inhibitory effect. As shown in Fig. 6 and 7A, removal of distamycin A from treated, SFV-infected cells after contact for as long as 3 hr resulted in rapid recovery of virus replication with a final yield of virus quantitatively similar to that obtained from untreated, infected cells. This reversibility of inhibition by distamycin A is similar to that observed with rifampin (Fig. 7B). Thus, these two antibiotics apparently exert their effects only when present 7 A DISTAMYCIN A 6f DISTAMYCIN / TREATED IL. / CD '1O FIG. 5. Inhibition of poxvirus replication by distamycin A and rifampin. Shope fibroma virus-infected BSC1 monolayers were treated with 25 tg of distamycin A per ml (A) or with rifampin (B), 1 jug/ml. The antibiotics were added at different time intervals in the virus growth cycle (), and the cultures were incubated to 48 hr postinfection. The virus yields in the treated infected cultures were determined ().

5 VOL. 1, 1972 ANTIPOXVIRUS ANTIBIOTICS 487 FIG. 6. Reversibility of distamycin A inhibition of Shopefibroma virus (SFV) replication.sfv-infected cultures were treated with 25,gg of distamycin A per ml (), and the virus progeny at different time periods after infection were determined. Untreated infected cultures were used as a control of the virus growth cycle (X). At 24 hr after infection, distamycin A was washed out from part of the antibiotic-treated infected cells, and the virus yield at different times was determined (A, reversed). in the environment during virus replication. Upon their removal from infected cultures, virus replication is resumed. DISCUSSION Schabel et al. (11) reported in 1953 that netropsin has a marked therapeutic effect on experimental vaccinia virus infection in intracerebrally inoculated mice. Verini and Ghione (13) reported that distamycin A, which resembles netropsin (Fig. 1), inhibited the development of vaccinia virus. These findings demonstrated that the two antibiotics have an antipoxvirus property and can interfere with virus-specific processes in the infected cell. Modifications of the distamycin A molecule like those in compounds II and XIII demonstrated (Verini et al., unpublished data) that the antipoxvirus activity of the antibiotic increased to the number of pyrrole residues (up to five), and that both the formyl group and the propion amidine function are essential for the biological activity of distamycin A. Studies on the mode of action of distamycin A (3, 8-1, 16) demonstrated that the antibiotic interacts with DNA molecules and inhibits both replication and transcription (3, 1) by DNA-dependent DNA and ribonucleic acid (RNA) polymerases, respectively. It was also demonstrated that distamycin A and netropsin form cross-links with A-T rich regions of DNA molecules (4, 8). The interaction of netropsin with A-T clusters causes stiffening and elongation of the DNA molecules which differs from the effect of distamycin A, probably owing to the second guanidino group of netropsin. The antibiotic congocidine, which closely resembles netropsin, might have a similar ability to interact with DNA. These properties of netropsin and distamycin A suggest the mode of antipoxvirus activity of the antibiotics. The analysis of vaccinia virus DNA (5) revealed a base composition with a high (64%) content of A + T. The ability of congocidine to inhibit not only poxvirus replication but also the residual virions present in infected cells after a 24-hr period of treatment might be explained by interaction of the antibiotic with the viral DNA genomes. It is assumed that congocidine and distamycin A interact with the genomes of the poxvirions, thus inhibiting the transcription by the A. DISTAMYCIN A REVERSED 5-~~~~~~~~~~~~(a _ * ~- - _ DISTAMYCIN A TREATED E - -- (25pg/mt) v 7 - B. R FAMPICIN ( o 6 REVERSED T M E ( hours) FIG. 7. Reversibility of distamycin A and rifampin inhibition of Shope fibroma virus (SFV) replication. SFV-infected cultures were treated with 25 isg of distamycin A per ml (A) or 1,ug of rifampin per ml (B). At different times after treatment (), the antibiotics were washed from the cultures, which were reincubated with fresh medium for an additional period of 48 hr. Infected untreated cultures () were used as a control. The virus yield at different time intervals after infection was determined (A).

6 488 BECKER, ASHER, AND ZAKAY-RONES ANTIMICROB. AG. CHEMOTHER. virions' associated RNA polymerase (7). The irreversibility of the inhibitory effect of congocidine, which differs from the inhibitory effect of distamycin A, might reflect the difference between the binding of the two antibiotics to the viral DNA. Further studies are in progress to determine the effect of these antibiotics on the poxvirus DNAdependent RNA polymerase. The inhibitory effect of distamycin A on poxvirus replication resembles that of rifampin (6, 12, 15). Both distamycin A and rifampin inhibit poxvirus replication, at a concentration of about 1 Ag/ml, only when present in the medium of the infected cells. Upon removal of the antibiotic from treated, infected cells, the replication of the virus is resumed. The interesting feature of the antipoxvirus activity of congocidine is its ability to inhibit the synthesis of poxvirus progeny irreversibly. Such an irreversible inhibitor of virus replication might have a potential value in the treatment of poxvirus infections. Further studies are needed to determine the therapeutic value of congocidine. ACKNOWLEDGMENTS This study was supported by a grant from Farmitalia, Milan, Italy. We are grateful to M. Ghione and Farmitalia for the generous supply of distamycin A, compounds II and XIII, and congocidine. We thank L. Silvestry and Gruppo Lepetit, Milan, Italy, for the gift of rifampin and their support. The capable assistance of Yafa Cohen is greatly appreciated. LITERATURE CITED 1. Arcamone, F., S. Penco, P. Orezzi, V. Nicolella, and A. Pirelli Structure and synthesis of distamycin A. Nature (London) 23: Casazza, A. M., and M. Ghione Therapeutic action of distamycin A on vaccinia virus infections in vivo. Chemotherapia 9: Chandra, P., C. Zimmer, and H. Thrum Effect of distamycin A on the structure and template activity of DNA in RNA-polymerase system. Fed. Eur. Biol. Soc. Letters 7: Finlay, A. C., F. A. Hochstein, B. A. Sobin, and F. X. Murphy Netropsin, a new antibiotic produced by a streptomyces. J. Amer. Chem. Soc. 73: Joklik, W. K The purification of four strains of poxvirus. Virology 18: Katz, E., P. Grimely, and B. Moss Reversal of anti-viral effects of rifampicin. Nature (London) 227: Kates, J. R., and B. R. McAuslan Poxvirus DNAdependent RNA polymerase. Proc. Nat. Acad. Sci. U.S.A. 58: Krey, A. K., and F. E. Hahn Studies on the complex of distamycin A with calf thymus DNA. Fed. Eur. Biol. Soc. Letters 1: Puschendorf, B., and H. Grunicke Effect of distamycin A on the template activity of DNA in a DNA polymerase system. Fed. Eur. Biol. Soc. Letters 4: Puschendorf, B., E. Petersen, H. Wolf, H. Werchau, and H. Grunicke Studies on the effect of distamycin A on the DNA dependent RNA polymerase system. Biochem. Biophys. Res. Commun. 43: Schabel, F. M., W. R. Laster, R. W. Brockman, and H. E. Skipper Observations on antiviral activity of netropsin. Proc. Soc. Exp. Biol. Med. 83: Subak-Sharpe, J. H., M. C. Timbury, and J. F. Williams Rifampicin inhibits the growth of some mammalian viruses. Nature (London) 222: Verini, M. A., and M. Ghione Activity of distamycin A on vaccinia virus infection in cell cultures. Chemotherapia 9: Werner, G. H., P. Ganter, and Y. de Ratuld Studies on the antiviral activity of distamycin A. Chemotherapia 9: Zakay-Rones, Z., and Y. Becker Anti-poxvirus activity of rifampicin associated with hydrazone sidechain. Nature (London) 226: Zimmer, C., and G. Luck Optical rotatory dispersion properties of nucleic acid complexes with the oligopeptide antibiotics distamycin A and Netropsin. Fed. Eur. Biol. Soc. Letters 1: