Platinum(II) and palladium(II) complexes of pyridine-2-carbaldehyde thiosemicarbazone as alternative antiherpes simplex virus agents.

The cytotoxicity and the antivirus activity of Pd(II) and Pt(II) complexes with pyridine-2-carbaldehyde thiosemicarbazone (HFoTsc) against HSV replication were evaluated on four HSV strains-two wt strains Victoria (HSV-1) and BJA (HSV-2) and two ACV(R) mutants with different tk gene mutations R-100 (TK(A), HSV-1) and PU (TK(N), HSV-2). The experiments were performed on continuous MDBK cells and four HSV 1 and HSV 2 strains were used, two sensitive to acyclovir and two resistant mutants. The five complexes of HFoTsc, [Pt(FoTsc)Cl], [Pt(FoTsc)(H(2)FoTsc)]Cl(2), [Pt(FoTsc)(2)], [Pd(FoTsc)(H(2)FoTsc)]Cl(2), and [Pd(FoTsc)(2)], were found to be effective inhibitors of HSV replication. The most promising, active, and selective anti-HSV agent was found to be complex [Pt(FoTsc)(H(2)FoTsc)]Cl(2). This complex could be useful in the treatment of HSV infections, since it is resistant to ACV mutants. PCR study of immediate early 300 bp ReIV Us1 region reveals that the complex [Pt(FoTsc)(H(2)FoTsc)]Cl(2) specifically suppressed wt HSV-1 genome 2 hours after the infection, not inducing apoptosis/necrosis on the 8 hours after virus infection. The target was found to be most probably the viral, instead of the host cell DNA.

Effect of metal complexes of acyclovir and its acetylated derivative on Herpes simplex virus 1 and Herpes simplex virus 2 replication.

The effect of zinc, nickel, cobalt and cadmium complexes of acyclovir (ACV) and its omicron-acetylated derivative (Ac-ACV) on the replication of wild type (wt) and ACV-resistant (ACV(R)) strains of Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2) was examined. According to cytotoxicity, these compounds followed the order Ni-ACV chloride > Cd-ACV 3 Ni-ACV nitrate > ACV = Zn-ACV nitrate = Ac-ACV = Zn-Ac-ACV > Zn-ACV chloride > Co-ACV. Besides Ac-ACV, the only active complexes in inhibiting virus replication were Zn-ACV nitrate and Zn-Ac-ACV, which effectively suppressed the growth of both wt and ACVR strains of HSV-1 and HSV-2. The most active and most selective inhibitor of the growth of ACVR strains of HSV-1 and HSV-2 was Ac-ACV; its EC50 and SI were 100 and 10 times higher than those of ACV, respectively. Zn-Ac-ACV was less active than Ac-ACV, obviously due to the stability of the complex. Zn-ACV nitrate was active against both wt and ACVR strains of HSV-1; its activity and selectivity were 100 and 75 times higher than those of ACV, respectively. Ac-ACV and Zn-Ac-ACV suppressed the pre-mitotic arrest caused by HSV-1 infection during the first 2 hrs of infection and later on restored the cell division.

Expression of cellular proteins Bcl-X(L), XIAP and Bax involved in apoptosis in cells infected with herpes simplex virus 1 and effect of pavine alkaloid (-)-thalimonine on virus-induced suppression of apoptosis.

The expression of three cellular proteins involved in the modulation of apoptosis, namely antiapoptotic Bcl-X(L) and XIAP and proapoptotic Bax, was investigated in cells infected with Herpes simplex virus 1 (HSV-1). To assess whether the regulation of apoptosis in virus-infected cells depends on strain specificity the wild-type (wt) strain Victoria and the mutant R-100 resistant to acyclovir (ACV) were used. In addition, the expression of Bcl-X(L), XIAP and Bax was studied in cells infected with HSV-1 and treated with pavine alkaloid (-)-thalimonine. Our previous work has demonstrated that (-)-thalimonine irreversibly inhibits the replication of wt HSV-1 in cultured cells. Our data showed that (-)-thalimonine down-regulates the expression of viral proteins U(L)17, VP11-12, VP22, VP24 and gamma1 34.5, and affects negatively the posttranslational processing of glycoproteins D (gD) and G (gG). As both gamma1 34.5 and glycoprotein D possess antiapoptotic activity, we investigated whether the antiviral effect of the alkaloid could also be due to its ability to suppress the antiapoptotic activity of the virus. Our results demonstrated that: (i) the virus induced overexpression of antiapoptotic proteins Bcl-X(L) and XIAP; (ii) (-)-thalimonine reduced their overexpression, and (iii) this effect was stronger with the acyclovir resistant mutant R-100 than with the wt virus. Taken together, these data suggest that: (i) the virus abolishes apoptosis by means of virus-induced up-regulation of cell-specific prosurvival proteins Bcl-X(L) and XIAP, and (ii) (-)-thalimonine, apart from affecting essential viral targets, inhibits the infectious progeny production via restoration of apoptosis during viral replication.

Antiviral activity of platinum (II) and palladium (II) complexes of pyridine-2-carbaldehyde thiosemicarbazone.

A heterocyclic compound, pyridine-2-carbaldehyde thiosemicarbazone (HFoTsc), and its six metal coordinated bound complexes, three with platinum (II) and three with palladium (II), were studied for their activity against herpes simplex virus 1 (HSV-1) infection in cultured cells. According to their cytotoxicity the compounds were divided into two groups. Group I (cytotoxic compounds) included all three palladium complexes and [Pt(HFoTsc)2] Cl2, with maximum non-toxic concentration (MNC) of 1-10 micromol/l and a 50% cytotoxic concentration (CC50) of 20-100 micromol/l. Group 2 (low cytotoxic compounds) with MNC of 100 micromol/l and CC50 of 548-5820 micromol/l included compounds in the following order: [Pt(HFoTsc)2] Cl2

Cell response to herpes simplex virus type 1 infection mediated by biphasic calcium-phosphate ceramics: in vitro approach.

Based on well-documented data showing that bioactive ions (such as Ca2+, PO4-, etc.) released by BCPC induce various cell responses and on the significance of herpes outbreaks in human pathology, we investigated whether BCPC can modify cell response to HSV-1 infection. The roles of some physical and chemical properties of ceramics were evaluated using three BCPC samples--French commercial macro-microporous (FR) and two Bulgarian microporous laboratory samples--one of which was modified with magnesium (BG and BG + Mg). Samples only washed in 0.9% NaCl were designated as nonconditioned while those resuspended in cell growth medium for 10 days after washing were designated as conditioned. Experiments were done on cells from the continuous MDBK line precultured on BCPC surfaces for different time intervals and thereafter HSV-1 infected. The yield of infectious virus progeny, measured as virus titers, was the parameter used to determine the cell response to HSV-1 infection mediated by BCPC as compared to that of the virus control, that is, virus yield in cells cultured without BCPC. The data obtained show that all three nonconditioned BCPC samples were able to modify cells to resist HSV-1 infection. The prolongation of the resistant state depended on the specific physical and chemical properties of the particular BCPC sample: as the data show that the conditioning procedure (1) increased the ability of BG + Mg to promote cell resistance, and (2) reduced the ability of FR samples to modify cells to resist HSV-1 infection. The data obtained show that apart from Ca2+ and PO4-, ions of biometals such as Mg2+ also are responsible for the induction and maintenance of cell resistance to HSV-1 infection.

Cytotoxicity of Co(III) Complexes of Arginine.

The cytotoxicity of four Co(III) complexes of arginine on nontumour MDBK cells and on two cell lines derived from transplantable tumors, LSCC-SF(Mc29) and LSR-SF (SR), was evaluated comparative!y. Based on the cytotoxic concentration required to inhibit cell surveillance by 50% cc(nabla') it was found that: (i) the cytotoxicity of complexes tested increases when the concentration decreased; (ii) the cell surveillance depends on both complex and cell specificities. The complex specificity was illustrated by the order 1 > 4 > 2 >/= 3 . The cell specific response was demonstrated by the fact that LSCC-SG (Mc29) cells were up to 60 times more sensitive to 1 while LSR-SF (SR) cells were up to 1000 times more sensitive to 2 as compared to MDBK cells. Furthermore, with the prolongation of action on nontumour cells the cytotoxicity of 4 decreased up to 300 times while for both tumour cells it was independent on the duration of action.

Zinc(II) Complexes - A New Group of Non-Mutagenic Herpes Simplex Virus Inhibitors.

Former studies showed that complexes of Zn(II) with picolinic and with aspartic acids, Zn(pic)(2) and Zn(asp)(2), are able to inhibit HSV infection in cultured cells by affecting key steps of virus replication. As these complexes are candidates for a novel class anti-HSV drugs, further studies on their mutagenicity are of particular interest. In the present paper we present data showing that Zn(pic)(2) and Zn(asp)(2) do not express mutagenic effect in both prokaryotic (Salmonella typhimurium) and eukaryotic (Saccharomices cerevisiae) test systems.

Increased Efficacy of Zinc Complexes With Picolinic and Aspartic Acids Against Herpes Simplex Virus (HSV) Infection When Combined With the Pavine Alkaloid (-)-Thalimonine.

Complexes of zinc with picolinic and aspartic acids inhibit key steps of HSV-1 replication affecting different virus-specific targets. As was recently demonstrated by us, the pavine alkaloid (-)-thalimonine irreversibly inhibits HSV-1 infection in cultured cells. The aim of the present study was the evaluation of the combined effect of zinc complexes and (-)-thalimonine on uninfected and HSV-1 infected cells. The data obtained have shown that zinc complexes and the alkaloid exert decreased cytotoxicity (antagonistic effect) and significantly increased anti-HSV-1 activity (synergistic effect) when applied in dual chess-board combinations as compared to the individual effects of compounds tested. These combinations are also effective against the infection caused by a resistant to acyclovir (ACV) HSV-1 mutant and the effect has been recognised as synergistic.

Effect of Copper Acyclovir Complexes on Herpes Simplex Virus Type 1 and Type 2 (HSV-1, HSV-2) Infection in Cultured Cells.

We have found that when copper, zinc or cobalt is bound to a suitable ligand, the appropriate complex exhibited a significant anti-HSV effect (Varadinova et al., 1993; 1996). Recently published data by Sagripanti et al. (1997) also show that the inhibition of HSV by copper was enhanced by reducing agents and that mechanism of the inactivation is similar as for copper-mediated DNA damage (Aruoma, et al. 1991; Dizdaroglu, et al., 1991; Toyokuni and Sagripanti, 1994). Therefore it was interesting to study the efect of Cu(ll) coordination compounds with acyclovir (ACV) on the replication of HSV in cultured cells. The experiments on cytotoxicity as well as on the activity of three different Cu-ACV complexes [Cu(ACV)(2)Cl(2)(H(2)O)(2)] = (A); [Cu(ACV)(2)(H(2)O)(3)](NO(3))(2).H(2)O = (B) and [Cu(ACV)(2)(H(2)O)(2)](NO(3))(2)] = (C) towards virus replication, with special attention on the growth of ACV-resistant strain R-100 were performed on MDBK cells. ACV was used as a reference compound. The following results were obtained: 1) Increased cell's viability in the presence of 20-40(g/ml ACV and decreased one in the presence of Cu-ACV complexes with relative level (A) >> (B) > (C); 2) Cu-ACV complexes are more cytotoxic than the ligand - ACV and the relative level is (C)>(B)>(A); 3) The anti-HSV effect of ACV can be modulated by copper at levels depending on the specificity of the particular virus strain: (i) for the ACV sensitive strain DA (HSV-1) - ACV ((A) > (C) > (B); (ii) for the ACV sensitive strain Bja (HSV-2) (A) > ACV > (C) > (B); (iii) for strain R-100 (ACV(R), TK(a)) - (A) > ACV > (C) > (B). This findings are consistent with previously published data and undoubtedly show that Cu-ACV complexes could be useful in the treatment of HSV infections, especially when the causative agent is a resistant to ACV mutant.

Effect of Complexes of Zinc, Cobalt and Copper With D-Aminosugars on the Replication of Herpes Simplex Virus Type 1 (HSV-1).

Our previous results show that Zn(pic)(2) and Zn(asp)(2) inhibit key steps of the replication of HSV-1. Anti-HSV effect of complexes of Co(II) with aminoacids Lys and Ser was also found. In the present study we describe the effect of complexes of Zn(II), Co(II) and Cu(II) with D-aminosugars on the replication of HSV-1 and on the infectivity of free virions. The experiments were done using primary rabbit kidney cells (r.k.), diploid human embryonal fibroblasts (F) and Vero cells. No differences in the toxicity of metal complexes on diploid cells- r.k. and F, were found. Neither metal complexes, nor ligands-galactosoxime and glucosoxime, influenced the viral replication. During 1-4h prolonged contact only Cu(Gl.NOH)(2) inactivated HSV-1 virions up to 90%. The results show that D-aminosugars are not suitable ligands for Zn(II), Cu(II) and Co(II) in respect of the inhibition of viral replication. However, only Cu(Gl.NOH)(2) was able to inhibit the infectivity of free virions.

Complexes of zinc with picolinic and aspartic acids inactivate free varicella-zoster virions.

Zn(II) picolinate and aspartate, Zn(pic)(2) and Zn(asp)(2), have been shown to inhibit key steps of the replication of HSV-1. In the present study we describe the effect of Zn(pic)(2) and Zn(asp)(2) on the replication of VZV and on the infectivity of free virions. The experiments are done using BHK-21 cells, a clinical isolate of VZV and Zn-complexes in concentration of 10 muM. When Zn-complexes are present during the whole period of infection, the yield of infectious virus progeny decreases up to 98%. The infectivity of VZV is completely restored after the removal of zinc. The virucidal effect is manifested at the 2nd h of contact, when 90% of the virions are inactivated. The results show that both Zn(pic)(2) and Zn(asp)(2) specifically inactivate free VZV virions with no effect on viral replication.

Effect of Complexes of Cobalt With Aminoacids on the Replication of Herpes Simplex Virus Type 1 (HSV-1).

Cobalt, being essential metal, influences different physiological and enzymatic functions. As cobalt does not accumulate in the body, Co-compounds have relatively low toxicity. The aim of the present study is the effect of complexes of Co(II) with aminoacids - lysine, arginine, histidine and serine on HSV-1 replication. No effect of [O(2)Co(his)(4)].nH(2)O and [O(2)Co(arg)(2)].nH(2)O on HSV-1 infection in vitro was found. Both, [O(2)Co(lys)(2)].nH(2)O and [O(2)Co(ser)(2)].nH(2)O suppress the attachement of HSV-1 particles onto target cells and the viral replication as well. Moreover, the properties of the particular Co-complex (charge, stability, structure) are manifestated by their virucidal effect. Thus, [O(2)Co(ser)(2)].nH(2)O irreversibly inhibits the infectious activity of free HSV-1 virions, while virucidal effect of [O(2)Co(lys)(2)].nH(2)O is completely reversible after the 2h of contact.

Mode of action of Zn-complexes on herpes simplex virus type 1 infection in vitro.

The influence of Zn-complexes with biologically active ligands like aminoacids: picolinic acid as Zn(pic)2 and asparaginic acid as Zn(asp)2 on HSV-1 infection in vitro was investigated. Studies on kinetics of Zn-ions using labeled 65Zn(pic)2 as a marker, show that at the 45th min Zn-ions are exposed onto or into cells. When cells were infected 60min after the treatment with 65Zn(pic)2, 1h later the activity of Zn-ions in the cell fraction decreased while in the medium increased. This result shows that soon after the infection cells expelled part of their Zn. If Zn(pic)2 or Zn(asp)2 are added at the end of the adsorption period, ICP4 and ICP8 are localized in the cytoplasm but not in the nucleus of infected cells, while the synthesis of gH is decreased. A sharply increased number of nucleocapsids with low electron density cores was also found.

Influence of T-actemodulin on herpes simplex virus infection.

The aim of the investigation was to study the direct influence of T-actemodulin (TAM) on the Herpes Simplex Virus (HSV) infection in vitro and in vivo. HSV-1 (strain Vic) and HSV-2 (strain Nissa) were used in the experiments in vitro. The experiments were performed using multicycle and singlecycle growth tests. Seventy-five patients suffering from HSV infections (labialis and genitalis) were locally treated by 0.04% TAM-containing hydrophilic gel or water-washable cream (applied every 3 h). The results showed that TAM: decreased the infectious virus titre; reduced the infectious virus yield at the end of the single cycle of the virus replication; prolonged the latent period; and exerted an irreversible inhibitory effect on the infectious virus titre when applied at immediate early intervals after the infection. No relapses were observed in 84% of the patients treated with TAM. Eight percent of the patients were with only reduced relapses and in 8% of the patients TAM had a mild effect. TAM exerted a preventive effect against the disease when the treatment was initiated in the period of paresthesia.

Influence of tick-borne encephalitis and West Nile viruses on the chromosomes of pig kidney cells.

Changes of the mitotic index (MI) were studied in pig kidney (PS) cells infected either with tick-borne encephalitis (TBE) virus strains Hypr and Ir-32, or with West Nile (WN) virus prototype strain, strains K-99 and E-13. The cell division was arrested by the infection (metaphase barrier). The effect of TBE virus strains was manifested by the appearance of lagging chromosomes while the action of WN virus strains by colchicine--like metaphases. The prototype TBE virus strain Hypr affected the chromosomes and mitotic apparatus of PS cells less markedly than did the freshly isolated Ir-32 strain.