Antiviral Effect of Natural and Semisynthetic Diterpenoids against Adenovirus Infection in vitro.

The emergence and re-emergence of viruses has highlighted the need to develop new broad-spectrum antivirals to mitigate human infections. Pursuing our search for new bioactive plant-derived molecules, we study several diterpene derivatives synthesized from jatropholones A and B and carnosic acid isolated from Jatropha isabellei and Rosmarinus officinalis, respectively. Here, we investigate the antiviral effect of the diterpenes against human adenovirus (HAdV-5) that causes several infections for which there is no approved antiviral therapy yet. Ten compounds are evaluated and none of them present cytotoxicity in A549 cells. Only compounds 2, 5 and 9 inhibit HAdV-5 replication in a concentration-dependent manner, without virucidal activity, whereas the antiviral action takes place after virus internalization. The expression of viral proteins E1A and Hexon is strongly inhibited by compounds 2 and 5 and, in a lesser degree, by compound 9. Since compounds 2, 5 and 9 prevent ERK activation, they might exert their antiviral action by interfering in the host cell functions required for virus replication. Besides, the compounds have an anti-inflammatory profile since they significantly inhibit the levels of IL-6 and IL-8 produced by THP-1 cells infected with HAdV-5 or with an adenoviral vector. In conclusion, diterpenes 2, 5 and 9 not only exert antiviral activity against adenovirus but also are able to restrain pro-inflammatory cytokines induced by the virus.

Synthesis, Antiviral and Cytotoxic Activity of Novel Terpenyl Hybrid Molecules Prepared by Click Chemistry.

Naturally occurring terpenes were combined by click reactions to generate sixteen hybrid molecules. The diterpene imbricatolic acid (IA) containing an azide group was used as starting compound for the synthesis of all the derivatives. The alkyne group in the terpenes cyperenoic acid, dehydroabietinol, carnosic acid γ-lactone, ferruginol, oleanolic acid and aleuritolic acid was obtained by esterification using appropriate alcohols or acids. The hybrid compounds were prepared by combining the IA azide function with the different terpene-alkynes under click chemistry conditions. The cytotoxic activity of the terpene hybrids 1⁻16 was assessed against Vero cells and tumour cell lines (HEP-2, C6 and Raw 264.7). Compounds 1, 2, 3 and 7 showed cytotoxic activity against the tested cell lines. The antiviral activity of the compounds was evaluated against HSV-1 KOS, Field and B2006 strain. For the pairs of hybrid compounds formed between IA-diterpene (compounds 3⁻8, except for compound 7), a moderate activity was observed against the three HSV-1 strains with an interesting selectivity index (SI ≥10, SI = CC50/CE50) for some compounds.

Antiviral action of synthetic stigmasterol derivatives on herpes simplex virus replication in nervous cells in vitro.

Polyfunctionalized stigmasterol derivatives, (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), inhibit herpes simplex virus type 1 (HSV-1) replication and spreading in human epithelial cells derived from ocular tissues. Both compounds reduce the incidence and severity of lesions in a murine model of herpetic stromal keratitis when administered in different treatment modalities. Since encephalitis caused by HSV-1 is another immunopathology of viral origin, we evaluate here the antiviral effect of both compounds on HSV-1 infected nervous cell lines as well as their anti-inflammatory action. We found that both stigmasterol derivatives presented low cytotoxicity in the three nervous cell lines assayed. Regarding the antiviral activity, in all cases both compounds prevented HSV-1 multiplication when added after infection, as well as virus propagation. Additionally, both compounds were able to hinder interleukin-6 and Interferon-gamma secretion induced by HSV-1 infection in Neuro-2a cells. We conclude that compounds 1 and 2 have exerted a dual antiviral and anti-inflammatory effect in HSV-1 infected nervous cell lines, which makes them interesting molecules to be further studied.

Effect of the potent antiviral 1-cinnamoyl-3,11-dihydroxymeliacarpin on cytokine production by murine macrophages stimulated with HSV-2.

The limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV-2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF-α, IFN-γ, NO, IL-6 and IL-10 were increased by CDM used alone or in combination with HSV-2. Besides, CDM not only synergized TNF-α production combined with IFN-γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV-2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina.

Therapeutic effect of meliacine, an antiviral derived from Melia azedarach L., in mice genital herpetic infection.

Since natural products are considered powerful sources of novel drug discovery, a partially purified extract (meliacine) from the leaves of Melia azedarach L., a plant used in traditional medicine in India for the treatment of several diseases, has been studied. Meliacine exhibits a potent antiviral effect against several viruses without displaying cytotoxicity. The purpose of the present study was to evaluate the therapeutic effect of intravaginal administration of meliacine in a mouse model of genital herpetic infection. BALB/c female mice were infected with MS or G strains of Herpes Simplex Virus type 2 and then treated with meliacine topically. An overall protective effect was observed. Animal survival increased, the severity of the disease was reduced, life span was extended and virus shedding in vagina fluids was diminished. In addition, meliacine reduced the amount of virus that migrated to the brain and vaginal fluids presented higher levels of IFN-gamma and TNF-alpha than untreated infected mice. These results indicate that meliacine could be an alternative therapeutic compound against HSV-2 genital infection.

The synergistic effect of IFN-alpha and IFN-gamma against HSV-2 replication in Vero cells is not interfered by the plant antiviral 1-cinnamoyl-3, 11-dihydroxymeliacarpin.

BACKGROUND: Recent studies have shown that gamma interferon (IFN-gamma) synergizes with IFN-alpha/beta to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. Since IFN response represents an early host defense event against viral infection and the fact that treatment with meliacine, a plant antiviral, ameliorate the severity of the herpetic infection in female mice infected intravaginally with HSV-2, we wanted to investigate whether the administration of meliacine to HSV-2 infected mice could altered the homoestasis of IFNs host response. For this purpose we studied the effect of the compound 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), which is the responsible for meliacine antiviral action, on the HSV-2 inhibition exerted by IFN alpha, IFN-gamma or their combination. RESULTS: We have found that like HSV-1, IFN-gamma synergizes with IFN-alpha to inhibit HSV-2 replication in Vero cells. While treatment with IFN-alpha or IFN-gamma alone has weak antiviral action, HSV-2 plaque formation, viral replication and the onset of viral CPE in Vero cells are synergistically inhibited by interferon combination. In addition, CDM treatment contributes to protect cells from virus cytopathic effect and causes a strong inhibition of HSV-2 titer. Moreover, the presence of CDM for 2 h before IFN induction, during the 16 h induction period, only for 24 h after infection or during the complete IFN treatment period, reduces virus yields in an additive way without affecting IFN antiviral action. CONCLUSION: The results reported here indicated that the presence of CDM did not alter the antiviral activity of IFN-alpha, IFN-gamma or the synergism exerted by their combination. As a result we can envision that the administration of CDM in vivo could not affect the biological activity of IFNs, which are so important mediators of the innate resistance to HSV-2 infection.

Antiherpes virus activities of new 6-19 carbon-bridged steroids and some synthetic precursors.

Three synthetic 6,19-carbon bridged steroids: 3beta,20beta-diacetyloxy-5alpha-chloro-19a(R)-hydroxy-6,19-methanopregnane, 3beta,20beta-diacetyloxy-5alpha-chloro-6,19-methanopregnane, 6,19-methanopregn-4-ene-3,20-dione and four synthetic precursors: 3beta,20beta-diacetyloxy-19-hydroxypregn-5-ene, 3beta,20beta-diacetyloxy-pregn-5-en-19-al, 3beta,20beta-diacetyloxy-19(E)-(methoxymethylidene)-pregn-5-ene and 20beta-acetyloxy-3beta-hydroxy-19(E)-(methoxymethylidene)-pregn-5-ene were tested against herpes virus replication in cell cultures. Several compounds were cytotoxic for stationary cells. Antiviral studies performed with all compounds against HSV-1 indicated a dose-dependent virus susceptibility with selectivity indexes (SI) values in the range 1.7-183.2. Selected compounds were also tested against HSV-2 and the SI values obtained were in the range of 31-273. Attempts to reveal the step of virus multiplication affected by pregnanes were performed with one compound. HSV-1 virus incubation with the compound did not alter the ability of virus particles to infect cells; moreover, neither virus adsorption nor penetration appeared to be affected. The drug must be present during at least the first 7 h of the virus cycle to inhibit more than 90% of virus production. All these results suggest that these novel molecules interfere with an intracellular step of virus multiplication, thus behaving like true antivirals.