Two synthetic steroid analogs reduce human respiratory syncytial virus replication and the immune response to infection both in vitro and in vivo.

HRSV is responsible for many acute lower airway infections and hospitalizations in infants, the elderly and those with weakened immune systems around the world. The strong inflammatory response that mediates viral clearance contributes to pathogenesis, and is positively correlated with disease severity. There is no specific effective therapy on hand. Antiviral synthetic stigmastanes (22S, 23S)-22,23-dihydroxystigmast-4-en-3-one (Compound 1) and 22,23-dihydroxystigmasta-1,4-dien-3-one (Compound 2) have shown to be active inhibiting unrelated virus like Herpes Simplex type 1 virus (HSV-1) and Adenovirus, without cytotoxicity. We have also shown that Compound 1 modulates the activation of cell signaling pathways and cytokine secretion in infected epithelial cells as well as in inflammatory cells activated by nonviral stimuli. In the present work, we investigated the inhibitory effect of both compounds on HRSV replication and their modulatory effect on infected epithelial and inflammatory cells. We show that compounds 1 and 2 inhibit in vitro HRSV replication and propagation and reduce cytokine secretion triggered by HRSV infection in epithelial and inflammatory cells. The compounds reduce viral loads and inflammatory infiltration in the lungs of mice infected with HRSV.

A synthetic stigmastane displays antiadenoviral activity and reduces the inflammatory response to viral infection.

Although human adenovirus (ADV) infections are mild and self-limited in immunocompetent individuals, they can be severe and life-threatening in immunocompromised patients. Despite their significant clinical impact, there are not currently approved antiviral therapies for ADV infections. On the other hand, in some cases, the immune response induced by ADV infection can cause tissue damage. Even more, in the case of adenovirus vectors used in gene therapy, host immunity generally antagonize viral efficacy. Therefore, the need for searching an effective and safe therapy is increasing. In this work, we describe the antiadenoviral activity of the synthetic stigmastane (22S, 23S)-22,23-dihydroxystigmast-4-en-3-one (Compound 1) with already reported antiviral and antiinflammatory activities against other viruses of clinical importance. Compound 1 displayed no virucidal activity and did not affect ADV entry to the cells. The compound inhibited viral replication and it also reduced cytokine secretion in epithelial and inflammatory infected cells. Thus, Compound 1 would be a promissory drug potentially useful against adenoviral infections as well as an adjuvant of adenoviral vectors in gene therapy.

Virucidal, antiviral and immunomodulatory activities of ß-escin and Aesculus hippocastanum extract.

OBJECTIVES: ß-Escin, one of the constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), inhibits NF-κB activation, which plays an important role in HSV-1 replication. The aim was to examine the antiherpetic activity of ß-escin and AH, as well as their effect on the activation of NF-κB and AP-1 and cytokine secretion in epithelial cells and macrophages. METHODS: Cell viability was evaluated using MTT assay, and antiviral and virucidal activity was determined by plaque assay. The effect on NF-κB and AP-1 signalling pathways activation was determined by a luciferase reporter assay, and cytokine production was measured by ELISA. KEY FINDINGS: ß-Escin and AH had virucidal and anti-HSV-1 activities, and the antiviral activity was discovered for other enveloped viruses (VSV and Dengue). Moreover, ß-escin and AH significantly reduced NF-κB and AP-1 activation and cytokine production in macrophages stimulated with HSV-1 and TLRs ligands. However, an enhanced activation of these pathways and an increase in the levels of pro-inflammatory cytokines in ß-escin and AH-treated HSV-1-infected epithelial cells were found. CONCLUSIONS: This study demonstrates virucidal and broad-spectrum antiviral activities for ß escin and AH. Besides, ß-escin and AH modulate cytokine production depending on the stimuli (viral or non-viral) and the cell type under study.

Synthetic stigmasterol derivatives inhibit capillary tube formation, herpetic corneal neovascularization and tumor induced angiogenesis: Antiangiogenic stigmasterol derivatives.

Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice. Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin. Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events.

Synthetic stigmastanes with dual antiherpetic and immunomodulating activities inhibit ERK and Akt signaling pathways without binding to glucocorticoid receptors.

BACKGROUND: We have previously shown that some synthetic hydroxylated stigmastanes derived from plant sterols inhibit in vitro HSV-1 replication in ocular cell lines and decrease cytokine production in stimulated macrophages, suggesting that these steroids might combine antiviral and immunomodulating properties. In this paper we report the synthesis of some analogs fluorinated at C-6 in order to study the effect of this modification on bioactivity. METHODS: The following methods were used: organic synthesis of fluorinated analogs, cytotoxicity determination with MTT assays, cytokine production quantification with ELISAs, glucocorticoid activity determination by displacement assays, immunofluorescence and transcriptional activity assays, studies of the activation of signaling pathways by Western blot, antiviral activity evaluation through virus yield reduction assays. RESULTS: We report the chemical synthesis of new fluorinated stigmastanes and show that this family of steroidal compounds exerts its immunomodulating activity by inhibiting ERK and Akt signaling pathways, but do not act as glucocorticoids. We also demonstrate that fluorination enhances the antiviral activity. CONCLUSIONS: Fluorination on C-6 did not enhance the anti-inflammatory effect, however, an increase in the in vitro antiviral activity was observed. Thus, our results suggest that it is possible to introduce chemical modifications on the parent steroids in order to selectively modulate one of the effects. GENERAL SIGNIFICANCE: This family of steroids could allow the development of an alternative treatment for ocular immunopathologies triggered by HSV-1, without the undesirable side effects of the currently used drugs.

Synthetic pregnenolone derivatives as antiviral agents against acyclovir-resistant isolates of Herpes Simplex Virus Type 1.

The conventional therapy for the management of Herpes Simplex Virus Type 1 (HSV-1) infections mainly comprises acyclovir (ACV) and other nucleoside analogues. A common outcome of this treatment is the emergence of resistant viral strains, principally when immunosuppressed patients are involved. Thus, the development of new antiherpetic compounds remains as a central challenge. In this work we describe the synthesis and the in vitro antiherpetic activity of a new family of steroidal compounds derived from the endogenous hormone pregnenolone. Some of these derivatives showed a remarkable inhibitory effect on HSV-1 spread both on wild type and ACV-resistant strains. The results also show that these compounds seem to interfere with the late steps of the viral cycle.

Small Molecules as Anti-TNF Drugs.

Tumor necrosis factor (TNF, TNF-α, cachectin) is a pleiotropic, proinflammatory cytokine with multiple biological effects, many of which are not yet fully understood. Although TNF was initially described as an anti-tumor agent more than three decades ago, current knowledge places it central to immune system homeostasis. TNF plays a critical role in host defense against infection, as well as an inhibitory role in autoimmune disease. However, TNF overproduction generates deleterious effects by inducing the transcription of genes involved in acute and chronic inflammatory responses including asthma, rheumatoid arthritis, Crohn's disease, and psoriasis. Direct inhibition of TNF by biologics, such as monoclonal antibodies and circulating TNF receptor constructs, has produced effective treatments for these disorders and validated the inhibition of this proinflammatory cytokine as an effective therapy. Unfortunately, these biological therapies suffer from several drawbacks, including high cost and the induction of autoantibody production. Thus, the development of small molecules able to modulate TNF production or signaling pathways remains a central challenge in Medicinal Chemistry. Considerable efforts have been made over the past two decades to develop such inhibitors, which could potentially be administered orally and would presumably be cheaper. This review is focused on the recent development of compounds that modulate the activity of this cytokine by acting at different levels, such as TNF expression, processing, binding to its receptors and direct inhibition. These approaches will be compared and discussed.

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.

Immunomodulatory activity of an anti-HSV-1 synthetic stigmastane analog.

Many viral infections are associated with the development of immunopathologies and autoimmune diseases, which are of difficult treatment and for which no vaccines are yet available. Obtaining compounds that conjugate both antiviral and immunomodulatory activities in the same molecule would be very useful for the prevention and/or treatment of these immunopathologies. The compound (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) displays anti-Herpes simplex virus type 1 activity in vitro and reduces the incidence of herpetic stromal keratitis (HSK) in mice, a chronic inflammatory syndrome induced by ocular HSV-1 infection. In the present study, compound 1 showed opposite immunomodulatory properties in vitro. It induced the release of pro-inflammatory cytokines in HSV-1-infected epithelial cells of ocular origin, and significantly reduced the production of these cytokines in LPS-activated macrophages. RNA microarrays revealed various overexpressed and repressed genes in compound 1 treated infected epithelial cells and activated macrophages, many of which are associated with innate immune responses and inflammatory processes. These immunomodulatory properties of compound 1, together with its previously reported antiviral activity, make it a potential drug for the treatment of HSK and many other immunopathologies of viral and non-viral origin.

A natural antiviral and immunomodulatory compound with antiangiogenic properties.

Meliacine (MA), an antiviral principle present in partially purified leaf extracts of Melia azedarach L., reduces viral load and abolishes the inflammatory reaction and neovascularization during the development of herpetic stromal keratitis in mice. 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), obtained from MA, displays anti-herpetic and immunomodulatory activities in vitro. We investigated whether CDM interferes with the angiogenic process. CDM impeded VEGF transcription in LPS-stimulated and HSV-1-infected cells. It proved to have neither cytotoxic nor antiproliferative effect in HUVEC and to restrain HUVEC migration and formation of capillary-like tubes. Moreover, MA inhibits LMM3 tumor-induced neovascularization in vivo. We postulate that the antiangiogenic activity of CDM displayed in vitro as a consequence of their immunomodulatory properties is responsible for the antiangiogenic activity of MA in vivo, which would be associated with the lack of neovascularization in murine HSV-1-induced ocular disease.

Cytotoxic tirucallane triterpenoids from Melia azedarach fruits.

The phytochemical investigation of the dichloromethane-soluble part of the methanol extract obtained from the fruits of Melia azedarach afforded one new tirucallane-type triterpene, 3-alpha-tigloylmelianol and three known tirucallanes, melianone, 21-beta-acetoxy-melianone, and methyl kulonate. The structure of the isolated compounds was mainly determined by 1D and 2D NMR experiments as well as HPLC-Q-TOF mass spectrometry. The cytotoxicity of the isolated compounds toward the human lung adenocarcinoma epithelial cell line A549 was determined, while no activity was observed against the phytonematode Meloidogyne incognita.

1-Cinnamoyl-3,11-dihydroxymeliacarpin delays glycoprotein transport restraining virus multiplication without cytotoxicity.

The 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), isolated from extracts of Melia azedarach L., displays antiviral and immunomodulating properties. CDM is the first reported tetranortriterpenoid responsible for the alkalinization of intracellular compartments affecting both, viral endocytic and exocytic pathways. Considering that viral glycoprotein synthesis is completely dependent upon cellular membrane trafficking, we questioned whether CDM might also interfere with the normal transport of cellular glycoproteins. This study demonstrates that CDM promoted a transient block in the transport of two cellular glycoproteins, the transferrin receptor (TfR) and TNF-alpha. Nevertheless, CDM did not affect the transferrin binding ability of TfR and did not impede the TNF-alpha secretion. On the other hand, CDM disturbed the intracellular localization of capsid, glycoprotein and tegument proteins simultaneously in the same HSV-1 infected cells. Besides, we show that concanamycin A and monensin provoke a permanent blockage of viral and cellular glycoproteins, in contrast to the delay observed after CDM treatment. Thus, the delay on glycoprotein transport caused by CDM would account for the strong inhibition on virus multiplication without interfering with the bioactivity of cellular glycoproteins.

A natural tetranortriterpenoid with immunomodulating properties as a potential anti-HSV agent.

Meliacine (MA), an antiviral principle present in partially purified leaf extracts of Melia azedarach L., prevents the development of herpetic stromal keratitis (HSK) in mice by diminishing the viral load in the eye and the severity of lesions caused by a virus-induced immunopathological reaction. The tetranortriterpenoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), obtained from MA purification, displays anti-herpetic activity and impedes nuclear factor kappaB (NF-kappaB) activation in HSV-1 infected conjunctival cells. To extend our understanding about CDM biological properties, we investigated its anti-HSV-1 activity as well as the effect on NF-kappaB activation and cytokine secretion induced by viral (HSV-1) and no-viral (LPS) stimuli, in corneal cells and macrophages. CDM exerted a potent anti-HSV-1 effect on corneal cells and inhibited NF-kappaB translocation to the nucleus, leading to a decrease in IL-6 production. Besides, CDM seemed to modulate IL-6 and TNF-alpha responses in macrophages, whether they were infected with HSV-1 or stimulated with LPS. However, CDM did not affect NF-kappaB activation in these cells, suggesting that an alternative NF-kappaB cell signaling pathway would be involved in the modulation of cytokine production. We conclude that, in addition to its antiviral effect, CDM would be acting as an immunomodulating compound which would be responsible for the improvement of murine HSK already reported.

Anti-herpetic and anti-inflammatory activities of two new synthetic 22,23-dihydroxylated stigmastane derivatives.

Stromal keratitis resulting from ocular infection with Herpes simplex virus type 1 (HSV-1) is a common cause of blindness. This report investigates the antiviral and anti-inflammatory properties of two new synthetic stigmastane analogs in the experimental model of HSV-1-induced ocular disease in mice. (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (1) and (22S,23S)-22,23-dihydroxystigmasta-1,4-dien-3-one (2) exhibited anti-HSV-1 activity in vitro and ameliorated the signs of murine herpetic stromal keratitis (HSK), although none of the compounds showed antiviral activity in vivo. We discuss that the improvement of HSK could be due to an immunomodulatory effect of both compounds.

The in vitro immunomodulatory activity of a synthetic brassinosteroid analogue would account for the improvement of herpetic stromal keratitis in mice.

Herpes simplex virus type 1 (HSV-1) induces an ocular chronic immunoinflammatory syndrome named herpetic stromal keratitis that can lead to vision impairment and blindness. We have reported that the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one, designated as 2, is a potent antiviral in vitro and reduces the incidence of murine herpetic stromal keratitis, although it does not exert an antiviral effect in vivo. In the present report, we investigated whether brassinosteroid 2 may play a role in the modulation of the response of epithelial and immune cells to HSV-1 infection. Compound 2 blocked HSV-1-induced activation of NF-kappaB by inhibiting its translocation to the nucleus of infected corneal and conjunctival cells in vitro, as well as significantly reduced the secretion of TNF-alpha in infected NHC cells. Conversely, IL-6 production was enhanced by compound 2 after HSV-1 infection in both cell types. The production of these cytokines was considerably reduced in a LPS-stimulated macrophage cell line after treatment with compound 2. In conclusion, brassinosteroid 2 would be playing a modulating effect as an inductor or inhibitor, depending on the cell type involved. The improvement of disease observed in mice could be a balance between both, the immunostimulating and immunosuppressive effects of brassinosteroid 2 in vivo.

Syntheses of immunomodulating androstanes and stigmastanes: comparison of their TNF-alpha inhibitory activity.

In a previous work our group showed that some synthetic stigmastanes may play a role in immune-mediated inflammation. In this paper we report the syntheses of a series of new steroidal compounds derived from dehydroepiandrosterone and stigmasterol, and the evaluation of their in vitro inhibitory activity of the TNF-alpha production by macrophages. A preliminary qualitative structure-activity relationship was established.

1-Cinnamoyl-3,11-dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-kappaB modulating properties.

We have reported the isolation of the tetranortriterpenoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) from partially purified leaf extracts of Melia azedarach L. (MA) that reduced both, vesicular stomatitis virus (VSV) and Herpes simplex virus type 1 (HSV-1) multiplication. CDM blocks VSV entry and the intracellular transport of VSV-G protein, confining it to the Golgi apparatus, by pre- or post-treatment, respectively. Here, we report that HSV-1 glycoproteins were also confined to the Golgi apparatus independently of the nature of the host cell. Considering that MA could be acting as an immunomodulator preventing the development of herpetic stromal keratitis in mice, we also examined an eventual effect of CDM on NF-kappaB signaling pathway. CDM is able to impede NF-kappaB activation in HSV-1-infected conjunctival cells and leads to the accumulation of p65 NF-kappaB subunit in the cytoplasm of uninfected treated Vero cells. In conclusion, CDM is a pleiotropic agent that not only inhibits the multiplication of DNA and RNA viruses by the same mechanism of action but also modulates the NF-kappaB signaling pathway.

In vitro and in vivo antiherpetic activity of three new synthetic brassinosteroid analogues.

Brassinosteroids are a novel group of steroids that appear to be ubiquitous in plants and are essential for normal plant growth and development. It has been previously reported that brassinosteroid analogues exert an antiviral activity against herpes simplex virus type 1 (HSV-1) and arenaviruses. In the present study, we report the chemical synthesis of compounds (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (2), (22S,23S)-5alpha-fluoro-3beta-22,23-trihydroxystigmastan-6-one (3), (22S,23S)-3beta,5alpha,22,23-tetrahydroxy-stigmastan-6-one (4) as well as their antiherpetic activity both in a human conjunctive cell line (IOBA-NHC) and in the murine herpetic stromal keratitis (HSK) experimental model. All compounds prevented HSV-1 multiplication in NHC cells in a dose dependent manner when added after infection with no cytotoxicity. Administration of compounds 2, 3, and 4 to the eyes of mice at 1, 2, and 3 days post-infection delayed and reduced the incidence of HSK, consisting mainly of inflammation, vascularization, and necrosis, compared to untreated, infected mice. However, viral titers of eye washes showed no differences among samples from treated and untreated mice. Since the decrease in the percentage of mice with ocular lesions occurred 5 days after treatment had ended, we suggest that brassinosteroids 2, 3, and 4 did not exert a direct antiviral effect in vivo, but rather may play a role in immune-mediated stromal inflammation, which would explain the improvement of the clinical signs of HSK observed.

Block of vesicular stomatitis virus endocytic and exocytic pathways by 1-cinnamoyl-3,11-dihydroxymeliacarpin, a tetranortriterpenoid of natural origin.

Previously, it has been shown that 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a natural compound isolated from leaf extracts of Melia azedarach L., inhibits the vesicular stomatitis virus (VSV) multiplication cycle when added before or after infection. Here, we have established that the lack of VSV protein synthesis in CDM pre-treated Vero cells is ascribed to the inhibition of an initial step during virus multiplication, although indirect immunofluorescence (IFI) studies confirmed that the binding and uptake of [(35)S]methionine-labelled VSV was not affected by CDM pre-treatment. Instead, our findings revealed that this compound impedes the uncoating of VSV nucleocapsids in pre-treated Vero cells, since the antiviral action of CDM was partially reversed by inducing VSV direct fusion at the plasma membrane, and VSV M protein fluorescence was confined to the endosomes, even 2 h post-internalization. Furthermore, CDM induced cytoplasmic alkalinization, as shown by acridine orange staining, consistent with the inhibition of virus uncoating. Although VSV proteins are synthesized when CDM is added after infection, IFI studies revealed that G protein was absent from the surface of infected cells and co-localized with a Golgi marker. Therefore, CDM inhibits the transport of G protein to the plasma membrane. Taken together, these findings indicate that CDM exerts its antiviral action on the endocytic and exocytic pathways of VSV by pre- or post-treatment, respectively.

An antiviral meliacarpin from leaves of Melia azedarach L.

Bioassay guided purification of the ethyl acetate extract of leaves of Melia azedarach led to the isolation of the limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin, which showed IC50 values of 6 microM and 20 microM for vesicular stomatitis (VSV) and herpes simplex (HSV-1) viruses, respectively. Its structure was established by spectroscopic methods.