5-O-Alkylated derivatives of 5-hydroxy-2'-deoxyuridine as potential antiviral agents. Anti-herpes activity of 5-propynyloxy-2'-deoxyuridine.

Alkylation of 5-hydroxyuridine or 5-hydroxy-2'-deoxyuridine with various activated alkylating agents in the presence of 1 equiv of NaOH gave a series of new nucleoside analogues which were evaluated for antiviral activity against vaccinia virus, herpes simplex-1 virus, and vesicular stomatitis virus in both primary rabbit kidney cells and human skin fibroblasts. One of these analogues, 5-propynyloxy-2'-deoxyuridine, was a potent inhibitor of herpes simplex virus. Structure-activity considerations suggest that the anti-herpes activity is dependent on the integrity of the acetylene group since substitution of phenyl, p-nitrophenyl, vinyl, carboxamido, or carboxyl for the triple bond led to diminished antiviral activity.     

Discovery of dihydro-alkyloxy-benzyl-oxopyrimidines as promising anti-influenza virus agents

A series of novel dihydro-alkyloxy-benzyl-oxopyrimidine derivatives were synthesized and evaluated for their activity against influenza virus in Madin-Darby canine kidney cells. Four dihydro-alkyloxy-benzyl-oxopyrimidine derivatives (4a1, 4a2, 4a3, and 4d1) showed potent activity against influenza virus. Among them, compound 4a3 was the most promising lead with broad activity against influenza A (antiviral EC(50) values of 9 and 18 μm for the A/H1N1 and A/H3N2 subtype, respectively) and influenza B viruses (EC(50) : 33 μm). The antiviral mechanism of action of these dihydro-alkyloxy-benzyl-oxopyrimidine derivatives must be quite different from that of the currently approved anti-influenza virus drugs that target the viral M2 or neuraminidase proteins. The dihydro-alkyloxy-benzyl-oxopyrimidine derivatives represent a new avenue for further optimization and development of novel anti-influenza virus agents.     

Novel pyrimidine and purine derivatives of L-ascorbic acid: synthesis and biological evaluation.

The novel pyrimidine derivatives 1-6 of 2,3-dibenzyl-4,5-didehydro-5, 6-dideoxy-L-ascorbic acid were synthesized by the condensation of pyrimidine bases with 5,6-diacetyl-2,3-dibenzyl-L-ascorbic acid (DDA). Both N-9 (7) and N-7 (8) regioisomers were obtained in the reaction of 6-chloropurine with 5-acetyl-6-bromo-2, 3-dibenzyl-L-ascorbic acid (ABDA), while the reaction of 6-(N-pyrrolyl)purine with ABDA afforded exclusively the N-9 isomer 9. Structures of all newly prepared compounds were deduced from the chemical shifts in (1)H and (13)C NMR spectra, as well as connectivities in 2D homo- and heteronuclear correlation spectra. An unambiguous proof of the structure and conformation of 7 was obtained by X-ray crystallographic analysis. Compounds 1-9 were found to exert cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), murine leukemia (L1210/0), murine mammary carcinoma (FM3A), and human T-lymphocytes (Molt4/C8 and CEM/0), as well as antiviral activities against varicella-zoster virus (TK(+)VZV and TK(-)VZV) and cytomegalovirus (CMV). The compound 6 containing a trifluoromethyl-substituted uracil ring exhibited marked antitumor activity. The N-7-substituted purine regioisomer 8 had greater inhibitory effects on the murine L1210/0 and human CEM/0 cell lines than the N-9 isomer 7. Compound 9 with the 6-purine-substituted pyrrolo moiety had a more pronounced selective cytostatic activity against human (Molt4/C8 and CEM/0) cell lines than murine (L1210/0 and FM3A/0) and human (MiaPaCa2, MCF7, HeLa, and Hep2) tumor cell lines and normal fibroblasts (Hef522). The compound 6 exhibited the most potent antiviral activities against TK(+)VZV, TK(-)VZV, and CMV, albeit at concentrations that were only slightly lower than the cytotoxic concentrations.     

The antiviral effect of human interferon alpha is dependent on phosphoinositide-derived messengers

Neomycin the putative blocker of membrane polyphosphoinositide hydrolysis, inhibited the antiviral activity of human interferon alpha, when tested on human quiescent fibroblasts challenged with vesicular stomatitis virus. The anti-interferon effect of neomycin could be correlated in terms of dose dependence for both neomycin (0.05-1 mM) and interferon (100-5,000 IU/ml). The results suggest that the antiviral activity of interferon alpha depends on diacylglycerol formation. Indeed, the synthetic diacylglycerol (50 microM) was as effective as 100 IU/ml interferon in inducing the antiviral state.     

Virucidal activity of hypericin against enveloped and non-enveloped DNA and RNA viruses.

Hypericin is a polycyclic anthrone first isolated from the plant St. Johnswort and was shown to have dramatic anti-retroviral activity against Friend leukemia virus and radiation leukemia virus in mice. Hypericin displayed marginal activity (IC50 = 6 micrograms/ml) against Moloney murine leukemia virus (Mo-MuLV) in vitro. Hypericin did not display selective antiviral activity against herpes simplex virus, influenza A, adenovirus, or poliovirus. The 50% cytotoxic concentration was approximately 25 micrograms/ml. When virus was incubated with hypericin before infecting cells, the drug was virucidal to all enveloped viruses tested (herpes simplex, influenza virus A, and Mo-MuLV) at concentrations of 1.56 micrograms/ml to 25 micrograms/ml. Hypericin was not virucidal to the non-enveloped viruses tested (adenovirus and poliovirus). These data indicate that the mechanism of viral inactivation for hypericin is dependent upon the presence of a viral lipid envelope. In vivo, hypericin (50 mg/ml) was effective against FLV or HSV-1 if incubated with the virus for 1 h at 37 degrees C before infecting mice, but was not effective if pre-incubated with virus for 1 h at 4 degrees C or if administered concurrently with virus.     

Antiviral effect of lithium chloride

Studies in human embryo fibroblasts infected with measles or herpes simplex virus showed a reduction in virus yield when cultures were pretreated with 1-10 mM lithium chloride doses. Maximum effect was obtained by a 1 h treatment with 10 mM lithium chloride, preceding viral infection by 19-24 hours. A specific antiviral effect against measles virus was manifest immediately after culture pretreatment. Intermittent treatment with 10 mM lithium chloride of cultures persistently infected with measles or herpes virus obtained from human myeloid K-562 cell line shows a reduction in the extracellular virus yield. In the K-562/herpes virus system, the culture treatment with lithium chloride and acyclovir (10 microM) has an additive inhibitory effect on virus production. The paper is focused on the mechanism of lithium chloride antiviral action and the expediency of lithium therapy in SSPE (subacute sclerosing panencephalitis).     

CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNβ-dependent and -independent way

Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.     

Dual antiviral activity of human alpha-defensin-1 against viral haemorrhagic septicaemia rhabdovirus (VHSV): inactivation of virus particles and induction of a type I interferon-related response

It is well known that human alpha-defensin-1, also designated as human neutrophil peptide 1 (HNP1), is a potent inhibitor towards several enveloped virus infecting mammals. In this report, we analyzed the mechanism of the antiviral action of this antimicrobial peptide (AMP) on viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Against VHSV, synthetic HNP1 possesses two antiviral activities. The inactivation of VHSV particles probably through interfering with VHSV-G protein-dependent fusion and the inhibition of VHSV replication in target cells by up-regulating genes related to the type I interferon (IFN) response, such as Mx. Neither induction of IFN-stimulated genes (ISGs) by HNP1 nor their antiviral activity against fish rhabovirus has been previously reported. Therefore, we can conclude that besides to acting as direct effector, HNP1 acts across species and can elicit one of the strongest antiviral responses mediated by innate immune system. Since the application of vaccine-based immunization strategies is very limited, the used of chemicals is restricted because of their potential harmful impact on the environment and no antimicrobial peptides from fish that exhibit both antiviral and immunoenhancing capabilities have been described so far, HNP1 could be a model molecule for the development of antiviral agents for fish. In addition, these results further confirm that molecules that mediate the innate resistance of animals to virus may prove useful as templates for new antivirals in both human and animal health.     

Nucleoside pools of acyclovir-treated herpes simplex type 1 infected cells

Nucleoside pools of herpes simplex type 1 (HSV-1)-infected and uninfected African green monkey kidney (GMK) cells and human fetal lung fibroblasts (HL) have been analysed with high-performance liquid chromatography (HPLC). The only nucleosides found in measurable amounts were deoxythymidine (dThd) and adenosine (Ado). The dThd pool seemed to be greater in GMK cells than in HL cells. dThd was also the only nucleoside excreted into the medium. HSV-1 infection reduced the dThd concentration of GMK cells. Addition of acyclovir (ACV) to HSV-1-infected GMK cells inhibited virus replication. This resulted in a dThd concentration similar to that of uninfected GMK cells. dThd added to HSV-1-infected GMK and HL cells reduced the antiviral action of ACV but not that of phosphonoformic acid (PFA). ACV is known to be activated mainly by HSV-induced deoxythymidine kinase (dTK), an enzyme which utilizes dThd as a substrate, while the action of PFA is independent of dTK. The low antiviral activity of ACV in GMK cells as compared to HL cells may be explained by the presence of high amounts of dThd in GMK cells.     

Effect of selected neuroleptic agents and stereo-isomeric analogues on virus and eukaryotic cells.

Chlorpromazine, cis(Z)-chlorprothixene (Truxal), and the non-neuroleptic trans(E)-chlorprothixene and trans(E)-flupenthixol were studied in vitro for possible antiviral effect on Herpes simplex virus 2 and for toxic effect on human diploid fibroblasts. Based on an enzyme-linked immunosorbent assay (ELISA) antiviral activity was demonstrated for all the compounds in the concentration range 0.39 micrograms/ml-25 micrograms/ml. A cell-toxic effect was shown in the higher concentration range for all the compounds except cis(Z)-chlorprothixene. A cell-stimulatory effect was also detected at the lower concentration range (about 3.13 micrograms/ml) for all compounds. Thus both cell stimulation and antiviral effect can be found for the same agent within the same concentration range. The results point to the possibility of creating different antiviral drugs--which would also include a cell-stimulatory activity--among psychopharmacological drugs and their stereoisomeric analogues.     

朱砂七总蒽醌体外抗单纯疱疹病毒的研究

目的研究朱砂七总蒽醌(ZSQ)体外对单纯疱疹病毒的抑制作用。方法运用单纯疱疹病毒I型(HSV-Ⅰ)和Ⅱ型(HSV-Ⅱ),以非洲绿猴肾细胞(Vero细胞)为宿主细胞,通过观察感染病毒后的细胞变性反应(CPE)和MTI比色法,检测ZSQ对单纯疱疹病毒的抑制作用。结果与病毒对照组相比,朱砂七总蒽醌各质量浓度组均能有效地保护感染HSV-I和HSV-Ⅱ病毒的Vero细胞,抑制单纯疱疹病毒的复制,使病毒导致的细胞病变减弱。ZSQ抗HSV-I病毒作用IC50为0.004 7g.L-1,抗病毒有效率达90.83%,治疗指数(TI)为16.62;总蒽醌抗HSV-Ⅱ病毒作用IC50为0.006 7g.L-1,抗病毒有效率达90.06%,治疗指数(TI)为11.66。结论 ZSQ在体外显示出明显的保护宿主细胞、抵抗单纯疱疹病毒感染的活性。     

Inhibition of herpes simplex virus infection by tannins and related compounds

Several chemically defined plant extracts were investigated for their antiviral action on herpes simplex virus (HSV-1, HSV-2)-infected African green monkey kidney cells and human adenocarcinoma cells, using a plaque formation assay. Among them, the monomeric hydrolyzable tannins, oligomeric ellagitannins and condensed tannins, having galloyl groups or hexahydroxydiphenoyl groups, had the most potent anti-HSV activity. Their 50% effective doses (0.03-0.1 microgram/ml) were by two-three orders of magnitude lower than their 50% cytotoxic doses (greater than 10 micrograms/ml). On the other hand, gallic acid, neutral polysaccharides, chemically modified (N,N-dimethylaminoethyl-, carboxymethyl-, and sulfated-) glucans, sialic acid-rich glycoproteins, and uronic acid-rich pine cone polysaccharide showed little or no activity. Using radiolabeled virus particles, we demonstrated that the anti-HSV effect of the tannins is due to inhibition of virus adsorption to the cells.     

Natural antiviral activity of mouse macrophages against encephalomyocarditis virus

Resident mouse peritoneal cells (PC) express a significant antiviral activity against encephalomyocarditis virus (EMCV) in vitro, as judged by decreased virus yield from infected mouse embryo fibroblasts (MEF). This natural antiviral activity of PC was not due either to enhanced lysis of virus-infected cells, as these were protected from lysis rather than destructed by PC, or to interferon (IFN) production, as no direct correlation between IFN and anti-EMCV activity was found. Among PC, macrophages (M phi) appear to be responsible for the anti-EMCV activity, which was indeed attributable to a Thy 1.2-negative, adherent mononuclear cell. Moreover, M phi-defective C3H/HeJ mice showed a significant impairment of anti-EMCV activity, whereas M phi of mice defective for natural killer (NK) activity (bg/bg, SJL/J) or for mature T cells (nu/nu) possessed an intact antiviral capacity.     

The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo

Treatment of human cytomegalovirus (HCMV) infections with any of the currently available antiviral agents is frequently associated with the occurrence of severe complications, seriously threatening the successful outcome of treatment. Therefore, the development of novel antiviral strategies is a challenging goal of current investigations. Previously, we reported that artesunate (ART) is an effective, non-cytotoxic inhibitor of HCMV in vitro. Here, we demonstrate that the efficacy of the antiviral effect of ART is augmented by co-treatment of HCMV-infected fibroblasts with ferrous iron, i.e. Ferrosanol, and/or the iron transfer-mediating molecule holo-transferrin. This could alleviate the HCMV-induced modulation of cell surface expression of adhesion molecule Thy-1, suggesting that ART might be able to prevent pro-inflammatory effects of infection. The iron-enhanced, antiviral effect of ART could also be demonstrated in cultured cells infected with rat cytomegalovirus. Experiments using the RCMV/rat model showed that both the viral DNA load and virus titers in the salivary glands from infected rats were significantly reduced upon treatment with ART. Furthermore, an additive antiviral effect for ART together with each one of conventional anti-HCMV drugs, i.e. ganciclovir, cidofovir or foscarnet, was detected in HCMV-infected fibroblasts. These findings might open new perspectives regarding the use of ART in clinical trials.     

扇贝裙边糖胺聚糖体外抗I型单纯疱疹病毒实验研究

目的研究扇贝裙边糖胺聚糖(glycosaminoglycan from Scallop Skirt,SS-GAG)体外抗单纯疱疹病毒Ⅰ型(HSV-Ⅰ)的作用。方法运用单纯疱疹病毒Ⅰ型(HSV-ⅠSM44株),并以非洲绿猴肾细胞(Vero细胞)为宿主细胞,通过观察病毒感染后的细胞变性反应(cytopathic effect,CPE)和运用MTT比色法,检测不同浓度药物SS-GAG对Ⅰ型单纯疱疹病毒是否有直接的灭活作用、对HSV-Ⅰ感染复制的抑制活性以及对HSV-Ⅰ感染细胞的综合作用等,并观察药物对Vero细胞的毒性作用。结果与病毒对照组相比,SS-GAG各浓度组(100、50、25mg.L-1)能有效地保护经HSV-Ⅰ感染的Vero细胞,使细胞活性增强(P<0.01);并减弱HSV-Ⅰ导致的病变效应,抑制病毒的复制。此作用随着药物浓度的增加而增强。但是SS-GAG对HSV-Ⅰ没有直接的灭活作用;SS-GAG在50~1600mg.L-1浓度范围内对Vero细胞无明显的细胞毒性。结论SS-GAG在体外实验系统中显示出明显的保护宿主细胞抵抗HSV-Ⅰ病毒感染的活性作用。     

SKI-1/S1P inhibition: a promising surrogate to statins to block hepatitis C virus replication

Hepatitis C virus (HCV) is often associated with steatosis, cirrhosis and hepatocellular carcinoma (HCC). Statins (HMG-CoAR inhibitors) have been shown to exert an antiviral effect in vitro, principally on replicon harboring cells, but the effect of their use alone in vivo remains controversial. In clinical trials, when used in combination with the standards of care (SOC), they led to an increased proportion of sustained virological responder (SVR). Here we investigated the implication of SKI-1/S1P, a master lipogenic pathways regulator upstream of HMG-CoAR, on different steps of HCV life cycle. We compared the HCV antiviral effect of the most potent SKI-1/S1P small molecule inhibitor (PF-429242) with a set of two statins on different steps of the viral life cycle, and showed that SKI-1/S1P inhibitor blocked HCVcc (strain JFH-1) RNA replication (EC(50)= 5.8 μM) more efficiently than statins. Moreover, we showed that PF-429242 could reduce lipid droplets accumulation in Huh7 cells. Interestingly, PF-429242 dramatically reduced infectious particles production (EC(90)= 4.8 μM). Such inhibition could not be achieved with statins. SKI-1/S1P activity is thus essential for viral production and its inhibition should be considered for antiviral drug development.     

Antiviral activity of aliphatic nucleoside analogues: structure-function relationship.

Of a series of 58 aliphatic nucleoside analogues, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA] proved to be the most active congener, when assayed for antiviral activity in primary rabbit kidney cell cultures challenged with either vaccinia or vesicular stomatitis virus. Whereas most analogues derived from substituted purine and pyrimidine bases and bearing various hydroxy- or amino-substituted alkyl chains did not show evidence of antiviral activity at a concentration of 2 mM, (S)-DHPA inhibited both vaccinia and vesicular stomatitis virus replication at 0.05-0.1 mM. For 9-[(RS)-2,3-diazidopropyl]adenine and some di- and trihydroxybutyl analogues of DHPA, viz., 9-[(2RS,3SR)-2,3-dihydroxybutylladenine, 9-[(RS)- or 9-[(S)-3,4-dihydroxybutyl]adenine, 9-[(2S,3R)-2,3,4-trihydroxybutyl]adenine, and 3-(adenin-9-yl)-(RS)-alanine, an antiviral effect was noted at a concentration of 0.5-1 mM.     

Effect of Selected Neuroleptic Agents and Stereo-Isomeric Analogues on Virus and Eukaryotic Cells

Abstract: Chlorpromazine, cis(Z)-chlorprothixene (Truxal^®), and the non-neuroleptic trans(E)-chlorprothixene and trans(-E)-flupenthixol were studied in vitro for possible antiviral effect on Herpes simplex virus 2 and for toxic effect on human diploid fibroblasts. Based on an enzyme-linked immunosorbent assay (ELISA) antiviral activity was demonstrated for all the compounds in the concentration range 0.39 μg/ml-25 μg/ml. A cell-toxic effect was shown in the higher concentration range for all the compounds except cis(Z)-chlorprothixene. A cell-stimulatory effect was also detected at the lower concentration range (about 3.13 μg/ml) for all compounds. Thus both cell stimulation and antiviral effect can be found for the same agent within the same concentration range. The results point to the possibility of creating different antiviral drugs - which would also include a cell-stimulatory activity - among psychopharmacological drugs and their stereo-isomeric analogues.     

Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral activity against coxsackievirus B3

Coxsackievirus B3 (CVB-3) is a major causative agent of chronic heart muscle infections. The present study describes a cell culture system with an ongoing virus infection to evaluate two novel inhibitory strategies, either individually or combined: (1) RNA interference (RNAi) to degrade cytoplasmatic CVB-3 RNA and (2) a vector-delivered soluble variant of the coxsackievirus-adenovirus receptor fused to a human immunoglobulin (sCAR-Fc), which inhibits cellular uptake of CVB-3. Both approaches were capable of inhibiting CVB-3 in persistently infected human myocardial fibroblasts. The antiviral effect of a single treatment lasted for up to one week and could be extended by repeated applications. Each of the single treatments initially reduced the virus titer by approximately 1-log, whereas the combination of both approaches resulted in 4-log inhibition and retained substantial antiviral activity at later time points, when the effect of sCAR-Fc or siRNAs alone had already disappeared. Further analysis revealed that sCAR-Fc protects cells from virus-induced lysis but does not diminish the virus load. Reduction of the virus titer was only achieved with additional destruction of viral RNA by RNAi. Taken together, combination of RNAi and a protein-based antiviral strategy was found to result in a strong synergistic inhibition of an ongoing virus infection.     

Drug Delivery Systems and Liver Targeting for the Improved Pharmacotherapy of the Hepatitis B Virus (HBV) Infection

In spite of the progress made in vaccine and antiviral therapy development, hepatitis B virus (HBV) infection is still the most common cause of liver cirrhosis and hepatocellular carcinoma, with more than 400 million people chronically infected worldwide. Antiviral therapy with nucleos(t)ide analogues and/or immunomodulating peptides is the only option to control and prevent the progression of the disease in chronic hepatitis B (CHB)-infected patients. So far, the current antiviral monotherapy remains unsatisfactory because of the low efficacy and the development of drug resistance mutants. Moreover, viral rebound is frequently observed following therapy cessation, since covalent closed circular DNA (cccDNA) is not removed from hepatocytes by antiviral therapy. First, this review describes the current pharmacotherapy for the management of CHB and the new drug candidates being investigated. Then, the challenges in the development of drug delivery systems for the targeting of antiviral drugs to the liver parenchyma are discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to eradicate the virus from the host are addressed.