In Vitro Studies on the Antiviral Activity of 1,3-Bis(2-Chloroethyl)-1-Nitrosourea

Chemotherapy experiments carried out in vitro demonstrated that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was active against lymphocytic choriomeningitis virus and had an equivocal antiviral effect on Semliki Forest, herpes simplex, and vaccinia viruses. No antiviral effect was observed with BCNU against western equine encephalomyelitis, polio, and parainfluenza (HA-1) viruses. Activity of the drug was determined by inhibition of viral-induced cytopathogenic effect in KB or chick embryo cells and by reduction of virus titer in cell culture supernatant fluid. Maximal activity against the viruses was observed when drug and virus were incubated together for 30 min prior to addition to cells; essentially no activity could be demonstrated if BCNU and virus were added to cells with no prior incubation.     

The Interferon-Inducible Chemokines MuMig and Crg-2 Exhibit Antiviral Activity In Vivo

MuMig (murine monokine induced by gamma interferon) and Crg-2 (cytokine responsive gene 2) are two murine chemokines of the CXC family that are induced by the interferons (IFNs): MuMig specifically by IFN-γ and Crg-2 by IFN-α, IFN-β, and IFN-γ. To investigate the biological roles of these chemokines, recombinant vaccinia viruses (rVVs) encoding either MuMig or Crg-2 were constructed. In vitro, the chemokine-encoding rVVs replicated to similar levels to the control virus. Athymic nude mice inoculated with 10⁵ PFU or less of VV-HA-Mig or VV-HA–Crg-2 resolved the infection successfully whereas mice given a similar dose of the control virus VV-HA-TK died from generalized infection. At higher doses, there was mortality in all groups but death was significantly delayed in mice infected with either chemokine-encoding rVV compared with those infected with the control virus. Virus-encoded MuMig and Crg-2 enhanced the cytolytic activity of NK cells and splenic cellularity by two- to threefold and resulted in significant increases in mononuclear cell infiltration in the livers of mice. Using specific neutralizing or depleting antibodies, we have established that the control of rVV replication in athymic nude mice, as a consequence of virus-expressed MuMig and Crg-2, requires NK cells and IFN-α, IFN-β, and IFN-γ.     

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.     

Synthesis and Antiviral Activity of 1,5-and 1,3-Dialkyl-1,2,4-triazole C-Nucleosides Derived from 1-(Chloroalkyl)-1-aza-2-azoniaallene Salts

Abstract

Reactions of α, α′-dichloroazo compounds 2 with SbCl₅ gave 1-(chloroalkyl)-1-aza-2-azoniaallene salts 3 as reactive intermediates. Cycloadditions of 3 with the ribofuranosyl cyanide 4 afforded the β-D-ribofuranosyl-1,2,4-triazolium salts 5, which rearranged spontaneously to salts 6. Hydrolysis of 6 gave the 1,2,4-triazole C-nucleosides 7, which yielded the free nucleosides 8 after deblocking. Analogously, 12 was prepared from the cycloaddition of 4 with the α-chloroazo compound 10 in the presence of SbCl₅. Deblocking of 12 with sodium methoxide afforded 13. Compounds 8a,b,e,f and 13 were tested against HIV-1, HIV-2, HSV-1 and HSV-2 and were found to be inactive.     

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)/interleukin-2 chemoimmunotherapy of murine L1210 leukemia

Summary We have developed a chemoimmunotherapy regimen for the treatment of L1210-cell-induced ascites tumors in mice using a combination of sub-toxic doses of interleukin-2 (IL-2) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is administered intraperitoneally 4 days after tumor implantation and followed 2 days later by single doses of human recombinant IL-2 for 3 consecutive days. An optimum survival of 84% was achieved using 1500 U IL-2. Reduced survival was observed when lower or higher IL-2 dosages were used. No therapy resulted when heat-inactivated IL-2 was used or when IL-2 was used without chemotherapy. Surviving animals were resistant to L1210 leukemia but not P815 mastocytoma tumor challenge suggesting the combined BCNU/IL-2 therapy stimulated tumor-specific immunity.     

1,3-Bis[N-sulfonyl-(1R,2S)-1,3-diphenyl-2-aminopropanol]benzene: an excellent ligand for titanium-catalyzed asymmetric AlPh3(THF) additions to aldehydes

Asymmetric AlPh(3) (THF) additions to a wide variety of aldehydes catalyzed by a titanium catalyst of 20 mol % 1,3-bis[N-sulfonyl-(1R,2S)-1,3-diphenyl-2-aminopropanol]benzene (1) are reported. The catalytic system works excellently for aromatic aldehydes bearing either an electron-donating or an electron-withdrawing substituent on the aromatic ring to afford secondary diaryl alcohols in excellent isolated yields of >or=95% and excellent enantioselectivities of >or=94% ee. The phenyl addition to cinnamaldehyde or 2-furylaldehyde gave corresponding secondary alcohols in 85% and 95% ee, respectively. For aliphatic aldehydes, increasing enantioselectivities of the addition products in terms of increasing steric sizes of aldehydes are observed, and this trend goes from the linear 1-pentanal (87% ee), the secondary cyclohexylaldehyde (95% ee) or the 2-methylpropanal (97% ee), to the tertiary 2,2-dimethylpropanal (99% ee).     

Induction of Antiviral Activity In Vivo and In Vitro by Human Placenta Ribonucleic Acid Treated with Nitrous Acid

Induction of antiviral activity and interferon by human placenta ribonucleic acid deaminated with sodium nitrite (NO₂-RNA) was studied in vitro and in vivo. (1) Viral multiplication in diploid cells from human kidney (HK cells) was depressed by pretreatment with NO₂-RNA, but not by pretreatment with the original placenta RNA. (2) NO₂-RNA showed an interferon-inducing activity in rabbits and mice. (3) NO₂-RNA sedimenting in 18 S and 28 S regions showed a higher antiviral activity than that sedimenting in 4 S region.     

PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG_20K-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG_20K-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.     

Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.The Coronaviridae are a group of enveloped positive-strand RNA viruses of the group Nidovirales. This group of viruses was not, until recently, of major concern as a matter of public health, although they were long recognized as important agents of serious disease in domestic and companion animals. The recent evidence of zoonotic transfer of this family of viruses from bats to animals such as palm civet cats and then to humans during the 2002-2003 outbreak greatly increased scientific interest in the Coronaviridae (7, 14, 19). The best-known coronavirus (CoV) is the causative agent of severe acute respiratory syndrome (SARS), termed the SARS-related coronavirus (SARS-CoV) (7, 14, 19). The lethal SARS outbreaks in China and Canada in 2002-2003 first brought SARS-CoV to public attention. The subsequent identification of two new human coronaviruses associated with acute respiratory infections in humans further illuminated the continuing potential threat that coronaviruses present to public health (31, 36).Infection with SARS-CoV results from the binding of SARS-CoV spike glycoprotein (S) to angiotensin-converting enzyme 2 (ACE2) on the surface of susceptible cells in the lung followed by viral fusion with host cell membranes and transfer of virion contents into the cell (12, 25, 27). The infection stimulates significant cytokine responses in lung tissue that, together with pathologies associated with rapidly replicating virus, cause damage to the airway epithelium and alveolar membranes resulting in edema, respiratory distress, and (in ∼10% of cases) death (5). Due to the proven threat from SARS-CoV infections and the possibility of future zoonotic transmission of coronaviruses, efforts have been initiated to identify agents that could either reduce infection or suppress the deleterious cytokine response to SARS-CoV infection (8, 29).The molecular physiology of the SARS-CoV life cycle and the host response to infection have provided numerous potential targets for chemotherapeutic intervention. In addition to vaccine development strategies, various research groups have targeted the SARS-CoV-specific main protease or viral attachment, entry, and fusion for intervention. SARS-CoV protease inhibitors which inhibit the enzyme at concentrations from 0.5 to 7 μM have been reported (2). The SARS-CoV papain-like protease (PLP) has also been successfully developed as a target for small-molecule antivirals, some of which are active in the 100 nM range (22). Viral entry inhibitors include SARS-CoV S glycoprotein heptad repeat peptides identified as potential inhibitors of viral fusion (3). Another broad-spectrum antiviral approach involves targeting the high-mannose oligosaccharides that are commonly found on viral surface glycoproteins. For example, carbohydrate-binding lectins, including Urtica dioica agglutinin (UDA), have been reported to bind to the SARS-CoV S protein and inhibit viral fusion and entry (33).The antiviral protein griffithsin (GRFT) was originally isolated from the red alga Griffithsia sp. based upon its activity against the human immunodeficiency virus (HIV) (17). This unique 12.7-kDa protein was shown to bind specifically to oligosaccharides on the surface of the HIV envelope glycoprotein gp120. GRFT was shown to possess three largely identical carbohydrate-binding domains orientated as an equatorial triangle and affording multivalent binding and thereby increasing potency (37) (Fig. ​(Fig.1).1). Due to GRFT''s ability to bind to specific oligosaccharides on envelope glycoproteins and block viral entry, it was hypothesized that GRFT might show broad-spectrum antiviral activity against other viruses, including SARS-CoV (38). Here we report the testing of GRFT for antiviral activity against a spectrum of coronaviruses, including SARS-CoV. In addition we present data on the specific binding interactions between GRFT and the SARS-CoV S protein. Finally, we evaluate the in vivo efficacy of intranasal administration of GRFT against infection with SARS-CoV in a lethal mouse model of pulmonary infection and explore the effects that GRFT treatment has on the induction of host cytokine response to SARS-CoV infection.Open in a separate windowFIG. 1.The amino acid sequence and carbohydrate binding domains of griffithsin. Griffithsin monomers contain three distinct, nonlinear, and uniform binding sites for monosaccharides such as mannose and glucose. The binding sites (red, blue, and yellow) are shown both in the amino acid sequence of griffithsin (A) and binding to the disaccharide maltose in a three-dimensional representation derived from the X-ray crystal structure (B).     

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione: Tautomery and coordination to copper(II)

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione (H₂L) was synthesized by azocoupling of diazonium salt of 2-hydroxyaniline with 1-phenylbutane-1,3-dione and characterized by IR, ¹H and ¹³C NMR spectroscopies and X-ray diffraction analysis. In solution, H₂L exists as a mixture of the enol-azo and hydrazone tautomeric forms and a decrease of temperature and of solvent polarity shifts the tautomeric balance to the hydrazone form. In the solid state, H₂L crystallizes from ethanol-water in the monohydrate hydrazone form, as shown by X-ray analysis. The dissociation constants of H₂L (pK₁ = 5.98 ± 0.04, pK₂ = 9.72 ± 0.03) and the stability constants of its copper(II) complex (log β₁ = 11.01 ± 0.07, log β₂ = 20.19 ± 0.08) were determined by the potentiometric method in aqueous-ethanol solution. The copper(II) complex [Cu₂(μ-L)₂]_n was isolated in the solid state and found by X-rays to be a coordination polymer of a binuclear core with a distorted square pyramidal metal coordination geometry.     

The Antiviral Activity of Chitosan (Review)

Data on the inhibitory effect of chitosan on viral infections in animals, plants, and microorganisms are reviewed. The effects of the physicochemical parameters and structure of chitosan on its antiviral activity are analyzed. Possible mechanisms of the inhibitory effect of chitosan on viral infections are discussed.     

清温解毒汤提取物体内抗病毒活性研究

目的:研究清温解毒汤提取物体内对流感病毒的防治作用,阐明该提取物体内抗病毒的作用机制。方法:选用NIH小鼠,于乙醚浅麻醉下经鼻滴入流感病毒,观察清温解毒汤提取物对感染病毒小鼠的保护作用,以及对小鼠肺指数的影响。结果:对感染病毒小鼠的保护作用实验显示,清温解毒汤提取物低、中、高剂量组的小鼠死亡率均较病毒对照组减少;清温解毒颗粒中、高剂量组能明显提高小鼠的平均存活天数(P<0.05)及延长动物的生命率;对小鼠的肺指数的影响实验则显示清温解毒汤提取物组动物的低、中、高剂量组动物肺指数与病毒对照组比较,虽无显著性差异(P>0.05),但存在明显的剂量依赖关系。结论:清温解毒汤提取物具有一定的抗流感病毒作用,其机制可能与其能够延长小鼠存活时间及抑制肺肿胀有关。     

Antiviral Activity of 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)Uracil against Thymidine Kinase Negative Strains of Varicella-Zoster Virus

Mechanism of antiviral activity of 1-β-d -arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) against the YSR strain of varicella-zoster virus (VZV), which is a mutant derived from the wild YS strain and is completely deficient in viral thymidine kinase (TK), was searched in comparison with antiviral activity of other thymidine analogues, guanosine analogue and thymidylate synthase (TS) inhibitor in human embryo lung fibroblast cells. Thymidine analogues, such as BV-araU, 5-iododeoxyuridine (IUDR), 1-β-d -arabinofuranosylthymine (araT), and guanosine analogue, such as 9-(2-hydroxyethoxymethyl)guanine (ACV), showed higher antiviral activity to the YS strain than to the YSR strain. Though, BV-araU also had the antiviral activity of a microgram level against the YSR strain. In contrast to these results, TS inhibitor, 5-fluorodeoxyuridine (FUDR), had higher antiviral activity to the YSR strain than to the YS strain. Highly synergistic antiviral activities of FUDR to the YS strain and the YSR strain were observed in combination with IUDR, araT, or ACV. However, weakly synergistic or additive inhibition to the YSR strain was shown in combination of BV-araU and FUDR, in spite of highly synergistic effect of this combination to the YS strain. The viral and cellular TS activity was partially inhibited by BV-araU monophosphate, but not by BV-araU. These results indicate that BV-araU is converted into BV-araU monophosphate by cellular TK, and the inhibition of TS activity by BV-araU monophosphate in the YSR strain-infected cells results in the suppression of viral replication.     

Significant In Vivo Anti-Inflammatory Activity of Pytren4Q-Mn a Superoxide Dismutase 2 (SOD2) Mimetic Scorpiand-Like Mn (II) Complex

Background

The clinical use of purified SOD enzymes has strong limitations due to their large molecular size, high production cost and immunogenicity. These limitations could be compensated by using instead synthetic SOD mimetic compounds of low molecular weight.

Background/Methodology

We have recently reported that two SOD mimetic compounds, the Mn^II complexes of the polyamines Pytren2Q and Pytren4Q, displayed high antioxidant activity in bacteria and yeast. Since frequently molecules with antioxidant properties or free-radical scavengers also have anti-inflammatory properties we have assessed the anti-inflammatory potential of Pytren2Q and Pytren4Q Mn^II complexes, in cultured macrophages and in a murine model of inflammation, by measuring the degree of protection they could provide against the cellular injury produced by lipopolisacharide, a bacterial endotoxin.

Principal Findings

In this report we show that the Mn^II complex of Pytren4Q but not that of Pytren2Q effectively protected human cultured THP-1 macrophages and whole mice from the inflammatory effects produced by LPS. These results obtained with two molecules that are isomers highlight the importance of gathering experimental data from animal models of disease in assessing the potential of candidate molecules.

Conclusion/Significance

The effective anti-inflammatory activity of the Mn^II complex of Pytren4Q in addition to its low toxicity, water solubility and ease of production would suggest it is worth taking into consideration for future pharmacological studies.     

Antifungal Activity In Vitro and In Vivo of Mesquite Extract (Prosopis glandulosa) Against Phytopathogenic Fungi

Fungi are the primary infectious agents in plant crops and many post-harvest fungal diseases of fruit and vegetables causing significant economic losses worldwide. Here, the antifungal effect of Prosopis glandulosa extract (PgE) against phytopathogenic fungi was evaluated. The effect with PgE (5, 4, 2, 1, 0%) as AI (%) and radial growth rate reduction (K_r %) were determined in vitro in Colletotrichum gloeosporoides, Fusarium oxysporum, Rhizopus oryzae and R. stolonifer (1 × 10⁵ spores/mL). The phytopathogenicity of fungal strains was performed under in vivo conditions (room temperature, 25–30 °C and refrigeration, ~ 4 °C) by fruit surface inoculation method on strawberries, tomatoes and carrots by recording the development of mycelial growth, necrosis, soft rot and dehydration symptoms showed on each fruit at 14 days. The extract (5%) showed the highest AI against C. gloesporioides (~ 96%), and F. oxysporum (~ 79%) and growth rate reduction of 74.92% and 64.82% respectively. Likewise, the extract controls the development of phytopathogenicity symptoms against C. gloesporioides and F. oxysporum in vivo conditions, nevertheless, was less efficiency against both Rhizopus species. The P. glandulosa extract represents an efficient, economical, and eco-friendly alternative to preserve the quality of the agricultural products and to increase their shelf life.     

Synthesis and Antiviral Activities of Enantiomeric 1-[2-(Hydroxymethyl) Cyclopropyl] Methyl Nucleosides

Abstract

Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100μM.     

Decomposition of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) in aqueous solution

BCNU, labelled with ¹⁴C in the chloroethyl groups, decomposes in neutral aqueous solution to release half of its radioactivity as volatile products. These have been identified by a combination of gas chromatography and mass spectrometry as vinyl chloride, acetaldehyde, dichloroethane, and chloroethanol. This set of products is consistent with the existence of chloroethylcarbonium ions as reactive intermediates which would produce the previously described substituted nucleotides.