RESUMO
There is an urgent need to provide effective anti-HIV microbicides to resource-poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin-N (rCV-N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV-N was isolated with a yield of 350 µg/g of dry seed weight. The observed amino acid sequence of rCV-N matched the expected sequence of native CV-N, as did the mass of rCV-N (11 009 Da). Purified rCV-N from soya is active in anti-HIV assays with an EC50 of 0.82-2.7 nM (compared to 0.45-1.8 nM for E. coli-produced CV-N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV-N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti-HIV microbicide development.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Transporte/biossíntese , Glycine max/genética , Engenharia de Proteínas , Sementes/genética , Fármacos Anti-HIV , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Sementes/metabolismo , Glycine max/metabolismoRESUMO
The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein's mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 min, but was cleared within 4h. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6h, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.
Assuntos
Antivirais/uso terapêutico , Proteínas de Bactérias/uso terapêutico , Proteínas de Transporte/uso terapêutico , Ebolavirus/efeitos dos fármacos , Lectinas/uso terapêutico , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Modelos Animais de Doenças , Ebolavirus/fisiologia , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Concentração Inibidora 50 , Injeções Subcutâneas , Lectinas/administração & dosagem , Lectinas/metabolismo , Lectinas/farmacologia , Fígado/virologia , Marburgvirus/efeitos dos fármacos , Proteínas de Membrana , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Soro/virologia , Baço/virologia , Análise de Sobrevida , Carga ViralRESUMO
The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect.
Assuntos
Proteínas de Bactérias/farmacologia , Proteínas de Transporte/farmacologia , Gammaretrovirus/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Lectinas/farmacologia , Lectinas de Plantas/farmacologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Gammaretrovirus/efeitos dos fármacos , Glicosilação , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Humanos , Proteínas de MembranaRESUMO
Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in â¼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Antivirais/administração & dosagem , Antivirais/química , Linhagem Celular , Clorófitas/química , Modelos Animais de Doenças , Genótipo , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Camundongos , Modelos Moleculares , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/química , Ligação Proteica , Conformação Proteica , Rodófitas/química , Proteínas do Envelope Viral/metabolismo , Carga Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.
Assuntos
Proteínas de Algas/farmacologia , Inibidores da Fusão de HIV/efeitos adversos , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Lectinas/farmacologia , Proteínas de Algas/genética , Proteínas de Algas/isolamento & purificação , Proteínas de Algas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colo do Útero/cirurgia , Colo do Útero/virologia , Citocinas/biossíntese , Avaliação Pré-Clínica de Medicamentos , Feminino , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Lectinas/genética , Lectinas/isolamento & purificação , Lectinas/metabolismo , Lectinas de Plantas , Ligação Proteica , Coelhos , Técnicas de Cultura de Tecidos , Transplante de Tecidos , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Compounds that stabilize p53 could suppress tumors providing a additional tool to fight cancer. Mdm2, and the human ortholog, Hdm2 serve as ubiquitin E3 ligases and target p53 for ubiquitylation and degradation. Inhibition of Hdm2 stabilizes p53, inhibits cell proliferation and induces apoptosis. Using HTS to discover inhibitors, we identified three new alkaloids, isolissoclinotoxin B, diplamine B, and lissoclinidine B from Lissoclinum cf. badium. Lissoclinidine B inhibited ubiquitylation and degradation of p53, and selectively killed transformed cells harboring wild-type p53, suggesting this compound could be used to develop new treatments.
Assuntos
Alcaloides/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Urocordados/química , Acridinas/química , Acridinas/isolamento & purificação , Acridinas/metabolismo , Alcaloides/química , Alcaloides/isolamento & purificação , Animais , Linhagem Celular Transformada , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Fenantrolinas/química , Fenantrolinas/isolamento & purificação , Fenantrolinas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitina/metabolismoRESUMO
The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC(50)] = approximately 1 micro g/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC(50) = 0.004 to 0.04 micro g/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N's interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.