RESUMO
Isolated from Hypericum species H. chinese L. var. salicifolium, biyouyanagin A was assigned structure 1a or 1b on the basis of NMR spectroscopic analysis. This novel natural product exhibited significant anti-HIV properties and inhibition of lipopolysaccharide-induced cytokine production. Described herein are the total syntheses of biyouyanagin A and several analogues (3-11), structural revision of biyouyanagin A to 2b, and the biological properties of all synthesized compounds. The total synthesis proceeded through cascade sequences that efficiently produced enantiomerically pure key building blocks 15b (ent-zingiberene) and 18 (hyperolactone C) and featured a novel [2 + 2] photoinduced cycloaddition reaction which occurred with complete regio- and stereoselectivity. Biological investigations with the synthesized biyouyangagins A (2-11) and hyperolactones C (12-16) revealed that the activity of biyouyanagin A most likely resides in its hyperolactone C structural domain.
Assuntos
Fármacos Anti-HIV/síntese química , Sesquiterpenos/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Furanos/síntese química , Furanos/química , Furanos/farmacologia , HIV/efeitos dos fármacos , Humanos , Hypericum/química , Concentração Inibidora 50 , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Linfócitos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Sensibilidade e Especificidade , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Compostos de Espiro , EstereoisomerismoRESUMO
The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T(90) values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between â¼40 to 49°C (nâ=â34). For select Envs (nâ=â10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T(90) (pâ=â0.029), though two 'outliers' were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1(ADA) was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1(JR-CSF). Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.
Assuntos
HIV-1/metabolismo , HIV-1/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Temperatura , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bacteriophage display technology to the HIV-1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58-amino-acid-residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosensor analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (KD) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the Ad5 (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp120 in a biosensor analysis and to gp160 transiently expressed in African-green-monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the Ad5 fibres showed any virus neutralization activity, suggesting that the affibody bound to a non-neutralizing site on gp120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670-30D and 697-30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120-binding affibody ligand indicates that future affibody-based strategies might evolve to complement antibody-based efforts for HIV-1 therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene-therapy-mediated efforts are discussed.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , Proteínas Recombinantes de Fusão/química , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Ligação Competitiva , Técnicas Biossensoriais , Antígenos CD4/química , Linhagem Celular , Chlorocebus aethiops , Dimerização , Mapeamento de Epitopos , Imunofluorescência , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , Humanos , Ligantes , Dados de Sequência Molecular , Testes de Neutralização , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
HIV-1 env based DNA vaccines are generally found to be poor B-cell immunogens. We examined the role of an N-glycan located in the V3 loop of HIV-1 (N306) that is known to modulate the immunogenicity of gp120. Here we describe intranasal immunizations with env (HIV-1 BRU) based genetic immunogens in combination with subcutaneous boosts of recombinant gp160 (rgp160) in mice. Immunization with DNA alone resulted in detectable IgA responses to rgp160 in both faeces and bronchoalveolar lavage (BAL) fluid, but the additional boosting increased the faecal IgA titres only. Protein boosting was required for induction of faecal IgA antibodies capable of neutralizing a homologous laboratory strain and a subtype B primary isolate. The B-cell response towards V3 loop peptides was not only directed against the homologous subtype B but also against the subtype F. In contrast to our previous observations on IgG, there were no differences in anti-gp160 IgA titres elicited by the N-glycan mutant and the wild-type immunogen. These results indicate that intranasal administration of plasmids containing env in combination with a subcutaneous boost proved to be an effective way of eliciting neutralizing mucosal IgA against HIV-1.