RESUMO
Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.
Assuntos
Antivirais/administração & dosagem , Proteínas do Capsídeo/genética , Cinamatos/administração & dosagem , Depsídeos/administração & dosagem , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/tratamento farmacológico , Salvia miltiorrhiza/química , Animais , Antivirais/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/química , Linhagem Celular , Cinamatos/farmacologia , Depsídeos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/metabolismo , Infecções por Enterovirus/virologia , Heparitina Sulfato/metabolismo , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , Camundongos , Mutação , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Ligação Proteica/efeitos dos fármacos , Eletricidade Estática , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/química , Fatores de Virulência/genética , Ácido RosmarínicoRESUMO
This study demonstrates that compounds 1-4 from an extract of Plectranthus amboinicus inhibit the binding of AP-1 to its consensus DNA sequence. Thymoquinone (5) was further identified as a nonpolar ingredient from the hexane extract of P. amboinicus to suppress the expression of lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α). We then synthesized 2-alkylidenyl-4-cyclopentene-1,3-diones as the designed biomimetics of thymoquinone, and found that compounds 8a, 8b and 8d were more potent TNF-α inhibitors.