RESUMO
Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISA-based high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.
Assuntos
Distrofia Muscular de Duchenne/tratamento farmacológico , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Biossíntese de Proteínas/efeitos dos fármacos , Utrofina/genética , Regiões 5' não Traduzidas/genética , Animais , Betaxolol/farmacologia , Betaxolol/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Sítios Internos de Entrada Ribossomal/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Distrofia Muscular de Duchenne/genética , Mioblastos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Pravastatina/farmacologia , Pravastatina/uso terapêutico , Biossíntese de Proteínas/genética , Regulação para Cima/efeitos dos fármacos , Utrofina/metabolismoRESUMO
Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.
Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Sítios Internos de Entrada Ribossomal , Florizina/análogos & derivados , Animais , Antibacterianos/farmacologia , Morte Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Flavonoides/farmacologia , Genes Reporter , Hepacivirus/efeitos dos fármacos , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal/genética , Luciferases/metabolismo , Camundongos Endogâmicos BALB C , Mutação/genética , Florizina/farmacologia , Florizina/uso terapêutico , Reprodutibilidade dos Testes , Replicação Viral/efeitos dos fármacosRESUMO
Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.