RESUMEN
Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential interest for exosome research and oncology.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Molécula de Adhesión Celular Epitelial/metabolismo , Exosomas/metabolismo , Dominios PDZ , Bibliotecas de Moléculas Pequeñas/farmacología , Sindecanos/metabolismo , Sinteninas/antagonistas & inhibidores , Apoptosis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Molécula de Adhesión Celular Epitelial/genética , Exosomas/genética , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Dominios y Motivos de Interacción de Proteínas , Sindecanos/genética , Células Tumorales CultivadasRESUMEN
Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA74, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.
Asunto(s)
Virus del Dengue/enzimología , Dengue/virología , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Metiltransferasas/análisis , Proteínas no Estructurales Virales/análisis , Antivirales/farmacología , Dengue/tratamiento farmacológico , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Metiltransferasas/metabolismo , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.
