RESUMEN
The therapeutic potential of microRNA-21 (miR-21) small-molecule inhibitors has been of particular interest to medicinal chemists. Moreover, the development of more facile screening methods is lacking. In the present study, two potential screening strategies for miR-21 small-molecule inhibitor including the stem-loop reverse transcription-quantitative PCR and dual luciferase reporter assay system were demonstrated and discussed in detail. A pmirGLO-miR21cswt plasmid and its two different mutants were constructed for dual luciferase reporter assay system. In addition, the sensitivity and specificity of these two methods were validated. Our results demonstrated that both strategies are decent choices for the screening of small-molecule inhibitors for miR-21 and possibly other miRNAs. Eventually, we applied our optimized strategy to discover and characterize several promising compounds such as azobenzene derivate A, enoxacin, and norfloxacin for their potential impact on intracellular miR-21 concentration.
Asunto(s)
Genes Reporteros/efectos de los fármacos , Luciferasas de Luciérnaga/antagonistas & inhibidores , MicroARNs/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos , Genes Reporteros/genética , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Células Tumorales CultivadasRESUMEN
MicroRNA-21, as an oncogenic miRNA, has caught great attention for medicinal chemists to develop its novel inhibitors for cancer therapy. In the present study, we designed 4-benzoylamino-N-(prop-2-yn-1-yl)benzamides as miR-21 inhibitor candidates on the basis of scaffold hopping. Eighteen compounds were synthesized. The inhibitory activities of synthesized compounds against the expression of miR-21 were evaluated using stem loop RT-qPCR and compound 1j was discovered as the most potent compound, which displayed a time and concentration dependent inhibition manner. In addition, various functional assays such as the expression of miR-21 target gene detected by Western blotting and the cell growth and apoptosis detected by flow cytometric analysis were checked in Hela (human epithelioid cervix carcinoma) and U-87 MG (human glioblastoma) cells to confirm its activity. The results indicate that compound 1j can enhance apoptosis, retard proliferation, and up-regulate PDCD4, a target protein of miR-21. In addition, the compound 1j does not influence the expression of multiple miRNAs and the genes that participate in miRNA universal biosynthesis pathway. These results strongly support the assumption that title compounds can serve as a small molecule inhibitor of miR-21.
Asunto(s)
Benzamidas/química , MicroARNs/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzamidas/metabolismo , Benzamidas/toxicidad , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , MicroARNs/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A series of [1,2,4]triazolo[1,5-a]pyridinylpyridines were synthesized and characterized. Their antiproliferative activities in vitro were evaluated by MTT against three human cancer cell lines including HCT-116, U-87 MG and MCF-7 cell lines. The SAR of target compounds was preliminarily discussed. The compounds 1c and 2d with potent antiproliferative activities were tested for their effects on the AKT and p-AKT(473). The anticancer effect of 1c was evaluated in mice bearing sarcoma S-180 model. The results suggest that the title compounds are potent anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Piridinas/farmacología , Triazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Células MCF-7 , Ratones , Modelos Moleculares , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Células Tumorales CultivadasRESUMEN
OBJECTIVE: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. METHODS: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) in the spleen. CONCLUSIONS: MTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.