RESUMEN
The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.
Asunto(s)
Anemia de Células Falciformes/sangre , Descubrimiento de Drogas/métodos , Hemoglobina Fetal/biosíntesis , Biosíntesis de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Talasemia beta/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Técnicas Biosensibles/métodos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Células K562 , Proteínas Luminiscentes/genética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Globinas beta/genética , Talasemia beta/tratamiento farmacológico , gamma-Globinas/genética , Proteína Fluorescente RojaRESUMEN
Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3'UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3'UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.