RESUMO
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.
Assuntos
Anemia Falciforme/sangue , Descoberta de Drogas/métodos , Hemoglobina Fetal/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Talassemia beta/sangue , Anemia Falciforme/tratamento farmacológico , Técnicas Biossensoriais/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Proteínas Luminescentes/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Globinas beta/genética , Talassemia beta/tratamento farmacológico , gama-Globinas/genética , Proteína Vermelha FluorescenteRESUMO
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.