RESUMO
Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.
Assuntos
Pirimidinas/química , Pirimidinas/farmacologia , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/química , Bactérias/citologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Cristalografia por Raios X , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Mononucleotídeo de Flavina/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Transferases Intramoleculares/genética , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Modelos Moleculares , Dados de Sequência Molecular , Pirimidinas/isolamento & purificação , Pirimidinas/uso terapêutico , RNA Bacteriano/genética , Reprodutibilidade dos Testes , Riboflavina/biossíntese , Riboswitch/genética , Especificidade por SubstratoRESUMO
Mice deficient in either platelet-derived growth factor (PDGF) B chain or PDGF receptor (PDGFR) beta lack mesangial cells. PDGF stimulates proliferation and migration of metanephric mesenchymal cells, from which mesangial cells are derived. Binding of PDGF to PDGFR-beta induces autophosphorylation at specific tyrosine residues and activates various effector proteins, including phosphatidylinositol-3-kinase (PI3-K). This study explored the role of PI 3-K and reactive oxygen species (ROS) in PDGF-mediated signaling using cells established from wild-type and PDGFR-beta -/- metanephric blastemas at 11.5 days post-conception. PDGF-induced effects that were dependent on PI3-K activation were determined using PDGFR-beta -/- cells made to express "add-back" mutant PDGFR-beta capable of binding PI3-K. We found that PDGF is mitogenic for mesenchymal cells expressing PDGFR-beta, and PI3-K is an important regulator of PDGF-induced DNA synthesis. Activation of ERK1/2 is partially dependent on PI3-K, and both the PI3-K and MEK-ERK1/2 pathways contribute to PI3-K-dependent mitogenesis. In addition, PDGF-induced DNA synthesis in wild-type cells was found to be dependent on ROS that are generated downstream of PI3-K activation. Using antisense oligonucleotides and small interfering RNA, we determined that the NAD(P)H oxidase Nox4 produces these ROS that activate Akt and the MEK-ERK1/2 mitogenic cascade. In conclusion, the present study demonstrates Nox4 involvement in PDGF-induced DNA synthesis in metanephric mesenchymal cells and provides the first evidence that PDGF-induced PI3-K activity enhances production of ROS by Nox4.