ABSTRACT
RNase L, a principal mediator of innate immunity to viral infections in higher vertebrates, is required for a complete IFN antiviral response against certain RNA stranded viruses. dsRNA produced during viral infections activates IFN-inducible synthetases that produce 5'-phosphorylated, 2',5'-oligoadenylates (2-5A) from ATP. 2-5A activates RNase L in a wide range of different mammalian cell types, thus blocking viral replication. However, 2-5A has unfavorable pharmacologic properties; it is rapidly degraded, does not transit cell membranes, and leads to apoptosis. To obtain activators of RNase L with improved drug-like properties, high-throughput screening was performed on chemical libraries by using fluorescence resonance energy transfer. Seven compounds were obtained that activated RNase L at micromolar concentrations, and structure-activity relationship studies resulted in identification of an additional four active compounds. Two lead compounds were shown to have a similar mechanistic path toward RNase L activation as the natural activator 2-5A. The compounds bound to the 2-5A-binding domain of RNase L (as determined by surface plasmon resonance and confirmed by computational docking), and the compounds induced RNase L dimerization and activation. Interestingly, the low-molecular-weight activators of RNase L had broad-spectrum antiviral activity against diverse types of RNA viruses, including the human pathogen human parainfluenza virus type 3, yet these compounds by themselves were not cytotoxic at the effective concentrations. Therefore, these RNase L activators are prototypes for a previously uncharacterized class of broad-spectrum antiviral agents.
Subject(s)
Antiviral Agents/metabolism , Endoribonucleases/metabolism , Enzyme Activators/metabolism , Immunity, Innate/physiology , Parainfluenza Virus 3, Human/metabolism , Adenine Nucleotides/metabolism , Animals , Antiviral Agents/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Dimerization , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Activators/pharmacology , Fluorescence Resonance Energy Transfer , Mice , Models, Molecular , Oligonucleotides/genetics , Oligoribonucleotides/metabolism , Parainfluenza Virus 3, Human/drug effects , Protein Binding , Protein Structure, Tertiary , Structure-Activity Relationship , Surface Plasmon Resonance , Virus Replication/drug effectsABSTRACT
Branched tris-DNA, in which two oligonucleotides of the same sequence and one other oligonucleotide of a different sequence are connected with a rigid central linker, was prepared chemically by using a DNA synthesizer. Two branched tris-DNA molecules with complementary DNA sequences form dimer and tetramer as well as linear and spherical oligomer complexes. The complex formation was studied by UV/thermal denaturation, enzyme digestion, gel electrophoresis, and AFM imaging.
