ABSTRACT
Aminoglycoside antibiotics are small-molecule drugs that bind RNA. The affinity and specificity of aminoglycoside binding to RNA can be increased through chemical modification, such as guanidinylation. Here, we report the binding of guanidinoneomycin B (GNB) to an RNA helix from the HIV-1 frameshift site. The binding of GNB increases the melting temperature (T(m)) of the frameshift-site RNA by at least 10 degrees C, to a point at which a melting transition is not even observed in 2 M urea. A structure of the complex was obtained by using multidimensional heteronuclear NMR spectroscopic methods. We also used a novel paramagnetic-probe assay to identify the site of GNB binding to the surface of the RNA. GNB makes major-groove contacts to two sets of Watson-Crick bases and is in van der Waals contact with a highly structured ACAA tetraloop. Rings I and II of GNB fit into the major groove and form the binding interface with the RNA, whereas rings III and IV are exposed to the solvent and disordered. The binding of GNB causes a broadening of the major groove across the binding site.
Subject(s)
Aminoglycosides/metabolism , HIV-1/genetics , RNA, Viral/metabolism , Aminoglycosides/chemistry , Aminoglycosides/genetics , Base Sequence , Binding Sites , Frameshifting, Ribosomal/genetics , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Nucleic Acid Conformation/drug effects , Nucleic Acid Denaturation/drug effects , RNA, Viral/chemistry , RNA, Viral/genetics , Substrate Specificity , Thermodynamics , Urea/pharmacologyABSTRACT
Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.
Subject(s)
Drug Delivery Systems , Heparan Sulfate Proteoglycans/metabolism , Membrane Glycoproteins/metabolism , Neomycin/pharmacokinetics , Protein Synthesis Inhibitors/pharmacokinetics , Animals , Biological Transport/drug effects , Biological Transport/physiology , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Guanidine/analogs & derivatives , Guanidine/chemical synthesis , Guanidine/pharmacokinetics , N-Glycosyl Hydrolases/pharmacokinetics , Neomycin/analogs & derivatives , Neomycin/chemical synthesis , Peptides/chemical synthesis , Peptides/pharmacokinetics , Plant Proteins/pharmacokinetics , Protein Binding/drug effects , Protein Synthesis Inhibitors/chemical synthesis , Ribosome Inactivating Proteins, Type 1 , SaporinsABSTRACT
Symmetrical homometallic dinuclear complexes of the type [(Ru(dpq)2)2(phen-SOS-phen)]4+, with a flexible 2-mercaptoethyl ether linker joining the two [Ru(dpq)2(phen)]2+-based sub-units, have DNA dissociation constants (Kd) in the nM range.
