Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analog, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 >M. NMR characterization of a soluble analog, 2, revealed a non-canonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important implications for RNA ligand design.

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Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.