Probing the <i>hras</i>-1^Y i-motif with small molecules

Abstract

Non-B DNA structures represent intriguing and challenging targets for small molecules. For example, the promoter of the <i>HRAS</i> oncogene contains multiple G-quadruplex and i-motif structures, atypical globular folds that serve as molecular switches for gene expression. Of the two, i-motif structures are far less studied. Here, we report the first example of small organic compounds that directly interact with the <i>hras</i>-1^Y i-motif. We use a small molecule microarray screen to identify drug-like small molecules that bind to the <i>hras</i>-1^Y i-motif but not to several other DNA or RNA secondary structures. Two different lead compounds, <b>1</b> and <b>2</b>, were discovered to have 7.4 ± 5.3 μM and 5.9 ± 3.7 μM binding affinity by surface plasmon resonance and similar affinity by fluorescence titration. A structure-activity relationship (SAR) was developed and two improved analogues of <b>2</b> demonstrated submicromolar binding affinities. Both compounds display pH-dependent binding, indicating that they interact with the DNA only when the i-motif is properly folded. Chemical shift perturbation shows that <b>1</b> alters the structure of the i-motif, while <b>2</b> has no effect on the i-motif conformation, indicating different modes of interaction.