Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Abstract

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.

Figure 1.

SMM Z score of G4s binding to (A) pyridosatin and (B) CLX. Horizontal line corresponds to ‘hit’ cutoff of 3. (C) Increase in melting temperature of a panel of G-quadruplexes in the presence of four equivalents of CLX (measured by circular dichroism). (D) Chemical structures of Chlorhexidine (top), Alexidine (middle) and Proguanil (bottom).

Figure 2.

(A) ¹H NMR of CLX and N-methyl-l-valine (non-binding control, peaks indicated with *) (Top), WaterLOGSY NMR of CLX and N-methyl-l-valine in the absence (middle) and presence (bottom) of KRAS G4. (B) ¹³C NMR of ¹³C-labeled CLX in the absence (red) and presence (blue) of KRAS G4. (C) ¹H NMR of imino peaks of KRAS G4 in the presence of 0–5 eq of CLX.

Figure 3.

Structure of KRAS G4 (PDB: 5I2V). Nucleic acids are colored based on magnitude of chemical shift perturbations of H8/H6 protons in the presence of 1 eq of CLX: 0–0.020 Δδ ppm (blue), 0.020–0.035 Δδ ppm (green), 0.035–0.050 Δδ ppm (yellow), 0.050–0.080 Δδ ppm (orange), >0.080 Δδ ppm (red). Indicated binding site for CLX (cyan) is the most consistent with chemical shift perturbations and docking studies.

Figure 4.

(A) Comparison of CLX activities across a panel of cancer cell lines from viability assay. The violin plot summarizes the distribution of log₁₀(AC₅₀) grouped by their respective KRAS mutational status and primary site. The P-values are calculated using Student's t-test. (B) Western blot analysis of KRAS protein levels in MDA-MB-231 cells after 48 and 72 h of treatment with CLX or PG. (C) Western blot analysis of MDA-MB-231 cells transiently transfected with HA-tagged KRAS plasmid (lacking a G4) and dosed with CLX or PG for 48 h.