Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Nucleic Acids Research, Apr 2018

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.

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Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Nucleic Acids Research Characterization of clinically used oral antiseptics as quadruplex-binding ligands David R. Calabrese 2 Katherine Zlotkowski 2 Stephanie Alden 2 William M. Hewitt 2 Colleen M. Connelly 2 Robert M. Wilson 2 Snehal Gaikwad 1 Lu Chen 0 Rajarshi Guha 0 Craig J. Thomas 0 Beverly A. Mock 1 John S. Schneekloth 0 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health , Bethesda, MD , USA 1 Laboratory of Cancer Biology and Genetics, National Cancer Institute , Bethesda, MD 20892-4258 , USA 2 Chemical Biology Laboratory, National Cancer Institute , Frederick, MD 21702-1201 , USA 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. INTRODUCTION Small molecules that bind to nucleic acids are powerful chemical tools that can control gene expression and have substantial potential as chemotherapeutics ( 1–5 ). While most modern medicinal chemistry focuses on protein targets, an increased understanding of the structure and function of non-coding nucleic acids suggests that oligonucleotides may be suitable targets for small molecules as well. Attempts to target B-DNA, for example with pyrroleimidazole polyamides ( 6,7 ), have resulted in compounds with remarkable selectivity but have yet to yield a clinically used drug. While it is unlikely that approved drugs will bind to B-DNA with selectivity, little is known about how such drugs interact with other comparatively rare alternatively folded nucleic acid structures. Interactions with these structures could have profound effects in a cellular context and could result in unanticipated pharmacological consequences. One structure that has received considerable interest as a small molecule target is the G-quadruplex (G4). G4s are non-B DNA structures with globular folds that occur in guanine-rich sequences. G4s are characterized by stacks of Hoogsteen-bonded guanine tetrads stabilized by central potassium ions and flanked by loop regions ( 8 ). DNA G4s have been identified in genome-wide structural probing studies using a G4-specific antibody ( 9 ), as well as in a chemical probing approach employing ss-DNA seq ( 10 ), and are estimated to exist in several thousand locations in the human genome. Furthermore, G4s have been implicated in regulating gene expression ( 11 ). Folded DNA G4s are enriched in nucleosome-depleted regions of the genome, occurring primarily in the promoter regions of both oncogenes and developmental genes that are normally silent in differentiated cells ( 9 ). Although the existence of DNA G4s is well established, the presence of folded G4 structures in RNA has proven to be controversial, with some studies confirming their existence ( 12 ) and others providing evidence that they are globally unfolded in vivo ( 13 ). However, RNA G4s have been implicated in inhibiting translation ( 14,15 ) or modulating telomere structure and function (16) and can be targeted by small molecules ( 17 ). Published by Oxford University Press on behalf of Nucleic Acids Research 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. By targeting G4s with small molecules, it is possible to control the expression of otherwise ‘undruggable’ oncogenic proteins, such as KRAS. KRAS, a prototypical oncogene, encodes for a small GTPase that influences cell growth and apoptosis ( 18 ). Single point mutations, commonly at codons 12, 13 or 61, cause KRAS to be constitutively active and trigger oncogenesis. Consequently, these mutations are found in a wide variety of tumors. While KRAS is an attractive drug target, small mol (...truncated)


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Calabrese, David R, Zlotkowski, Katherine, Alden, Stephanie, Hewitt, William M, Connelly, Colleen M, Wilson, Robert M, Gaikwad, Snehal, Chen, Lu, Guha, Rajarshi, Thomas, Craig J, Mock, Beverly A, Schneekloth, John S. Characterization of clinically used oral antiseptics as quadruplex-binding ligands, Nucleic Acids Research, 2018, pp. 2722-2732, Volume 46, Issue 6, DOI: 10.1093/nar/gky084