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)