Chalcones bearing a 3,4,5-trimethoxyphenyl motif are capable of selectively inhibiting oncogenic K-Ras signaling

Article abstract of DOI:10.1016/j.bmcl.2020.127144

Ras proteins are small GTPases which regulate cellular proliferation, differentiation, and apoptosis. Constitutively active mutant Ras are expressed in ~15–20% human cancers, and K-Ras mutations account for ~85% of all Ras mutations. Despite the significa

Full text of DOI:10.1016/j.bmcl.2020.127144

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chalcones bearing a 3,4,5-trimethoxyphenyl motif are capable of selectively
inhibiting oncogenic K-Ras signaling
Sarah E. Kovara, Cody Fourmanb, Christine Kinstedtb, Brandon Williamsb, Christopher Morrisb,
⁎
⁎
Kwang-jin Choa, , Daniel M. Ketchab,
a Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA
b Department of Chemistry, Wright State University, Dayton, OH 45435, USA
A R T I C L E I N F O
A B S T R A C T
InChIKeys:
Ras proteins are small GTPases which regulate cellular proliferation, differentiation, and apoptosis.
Constitutively active mutant Ras are expressed in ~15–20% human cancers, and K-Ras mutations account for
~85% of all Ras mutations. Despite the significance of Ras proteins in refractory cancers, there is no anti-Ras
drug available in clinic. Since K-Ras must interact with the plasma membrane (PM) for biological activity,
inhibition of the K-Ras/PM interaction is a tractable approach to block oncogenic K-Ras activity. Here, we
discovered chalcones 1 and 8 exhibit anti-K-Ras activity, and show that the compounds mislocalize K-Ras from
the PM and block oncogenic K-Ras signal output. Also, 1 inhibits the growth of K-Ras-driven human cancer cells.
Our data suggest that 1 could be a promising starting point for developing anti-K-Ras cancer drug.
CTWCGYDTHCEYSA-BQYQJAHWSA-N
HORVIDXMQQVQAS-RUDMXATFSA-N
BPHYOXFAYJPCCZ-BQYQJAHWSA-N
LGVZQFTZVRFJMS-BQYQJAHWSA-N
WXPAWDMWSJYTRJ-RUDMXATFSA-N
GHQYAKZDBZTTSS-RUDMXATFSA-N
XJYIPYGBDWACTE-BJMVGYQFSA-N
VIFRKOWCKUMHOG-IZZDOVSWSA-N
Keywords:
K-Ras
Trimethoxy chalcone
Plasma membrane
Mislocalization
K-Ras-driven cancer
Ras proteins are small, membrane-bound GTPases that oscillate
between active GTP-bound and inactive GDP-bound states through in-
teraction with guanine nucleotide exchange factors (GEFs) and GTPase-
activating proteins (GAPs), respectively.1 Ras proteins stimulate mul-
tiple downstream effectors which regulate cell proliferation, differ-
entiation, survival and apoptosis.2 Four Ras isoforms, K-Ras4A and K-
Ras4B (both encoded by KRAS), as well as H-Ras and N-Ras are ubi-
quitously expressed in mammalian cells and must interact primarily
with the inner leaflet of the plasma membrane (PM) for conducting
signal transduction.2 For this requisite stable PM interaction, Ras un-
dergoes a series of posttranslational modification at the C-terminal
CAAX motif (where C = Cys, A = aliphatic amino acids, and X = Met
or Ser). First, the Cys is farnesylated by a cytosolic farnesyltransferase,
which allows Ras to bind to the cytosolic leaflet of the ER. RCE1 (Ras
converting CAAX endopeptidase 1) then cleaves the AAX tripeptide,
followed by the methylation of the now C-terminal farnesylated Cys by
ICMT (isoprenylcysteine carboxyl methyltransferase).3–5 N-, H-, and K-
Ras4A (the alternative splicing variant of K-Ras) are further modified
with addition of palmitic acids on one or two other Cys near the
farnesylated Cys, allowing Ras to interact with the PM. For K-Ras4B
(hereafter referred to as K-Ras), a polybasic domain made of six lysine
residues near the farnesylated Cys allows K-Ras to interact with anionic
phospholipids in the PM through electrostatic interaction.6,7
Constitutively active mutant Ras are found in ~15–20% of all
human cancers.8 Of those, oncogenic mutant K-Ras is expressed in
~98% of pancreatic, 45% of colorectal, and 30% of all lung cancers, but
as of yet there are no anti-K-Ras therapies available in clinic.9,10 One
approach for blocking oncogenic K-Ras activity is to dissociate K-Ras
from the PM, since K-Ras must interact primarily with the PM.9–11
Removal of oncogenic K-Ras from the PM inhibits the growth of K-Ras-
driven cancer cells in vitro and in vivo.12–14 Therefore, disrupting K-Ras/
PM interaction is a tractable target for blocking oncogenic K-Ras ac-
tivity.
Recently, we performed a cell-based high content screening to
identify novel compounds capable of dissociating K-Ras from the PM.
Identification and characterization of such compounds will provide
deeper insights into K-Ras trafficking to and interaction with the PM,
which might offer a starting point for the development of novel anti-K-
⁎ Corresponding authors.
E-mail addresses: kwang-jin.cho@wright.edu (K.-j. Cho), daniel.ketcha@wright.edu (D.M. Ketcha).
https://doi.org/10.1016/j.bmcl.2020.127144
Received 20 December 2019; Received in revised form 23 March 2020; Accepted 26 March 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:SarahE.Kovar,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127144

S.E. Kovar, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Ras cancer therapies. Given the historical and ongoing challenges as-
sociated with anti-K-Ras drug discovery efforts, it is not surprising that
there has been a strong reliance on the examination of large compound
libraries. From a practical standpoint, hits obtained from such re-
positories might include a number of unrelated chemical classes, or
complex core structures which may present no obvious starting-off
point for library expansion by rational drug design. Moreover, since the
target responsible for inducing disruption of K-Ras PM localization is
largely unknown, molecules of so-called privileged classes warrant at-
tention as they afford the advantages of impacting a number of po-
tentially relevant biological pathways along with the expected benefit
of high hit rate per library size.15,16
analogue was also included.24,25 To complete this library based upon
the 3,4,5-trimethoxy pattern on ring B, most of the remaining com-
pounds in this study included functionalities running the gamut of EW
(-F, -Br) to ED (-OCH3, -N(CH3)2).19,26,27,28 Notable exceptions entail
the 2-azachalcone derivative 3,29 which was included due to the elec-
tron deficient nature of the pyridine ring as well as to identify any ef-
fects which might be attributable to metal binding by chelation to the
proximate ring nitrogen and carbonyl group of the 2-pyridyl ketone.
Additionally, as many of the biological effects of chalcones have been
ascribed to the Michael-acceptor properties of the α,β-unsaturated
carbonyl system linking the two aromatic rings, the 2,6-difluoro moi-
eties of compounds 4 and 10 were designed for the putative enhanced
acceptor abilities due to orthogonality induced by such bis-ortho sub-
stituents.30 Finally, although not strictly axiomatic, it has often been
noticed that so-called “reverse chalcones” wherein the carbonyl and
ethylene groups are interchanged, display similar biological activities
as the original. Thus, the 3′,4′,5′-ring A analog of compound 1 (com-
pound 9)19 was included, especially as this substrate was apparently
devoid of tubulin binding effects.31
Thus, in lieu of such large exploratory compound libraries, it was
reasoned that if an easily synthesized, structurally simple privileged
scaffold were to be employed, upon identification of a putative phar-
macophore on either ring, rapid diversification and structure activity
relationships (SAR) might be easily developed about that core structure.
The chalcone privileged scaffold (1,3-diaryl-2-propen-1-one),17 con-
sisting of two aromatic rings A and B linked by a conjugated carbonyl
system, served as an excellent starting point, allowing for a highly-
optimizable class of compounds that boasts facile synthesis and a wide
range of biological activities. Design considerations involved in the
selection of a small exploratory panel for this study centered on the
inclusion of functionalities shown to be of importance to the anticancer
properties of chalcones, broadly defined.18 Of these, the trimethox-
yphenyl motif is probably the most common pharmacophore in-
vestigated for anticancer properties, with the 3′,4′,5′-pattern on ring A
notably associated with cytotoxic/antiproliferative effects arising from
tubulin interaction.19,20 So as to minimize any possible confounds at-
tributable to this mechanism of action in this initial screening set, it was
decided to focus upon chalcones featuring the inverse substitution
pattern, e.g., 3,4,5-trimethoxy on ring B (Table 1).
Chalcones 1–10 were prepared by base-catalyzed Claisen-Schmidt
condensation utilizing commercially available benzaldehydes and aryl
methyl ketones with ethanol as the solvent and aqueous NaOH (10%) as
the base (Scheme 1). Characterization of compounds were accom-
plished by GC/MS and 1H and 13C NMR analysis with acceptable pu-
rities > 96%. The conjugated carbonyl system of chalcones was verified
to be the trans-isomers in all cases by 1H NMR wherein two doublets
with coupling constants from 15.5 to 16.1 Hz were observed. Melting
points and NMR spectra of known compounds were compared to those
previously published in literature; however, due to questionable NMR
data existing in some previous publications, all characterization data
and full spectra are provided in the Supplementary Data. To the best of
our knowledge, this is the first time chalcones 4 and 10 have been re-
ported in literature, and their identities were verified by high-resolution
mass spectrometry.
Though generally less studied and regarded as having less cytotoxic
potential when positioned on the B-ring,21 we were intrigued by the
report that the 4′-nitro-3,4,5-trimethoxyphenyl derivative 122 exhibited
anti-inflammatory, antioxidant and anticancer cancer properties.23 The
nontoxic nature of this substrate, in addition to its multitarget potential,
provided the impetus for the development of this initial flight of chal-
cones for the mislocalization of K-Ras. Moreover, to eliminate any
conjecture that the strongly electron-withdrawing (EW) 4′-nitro group
might be bio-reductively converted to the corresponding electron-do-
nating (ED) 4′-amino derivative 2 under the screening conditions, this
To examine the effect of chalcone compounds on K-Ras interaction
with the PM, we performed quantitative confocal microscopy. Madin-
Darby kidney (MDCK) cells stably co-expressing green fluorescent
protein (GFP)-tagged oncogenic mutant K-Ras (K-RasG12V) and
mCherry-CAAX, a generic endomembrane marker32,33 were treated
with different concentrations of chalcone compounds for 48 h, and cells
were fixed and imaged by a confocal microscope. To quantitate the
extent of K-RasG12V dissociation from the PM, we used Manders
Table 1
The IC50 and Emax values of chalcones synthesized from Scheme 1 for K-Ras dissociation from the PM.
Compound
X
R1
R2
IC50 (µM)
S.E.M.
Emax
S.E.M.
1
2
CeH
CeH
N
4′-NO2
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
4-NO2
7.01
9.17
52.97
8.20
15.90
24.64
8.29
7.42
8.20
7.49
0.92
3.12
2.96
1.80
8.23
2.39
2.19
0.53
2.87
2.69
0.57
0.54
0.45
0.45
0.39
0.47
0.45
0.58
0.46
0.45
0.04
0.03
0.02
0.03
0.02
0.02
0.05
0.03
0.02
0.01
4′-NH2
3
H
4
CeH
CeH
CeH
CeH
CeH
CeH
CeH
2′,6′-F2
4′-F
5
6
4′-Br
7
4′-OCH3
4′-N(CH3)2
3′,4′,5′-OCH3
3′,4′,5′-OCH3
8
9
10
2,6-F2
IC50: 50% inhibitory concentration for K-RasG12V PM dissociation.
max: Maximal effects elicited by the compounds.
S.E.M.: Standard error of mean from three independent experiments.
E
2

S.E. Kovar, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 1. Reagents: ethanol, aqueous NaOH
(10%).
Fig. 1. 1 and 8 are most potent and effective at dissociating K-Ras from the PM. MDCK cells stably co-expressing GFP-K-RasG12V or -H-RasG12V with mCherry-CAAX
were treated with chalcone compounds for 48 h. Cells were fixed with 4% paraformaldehyde (PFA) and imaged by confocal microscopy. (A and B) Representative
images are shown after treatment with 50 µM of 1 or 8. GFP-RasG12V dissociated from the PM is indicated by arrowheads. Values in merged panels represent the
fraction of mCherry-CAAX co-localized with GFP-RasG12V calculated by Manders coefficient. (C and D) IC50 of 1 and 8 for K-RasG12V and H-RasG12V were
estimated from the dose–response plots from three independent experiments.
coefficient, which calculates the fraction of mCherry-CAAX colocalizing
with GFP-K-RasG12V. The greater the value of the Manders coefficient,
the more extensive the displacement of K-RasG12V from the PM.33,34
The potency (IC50) and efficacy (Emax) of each compound were further
derived from the Manders coefficient values. Our data show that
compounds 1 and 8 are most potent and effective at dissociating K-Ras
from the PM, indicated by the lowest IC50 with the highest Emax values,
respectively (Table 1 and Fig. 1A and B). These data suggest that 3,4,5-
trimethoxy moiety on the B ring of chalcones might represent a possible
pharmacophore for K-Ras PM mislocalization. Though further work
remains to establish a reliable SAR, it is interesting to note that the
trends observed roughly parallel those reported in the publication that
inspired the choice of the prototype compound 1.23 For instance, while
the most active analogue in both instances possess an EW 4′-nitro
substituent, the next most active entity had a strongly ED substituent at
that site (e.g., dimethylamino derivative 8 here but –OCH3 in the
Bandgar publication).23 The lower activity of the 4′-amino analogue 2
relative to 1 suggests that the biological activity of the 4′-nitro deri-
vative is not an artifact due to adventitious reduction under the con-
ditions of the assay. In the case of halogens at the C4′-position, a slight
size effect is noticeable in both cases in that the smaller fluoro sub-
stituent is more active than the corresponding bromo atom in this
study, whereas in the earlier publication a chloro group was found
superior to a bromo atom.23 These comparisons are not meant to imply
any similarity in mechanism of action or cancer type, but merely to note
possible trends in the anticancer properties of 3,4,5-trimethoxyphenyl
chalcones.
Since dissociation of Ras from the PM blocks Ras signal transduc-
tion,33–36 we further examined the effect of 1 on oncogenic Ras signal
output. MDCK cells stably expressing GFP-K-RasG12V or -H-RasG12V
were treated with 1 for 48 h, and cell lysates were immunoblotted with
anti-phospho-ERK and Akt (Ser473) antibodies. The levels of phos-
phorylated ERK and Akt are measured to study Ras activity since active
Ras induces phosphorylation of ERK and Akt.2 Our data show that 1
significantly reduced the phosphorylation of ERK and Akt in K-
RasG12V-expressing cells, but not in H-RasG12V-expressing cells
(Fig. 2A–D). Together with confocal microscopy data, our data suggest
that 1 selectively dissociates the PM interaction and signal transduction
of oncogenic mutant K-Ras.
To further characterize the effect of 1 on oncogenic K-Ras signaling,
we performed cell proliferation assay on human pancreatic ductal
adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) cells
harboring oncogenic mutant K-Ras. For control cells, we used human
cancer cells expressing wild-type (WT) K-Ras (BxPC3 for PDAC, and
H1299, H1975 and H522 for NSCLC). Our data show that 1 sig-
nificantly inhibited the growth of PDAC cells expressing oncogenic K-
Ras (Fig. 2F and H). For NSCLC cells, 1 had a greater inhibitory activity
on H441 and H358 cells over cells expressing WT K-Ras (Fig. 2E and G).
A549 however, showed no growth inhibition, albeit it expressed on-
cogenic mutant K-Ras. The growth of cancer cells expressing WT K-Ras
was not inhibited by 1 (Fig. 2E–H). Previous studies reported that
growth of H441 and H358, but not A549 cells, and PDAC cells har-
boring oncogenic mutant K-Ras tested in our experiment is highly de-
pendent on oncogenic K-Ras activity, a phenomenon called K-Ras ad-
diction.37,38 Taken together, our data suggest that 1 inhibits growth of
K-Ras-addicted human cancer cells.
To examine the effect of substrates 1 and 8 on the PM interaction of
other Ras isoforms, MDCK cells stably co-expressing GFP-tagged onco-
genic mutant H-Ras (H-RasG12V) and mCherry-CAAX were treated
with 1 or 8 for 48 h, and cells were fixed and imaged using a confocal
microscope. Our data show that 1 and 8 did not mislocalize H-RasG12V
from the PM, suggesting the effects of these compounds are K-Ras-
specific (Fig. 1C and D).
Protein kinase C (PKC) can phosphorylate K-Ras at Ser181, and the
phosphorylated K-Ras is redistributed from the PM to other cellular
membranes.39 To examine whether 1 dissociates K-Ras from the PM
through activating PKC, we measured the level of phosphorylated
myristoylated alanine-rich C-kinase substrate (MARCKS),
a PKC
3

S.E. Kovar, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 2. Compound 1 inhibits oncogenic K-Ras signaling and growth of K-Ras-driven cancers. MDCK cells stably expressing GFP-K-RasG12V or -H-RasG12V were
treated with 1 for 48 h, and cell lysates were immunoblotted with anti-ppERK or -pAkt (S473) antibodies. (A and B) Representative blots from three independent
experiments are shown. Total ERK and Akt were used as loading controls. (C and D) The graphs show the means
S.E.M. from three independent experiments.
Significant differences between control (DMSO-treated) cells and 1-treated cells were assessed using a one-way ANOVA test (* p < 0.05; N.S. – not significant). A
panel of pancreatic ductal adenocarcinoma (PDAC) (E) and non-small cell lung cancer cells (NSCLC) (F) were plated on a 96-well plate and treated with various
concentrations of 1 for 4 days. Complete growth medium with the drug was replaced every 24 h. Cell proliferation was analyzed using CyQuant proliferation assay
kit. The graph shows the mean cell proliferation
S.E.M. from three independent experiments relative to that for the control cells (DMSO-treated). Cell lines
expressing oncogenic mutant K-Ras or wild-type K-Ras are shown in red and black, respectively. (G and H) The graphs represent the mean cell proliferation
S.E.M.
relative to that for the control cells (DMSO-treated) after treatment with 50 µM for 4 days. Open and closed bars represent cancer cells expressing wild-type K-Ras and
oncogenic K-Ras, respectively. Significant differences between 1-treated and control cells were assessed using Mann-Whitney U test (* p < 0.05, ** p < 0.001, N.S.
– not significant).
Fig. 3. 1 dissociates K-Ras from the PM through
PKC-mediated K-Ras phosphorylation. H358,
a
NSCLC cell line (A) or MDCK cells stably expressing
GFP-K-RasG12V (B) were treated with various con-
centrations of 1 for 48 h, and cell lysates were im-
munoblotted for phosphorylated MARCKS (p-
MARCKS) and quantified (means
S.E.M.).
Representative blots from three independent ex-
periments are shown. An GAPDH blots were used as
loading controls. (C) MDCK cells co-expressing GFP-
K-RasG12V S181A and mCherry-CAAX were treated
with 50 μM 1 for 48 h and images by confocal mi-
croscopy. Representative images of GFP-K-RasG12V
S181A from three independent experiments are
shown. Inserted values represent the fraction of
mCherry-CAAX co-localized with GFP-K-RasG12V
S181A calculated by Manders coefficient.
substrate. A NSCLC cell line, H358, of which growth was highly sen-
sitive to 1 (Fig. 2F and H) or MDCK cells stably expressing GFP-K-
RasG12V were treated with various concentrations of 1 and phos-
phorylated MARCKS, an indicator of PKC activity was measured. Our
data show that 1 increases MARCKS phosphorylation in a dose-
dependent manner (Fig. 3). To further validate our data, we mutated K-
Ras Ser181 residue to Ala (K-RasG12V S181A), which is insensitive to
PKC-mediated phosphorylation, and examined the effect of 1 in the PM
localization of K-RasG12V S181A. Our data show that K-RasG12V
S181A is not dissociated from the PM after 1 treatment, suggesting 1
4

S.E. Kovar, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
dissociates K-Ras from the PM through phosphorylating Ser181 residue.
Taken together, our data suggest that 1 dissociates oncogenic K-Ras
from the PM by PKC-mediated phosphorylation at Ser181. This in turn,
results in inhibition of oncogenic K-Ras activity and the growth of K-
Ras-addicted human cancer cells.
References
1. Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimen-
sions. Science. 2001;294(5545):1299–1304.
2. Hancock JF. Ras proteins: different signals from different locations. Nat Rev Mol Cell
Biol. 2003;4(5):373–384.
KRAS is one of the top ten genes mutated in human cancers har-
boring loss-of-function mutations for seven of the PKC isozymes,40
suggesting that PKC may suppress oncogenic K-Ras signaling such that
loss of PKC would be required for K-Ras to exert its full oncogenic
potential.40 PKC isozymes comprise three classes: conventional (α, β,
γ), novel (δ, ε, η, θ), and atypical (ζ, ι). Several studies have reported
that loss of PKCδ is found in cancers harboring oncogenic K-Ras. PKCδ
protein levels are lower in endometrial cancer cells harboring onco-
genic K-Ras than that of WT K-Ras.41 Also, total PKC activity is sig-
nificantly lower in human colorectal cancers compared to normal mu-
cosa because of decreased PKCβ and PKCδ.42 Moreover, approximately
40% of PKCδ loss-of-function mutations found in a large panel of
human cancers are in pancreatic cancers,40 and patients with lung
adenocarcinomas harboring oncogenic mutant K-Ras show an increased
overall survival rate when they also have higher PKCδ mRNA levels.43
These studies suggest that PKCδ may have an anti-cancer activity in
cancers expressing oncogenic mutant K-Ras. Taking these studies to-
gether with our data, we propose that 1 has anti-K-Ras activity through
stimulating PKCδ. The exact molecular mechanism of 1-mediated PKCδ
stimulation needs to be further elucidated.
3. Hancock JF, Magee AI, Childs JE, Marshall CJ. All ras proteins are polyisoprenylated
but only some are palmitoylated. Cell. 1989;57(7):1167–1177.
4. Gutierrez L, Magee AI, Marshall CJ, Hancock JF. Post-translational processing of
p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteo-
lysis. EMBO J. 1989;8(4):1093–1098.
5. Clarke S, Vogel JP, Deschenes RJ, Stock J. Posttranslational modification of the Ha-
ras oncogene protein: evidence for a third class of protein carboxyl methyl-
transferases. Proc Natl Acad Sci USA. 1988;85(13):4643–4647.
6. Zhou Y, Prakash P, Liang H, Cho KJ, Gorfe AA, Hancock JF. Lipid-sorting specificity
encoded in K-Ras membrane anchor regulates signal output. Cell. 2017;168(1–2)
239–251 e216.
7. Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, Grinstein S. Membrane phosphati-
dylserine regulates surface charge and protein localization. Science.
2008;319(5860):210–213.
8. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer.
Cancer Res. 2012;72(10):2457–2467.
9. Papke B, Der CJ. Drugging RAS: know the enemy. Science.
2017;355(6330):1158–1163.
10. Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ. Drugging the undruggable RAS:
mission possible? Nat Rev. 2014;13(11):828–851.
11. Gorfe AA, Cho KJ. Approaches to inhibiting oncogenic K-Ras. Small GTPases.
2019:1–10.
12. van der Hoeven D, Cho KJ, Zhou Y, et al. Sphingomyelin Metabolism Is a Regulator of
K-Ras Function. Mol Cell Biol. 2018;38(3).
13. Zimmermann G, Papke B, Ismail S, et al. Small molecule inhibition of the KRAS-
PDEdelta interaction impairs oncogenic KRAS signalling. Nature.
2013;497(7451):638–642.
A previous study demonstrated the effect of chalcones on cancer cell
lines harboring oncogenic K-Ras, where a class of indolyl-tetralone
chalcones induced apoptosis of A549 by cell cycle blockage.44 Although
the two sets of compounds are not strictly comparable, it is interesting
to note that a chalcone with the 3,4,5-trimethoxy motif on ring B lacked
any inhibition in that study. Here, we show that compound 1 inhibits
the growth of K-Ras-addicted human cancers, but not A549, which does
require oncogenic mutant K-Ras activity for its growth.37 Taken to-
gether, we propose that unlike the indolyl-tetralone chalcones, our
compounds are specifically targeting oncogenic K-Ras, and that the
mechanism of cell death induced by indolyl-tetralone chalcones is likely
independent of K-Ras inhibition. Therefore, we can conclude that
chalcones with the 3,4,5-trimethoxy moiety are a novel class of com-
pounds for oncogenic mutant K-Ras.
14. Leung ELH, Luo LX, Liu ZQ, et al. Inhibition of KRAS-dependent lung cancer cell
growth by deltarasin: blockage of autophagy increases its cytotoxicity. Cell Death Dis.
2018;9(2):216.
15. Kim J, Kim H, Park SB. Privileged structures: efficient chemical “navigators” toward
unexplored biologically relevant chemical spaces. J Am Chem Soc.
2014;136(42):14629–14638.
16. Evans BE, Rittle KE, Bock MG, et al. Methods for drug discovery - development of
potent, selective, orally effective cholecystokinin antagonists. J Med Chem.
1988;31(12):2235–2246.
17. Zhuang CL, Zhang W, Sheng CQ, Zhang WN, Xing CG, Miao ZY. Chalcone: a privi-
leged structure in medicinal chemistry. Chem Rev. 2017;117(12):7762–7810.
18. Mahapatra DM, Bharti SK, Asati V. Anti-cancer chalcones: structural and molecular
target perspectives. Eur J Med Chem. 2015;98:69–114.
19. Edwards ML, Stemerick DM, Sunkara PS. Chalcones – a new class of antimitotic
agents. J Med Chem. 1990;33(7):1948–1954.
20. Ducki S. The development of chalcones as promising anticancer agents. Idrugs.
2007;10(1):42–46.
21. Bhat BA, Dhar KL, Puri SC, Saxena AK, Shanmugavel M, Qazi GN. Synthesis and
biological evaluation of chalcones and their derived pyrazoles as potential cytotoxic
agents. Bioorg Med Chem Lett. 2005;15(12):3177–3180.
In summary, we identified that chalcone-based compounds 1 and 8
selectively dissociate oncogenic K-Ras from the PM through PKC-
mediated phosphorylation. Upon further investigation, we determined
1 capable of inhibiting oncogenic K-Ras signal output, as well as se-
lectively inhibiting the growth of K-Ras-addicted cancers. Our data
suggest that 1 could be a great starting point to develop anti-K-Ras
therapies. Future work will include thoroughly exploring the efficacy of
the trimethoxy pharmacophore towards the association of oncogenic K-
Ras and the PM in greater detail.
22. Gupta RA, Kaskhedikar SG. Synthesis, antitubercular activity, and QSAR analysis of
substituted nitroaryl analogs: chalcone, pyrazole, isoxazole, and pyrimidines. Med
Chem Res. 2013;22(8):3863–3880.
23. Bandgar BP, Gawande SS, Bodade RG, Totre JV, Khobragade CN. Synthesis and
biological evaluation of simple methoxylated chalcones as anticancer, anti-in-
flammatory and antioxidant agents. Bioorg Med Chem. 2010;18(3):1364–1370.
24. Pati HN, Holt HL, LeBlanc R, et al. Synthesis and cytotoxic properties of nitro- and
aminochalcones. Med Chem Res. 2005;14(1):19–25.
25. Ahmed FF, Abd El-Hafeez AA, Abbas SH, Abdelhamid D, Abdel-Aziz M. new 1,2,4-
triazole-chalcone hybrids induce caspase-3 dependent apoptosis in A549 human lung
adenocarcinoma cells. Eur J Med Chem. 2018;151:705–722.
26. Rojas J, Paya M, Dominguez JN, Ferrandiz ML. The synthesis and effect of fluori-
nated chalcone derivatives on nitric oxide production. Bioorg Med Chem Lett.
2002;12(15):1951–1954.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
27. Vaidya SS, Vinaya H, Mahajan SS. Microwave-assisted synthesis, pharmacological
evaluation, and QSAR studies of 1,3-diaryl-2-propen-1-ones. Med Chem Res.
2012;21(12):4311–4323.
28. Custodio JMF, Faria ECM, Sallum LO, et al. The influence of methoxy and ethoxy
groups on supramolecular arrangement of two methoxy-chalcones. J Braz Chem Soc.
2017;28(11):2180–2191.
Acknowledgment
29. Tran TD, Nguyen TTN, Do TH, Huynh TNP, Tran CD, Thai KM. Synthesis and anti-
bacterial activity of some heterocyclic chalcone analogues alone and in combination
with antibiotics. Molecules. 2012;17(6):6684–6696.
This work was supported by National Cancer Institute Grant
R00CA188593 to K.-J. C.
30. Batt DG, Goodman R, Jones DG, et al. 2'-Substituted chalcone derivatives as in-
hibitors of interleukin-1 biosynthesis. J Med Chem. 1993;36(10):1434–1442.
31. Salum LB, Altei WF, Chiaradia LD, et al. Cytotoxic 3,4,5-trimethoxychalcones as
mitotic arresters and cell migration inhibitors. Eur J Med Chem. 2013;63:501–510.
32. Choy E, Chiu VK, Silletti J, et al. Endomembrane trafficking of ras: the CAAX motif
targets proteins to the ER and Golgi. Cell. 1999;98(1):69–80.
Appendix A. Supplementary data
33. Cho KJ, Park JH, Piggott AM, et al. Staurosporines disrupt phosphatidylserine traf-
ficking and mislocalize Ras proteins. J Biol Chem. 2012;287(52):43573–43584.
34. Cho KJ, Casteel DE, Prakash P, et al. AMPK and endothelial nitric oxide synthase
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2020.127144. These data include MOL files and
InChiKeys of the most important compounds described in this article.
5

S.E. Kovar, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
signaling regulates K-Ras plasma membrane interactions via cyclic GMP-dependent
switch on K-Ras that promotes its association with Bcl-XL on mitochondria and in-
protein kinase 2. Mol Cell Biol. 2016;36(24):3086–3099.
35. Cho KJ, van der Hoeven D, Zhou Y, et al. Inhibition of acid sphingomyelinase de-
pletes cellular phosphatidylserine and mislocalizes k-ras from the plasma membrane.
Mol Cell Biol. 2016;36(2):363–374.
duces apoptosis. Mol Cell. 2006;21(4):481–493.
40. Antal CE, Hudson AM, Kang E, et al. Cancer-associated protein kinase C mutations
reveal kinase's role as tumor suppressor. Cell. 2015;160(3):489–502.
41. Reno EM, Haughian JM, Dimitrova IK, Jackson TA, Shroyer KR, Bradford AP.
Analysis of protein kinase C delta (PKC delta) expression in endometrial tumors. Hum
Pathol. 2008;39(1):21–29.
36. Willumsen BM, Christensen A, Hubbert NL, Papageorge AG, Lowy DR. The p21 ras C-
terminus is required for transformation and membrane association. Nature.
1984;310(5978):583–586.
42. Craven PA, DeRubertis FR. Loss of protein kinase C delta isozyme immunoreactivity
in human adenocarcinomas. Dig Dis Sci. 1994;39(3):481–489.
37. Singh A, Greninger P, Rhodes D, et al. A gene expression signature associated with
“K-Ras addiction” reveals regulators of EMT and tumor cell survival. Cancer Cell.
2009;15(6):489–500.
43. Ohm AM, Tan AC, Heasley LE, Reyland ME. Co-dependency of PKCdelta and K-Ras:
inverse association with cytotoxic drug sensitivity in KRAS mutant lung cancer.
Oncogene. 2017;36(30):4370–4378.
38. Hayes TK, Neel NF, Hu C, et al. Long-term ERK inhibition in KRAS-mutant pancreatic
cancer is associated with MYC degradation and senescence-like growth suppression.
Cancer Cell. 2016;29(1):75–89.
44. Wang YQ, Hedblom A, Koerner SK, et al. Novel synthetic chalcones induce apoptosis
in the A549 non-small cell lung cancer cells harboring a KRAS mutation. Bioorg Med
Chem Lett. 2016;26(23):5703–5706.
39. Bivona TG, Quatela SE, Bodemann BO, et al. PKC regulates a farnesyl-electrostatic
6