Cancers: 3D Pharmacophore-Based Discovery of Novel K 10.1 Inhibitors With Antiproliferative Activity

cancers
Article
3D Pharmacophore-Based Discovery of Novel KV10.1 Inhibitors
with Antiproliferative Activity
Žan Toplak 1 , Louise Antonia Hendrickx 2 , Špela Gubič 1 , Štefan Možina 1 , Bojana Žegura 3,4 , Alja Štern 3,4 ,
Matjaž Novak 3,4 , Xiaoyi Shi 5 , Steve Peigneur 2 , Jan Tytgat 2 , Tihomir Tomašič 1 , Luis A. Pardo 5, *
and Lucija Peterlin Mašič 1, *
1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia;

zan.toplak@ffa.uni-lj.si (Ž.T.); spela.gubic@ffa.uni-lj.si (Š.G.); stefan.mozina@ffa.uni-lj.si (Š.M.);
tihomir.tomasic@ffa.uni-lj.si (T.T.)
2 Toxicology and Pharmacology, Campus Gasthuisberg, University of Leuven, Onderwijs en Navorsing 2,
Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium; louise.hendrickx@kuleuven.be (L.A.H.);
steve.peigneur@kuleuven.be (S.P.); jan.tytgat@kuleuven.be (J.T.)
3 Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111,
1000 Ljubljana, Slovenia; bojana.zegura@nib.si (B.Ž.); alja.stern@nib.si (A.Š.); matjaz.novak@nib.si (M.N.)
4 University of Ljubljana, Kongresni trg 12, 1000 Ljubljana, Slovenia
5 AG Oncophysiology, Max-Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3,
37075 Göttingen, Germany; shi@em.mpg.de
* Correspondence: pardo@em.mpg.de (L.A.P.); lucija.peterlin@ffa.uni-lj.si (L.P.M.)
Simple Summary: A novel structural class of inhibitors of the voltage-gated potassium channel

KV 10.1 was discovered by a ligand-based drug design method using a 3D pharmacophore model.
The virtual screening hit compound ZVS-08 inhibited the channel in a voltage-dependent manner
Citation: Toplak, Ž.; Hendrickx, L.A.;
consistent with the action of a gating modifier. Structure–activity relationship studies revealed a
Gubič, Š.; Možina, Š.; Žegura, B.;
Štern, A.; Novak, M.; Shi, X.;
nanomolar KV 10.1 inhibitor that is selective for some KV and NaV channels but exhibits significant
Peigneur, S.; Tytgat, J.; et al. 3D inhibition of the hERG channel. KV 10.1 inhibitor 1 inhibited the growth of the MCF-7 cell line
Pharmacophore-Based Discovery of expressing high levels of KV 10.1 and low levels of hERG more potently than the Panc1 cell line (no
Novel KV 10.1 Inhibitors with KV 10.1 and high hERG expression). Moreover, the KV 10.1 inhibitor 1 induced significant apoptosis
Antiproliferative Activity. Cancers in tumour spheroids of Colo-357 cells. This study may provide a basis for the use of computational
2021, 13, 1244. https://doi.org/ drug design methods for the discovery of novel KV 10.1 inhibitors as new promising anticancer drugs.
10.3390/cancers13061244
Abstract: (1) Background: The voltage-gated potassium channel KV 10.1 (Eag1) is considered a
Academic Editor: Jason Roszik
near- universal tumour marker and represents a promising new target for the discovery of novel
anticancer drugs. (2) Methods: We utilized the ligand-based drug discovery methodology using 3D
Received: 29 January 2021
pharmacophore modelling and medicinal chemistry approaches to prepare a novel structural class of
Accepted: 10 March 2021
KV 10.1 inhibitors. Whole-cell patch clamp experiments were used to investigate potency, selectivity,
Published: 12 March 2021
kinetics and mode of inhibition. Anticancer activity was determined using 2D and 3D cell-based
models. (3) Results: The virtual screening hit compound ZVS-08 discovered by 3D pharmacophore
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in modelling exhibited an IC50 value of 3.70 µM against KV 10.1 and inhibited the channel in a voltage-
published maps and institutional affil- dependent manner consistent with the action of a gating modifier. Structural optimization resulted
iations. in the most potent KV 10.1 inhibitor of the series with an IC50 value of 740 nM, which was potent on
the MCF-7 cell line expressing high KV 10.1 levels and low hERG levels, induced significant apoptosis
in tumour spheroids of Colo-357 cells and was not mutagenic. (4) Conclusions: Computational
ligand-based drug design methods can be successful in the discovery of new potent KV 10.1 inhibitors.
Copyright: © 2021 by the authors. The main problem in the field of KV 10.1 inhibitors remains selectivity against the hERG channel,
Licensee MDPI, Basel, Switzerland. which needs to be addressed in the future also with target-based drug design methods.
This article is an open access article
distributed under the terms and Keywords: KV 10.1; ion channels; hERG; pharmacophore modelling; virtual screening; antiprolifera-
conditions of the Creative Commons tive activity
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Cancers 2021, 13, 1244. https://doi.org/10.3390/cancers13061244 https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 1244 2 of 24
1. Introduction
The voltage-gated potassium channel KV 10.1 (Eag1) is a member of the ether-à-go-go
family, which also includes the erg (eag-related gene, KV 11) and elk (eag-like K+ channel,
KV 12) subfamilies [1]. Like other KV channels, KV 10.1 is a tetramer with a transmembrane
region consisting of a voltage-sensor domain (VSD) with four alpha-helical segments (S1–
S4) and segments S5 and S6 forming the ion-conducting pore (Figure 1). Positively charged
residues in the VSD respond to membrane depolarisation by a movement that translates
into S5 and S6 helices opening the pore to K+ flux that drives the membrane voltage toward
the negative K+ equilibrium potential [2].
In healthy tissues, KV 10.1 is almost undetectable outside the central nervous system,
although it is highly expressed in over 70% of human cancers, regardless of tumour type [3].
On this basis, KV 10.1 is considered a nearly universal tumour marker and represents a
promising new target for new anticancer drug discovery [4]. The channel is involved in
cell cycle control and cell proliferation, migration, angiogenesis and resistance to hypoxia
of cancerous cells [5]. The mechanisms of cancer progression are complex with changes
in membrane potential control and non-canonical effects of overexpressed KV 10.1 that
impact the Ca2+ signalling and microtubule dynamics [6]. In vitro and in vivo animal
experiments showed that inhibiting the KV 10.1 channel leads to the reduction of tumour
progression [7–11]. Therefore, inhibition of the KV 10.1 could increase the survival of pa-
tients with the fibrosarcoma [12], ovary carcinoma [13], glioblastoma [14], acute lymphoid
leukaemia [15], gastric [16], head and neck [17] and colon cancers [18], all entities where
a correlation between KV 10.1 and poorer outcome has been documented. Importantly,
treatment with KV 10.1 inhibitors of brain metastasis patients improved survival in a retro-
spective study [14]. However, to date, no KV 10.1-specific small-molecule inhibitors have
been reported, as most of them also inhibit voltage-gated sodium channels or, most impor-
tantly, the cardiac hERG channel, thus posing the risk of cardiac side effects [4]. Recent
studies have pointed to a particular structural difference between KV 10.1 and hERG chan-
nels, suggesting the possibility of selective targeting of KV 10.1 in cancer therapies [19]. This
now opens exciting possibilities for further exploration using the published cryo-electron
microscopy structures of KV 10.1 and hERG [2,20].
To date, only a handful of different small molecules have been reported to inhibit
KV 10.1 (Figure 1). The identification of KV 10.1 inhibitors is mostly based on the already
known inhibitory activity at the structurally similar hERG channel and/or on the anti-
tumour properties of the small molecules. The anti-tumour activity of natural products has
led to the discovery of their blocking of Kv10.1, but to our knowledge, no other ligand- or
structure-based drug design approach has been used to discover new KV 10.1 inhibitors.
Cancers 2021, 13, 1244 3 of 24
Cancers 2021, 13, x FOR PEER REVIEW 3 of 25
Figure
Figure 1.
1. Opposite
Opposite two subunits of
two subunits of K
KV10.1 with voltage-sensor domain (VSD) in orange, pore domain and central cavity
V 10.1 with voltage-sensor domain (VSD) in orange, pore domain and central cavity in
in yellow, selectivity filter in red box. Structures of known ligands binding to the VSD and the central cavity of the pore
yellow, selectivity filter in red box. Structures of known ligands binding to the VSD and the central cavity of the pore domain.
domain. The structure (PDB: 5k7l) in the figure represents Kv10.1 with the pore domain in the closed conformation due
The structure (PDB: 5k7l) in the figure represents Kv10.1 with the pore domain in the closed conformation due to calmodulin
to calmodulin binding and VSD in the upward/conductive conformation. TBA: tetrabutylammonium: TEA: tetrae-
binding and VSDThe
thylammonium. in the upward/conductive
structure conformation.
of KV10.1 was prepared using TBA:
PyMOLtetrabutylammonium:
[21]. TEA: tetraethylammonium. The
structure of KV 10.1 was prepared using PyMOL [21].
Due to the high similarity between KV10.1 and hERG, particularly in the putative
Due to the high similarity between KV 10.1 and hERG, particularly in the putative
drug binding sites in the central cavities of the channels, achieving selective inhibition of
drug binding sites in the central cavities of the channels, achieving selective inhibition of
KV10.1 with pore-blocking small molecules presents a difficult hurdle. A VSD modulation
KV 10.1 with pore-blocking small molecules presents a difficult hurdle. A VSD modulation
approach could potentially circumvent this problem [20]. To date, only four types of small
approach could potentially circumvent this problem [20]. To date, only four types of
molecules are known to bind to the VSD, namely, mibefradil, natural bromotyrosine pur-
small molecules are known to bind to the VSD, namely, mibefradil, natural bromotyrosine
purealidin I analogues, amiodarone and 20(S)-ginsenoside Rg3 (Figure 1). The exact bind-
purpurealidin I analogues, amiodarone and 20(S)-ginsenoside Rg3 (Figure 1). The exact
ing sites sites
binding of these inhibitors
of these are still
inhibitors are unknown,
still unknown,but recently published
but recently cryo-electron
published mi-
cryo-electron
croscopy structures of both K V10.1 and hERG offer the possibility of using the VSD as a
microscopy structures of both KV 10.1 and hERG offer the possibility of using the VSD as a
binding
binding region
region for
for new
newmodulators
modulators [2,20,22,23].
[2,20,22,23]. InInthis
thiswork,
work,we
wedescribe
describethe
theligand-based
ligand-based
drug discovery of a new structural class of K V10.1 inhibitors using 3D pharmacophore
drug discovery of a new structural class of KV 10.1 inhibitors using 3D pharmacophore
modelling,
modelling,structural
structuraloptimisation
optimisationof ofthe
thevirtual
virtualscreening
screening hit
hit towards
towards potent
potent nanomolar
nanomolar
KKV10.1 inhibitors, selectivity profiling against hERG and some selected ion channels, mu-
V 10.1 inhibitors, selectivity profiling against hERG and some selected ion channels,
tagenicity
mutagenicitystudy, andand
study, antiproliferative
antiproliferativeactivity in 2D
activity andand
in 2D 3D 3D
cellcell
based models.
based models.
2.
2. Materials
Materialsand
andMethods
Methods
2.1.
2.1. Virtual
VirtualCompound
CompoundLibrary
LibraryPreparation
Preparation
Three
Three compound
compound libraries
libraries were
were prepared:
prepared: (1)(1)aa selected
selected set
set of
of purpurealidin
purpurealidin ana-ana-
logues
logues as KV 10.1 active compounds, (2) a calculated set of decoy molecules, and (3) aa
as K V10.1 active compounds, (2) a calculated set of decoy molecules, and (3)
merged
merged set of commercially
commercially available
availablecompounds
compoundsavailable
availableforfor
hithit identification
identification viavia vir-
virtual
tual screening
screening withwith prioritised
prioritised ligand-based
ligand-based pharmacophore
pharmacophore model.For
model. Forthe
theK KVV10.1 actives
actives
library,
library, structures
structuresof of11
11purpurealidin
purpurealidinI Ianalogues
analogueswerewereretrieved
retrievedfromfroma scientific publi-
a scientific pub-
lication
cation of of Moreels
Moreels et al.
et al. [22].
[22]. A second
A second library
library of 551ofdecoy
551 decoy molecules
molecules was generated
was generated based
based
on eachonofeach
the 11of the
KV10.111 Kinhibitors
V 10.1 inhibitors by submitting
by submitting their structures
their structures to the DUDE
to the DUDE decoy
decoy online generator [24], which resulted in 50 decoy molecules
online generator [24], which resulted in 50 decoy molecules per compound with similar per compound with
similar
1D 1D physicochemical
physicochemical propertiesproperties but dissimilar
but dissimilar 2D topology
2D topology in comparison
in comparison to the 11toactive
the 11
active compounds.
compounds.
A third library
A libraryofof556,000
556,000 compounds
compounds from commercial
from commercial providers was prepared
providers based
was prepared
on theon
based diversity sets from
the diversity Asinex,
sets from ChemBridge,
Asinex, ChemBridge,Enamine,
Enamine, KeyOrganics,
KeyOrganics, LifeChemicals,
LifeChem-
icals, Maybridge, and Vitas-M. Libraries were downloaded in SDF format, merged, and
Cancers 2021, 13, 1244 4 of 24
Maybridge, and Vitas-M. Libraries were downloaded in SDF format, merged, and dupli-
cates removed using the LigandScout Database Merger and Duplicates Remover nodes as
implemented in the Inte:Ligand Expert KNIME Extensions [25].
For each of the three libraries, a maximum of 200 conformations were generated for
each molecule using LigandScout’s iCon algorithm [26,27] with the default “BEST” settings
(Max. number of conformers per molecules: 200, Timeout (s): 600, RMS threshold: 0.8,
energy window: 20.0, max. pool size: 4000, max. fragment build time: 30). Each library
was saved in LDB (LigandScout database format) using LigandScout’s idbgen algorithm
with default settings.
2.2. Ligand-Based Pharmacophore Modelling

Eleven purpurealidin-based KV 10.1 inhibitors were used for creation of ten ligand-
based pharmacophore models in LigandScout 4.3 Expert. The models were generated
using the following ligand-based pharmacophore creation settings: Scoring function: phar-
macophore fit and atom overlap; Pharmacophore type: Merged feature pharmacophore;
Number of omitted features for merged pharmacophore: 4; partially matching features
optional, threshold (%): 10.0; Feature tolerance scale factor: 1.0; Maximum number of
result pharmacophores: 10. Creation of an exclusion volumes coat around the alignment of
the ligands was also enabled for each of the models. All ligands of the training set were
automatically aligned to the generated pharmacophore models. The resulting ligand-based
pharmacophore models were tested for their performance in the distinguishing the active
and decoy molecules. The best performing model was selected for virtual screening to
identify new KV 10.1 inhibitors.
2.3. Virtual Screening

Best final ligand-based pharmacophore model was used for virtual screening of the
library of 556,000 commercially available compounds. The following settings were used
for virtual screening: Scoring function: Pharmacophore-Fit; Screening mode: Match all
query features; Retrieval mode: Get best matching conformation; Max. number of omitted
features: 0, Check exclusion volumes: Checked. The virtual screening identified 18 virtual
screening hits that were then visually inspected and nine of them where selected and
purchased for in vitro screening against Kv10.1 using whole-cell patch clamp.
2.4. Chemistry
The synthesis of the virtual screening hit ZVS-08 and its analogues is shown in
Figures 2–6. Synthesis of differently substituted 4-(phenylamino)phenols 15, 18, 21 and
26 was carried out using four different approaches. o-Nitro-p-trifluoromethyl-substituted
phenol 15 was prepared in two steps (Figure 2). First, the nitro group was introduced
by nitration of 13 with concentrated nitric and sulphuric acid to give 14, followed by the
addition of p-aminophenol at high temperature to yield 15 [28]. The nitro-substituted
compound 18 was prepared by coupling p-aminophenol and 17 with the addition of base
at reflux (Figure 3). For the synthesis of compounds without the electron-withdrawing
nitro group, palladium-catalysed amination with Pd(dba3 )2 was used for compound 21
(Figure 4), while CuO and Cu were used as catalysts for the synthesis of 26 (Figure 6) [29].
The carboxylic acid 26 was converted to the methyl ester 27 using thionyl chloride in
anhydrous MeOH (Figure 6). Ethers 1 (Figure 2) and 4 (Figure 3) were formed from 15
and 18 using 3-dimethylamino-1-propyl chloride hydrochloride and Cs2 CO3 as base. The
preparation of epoxides 16, 19, 22, 24 and 28 was carried out using (±)-epichlorohydrin and
K2 CO3 as base [30]. The synthesis of 12 by Ritter reaction (Figure 2) was carried out using
acetonitrile in the presence of BF3 OEt2 [31]. For epoxide opening, morpholine was used to
give 2 and 8; aniline was used to yield compound 5 and dimethylamine for compounds
ZVS-08, 3, 6, 7 and 9. Reduction of nitro group of ZVS-08 by catalytic hydrogenation gave
compound 10 (Figure 2). The methyl ester of 9 was hydrolysed with 1 M sodium hydroxide
to give compound 11 (Figure 6). Synthetic procedures, analytical data, chemicals, reagents,
Cancers
Cancers2021,
2021,13,
13,x1244
FOR PEER REVIEW 5 5ofof2524

ide to give compound 11 (Figure 6). Synthetic procedures, analytical data, chemicals, rea-
and equipment used for organic synthesis and analysis are written in Supplementary
gents, and equipment
Materials used for organic synthesis and analysis are written in Supplemen-
ide to giveunder the Chemistry
compound 11 (Figuresection.
6). Synthetic procedures, analytical data, chemicals, rea-
tary Materials under the Chemistry section.
gents, and equipment
ide to give compound used for organic
11 (Figure synthesis
6). Synthetic and analysis
procedures, are written
analytical data, in Supplemen-
chemicals, rea-
tary Materials
gents, under the
and equipment Chemistry
used section.
for organic synthesis and analysis are written in Supplemen-
tary Materials under the Chemistry section.
Figure 2. Reagents, solvents and conditions: (a) H2SO4, HNO3, 80 °C, 1 week; (b) 4-aminophenol, isopropanol, 80 °C, 40 h;
Figure 2. Reagents, solvents and conditions: (a) H2 SO4 , HNO3 , 80 ◦ C, 1 week; (b) 4-aminophenol, isopropanol, 80 ◦ C, 40 h;
(b) 4-aminophenol, NaHCO3, EtOH, 100 °C, 20 h; (c) 3-dimethylamino-1-propyl chloride hydrochloride, Cs2CO3, DMF,
(b) 4-aminophenol,
Figure 2. Reagents, NaHCO , EtOH,
solvents 3and 100 ◦ C,(a)
conditions: 20 Hh;2SO
(c)43-dimethylamino-1-propyl chloride hydrochloride,
, HNO3, 80 °C, 1 week; (b) 4-aminophenol, Cs2 CO
isopropanol, 3 , DMF,
80 °C, 40 h;
100 °C,
◦
3 h; (d) (±)-epichlorohydrin, MeCN, K 2CO3, 80 °C, 16 h; (e) aniline, MeOH, r.t., 40 h; (f) morpholine, MeOH, r.t.,
◦
(b) 4-aminophenol, ± NaHCO , EtOH, 100 °C, 20 h; (c) 3-dimethylamino-1-propyl chloride hydrochloride, Cs CO 3, DMF,
36–48 h; (g) Me2NH × HCl, MeOH, 70 °C, 24 h; (h) 20% wt Pd/C, H2, MeOH, r.t., 2 h; (i) BF3OEt2, MeCN, 80 °C, 16 h. 40r.t.,
100 C, 3 h; (d) ( )-epichlorohydrin, MeCN, K CO , 80 C, 16 h; (e) aniline, MeOH, r.t., 40 h; (f) morpholine, MeOH,
3 2
Figure 2. Reagents, solvents and conditions: (a)
2 H 2 SO
3 4 , HNO 3 , 80 °C, 1 week; (b) 4-aminophenol, isopropanol, 80 °C, h;
◦ C, 24K
100
36–48 °C,
h; 3(g)
h; Me
(d)2(±)-epichlorohydrin,
(b) 4-aminophenol, NH × HCl, MeOH,
NaHCO 3, EtOH,
MeCN,
70100 °C, h;2CO
20 h;3,20%
(h) 80 3-dimethylamino-1-propyl
(c) °C,
wt 16
Pd/C,h; (e)
H2aniline,
, MeOH, MeOH, r.t.,
(i)40
r.t., 2chloride
h; BFh; Cs◦2MeOH,
(f) morpholine,
3 OEt2 , MeCN, 80
hydrochloride, C,
CO163, h.
r.t.,
DMF,
36–48
100 °C, h;3(g)
h; Me
(d)2NH × HCl, MeOH, 70MeCN,
(±)-epichlorohydrin, °C, 24 h;K2(h)
CO20% wt Pd/C, H2, MeOH, r.t., 2 h; (i) BF3OEt2, MeCN, 80 °C, 16 h.
3, 80 °C, 16 h; (e) aniline, MeOH, r.t., 40 h; (f) morpholine, MeOH, r.t.,
36–48 h; (g) Me2NH × HCl, MeOH, 70 °C, 24 h; (h) 20% wt Pd/C, H2, MeOH, r.t., 2 h; (i) BF3OEt2, MeCN, 80 °C, 16 h.
Figure 3. Reagents, solvents and conditions: (a) 4-aminophenol, NaHCO3, EtOH, 100 °C, 20 h; (b) (±)-epichlorohydrin,
MeCN, K2CO3, 80 °C, 16 h; (c) morpholine, MeOH, r.t., 36–48 h; (d) Me2NH × HCl, MeOH, 70 °C, 24 h; (e) 3-dimethylamino-
Figure 3.3. Reagents,
Reagents, solvents
solvents andandconditions:
conditions: (a)4-aminophenol,
4-aminophenol,NaHCO NaHCO3,, EtOH,
EtOH, 100
100 ◦°C, 20h;h;(b)
(b)(±(±)-epichlorohydrin,
Figure
1-propyl chloride hydrochloride, Cs2CO3, DMF,(a)100 °C, 3 h. 3 C, 20 )-epichlorohydrin,
MeCN,
Figure KK 2CO3, 80 °C,
3.2 CO
Reagents, ◦ 16 h; (c) morpholine, MeOH, r.t., 36–48 h; (d) Me 2NH × HCl, MeOH, 70 °C,◦ 24 h; (e) 3-dimethylamino-
MeCN, 3 , 80 C,solvents and conditions:
16 h; (c) morpholine, (a) 4-aminophenol,
MeOH, r.t., 36–48 h; (d) MeNaHCO
2 NH ×3, HCl,
EtOH, 100 °C,
MeOH, 70 20C,h;
24(b) (±)-epichlorohydrin,
h; (e) 3-dimethylamino-
1-propyl
MeCN, K chloride
2CO 3 , 80 hydrochloride,
°C, 16 h; (c) Cs2CO3, DMF,
morpholine, MeOH, 100 °C,36–48
r.t.,
◦ 3 h. h; (d) Me2NH × HCl, MeOH, 70 °C, 24 h; (e) 3-dimethylamino-
1-propyl chloride hydrochloride, Cs2 CO3 , DMF, 100 C, 3 h.
1-propyl chloride hydrochloride, Cs2CO3, DMF, 100 °C, 3 h.
Figure 4. Reagents, solvents, and conditions: (a) 4-aminophenol, BrettPhos, Pd(dba3)2, NaOt-Bu, 90 °C, 12 h; (b) (±)-epichlo-
rohydrin, MeCN, K2CO3, 80 °C, 16 h; (c) Me2NH × HCl, MeOH, 70 °C, 24 h.
Figure 4. Reagents, solvents, and conditions: (a) 4-aminophenol, BrettPhos, Pd(dba3)2, NaOt-Bu, 90 °C, 12 h; (b) (±)-epichlo-
rohydrin,
Figure 4. MeCN, K2CO3, 80 °C,
4. Reagents, 16 h; (c) Me2NH × HCl, MeOH, 70 °C, 24 h.Pd(dba3)2, NaOt-Bu, 90 °C, 12◦h; (b) (±)-epichlo-
Figure Reagents,solvents,
solvents,and
andconditions: (a)(a)
conditions: 4-aminophenol, BrettPhos,
4-aminophenol, BrettPhos, Pd(dba3 )2 , NaOt-Bu, 90 C, 12 h; (b) (±)-
rohydrin, MeCN, K2CO3, 80 °C, 16 h; (c) Me2NH × HCl, MeOH, 70 °C, 24 h.
epichlorohydrin, MeCN, K2 CO3 , 80 ◦ C, 16 h; (c) Me2 NH × HCl, MeOH, 70 ◦ C, 24 h.
Cancers 2021, 13, 1244 6 of 24
Figure 5.
5. Reagents,
Reagents,solvents,
solvents,and
andconditions: (a)(a)
(±)-epichlorohydrin, MeCN,
(±)-epichlorohydrin, K2CO
K32,CO
80 °C, 16◦ h; (b) Me2NH × HCl, MeOH,
Figure conditions: MeCN, 3 , 80 C, 16 h; (b) Me2 NH × HCl,
70 °C, 24 h.◦
MeOH,5.70
Figure C, 24 h.solvents, and conditions: (a) (±)-epichlorohydrin, MeCN, K2CO3, 80 °C, 16 h; (b) Me2NH × HCl, MeOH,
Reagents,
70 °C, 24 h.
Figure 6. Reagents, solvents, and conditions: (a) 4-aminophenol, K2CO3, CuO, Cu, 2-ethoxyethanol, 130 °C, 16 h; (b) thionyl
chloride, anhydrous MeOH, 70 °C, overnight; (c) (±)-epichlorohydrin, MeCN, K2CO3, 80 °C, 16 h; (d) NaOH, ◦ C,16
MeOH, water,
Figure
Figure 6.6.Reagents,
Reagents,solvents,
solvents,and
andconditions:
conditions: (a)
(a)4-aminophenol,
4-aminophenol, K
K22CO
CO33, ,CuO,
CuO,Cu,
Cu,2-ethoxyethanol,
2-ethoxyethanol,130 130°C, 16h;
h;(b)
(b)thionyl
thionyl
r.t., 12 h.
chloride,
chloride, anhydrous MeOH, 70 C, overnight; (c) (±)-epichlorohydrin, MeCN, K2 CO3 , 80 C, 16 h; (d) NaOH, MeOH,water,
anhydrous MeOH, 70 °C,
◦ overnight; (c) (±)-epichlorohydrin, MeCN, K 2CO3, 80 °C,
◦ 16 h; (d) NaOH, MeOH, water,
r.t.,
r.t., 12
12 h.
h. 2.5. Electrophysiological Recordings
2.5. Stage V-VI oocytes were isolated by partial ovariectomy from Xenopus laevis frogs
2.5.Electrophysiological
ElectrophysiologicalRecordings Recordings
(African
Stage clawed
V-VI frog). Mature
oocytes were female frogs
isolated by were purchased
partial ovariectomy fromfrom CRB Xénopes
Xenopus (Rennes,
laevis frogs
France) Stage
and V-VI
were oocytes
housed were
in the isolated
Aquatic by partial(KU
Facility ovariectomy
Leuven) in from Xenopuswith
compliance laevisthefrogs
reg-
(African
(African of clawed
clawed frog). Mature
frog). Mature female
female frogs were
frogs were the purchased
purchased from
from CRB Xé n
CRB Xénopes opes (Rennes,
(Rennes,
ulations
France) and the
were European
housed Union
inin
thethe (EU)
Aquatic concerning
Facility (KU(KU welfare
Leuven) of laboratory
in compliance animals
with with as
the reg-de-
France) and
clared inofDirective were housed
2010/63/EU. Aquatic
The concerning Facility
use of Xenopus Leuven)
laevis was in compliance
approved animals
by the Animal the
ulations the European Union (EU) the welfare
regulations of the European Union (EU) concerning the welfare of laboratory animals as of laboratory as de-
Ethics in
clared Committee of the KU Leuven (Project nr. P186/2019). After anesthetising the frogs
declared Directive
in Directive 2010/63/EU.
2010/63/EU. TheThe use useof of
Xenopus
Xenopus laevis
laeviswaswasapproved
approvedby bythetheAnimal
Animal
by
Ethicsa 15-min submersion in 0.1% tricaine methanesulphonate (pH 7.0), the oocytes were
EthicsCommittee
Committeeof ofthe
the KU
KU Leuven
Leuven (Project
(Project nr.nr. P186/2019).
P186/2019).After Afteranesthetising
anesthetisingthe thefrogs
frogs
collected.
by The isolated oocytes were then washed with a 1.5 mg/mL collagenase solution
byaa15-min
15-minsubmersion
submersionin in 0.1%
0.1% tricaine
tricaine methanesulphonate
methanesulphonate(pH (pH7.0),
7.0),the
theoocytes
oocyteswere were
for 2
collected.h to remove the follicle layer.
collected.The Theisolated
isolatedoocytes
oocyteswere werethen then washed
washed with with aa 1.51.5 mg/mL
mg/mLcollagenase
collagenasesolutionsolution
for Ion channels (KV1.x, KVlayer.
2.1, KV4.2, NaV1.4, NaV1.5) were expressed in Xenopus laevis
for22hhto toremove
removethe thefollicle
follicle layer.
oocytes,
Ion by linearisation of the plasmids and subsequent in vitro transcription using a
Ionchannels
channels (K (KVV1.x,
1.x,KKVV2.1,
2.1,KKVV4.2,4.2,NaNaVV 1.4,
1.4,Na Na V1.5)
V 1.5)were
wereexpressed
expressedininXenopus
Xenopuslaevis laevis
commercial
oocytes, T7 or SP6 mMESSAGE mMACHINE transcription kit (Ambion, Carlsbad,
oocytes,by bylinearisation
linearisationofofthe theplasmids
plasmidsand andsubsequent
subsequentininvitro vitro transcription
transcription using using aa
CA, USA). Defolliculated
commercial Xenopus oocytes were then injected with 20–50 nL of Carlsbad,
the cRNA
commercial T7 or or SP6SP6mMESSAGE
mMESSAGE mMACHINE
mMACHINE transcription
transcription kitkit (Ambion,
(Ambion, Carlsbad, CA,
at
CA, a concentration
USA). of
Defolliculated 1 ng/nL using
Xenopus a micro-injector
oocytes were then (Drummond
injected with Scientific1,
20–50 Broomall, PA,
USA). Defolliculated Xenopus oocytes were then injected with 20–50 nLnL of of
thethe cRNA
cRNA at
USA).
at The oocytes
a aconcentration
concentration ofof1were
1ng/nL
ng/nL incubated in a solution (Drummond
using a micro-injector
micro-injector containing
(Drummond (in mM): NaCl,
Scientific1,
Scientific1, 96; KCl,
Broomall,
Broomall, PA,2;
PA,
CaCl
USA).
USA).The2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4), supplemented with 50 mg/L gentamycin sul-
Theoocytes
oocyteswere wereincubated
incubatedin inaa solution
solutioncontaining
containing(in (inmM):
mM):NaCl,NaCl,96; 96;KCl,
KCl,2;2;
phate
CaCl and
CaCl2,2 ,1.8; 90
1.8;MgClMgClmg theophylline.
2, 22, and
2 and HEPES,
HEPES, 5After
5 (pH
(pH ex
7.4),vivo
7.4), translation, with
supplemented
supplemented thewith ion channels
50 mg/L
50 mg/L are correctly
gentamycin
gentamycin sul-
inserted
sulphate
phate andin and
90themg
90cell
mg membrane
theophylline.
theophylline. ofAfter
the oocytes.
After ex ex vivo
vivo translation,the
translation, theion ionchannels
channelsare arecorrectly
correctly
inserted
inserted Two-electrode
inthe
in thecell voltage-clamp
cellmembrane
membrane ofthe
of therecordings
oocytes. were performed at room temperature (18–
oocytes.
22 °C) using a Geneclamp
Two-electrode
Two-electrode 500 amplifier
voltage-clamp
voltage-clamp recordings(Molecular
recordings werewere Devices,
performed
performed San
at atJose,
room CA, USA) controlled
temperature
room temperature (18–22 ◦ C)
(18–
by
using
22 a pClamp
a Geneclamp
°C) using data
a Geneclampacquisition
500 amplifier system
500 amplifier (Axon
(Molecular Instruments1,
(MolecularDevices, Union
San Jose,
Devices, San Jose, City,
CA, CA, CA,
USA)USA) USA).
controlled Whole
controlled by a
cell
by a currents
pClamp pClamp datadatafrom oocytessystem
acquisition
acquisition were recorded
system (Axon (Axon 1–7 days afterUnion
Instruments1,
Instruments1, injection.
Union City, The
CA,bath
City, USA).
CA, solution
USA).Whole com-
Whole cell
position
currents
cell currents wasfrom
from either
oocytes ND96 (in
werewere
oocytes mM):
recordedrecorded NaCl,
1–7 days
1–796;after
KCl,
days 2; CaCl
injection.
after , 1.8;bath
2The
injection. MgCl
The 2, 2 and
solution
bath HEPES,
composition
solution com-5
(pH either
was
position 7.5)wasorND96
calcium-
either(inND96 free (in
mM): ND96
NaCl,mM): supplemented
96; KCl, 2;96;
NaCl, CaCl
KCl,with
2 , 1.8;
10 mM
MgCl
2; CaCl ,BaCl
2 and
2, 21.8;
2. HEPES,
MgCl Voltage
2, 2 and 5and
(pH current
HEPES,7.5) or5
electrodes
calcium- were
free ND96filled with
supplementeda 3 M solution
with 10 of
mM KCl
BaCl
(pH 7.5) or calcium- free ND96 supplemented with2 10 mM BaCl 2. Voltage and currentin . H 2O. Resistances
Voltage and currentof both electrodes
electrodes were
were kept were
electrodes between filled 0.5with
and a1.5 3M MΩ. solution of KCl in H 2O. Resistances of both electrodes
The elicited K
were kept between 0.5 and 1.5 MΩ.v1.x, Kv2.1, and Kv4.2 currents were filtered at 0.5 kHz and sampled at
2 kHz, TheNa V1.4, and Nav1.5 currents were filtered at 1 kHz and sampled at 20 kHz using a
elicited Kv1.x, Kv2.1, and Kv4.2 currents were filtered at 0.5 kHz and sampled at
four-pole low-pass
2 kHz, NaV1.4, and Na Bessel filter. Leak subtraction was performed using a -P/4 protocol.
v1.5 currents were filtered at 1 kHz and sampled at 20 kHz using a
four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol.
Cancers 2021, 13, 1244 7 of 24
filled with a 3 M solution of KCl in H2 O. Resistances of both electrodes were kept between
0.5 and 1.5 MΩ.
The elicited Kv 1.x, Kv 2.1, and Kv 4.2 currents were filtered at 0.5 kHz and sampled at
2 kHz, NaV 1.4, and Nav 1.5 currents were filtered at 1 kHz and sampled at 20 kHz using a
four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. For
the electrophysiological analysis of the compounds, a number of protocols were applied
from a holding potential of −90 mV. Currents for KV 1.x, KV 2.1, and KV 4.2 were evoked by
0.5 s depolarising pulses to 0 mV, followed by 0.5 s repolarising pulses to −50 mV. Currents
for NaV 1.4 and NaV 1.5 were evoked by 100 ms depolarising pulses to 0 mV.
Whole-cell patch clamp experiments were performed on stably transfected HEK-
293 cells [32,33]. The cells were grown on fibronectin-coated glass coverslips in DMEM
supplemented with 10% FCS and 300 µg/mL Zeocin. Experiments were performed at
room temperature under continuous perfusion. The extracellular solution contained (mM)
160 NaCl, 2.5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes/NaOH, pH 7.4, 8 glucose. For the exper-
iments determining conductance–voltage relationships, tail currents were made visible
by substituting 60 mM NaCl by KCl (162.5 mM KCl, 100 NaCl). Pipettes were pulled
from #1 glass capillaries (WPI) to resistances of 1–3 MΩ when loaded with the intracellular
solution (100 KCl, 45 N-methyl-D-glucamine, 10 1,2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -
tetraacetic acid (BAPTA), 10 Hepes pH 7.35). Data acquisition was performed using a
EPC10 Plus amplifier (HEKA Elektronik) and Patch Master software. Drug application
was performed using an 8-channel fast solution exchanger (ALA). All compounds were
dissolved in DMSO to a final concentration of 10 mM. 0.5% DMSO in extracellular solution
served as control. KV 10.1 currents were elicited by 500 ms depolarisations to +40 mV under
continuous perfusion with the compound-containing or control solution. hERG current
amplitudes were measured as the peak tail current during a 4 s repolarization to −40 mV
after a depolarising pulse to +20 mV lasting for 2 s.
2.6. Statistical Analysis

All electrophysiological data are presented as means ± SEM of n ≥ 3 independent ex-
periments unless otherwise indicated. Oocyte data were analysed using pClamp Clampfit
10.4 (Molecular Devices, Downingtown, PA, USA) and OriginPro 9 (Originlab, Northamp-
ton, MA, USA) or GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA,
USA). Patch-clamp data analysis was performed using FitMaster (HEKA Electronics (Lud-
wigshafen, Germany)), IgorPro (Wavemetrics, Inc, Lake Oswego, OR, USA) and Prism v.8.
The Dunnett test and one-way ANOVA were performed to calculate the significance of the
induced inhibition compared to the control (p < 0.05).
2.7. Cell Culture

Cell lines MCF-7 (DSMZ ACC 115), PANC-1 (DSMZ ACC 783), and hTERT-RPE1
(ATCC CRL 4000) were grown in the media recommended by the cell provider in a hu-
midified atmosphere at 5% CO2 and 37 ◦ C. For cell growth experiments, 104 cells/well
were seeded in 96-well flat bottom culture plates (Corning) and allowed to grow for 24 h.
The compounds were then added in the corresponding growth medium, and the plate was
imaged every hour for 72 h (two images per well and eight wells per condition in two
independent experiments for compound 8 and five for ZVS-08 and compound 1) in an
Incucyte system (Sartorius/Essen biosciences). Culture confluence was determined in the
phase contrast images and is expressed as percent confluence per well. The growth was
then normalised to the value in the absence of compounds.
Spheroids were cultured in round bottom ultra-low attachment 96-well plates (Corn-
ing) at a density of 5000 cells/well in 2% Matrigel (Corning) and centrifuged at 1000× g
for 10 min. Spheroid formation was monitored continuously in the Incucyte system and
treatments were added once spheroids were formed. As apoptosis sensitizer, 8.9 µM
cycloheximide was added to controls and treated spheroids. Caspase 3/7 green reagent
(Sartorius; 1:1000) was used as recommended by the manufacturer. Caspase 3/7 reagent
Cancers 2021, 13, 1244 8 of 24
substrate crosses the cell membrane and its cleavage by activated caspase-3/7 results in
the release of a DNA dye and fluorescent staining of nuclear DNA.
2.8. Evaluation of Mutagenic Activity with Salmonella/Microsomal Reverse Mutation Assay

Mutagenicity of compounds ZVS-08 and 8 was tested with the Salmonella/microsomal
reverse mutation (Ames) assay on Salmonella typhimurium strains TA98 and TA100 without
and with external metabolic activation according to the OECD guidelines (OECD TG 471)
with minor modifications. Briefly, 100 µL of ZVS-08 and 8 (0.158, 0.5, 1.58, 5, 15.8, and
50 mg/mL corresponding to the final concentration of 0.0158, 0.05, 0.158, 0.5, 1.58, and
5 mg/plate, respectively), 100 mL bacterial culture of S. typhimurium (overnight culture
at 37 ◦ C), and 500 mL of phosphate buffer (for assays without metabolic activation) or
10% S9 mix (for assays with metabolic activation) were added to 2 mL of molten top agar
(supplemented with histidine/biotin, final concentration 0.05 mM), gently mixed and
poured onto minimal agar plates. The number of His+ revertants was counted after 48 h
(TA100) and 72 h (TA98) of incubation at 37 ◦ C. Three plates were used per treatment
point. The mutagenic potential of the compounds was expressed as an induction factor (IF),
where IF = (number of revertants in the presence of the compound)/(number of revertants
in solvent control). Additionally, each plate was checked for possible toxic effects of
tested compounds. The concentration is considered as cytotoxic when the microcolony
lawn is reduced and is classified as moderately (distinguished by a marked thinning of
the bacterial lawn that may result in a pronounced increase in the size of microcolonies
compared to the solvent control plate), extremely reduced (distinguished by an extreme
thinning of the bacterial background lawn that may result in an increase in the size of
the microcolonies compared to the solvent control plate such that the microcolony lawn
is visible to the unaided eye as isolated colonies) or absent (distinguished by a complete
lack of any bacterial background lawn over greater than or equal to 90% of the plate).
4-nitroquinoline- N-oxide (4-NQNO; 0.25 µg/plate) for TA98 and sodium azide (NaN3 ;
0.25 µg/plate) for TA100 were used as positive controls without S9 metabolic activation,
while benzo[a]pyrene (B[a]P; 2.5 µg/plate) was used as positive controls for both bacterial
strains with S9 metabolic activation.
3. Results
3.1. Ligand-Based Identification of Novel KV 10.1 Inhibitors through in Silico Screening
The discovery of KV 10.1 inhibitors has been limited to reports of known compounds
or drugs that bind to this channel mostly as an off-target (Figure 1). Even though the three-
dimensional structure of KV 10.1 has been determined by cryo-electron microscopy [2],
the actual binding sites of small molecules in KV 10.1 remain mostly unknown. There-
fore, structure-based design and virtual screening are rarely used to identify new KV 10.1
inhibitors. Limitations of structure-based in silico approaches in the design of KV 10.1
inhibitors can be overcome by ligand-based methods, such as 3D similarity search and
pharmacophore modelling.
The latter method can be used to build ligand-based pharmacophore models (LBPM)
based on a set of active ligands. The pharmacophore model consists of a set of interactions
aligned in 3D space and is represented by a collection of pharmacophore features with
defined properties, such as aromatic ring, hydrogen bond donors and acceptors, hydropho-
bic, negatively and positively charged groups, and halogen bonds features. The known
active compounds are defined as a training set and aligned in 3D space, followed by the
generation of a pharmacophore model.
One of the most important requirements for generating ligand-based pharmacophore
models is that the ligands have the same mode of action and bind in the same orientation
in the binding site. Therefore, to generate and validate the LBPM, we used a set of 11
purpurealidin I analogues that showed KV 10.1 inhibition [22]. This is the only series of
analogues that was evaluated for inhibition of KV 10.1. They also displayed antiproliferative
effects in cancer cell lines with high KV 10.1 expression. Based on competition experiments
Cancers 2021, 13, 1244 9 of 24
with mibefradil, a suggested binding site for these compounds is in the VSD of KV 10.1,
although its exact location remains to be elucidated experimentally [22]. The LBPM method
circumvents this limitation as the model is based solely on the alignment of the ligands in
the training set. Due to the similarity between the ligands, we assumed that the binding
site is shared among the active purpurealidin analogues, which is necessary for a valid
LBPM generation. The higher diversity between KV 10.1 and hERG channels in the VSD
than in the pore of the channels highlights purpurealidin analogues as excellent starting
points for identifying novel KV 10.1 inhibitors [34].
In virtual screening using LBPM as a query, the ligands in the screening library are
clustered into active and decoy (inactive) datasets, and then, their pharmacophore fit scores
are calculated and ranked in the hitlist from highest to lowest score. Potentially active com-
pounds have high pharmacophore fit scores, while decoys poorly fit the pharmacophore
model and receive low scores. Screening results can be visualised by Receiver Operating
Characteristic (ROC) plots, where the rate of true positives (active compounds) is on the
y-axis and the rate of false positives (decoys) is on the x-axis. If the ROC plot curve follows
the diagonal line, it means that the model is performing poorly and cannot distinguish
between decoy and active compounds. A ROC curve plotted above the diagonal represents
a pharmacophore model that can detect active compounds. In addition, the enrichment fac-
tor (EF) represents the number of active compounds identified by a query pharmacophore
model, as opposed to the number of compounds hypothetically found to be active when
randomly screened. Thus, an enrichment factor of 1 means that the ligands are randomly
sorted. In contrast, high enrichment factors mean that only a small fraction of the hitlist
needs to be screened in vitro to identify a high number of active compounds.
To evaluate the performance of LBPM before running the real virtual screening cam-
paign, it needs to be trained and validated. Ideally, the model will find a high number
of active ligands from the training set and no ligands from the decoy set. Ligand-based
pharmacophore modelling was performed in LigandScout 4.3 Expert [35,36]. Ten 3D LBPM
with the best alignments were generated and scored with pharmacophore alignment scores.
Exclusion volume features (Figure 7A, grey spheres) were automatically added to each
model based on the shape of the ensemble of aligned molecules. These features repre-
sent restricted space and are important to increase the selectivity of a model for virtual
screening campaigns.
An initial pharmacophore model was constructed based on the alignment of the 11
most potent purpurealidin-based KV 10.1 inhibitors and is shown in Figure 7A [22]. It
consisted of 14 pharmacophore features, 12 of which were essential (four hydrophobic
features, marked as yellow spheres; two hydrogen bond acceptors, marked as red spheres;
one hydrogen bond donor, marked as a green arrow; two aromatic features, marked as
blue discs; one positive ionisable feature, marked as a blue star; and two halogen bond
features, marked as pink arrows), and two were marked as optional (dashed red circle
for one hydrogen bond acceptor and dashed pink arrow for one halogen bond). For each
active compound in the dataset, 50 decoys were generated using the DUD-E server [24].
The DUD-E database calculates decoys based on the given set of KV 10.1 inhibitors, which
means that some of the decoys could still inhibit KV 10.1 as they are similar to the active
molecules. However, this is highly unlikely. This initial LBPM was tested with a library of
active and decoy molecules. The performance of this model was very good in terms of the
enrichment factor of 51.1, finding 9 out of 11 active compounds from the dataset and no
decoy ligands (Figure 7B). However, it was too restrictive and performed poorly in virtual
screening of commercially available compounds as no hits were identified. Therefore,
our next step was to simplify the original LBPM while retaining its favourable screening
properties, with the model able to distinguish between active and inactive compounds.
Cancers 2021, 13, 1244 10 of 24
Figure
Figure 7. (A)
7. (A) Initial
Initial ligand-based
ligand-based pharmacophore
pharmacophore model
model for
for K KV10.1 inhibitors based on a library of
V 10.1 inhibitors based on a library of
purpurealidin analogues. Common chemical features of the
purpurealidin analogues. Common chemical features of the aligned molecules aligned molecules
includeinclude 4 hydro-
4 hydrophobic
phobic features (yellow spheres), 3 hydrogen bond acceptors (red spheres), 1 hydrogen bond do-
features (yellow spheres), 3 hydrogen bond acceptors (red spheres), 1 hydrogen bond donor (a green
nor (a green arrow), 2 aromatic features (blue discs), 1 positive ionisable feature (blue star), 3 halo-
arrow), 2 aromatic features (blue discs), 1 positive ionisable feature (blue star), 3 halogen bond
gen bond features (pink arrows), and exclusion volumes (grey spheres) that define restricted re-
features
gions(pink
based arrows), and exclusion
on the shape volumes
of the aligned (grey spheres)
molecules. thatfeatures
Optional define restricted
are marked regions based (C)
as dashed. on
the Final
shapeligand-based
of the alignedpharmacophore
molecules. Optional features are marked as dashed. (C) Final ligand-based
model. Common chemical features of the aligned molecules
pharmacophore model.bond
include 2 hydrogen Common chemical
acceptors features of
(red spheres), the aligned
1 hydrogen molecules
bond donor (ainclude 2 hydrogen
green sphere), 2 aro-
bond acceptors
matic features(red spheres),
(blue discs), 1and
hydrogen bond
1 positive donorfeature
ionisable (a green sphere),
(blue star).2Grey
aromatic features
spheres (blue
for exclusion
volumes
discs), are not shown.
and 1 positive ionisableResulting ROC star).
feature (blue plot (curve shown for
Grey spheres in blue) for (B)
exclusion initial and
volumes (D)shown.
are not final
LBPMROC
Resulting fromplot
virtually
(curvescreening
shown in562 compounds
blue) (11 and
for (B) initial KV10.1
(D)active compounds
final LBPM and 551 screening
from virtually generated
562 decoys)
compoundswith (11
the Kligand-based pharmacophore model. TP = true positives; FP = false positives;
V 10.1 active compounds and 551 generated decoys) with the ligand-based
AUC = area under the curve; EF = enrichment factor.
pharmacophore model. TP = true positives; FP = false positives; AUC = area under the curve; EF =
enrichment factor.
An initial pharmacophore model was constructed based on the alignment of the 11
Through
most potentseveral rounds of simplification,
purpurealidin-based we obtained
KV10.1 inhibitors our final
and is shown in LBPM,
Figure 7Awhich
[22].was
It con-
used for virtual screening. Specifically, we eliminated both optional
sisted of 14 pharmacophore features, 12 of which were essential (four hydrophobic fea- pharmacophore
features
tures,and removed
marked halogen
as yellow bond and
spheres; twohydrophobic
hydrogen bond features. In addition,
acceptors, marked weasconverted
red spheres;
the one
directional
hydrogen hydrogen
bond donor,bondmarked
donor feature to a arrow;
as a green non-vector feature. Our
two aromatic final marked
features, model as
(Figure 7C) consisted of six pharmacophore features: two aromatic,
blue discs; one positive ionisable feature, marked as a blue star; and two halogen one hydrogen bond bond
donor, two hydrogen bond acceptors, and one positive ionisable feature. The
features, marked as pink arrows), and two were marked as optional (dashed red circle for performance
of the
onemodel in the
hydrogen validation
bond acceptor screen was optimal
and dashed as it was
pink arrow for able
one to find allbond).
halogen activeFor
ligands
each ac-
andtive
no decoy
compound in the dataset, 50 decoys were generated using the DUD-E serverWhen
compounds, resulting in a high enrichment factor of 51.1 (Figure 7D). [24]. The
thisDUD-E
model was used incalculates
database the virtualdecoys
screening campaign,
based on the it was able
given to K
set of identify 18 potentially
V10.1 inhibitors, which
active compounds from the library of 556,000 diverse commercially
means that some of the decoys could still inhibit KV10.1 as they are similar available compounds.
to the active
Themolecules.
in silico hits were visually
However, this isinspected and nineThis
highly unlikely. compounds (Tablewas
initial LBPM 1) were
testedselected
with a and
library
purchased from the commercial vendors.
of active and decoy molecules. The performance of this model was very good in terms of
the enrichment factor of 51.1, finding 9 out of 11 active compounds from the dataset and
no decoy ligands (Figure 7B). However, it was too restrictive and performed poorly in
ure
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Cancers 2021, 13, 1244 11 of 24
tially
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commercially
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available
(Table
(Table 1)
1)1) were
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1)were
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pounds.
pounds. The
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in
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were
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visually
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inspected
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and nine
nine
diverse nine
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commercially
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compounds (Table 1)
available
(Table
(Table 1)
1) were
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were
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selected
pounds.
selected
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andThe
and purchased
in
purchasedsilico
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hits
from
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vendors.
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selected
selected
pounds.
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andThe
and
and purchased
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in silico hits
purchased
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from
from
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thethe
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vendors.
inspected and nine compounds (Table 1) were
vendors.
vendors.
selected
selected and purchased
andofpurchased from the commercial
from the commercial vendors.
vendors. fitfitscores
Table
Table 1. 1.List
Listof screening
screening compounds
compounds with with pharmacophore
pharmacophore scoresandandactivity
activity ononKVK V 10.1
10.1 at at
Table
Table
Table
Table 1.1.1.
1.List
List
List ofof
List
of ofscreening
screening
screening
screening compounds
compounds
compounds
compounds with
with
with
with pharmacophore
pharmacophore
pharmacophore
pharmacophore fit
fit
fit
fit scores
scores
scores
scores and
andand
and activity
activity
activity
activity on
on KKVK
on
on V10.1
K V10.1atat
V10.1
10.1 atat
compound
Table
Table
compound
compound 1.
1. concentration
List
List of
of screening
screening
concentration
concentration of 50 µM.
compounds
compounds
ofof5050 with
μM. with
μM. pharmacophore
pharmacophore fit
fit scores
scores and
and activity
activity on
on K
K 10.1
VV10.1 at
at
compound
Table
compound
compound 1. concentration
List of screening
concentration
concentration of of
of5050
50 μM.
compounds
μM.
μM. with pharmacophore fit scores and activity on K V10.1 at
Table 1.
compound
compound List of screening
concentration
concentration compounds
of
of 50
50 μM.
μM. with pharmacophore fit scores and activity on K V 10.1 at
compound concentration of 50 μM. Pharmacophore Fit Effect on
compound concentration of 50 μM.
Compound IDID Structure Pharmacophore
Pharmacophore
Pharmacophore Fit Effect
Fit
Fit Effecton
Effect onKv10.1
on Kv10.1
Kv10.1
Kv10.1 (%(%(%
(%
Compound
Compound
Compound ID
ID Structure
Structure
Structure Pharmacophore
Pharmacophore
Pharmacophore
Pharmacophore
Score Fit
FitFit
Fit Effect
Effect
Effect
Effect onon
on
on Kv10.1
Kv10.1
Kv10.1
Kv10.1
Inhibition) (%(%
(%
(%
CompoundID
Compound
Compound
Compound ID ID Structure
StructureStructure Score Pharmacophore
Score
Score Inhibition)
Fit Effect
Effect onKv10.1
Inhibition)
Inhibition)Kv10.1(% (%
CompoundID
Compound ID
ID
Structure
Structure
Structure
Pharmacophore
Score
Score
Score
Score Fit on
Inhibition)
Inhibition)
Inhibition)
Inhibition)
Score
Score Inhibition)
Inhibition)
ZVS-01
ZVS-01
ZVS-01 66.4900
66.4900
66.4900 − −1.13
1.13
−1.13 ±±±4.504.50(3)
4.50 (3)
ZVS-01
ZVS-01
ZVS-01
ZVS-01
ZVS-01
66.4900
66.4900
66.4900
66.4900
66.4900
−1.13
−1.13
−1.13
−1.13
−1.13 ±±±±4.50
±
4.50
4.50
4.50
4.50
(3)
(3)(3)
(3)
(3)
(3)
ZVS-01
ZVS-01 66.4900
66.4900 −1.13±±4.50
−1.13 4.50(3)(3)
ZVS-02
ZVS-02
ZVS-02 66.4745
66.4745
66.4745 5.03
5.03
5.03 ±
±±2.87 2.87
±2.87
2.87 (3)(3)
(3)
ZVS-02
ZVS-02
ZVS-02
ZVS-02
ZVS-02
66.4745
66.4745
66.4745
66.4745
66.4745
5.03
5.03
5.03 ±±
5.03
5.03
2.87
±±±2.87
2.87
2.87
(3)
(3)(3)
(3)
(3)
ZVS-02
ZVS-02 66.4745
66.4745 5.03 2.87
5.03 ± 2.87 (3) (3)
ZVS-03
ZVS-03
ZVS-03 66.4421
66.4421
66.4421 −1.14±±±3.47
−1.14
−1.14 3.47(5)
3.47 (5)
(5)
ZVS-03
ZVS-03
ZVS-03
ZVS-03
ZVS-03 66.4421
66.4421
66.4421
66.4421
66.4421 −1.14
1.14±±±
−1.14
− −1.14
−1.14 3.47
±3.47
±±3.47
3.47
3.47(5)(5)
(5)(5)
(5)
ZVS-03
ZVS-03 66.4421
66.4421 −1.14 3.47
−1.14 ± 3.47 (5) (5)
ZVS-04
ZVS-04
ZVS-04
ZVS-04
66.0596
66.0596
66.0596
66.0596 −
−0.86
−0.86
−0.86
0.86±± ±±0.75
0.75(5)
±±0.75
0.75
0.75
(5)
(5)
(5)
ZVS-04
ZVS-04
ZVS-04
ZVS-04 66.0596
66.0596
66.0596
66.0596 −0.86
−0.86
−0.86
−0.86 ±± 0.75
0.75
0.75 (5)
(5)(5)
(5)
ZVS-04
ZVS-04 66.0596
66.0596 −0.86±±0.75
−0.86 0.75(5)
(5)
ZVS-05
ZVS-05
ZVS-05 65.9545
65.9545
65.9545 8.84
8.84
8.84 ±±4.92
±±4.924.92
4.92 (6)
(6)
(6)
ZVS-05
ZVS-05
ZVS-05
ZVS-05
ZVS-05
65.9545
65.9545
65.9545
65.9545
65.9545
8.84
8.84
8.84 ±±
8.84
8.84 ±±
4.92
4.92
4.92
4.92
(6)
(6)
(6)(6)
(6)
ZVS-05
ZVS-05 65.9545
65.9545 8.84±±4.92
8.84 4.92(6)
(6)
ZVS-06
ZVS-06
ZVS-06 65.8501
65.8501
65.8501 −7.50±±±4.65
−7.50
−7.50 4.65(4)
4.65 (4)
(4)
ZVS-06
ZVS-06
ZVS-06
ZVS-06
ZVS-06 65.8501
65.8501
65.8501
65.8501
65.8501 −7.50
7.50±±±
−7.50
− −7.50
−7.50 ±
± 4.65
4.65
4.65
4.65
4.65 (4)
(4)
(4)
(4)
(4)
ZVS-06
ZVS-06 65.8501
65.8501 −7.50±±4.65
−7.50 4.65(4)
(4)
ZVS-07
ZVS-07
ZVS-07 65.8019
65.8019
65.8019 4.68±±±1.63
4.68
4.68 1.63(5)
1.63 (5)
(5)
ZVS-07
ZVS-07
ZVS-07
ZVS-07
ZVS-07
65.8019
65.8019
65.8019
65.8019
65.8019
4.68
4.68
4.68
4.68 ±±±±1.63
4.68 ± 1.63
1.63
1.63
1.63
(5)
(5)(5)
(5)
(5)
ZVS-07
ZVS-07 65.8019
65.8019 4.68±±1.63
4.68 1.63(5)(5)
ZVS-08
ZVS-08
ZVS-08 65.7908
65.7908
65.7908 80.27±±±0.63
80.27
80.27 0.63(3)
0.63 (3)
(3)
ZVS-08
ZVS-08
ZVS-08
ZVS-08 65.7908
65.7908
65.7908
65.7908 80.27
80.27
80.27
80.27 ± ±±0.63
± 0.63(3)(3)
0.63
0.63 (3)
(3)
ZVS-08
ZVS-08
ZVS-08 65.7908
65.7908
65.7908 80.27
80.27
80.27 ±±± 0.63
0.63
0.63 (3)
(3)(3)
ZVS-09
ZVS-09
ZVS-09 65.1035
65.1035
65.1035 0.15±±±1.67
0.15
0.15 1.67(4)
1.67 (4)
(4)
ZVS-09
ZVS-09
ZVS-09
ZVS-09 65.1035
65.1035
65.1035
65.1035 0.15
0.15 ± ±±1.67
0.15
0.15 ± 1.67
1.67
1.67(4)(4)
(4)
(4)
ZVS-09
ZVS-09
ZVS-09 65.1035
65.1035
65.1035 0.15
0.15
0.15 ±±± 1.67
1.67
1.67 (4)
(4)
(4)
Compound ID: screening name of the compound; Pharmacophore fit score: scoring function calcu-
Compound
Compound ID:
ID: screening
screening name
name ofof
ofthe
the compound;
compound; Pharmacophore
Pharmacophore fit score:
fit score:scoring
scoring function
function calcu-
calcu-
Compound
Compound
Compound
Compound ID:ID:
ID:
ID: screening
screening
screening
screening name
namename
name of of
of the
the the
the compound;
compound;
compound;
compound; Pharmacophore
Pharmacophore
Pharmacophore
Pharmacophore fitfit
fit
fit score:
score:
score:
score: scoring
scoring
scoring
scoring function
function
function
function calcu-
calcu-
calcu-
calcu-
lated
lated
lated fromthe
from
from
Compound the
theID: alignment
alignment
alignment
screening ofof
ofthethepharmacophore
the
name pharmacophore
pharmacophore
of the compound; features
features
features andthe
and
and
Pharmacophore thefeature
the feature
feature
fit RMS
RMS
RMS
score: deviation.
deviation.
deviation.
scoring Inhibition
Inhibition
Inhibition
function calcu-
lated
lated
lated
Compoundfrom
from
Compound
lated fromthe
from the
ID: ID:
the
the alignment
alignment
screening
alignment
alignment
screening ofof
name the
of
of the
name
of the
thethepharmacophore
pharmacophore
of the compound;
pharmacophore
pharmacophore
compound; features
features
features
features
Pharmacophore andand
Pharmacophore
and
and the
fit the
the
the
score:feature
feature
feature
feature
scoring RMS
RMS
fit score:RMS
RMS deviation.
deviation.
scoring
deviation.
deviation.
function calculated Inhibition
Inhibition
function calcu-
Inhibition
Inhibition
from the
data
data
data
lated are
are
are means
means
means
from the ± ±
± standard
standard
standard
alignment of error
error
error
the for
for
for thethe
the number
number
number
pharmacophore ofof
of independent
independent
independent
features and the experiments
experiments
experiments
feature RMS indicated
indicated
indicated in
deviation. in
in paren-
paren-
paren-
Inhibition
data
data
lated
data
data are
are
alignmentfrom
are
are means
means
of the
means
means ±
±± standard
the±pharmacophore
standard
alignment
standard
standard oferror
errorthe
error for
for
features
error the
for
for the
and
the
the number
number
pharmacophore ofof
the feature
number
number of
of independent
independent
features
RMS and the
deviation.
independent
independent experiments
experiments
feature RMS
Inhibition
experiments
experiments dataindicated
indicated
are means
indicated
indicated inin
deviation. in
in paren-
paren-
±Inhibition
standard
paren-
paren-
theses.
theses.
theses.
dataforare means ± of
standard error for the number of independent experiments indicated in paren-
theses.
theses.
error
data arethe
theses.
theses.
number
means independent
± standard error experiments
for the number indicated in parentheses.
of independent experiments indicated in paren-
theses.
theses.
3.2. Patch-Clamp Screening of Virtual Screening Hit Compounds
The nine selected in silico hits were tested by manual patch-clamp on a HEK293
cell line stably expressing KV 10.1 [33] at a compound concentration of 50 µM (Table 1).
Compound activity was tested by determining the current amplitude elicited by repetitive
stimuli to +40 mV from a holding potential of −80 mV. The most potent inhibitor was hit
compound ZVS-08 and after its resynthesis an IC50 value of 3.70 ± 1.01 µM was determined
against KV 10.1. The hit compound ZVS-08 represents a new structural class of KV 10.1
inhibitors with diarylamine structure and has a unique structure among the acquired
virtual screening hits. The alignment of ZVS-08 in the pharmacophore model used in
the virtual screening shows that all pharmacophore features of the model are fulfilled
(Figure 8). The most notable change from the purpurealidin analogues is the replacement
of the hydrogen bond acceptor group of the amide bond with the oxygen atom of the nitro
group of ZVS-08.
compound ZVS-08 and after its resynthesis an IC50 value of 3.70 ± 1.01 μM was determined
against KV10.1. The hit compound ZVS-08 represents a new structural class of KV10.1 in-
hibitors with diarylamine structure and has a unique structure among the acquired virtual
screening hits. The alignment of ZVS-08 in the pharmacophore model used in the virtual
screening shows that all pharmacophore features of the model are fulfilled (Figure 8). The
Cancers 2021, 13, 1244 12 of 24
most notable change from the purpurealidin analogues is the replacement of the hydrogen
bond acceptor group of the amide bond with the oxygen atom of the nitro group of ZVS-
08.
Figure8.
Figure 8. Alignment
Alignmentof ofthe
theidentified
identifiedpotent
potentKK V10.1
V 10.1 hitcompound
hit compound ZVS-08
ZVS-08 with
with thethe final
final ligand-
ligand-based
based pharmacophore
pharmacophore model
model used forused forscreening
virtual virtual screening
(middle),(middle),
structuresstructures ofactive
of the most the most active
purpurealidin
purpurealidin
analogue (right)analogue (right)
and virtual and virtual
screening screeningZVS-08
hit compound hit compound
(left). ZVS-08 (left).
3.3.
3.3. Characterisation
Characterisation of
of the
the K
KVV10.1 Inhibition by ZVS-08
The
The inhibition
inhibition by by ZVS-08
ZVS-08 was was characterised
characterised in in more
more detail
detailby bypatch
patchclamp.
clamp. MostMost
hERG
hERG blockers
blockers bind
bind to aa hydrophobic
hydrophobic cavity
cavity in the inner mouth of the channel pore [20], [20],
and
and some
some well-characterised
well-characterisedcompounds
compoundsuse usethe
theequivalent
equivalentstructure
structurein inKKVV10.1
10.1 (e.g.,
(e.g., [32])
[32])
to
to interfere
interfere with
with ion
ion permeation.
permeation. The The electrophysiological
electrophysiological behaviour
behaviourof ofour
ourhit
hit compound
compound
ZVS-08
ZVS-08was wassimilar
similarto to
thatthat
of astemizole, a well-established
of astemizole, open-channel
a well-established blockerblocker
open-channel of KV 10.1 of
with an IC
KV10.1 with in the range of 200 nM [33]. The inhibition was concentration-dependent
50an IC50 in the range of 200 nM [33]. The inhibition was concentration-depend-
(Figure 9A,B),
ent (Figure withwith
9A,B), an apparent IC50 of
an apparent IC3.70 3.70(Table
50 of µM 2). The
µ M (Table 2).onset of inhibition
The onset was slow
of inhibition was
and required minutes of continued stimulation (Figure 9C), which
slow and required minutes of continued stimulation (Figure 9C), which would be com- would be compatible
with an with
patible intracellular bindingbinding
an intracellular site. Moreover, the inhibition
site. Moreover, was not was
the inhibition homogeneous during
not homogeneous
the stimulus.
during CurrentCurrent
the stimulus. amplitude decreased
amplitude along thealong
decreased stimulus in the presence
the stimulus of ZVS-08,
in the presence of
resulting in apparent inactivation and smaller amplitude at the end of the
ZVS-08, resulting in apparent inactivation and smaller amplitude at the end of the pulse pulse than in the
beginning
than in the(Figure 9A,D).
beginning The effect
(Figure 9A,D).was
Thenot cumulative,
effect and the nextand
was not cumulative, stimulus
the next(2.3 s later)
stimulus
showed a similar peak current and decay, indicating that the compound
(2.3 s later) showed a similar peak current and decay, indicating that the compound is not is not trapped in
the closed channel. The apparent inactivation was more pronounced
trapped in the closed channel. The apparent inactivation was more pronounced at more at more depolarised
potentials
depolarised (Figure 9D). Additionally,
potentials to the changestointhe
(Figure 9D). Additionally, inactivation
changes inof KV 10.1, ZVS-08
inactivation also
of KV10.1,
noticeably slowed down deactivation of the channel (Figure 9E), again
ZVS-08 also noticeably slowed down deactivation of the channel (Figure 9E), again indi- indicating that
the compound needs to unbind before the channels can deactivate. The inhibition was
cating that the compound needs to unbind before the channels can deactivate. The inhibi-
slightly voltage dependent (Figure 9C). In the presence of ZVS-08, the conductance–voltage
tion was slightly voltage dependent (Figure 9C). In the presence of ZVS-08, the conduct-
relationship was well described by a Boltzmann function with a blocking component
ance–voltage relationship was well described by a Boltzmann function with a blocking
(Figure 9F). The activation was shifted by approximately −25 mV (semi-maximal activation
component (Figure 9F). The activation was shifted by approximately −25 mV (semi-max-
at −29.88 ± 1.8 mV in the presence and 4.39 ± 1.9 mV in the absence of ZVS-08), compatible
imal activation at −29.88 ± 1.8 mV in the presence and 4.39 ± 1.9 mV in the absence of ZVS-
with the action of a gating modifier.
08), compatible with the action of a gating modifier.
To clarify the mechanism of inhibition by ZVS-08, we compared the compound to
two well characterised inhibitors: astemizole, which behaves as an open channel blocker
binding to the promiscuous intracellular hydrophobic pocket in the channel family, and
mibefradil, which acts from the extracellular side and modifies channel gating. To test
if ZVS-08 shares binding site with any of the two inhibitors, we performed competition
experiments by determining if the presence of astemizole or mibefradil altered the effect of
ZVS-08 on Kv10.1 current. The results are summarised in Figure 10. The degree of inhibition
at the concentrations tested was independent of the presence of 500 nM astemizole or 1 µM
mibefradil, indicating that the compounds do not compete for the same binding site.
3.4. Analogues of the Hit Compound ZVS-08 and Structure–Activity Relationships

Analogues of the hit compound ZVS-08 were designed and synthesised to increase
potency and selectivity and to investigate structure–activity relationships (SAR) important
for KV 10.1 inhibition. The hit compound ZVS-08 was modified at four different positions
(Figure 11) to provide a focused library of compounds screened for KV 10.1 (Table 3) and
selected compounds for hERG inhibition (Table 2).
Cancers 2021, 13, 1244 13 of 24
A B C
3×10-9
Steady-state current amplitude (A)

1.0
Relative current amplitude

2×10-9
0.5
1×10-9
0.0 0
-8 -7 -6 -5 -4 0 1 2 3
Time (min)
log (ZVS-08) (M)
D E F
1.0
Normalized conductace
Control
0.5
ZVS-08
0.0
-100 -50 0 50 100 150
Vm (mV)
Figure
Figure9.9.(A)(A)Inhibition
Inhibition by different concentrations
by different concentrationsofofZVS-08
ZVS-08hithit compound.
compound. (B)(B) Dose–response
Dose–response (data(data
fromfrom 3–10 inde-
3–10 independent
pendent determinations, mean ± SEM). (C,D) Current–voltage relationship in the absence (upper traces)
determinations, mean ± SEM). (C,D) Current–voltage relationship in the absence (upper traces) or presence of 5 µM ZVS-08 or presence of 5
µ M ZVS-08 (lower traces) obtained by depolarisation from a holding potential of −80 mV to potentials ranging between
(lower traces) obtained by depolarisation from a holding potential of −80 mV to potentials ranging between −60 and
−60 and +100 mV. (E) The traces corresponding to depolarisation to +40 mV are superimposed for easier comparison. The
+100 mV. (E) The traces corresponding to depolarisation to +40 mV are superimposed for easier comparison. The inset
inset shows the normalised tail currents to highlight the slower deactivation in the presence of ZVS-08. (F) Conductance–
showsrelationship
voltage the normalised tail currents
in control (blacktosymbols)
highlightand
theinslower deactivation
the presence in the presence
(red symbols) ofZVS-08,
of 5 µ M ZVS-08.obtained
(F) Conductance–voltage
by biexponen-
relationship
tial in current
fit to the tail control (at
(black
−80 symbols) and in the presence
mV) and extrapolation to time(red
0. Thesymbols) of 5represent
solid lines µM ZVS-08,the obtained
result of abyfitbiexponential
using a Boltz-fit
to the
mann distribution (at −a80
tail currentwith mV)atand
block extrapolation
positive voltages.to time 0. The solid lines represent the result of a fit using a Boltzmann
distribution with a block at positive voltages.
To clarify the mechanism of inhibition by ZVS-08, we compared the compound to
two well characterised inhibitors: astemizole, which behaves as an open channel blocker
Table 2. Potencies of compounds ZVS-08, 1 and 8 against KV 10.1 and hERG.
binding to the promiscuous intracellular hydrophobic pocket in the channel family, and
Compound ID mibefradil, which acts from the extracellular
IC50 Kv10.1 (µM) side and modifiesICchannel
50 hERG gating.
(µM) To test if
ZVS-08 ZVS-08 shares binding site with any of the two
3.70 µM (95% CI: 2.08 µM to 6.47 µM) inhibitors, we performed competition ex-
0.194 µM (95% CI: 0.047 µM to 0.761 µM)
1 periments by determining if the presence of
0.740 µM (95% CI: 490 nM to 1.17 µM) astemizole or mibefradil altered the effect
0.207 µM (95% CI: 0.113 µM to 0.395 µM)of
8 ZVS-08 on Kv10.1 1.01current.
µM (95%The results
CI: 796 nM toare summarised
1.30 µM in µM
0.156 Figure
(95%10.
CI:The degree
0.070 µM to of inhibi-
0.373 µM)
tion at the concentrations tested was independent of the presence of 500 nM astemizole or
1 µ M mibefradil, indicating that the compounds do not compete for the same binding site.
Cancers 2021, 13, 1244 14 of 24
Figure 10. Competition results of ZVS-08 (red circles) with astemizole (known pore blocker, black
square) on KV10.1 (A,B) and mibefradil (gating modifier, black triangle) (C,D). (B,D) represent the
same dataset as (A,C), but each series is plotted on a different axis to visualise that the dose–re-
sponse is unchanged in the presence of the competitor. The fits resulted in the same IC 50 for the
presence and absence of the competitor (1.69 µ M for A and B and 1.47 µ M for (C,D)). Current am-
plitudes were measured in all cases at the end of a 500 ms depolarising pulse to +40 mV.
square)
3.4. on KV10.1 the
Analogues (A,B) and mibefradilZVS-08(gating modifier, black triangle) (C,D). (B,D) represent the
square) on KV 10.1of(A,B) Hit
andCompound
mibefradil (gating and Structure–Activity
modifier, black triangle) Relationships
(C,D). (B,D) represent the
same dataset as (A,C), but each series is plotted on a different axis to visualise that the dose–re-
same Analogues
dataset
sponse is unchanged of but
as (A,C), the hitpresence
each
in the compound theZVS-08
series isofplotted wereThedesigned
on a different
competitor. and
axisresulted
fits to visualisesynthesised
in thethat
same to increase
theICdose–response
50 for the
ispotency
unchanged andin selectivity
the presence and
of to
the investigate
competitor. Thestructure–activity
fits resulted in the
presence and absence of the competitor (1.69 µ M for A and B and 1.47 µ M for (C,D)). relationships
same IC 50 for the(SAR)
Current am-im-
presence
portant
andplitudes
absenceforof K
were V10.1
the inhibition.
measured in all
competitor (1.69 The
cases
µM hitAend
at for
the compound
ofBaand
and msZVS-08
500 1.47 µM forwas
depolarising modified
pulse
(C,D)). to +40at
Current mV.four different
amplitudes were
positionsin(Figure
measured all cases11) to provide
at the end of a a 500focused library ofpulse
ms depolarising compounds
to +40 mV.screened for KV10.1 (Table
3)3.4.
andAnalogues
selectedofcompounds
the Hit Compound ZVS-08
for hERG and Structure–Activity
inhibition (Table 2). Relationships
Analogues of the hit compound ZVS-08 were designed and synthesised to increase
potency and selectivity and to investigate structure–activity relationships (SAR) im-
portant for KV10.1 inhibition. The hit compound ZVS-08 was modified at four different
positions (Figure 11) to provide a focused library of compounds screened for KV10.1 (Table
3) and selected compounds for hERG inhibition (Table 2).
Figure 11. Design of the hit compound ZVS-08 analogues.


Compound
Table 2.
2. Potencies
Compound ID
of
of compounds
PotenciesID compoundsIC
IC 50 Kv10.1
50
ZVS-08, 11 and(μM)
88 against
against K
KVVVV10.1
10.1 and
IC50
and hERG.50 hERG (μM)
50
Table
Table 2. Potencies of compounds 50 Kv10.1
ZVS-08,
50
ZVS-08, and
1 and(μM)
8 against K 10.1 and IC
hERG.
hERG.50 hERG (μM)
Compound
Table 2. Potencies
Compound ID
of
ID compounds
3.70 µ MIC 50 Kv10.1
ZVS-08,
(95%
IC 50 CI:1
Kv10.1and(μM)
2.08 8
(μM)µagainst
M to K
6.47V10.1 and
0.194 µ IC
hERG.
M
IC hERG
(95%
50
50 CI:(μM)
hERG 0.047 µ M to
(μM)
Compound ID
ZVS-08 ID IC50 Kv10.1 (μM)
3.70 µ MIC (95% CI: 2.08 IC50
µ M to 6.47 0.194 µ IC hERG (μM)
M 50(95% CI:(μM)
0.047 µ M to
Compound
Compound
ZVS-08 ID 3.70 µµ M IC 50 Kv10.1
(95% Kv10.1
CI: (μM)
(μM)
2.08 µµ M to 6.47 0.194 IC
µµIC
M hERG
hERG
(95% CI: (μM)
0.047
0.047 µµµMM to
50
Compound
ZVS-08 ID 3.70 M
3.70 µ MIC IC
(95%
50
(95%
50 Kv10.1
µ M)
CI:
µCI: (μM)
2.08
2.08
M)2.08 M to 6.47 0.194
µ M to 6.47 0.194 µIC M 50 hERG
0.761
(95%
50
M 50(95%
500.761 µ
CI:
CI:
µ M)(μM)
M)0.047 M to
to
Compound
ZVS-08 ID 3.70 µ M 50 Kv10.1
(95% CI: (μM)
µ M to 6.47 0.194 µ M hERG
(95% CI: (μM)
0.047 µ M to
ZVS-08 3.70
0.740
3.70 µ
µ µM
M M(95%
(95%
(95% CI:
µ
CI:
µ M)
CI:
M)2.08
490
2.08 µ
µnM
MM to
to
to 6.47
1.17
6.47 0.194
0.207
0.194 µµ MM (95%
0.761
(95%
0.761 CI:
µ
CI:
µ M)
M)0.047
0.113
0.047 µ
µ M
M to
to
ZVS-08
ZVS-08 0.740
3.70 µµM M(95%
(95%µ µCI:
CI:M)2.08
490 µnM M to 6.47
1.17 0.207
0.194 µ M (95% 0.761CI:
µ M)0.113
0.047 µ M to
11
ZVS-08
ZVS-08 0.740
0.740 µµ M
M (95%
(95% µ
µ
µ
M)
M)
CI:
M)
CI:
M) 490
490 nM
nM to
to 1.17
1.17 0.207
0.207 µµ M
M
0.761
0.761
(95%
0.395
0.761
(95%
µµ
CI:
µ
CI:
µ
M)
M)
M)
M)0.113
0.11315µµ M
M24to
µof to
Cancers 2021, 13, 1244 11 0.740 µ M (95%µCI: 490 nM to 1.17 0.207 µ M (95%
M)490 0.395 CI:
0.761CI:
µ M)0.113 M to
1 0.740
0.740
1.01 µ
µ
µ M
MM (95%
(95%
(95% µCI:
CI:
M)
CI: 490
796 nM
nM
nM to
to
to 1.17
1.17
1.30 0.207
0.207
0.156 µµ MM (95%
(95%
0.395 CI:
µ M)0.113
0.113
0.070 µµ MM to
to
118 0.740 µ M (95% µCI:
M) 490 nM to 1.17 0.207 µ M (95%
0.395CI:
µ M)0.113 µ M to
1.01 µµM
0.740 (95%µµ
M(95% M)796
CI:
CI: 490nM nMto to1.30
1.17 0.156
0.207 µ M (95% 0.395CI:
µ M)0.070
0.113 µ M to
811 1.01
1.01 µµµMM (95% µM)
(95% µµCI:M)
M796
M)
CI: 796 nM
nM toto 1.30
1.30 0.156
0.156 µµµM
0.395
0.395
M (95%
µµM)
0.373CI:
0.395
(95% M)
µ M)
CI:M)0.070
0.070 µµµMM to
to
88 1.01 M (95% CI:
µ
µ M 796 nM to 1.30 0.156
M)796 nM to 1.30 0.156 µ M (95% M (95%
0.373 CI:
µ 0.070
0.395CI: 0.070 M to
8 1.01
1.01 µµMM (95%
(95% CI:
CI:
µ M 796 nM to 1.30 0.156 µ M (95%
0.373 CI:
µ M)0.070 µµM M to
to
88 1.01 µ M (95% CI: µM
µ M796 nM to 1.30 0.156 µ M (95% 0.373CI:
0.373 µ M)
µ M)0.070 µ M to
Table 8
3. Structures andand 1.01 µofMthe
potencies (95% CI:
designed
µ M 796 nM
and to 1.30 0.156
synthesised ZVSZVS µM
-08 (95%
analogues
0.373 CI: 0.070 µ M to
1–12.
µµM)
Table
Table 3. Structures
8 potencies of the µ
µ M
designed
M and synthesised -08 0.373
analogues
0.373 µ M)
M) 1–12.
Table 3.3. Structures
Structures and
and potencies
potencies of of the
the designed
designed
µM and
and synthesised
synthesised ZVS
ZVS -08
-08 0.373 µ M)1–12.
analogues
analogues 1–12.
Table 3.
3. Structures
TableCompound
Structures and
and potencies
potencies of
of the designed
designed and
theStructure and synthesised
synthesised ZVS
ZVS -08 analogues
-08Inhibition
analoguesat 1–12.
1–12.
Table 3. Structures
Compound IDand
ID potencies of the designed
Structure and synthesised
%% ZVS -08
ofofKv10.1
Kv10.1 analogues
Inhibition 1–12.
10atµM
10 μM
μM
Table
Table 3. Structures
Compound
3. Structures and
StructuresID potencies
and potencies of
potencies of the
of the
the designed
Structure and
designed and synthesised ZVS
% ofZVS
and synthesised
synthesised -08
Kv10.1
ZVS analogues
Inhibition
-08 analogues
analogues 1–12.
at 10
1–12.
Table 3.
Compound and
ID designed
Structure % of -08
Kv10.1 Inhibition 1–12.
at 10 μM
TableCompound
3. StructuresID
Compound and potencies of theStructure
ID designed and synthesised
Structure % of
% ofZVS -08 analogues
Kv10.1
Kv10.1 Inhibition
Inhibition 1–12.
at 10
at 10 μM
μM
Compound
Compound 111 ID
ID Structure
Structure %
% of
of Kv10.1
Kv10.1
87.32 Inhibition
Inhibition
87.32
± ± 4.60
4.60 (7) (7) at
at 10
10 μM
μM
Compound 1 ID Structure % of Kv10.1 Inhibition
87.32 ± 4.60 (7)
87.32 at 10 μM
± 4.60 (7) at 10 μM
Compound 11 ID Structure % of Kv10.1 Inhibition
87.32
1 87.32 ±±
87.32 ± 4.60
4.60 (7)
4.60 (7)
(7)
11 87.32
87.32 ±
± 4.60
4.60 (7)
(7)
1 87.32 ± 4.60 (7)
1 87.32 ±±26.80
4.60 (7)
222 23.93
23.93 ± 26.80
23.93 ± 26.80 (4)
(4) (4)
22 23.93
23.93 ±± 26.80
26.80 (4)
(4)
2 23.93 ± 26.80 (4)
22 23.93
23.93 ± ± 26.80
26.80 (4)
(4)
2 23.93 ± 26.80 (4)
23 23.93
22.08 ± 26.80 (4)
33 22.08
22.08 ±± 33.52
± 33.52
33.52 (4)
(4) (4)
33 22.08
22.08 ±± 33.52
33.52 (4)
(4)
3 22.08 ± 33.52 (4)
33 22.08
22.08 ± ± 33.52
33.52 (4)
(4)
3 22.08 ± 33.52 (4)
34 22.08
36.63 ±± 33.52
± 19.89
19.89 (7) (4)
(7)
4
44 36.63
36.63 ± 19.89 (7)
4 36.63
36.63 ±± 19.89
19.89 (7)
(7)
4 36.63 ± 19.89 (7)
44 36.63
36.63 ± ± 19.89
19.89 (7)
(7)
4 36.63 ± 19.89 (7)
4 36.63
2.53 ±±±11.73
19.89(3) (7)
55 2.53 11.73 (3)
555 2.53
2.53 ±± 11.73
± 11.73
2.53 (3) (3)
11.73 (3)
5 2.53 ± 11.73 (3)
55 2.53
2.53 ±± 11.73
11.73 (3)
(3)
5 2.53 ± 11.73 (3)
5 2.53 ±±11.73
28.38(3)
66 57.48
57.48 ± 28.38 (3)
(3)
6666 57.48
57.48
57.48 ±±
± 28.38 28.38
28.38 (3)
(3)
(3) (3)
66 57.48 ± 28.38
6 57.48
57.48 ± ± 28.38
28.38 (3)
(3)
57.48 ± 28.38 (3)
6 57.48
20.96 ±± 28.38 (3)
77 20.96 ± 30.27
30.27 (7) (7)
77 20.96
20.96 ±± 30.27
30.27 (7)
(7)
7 20.96
20.96 ± 30.27
± 30.27 (7)
(7)(7)
777 20.96
20.96 ± ± 30.27
30.27 (7)
7 20.96 ± 30.27 (7)
7 20.96 ± 30.27 (7)
8 8 81.20 ±± 4.97
81.20 4.97 (3)(3)
88 81.20
81.20 ±± 4.97
4.97 (3)
(3)
8 81.20
± 4.97± 4.97 (3)
88 81.20
81.20
81.20 ±
± 4.97
(3)(3)
4.97 (3)
8 81.20 ± 4.97 (3)
8 81.20 ± 4.97 (3)
9 9 2.00 ±± 7.45
2.00 7.45 (4)
(4)
99 2.00
2.00 ±± 7.45
7.45 (4)
(4)
9 2.00 ± 7.45 (4)
999 2.00
2.00
2.00 ±± 7.45
± 7.45 (4)(4)
7.45 (4)
9 2.00 ± 7.45 (4)
10 9 2.00 ±± 7.45
57.05 21.71(4) (2)
10 57.05 ± 21.71 (2)
10
10 57.05
57.05 ±± 21.71
21.71 (2)
(2)
10 57.05 ± 21.71 (2)
10
10 57.05
57.05 ±± 21.71
21.71 (2)
(2)
10
10 57.05 ± 21.71
57.05 ± 21.71 (2) (2)
10
11 57.05
37.67±±±21.71
7.13 (2) (2)
(2)
11 37.67 7.13
11
11 37.67
37.67 ± ± 7.13
± 7.13
7.13 (2)(2)
(2)
11 37.67
11
11 37.67
37.67 ±±± 7.13
7.13 (2)(2)
11
11 37.67
37.67 ± 7.13 7.13 (2)
(2) (2)
11
12 37.67
16.60 ±38.36
7.13
12 16.60 ±± 38.36 (2)
(2)
12
12 16.60
16.60 ±± 38.36
38.36 (2)
(2)
12 16.60 ± 38.36 (2)
Inhibition 12
12
data are means ± standard error (or range) for the number of 16.60
16.60 ±± 38.36
independent 38.36 (2)
(2)
experiments
Inhibition 12
Inhibition data 16.60 ± 38.36 (2)
Inhibition 12 are
data
12
data
are means
are
means ±± standard
standard error
error (or
(or range)
range) for
for the
the number
number of
of independent
independent
16.60 ± 38.36
16.60 ± 38.36 (2)experiments
experiments
(2)
indicated
indicated
Inhibition
indicated
in
Inhibitionin
in are means
means ±±
parentheses.
data
parentheses.
data are means
parentheses. ± standard
standard error
standard error (or
error (or range)
(or range) for
range) for the
for the number
the number of
number of independent
of independent experiments
experiments
Inhibition
indicated
Inhibition
indicated data
indata
in are
are means
parentheses.
means
parentheses. ±± standard
standard error
error (or
(or range)
range) for
for the
the number
number of
of independent
experiments
Inhibition
indicated indata are means ± standard error (or range) for the number of independent experiments
parentheses.
Inhibition
indicated
Inhibition in
data data are means
parentheses.
are means
Modifications
indicated in ±
parentheses. ± standard
instandard
the error
error (or
aromatic (or for
range)
portion range) for thewere
oftheZVS-08
number
ZVS-08 number
of of independent
independent
performed theexperiments
experiments
on the indicated in
phenyl ring
ring
indicated in parentheses.
Modifications in the aromatic portion of were performed on phenyl
indicated
parentheses. in parentheses.
Modifications in the aromatic portion of ZVS-08 were performed
bearing ortho-nitro and para-trifluoromethyl substituents. One or both groups were sys- on the phenyl ring
Modifications
bearing
bearing ortho-nitro and
ortho-nitro
Modifications in
and
in the
the aromatic portion
portion of
para-trifluoromethyl
aromatic ofsubstituents.
ZVS-08 were
ZVS-08 wereOne
substituents. performed
One or both
or
performedbothon on the phenyl
groups
groups
the phenyl ring
were sys-
were sys-
ring
Modifications
bearing ortho-nitro
Modifications
tematically
bearing removed
ortho-nitro inand
in
and the
the
in aromatic
compounds portion
aromatic portion
3, 6
para-trifluoromethyl and of
of7 ZVS-08
to were
substituents.
ZVS-08 were
analyse the
One or
performedboth
influence
One or both on
ofon the
groups
the
both
groupsphenyl
were
phenyl ring
sys-
ring
substituents
were sys-
Modifications
tematically
bearing removed
ortho-nitro in inthe
and aromatic portion
compounds 3, 6 and
para-trifluoromethyl of7 substituents.
ZVS-08
to were
analyse the performed
influence
One or both ofon the substituents
both
groups phenyl
were ring
sys-
Modifications
Modifications
bearing ortho-nitro
tematically
bearing removed
ortho-nitroinKinthe
and
and the
in aromatic
aromatic
compounds portion
portion
3,
para-trifluoromethyl6 and of7substituents.
ZVS-08
of ZVS-08
to were
were
analyse the
performed
One or on
both
influence
One or both on
ofthe the
groups
both
groups phenyl
phenylwere ring
ring
sys-
substituents
were sys-
on
on the potency
tematically
bearing of
removed
ortho-nitro
theortho-nitro
potency
tematically removed KVVand
ofand V 10.1
V10.1
in inhibition.
in compounds
compounds The
inhibition. The
3, nitro
3, 66 and
and
nitro group
group
to was
77substituents.
to analyse
analyse
was replaced
the
One
replaced
the by
influence
orby a
both
influence aof methyl
of groups
methyl
both ester
were
ester group
both substituents
substituents
sys-
group
bearing
bearing ortho-nitro
tematically
on the removed
potency of K and
in para-trifluoromethyl
in compounds 3,
3, 666 and 77substituents.
substituents.
to One
to analyse theOne
or orby
both both
groups
influence aaof groups
were
both were sys-
system-
substituents
tematically
on the
tematically removed
potency of
removed K V10.1
V 10.1
in inhibition.
compounds
inhibition.
compounds The
The
3, nitro
and
nitro
and 7 group
group
to was
analyse
was
analyse replaced
the influence
replaced
the by
influence methyl
of both
methyl
of both ester
ester group
substituents
group
substituents
on the potency
tematically
atically of
removed K V10.1
in inhibition.
compounds The
3, 6 nitro
and 7 group
to was
analyse replaced
the by
influence a methyl
of both ester group
substituents
on
on theremoved
on the
the potencyin
potency
potency ofcompounds
of
of
KKVVV10.1
K 10.1
3, 6 and
10.1 inhibition.
inhibition.
inhibition.
The
The
The
7 to analyse
nitro
nitro
nitro
group
group
group
the influence
was
was
was
replaced
replaced
replaced
ofbyboth
by
by
substituents
aa methyl
methyl
a methyl
ester on
ester group
ester group
group
theon the potency
potency of KVVinhibition.
of KV 10.1 10.1 inhibition. The nitro
The nitro groupgroup was replaced
was replaced by a methyl
by a methyl ester ester
groupgroup
in
compound 9, an amino group in compound 10, and a carboxylic acid group in compound
11, with the aim of removing the nitro group, which is sometimes associated with genotox-
icity [37]. The basic dimethylamino group was replaced by four different amines (R3 in
orange, Figure 11) to evaluate the importance of the cationic centre. Specifically, the basicity
of the amine was reduced by introducing the aniline moiety (5) or removed by forming the
acetamide (12). In addition, the aniline moiety in 5 was also introduced to assess whether
the compound could be further extended in this part of the molecule and whether the
Cancers 2021, 13, 1244 16 of 24
formation of additional cation–π or π–π interactions was possible. The morpholino moiety
in 2 and 8 was incorporated to maintain the basic character of the parent compound ZVS-08,
but to further increase the polarity of the compound. The aliphatic chain between the
basic centre and the aromatic ring of ZVS-08 (R4 in green, Figure 11) was also modified.
The hydroxy group was removed to generate compounds 1 and 4, which differ by the
substitution of the aromatic ring.
Based on the study of structure–activity relationships of ZVS-08 analogues, we identi-
fied structural parts important for KV 10.1 inhibition of this new structural type of KV 10.1
inhibitors (Table 3). It is concluded that the two most potent KV 10.1 inhibitors, 1 and
8, contain a combination of disubstituted phenyl ring with both ortho-nitro and para-
trifluoromethyl groups and basic moieties such as dimethylamino group in compound
Cancers 2021, 13, x FOR PEER REVIEW and morpholino group in compound 8. Both optimised compounds 1 and 8 exhibited
1 17 of 25
improved potencies compared to the hit compound ZVS-08 with the IC50 values of 740 nM
and 1.01 µM, respectively (Figure 12A, Table 2).
Figure 12. Cont.

Cancers 2021, 13, 1244 17 of 24
Figure 12. (A) Dose–response curves of compounds 1 and 8 for KV 1.10 inhibition. The red trace has
been included as reference and corresponds to ZVS-08 (from Figure 9B). The IC50 values from the fit
Figure 12. (A) Dose–response curves of compounds 1 and 8 for KV1.10 inhibition. The red trace has
were 740 nM (95% CI 490 nM to 1.17 µM, N = 7–9) for compound 1 and 1.01 µM (95% CI: 796 nM to
been included as reference and corresponds to ZVS-08 (from Figure 9B). The IC50 values from the
1.30740
fit were µM,nMN(95%
= 4–7)
CIfor
490compound (B) Representative
nM to 1.178.µM, Kv10.1 current
N = 7–9) for compound 1 and traces
1.01 µMobtained in 796
(95% CI: the presence
of different concentrations of Compound 1. Scale bars: 1 nA, 50 ms. The cell was
nM to 1.30 µM, N = 4–7) for compound 8. (B) Representative Kv10.1 current traces obtained in thedepolarized to
+40 mV for 500 ms. No leak subtraction was performed. (C) Representative traces obtained in the
presence of different concentrations of Compound 8. Scale bars: 1 nA, 50 ms. (D) Dose–response
curves of compounds ZVS-08, 1 and 8 for hERG inhibition. The IC50 values from the fit were
0.194 µM (95% CI 0.047 µM to 0.761 µM, N = 2–7) for ZVS-08, 0.207 µM (95% CI 0.113 µM to 0.395 µM,
N = 3–8) for compound 1 and 0.156 µM (95% CI: 0.070 µM to 0.373 µM, N = 3–8) for compound
8. (E) Representative traces of hERG currents obtained in the presence of different concentrations
of Compound 1. Scale bars: 150 pA, 1 s. Only the end of the response at +20 mV and the tail
current at −40 mV are represented. (F) Representative traces obtained in the presence of different
concentrations of Compound 8. Scale bars: 150 pA, 1 s.
Disubstituted ortho-nitro and para-trifluoromethyl compounds with a weaker basic

centre (5) or without it (12) were less potent. A similar effect of replacing the basic centre
was observed with purpurealidin analogues [22]. The disubstituted compound in which
the nitro group was replaced by the amino group in compound 10 was less potent. The
unsubstituted compound 3 was almost inactive, indicating the importance of the disubsti-
tuted ortho-nitro and para-trifluoromethyl phenyl moiety in this structural type of KV 10.1
inhibitors. Compounds 9 and 11, in which the nitro group was exchanged with the methyl
ester and carboxylic acid groups, were also inactive. The analogue of ZVS-08 without the
nitro group and retaining the para-trifluoromethyl group (6) was also less potent. All three
ortho-nitro compounds (2, 4 and 7) without the para-trifluoromethyl group and with basic
groups, showed almost the same inhibitory activity on KV 10.1. Compounds 1 and 4 lack
the hydroxyl group in the aliphatic part of the molecule, but this modification was not
essential for the inhibitory potency against KV 10.1. In general, the aliphatic amine plays an
important role in binding.
Compounds ZVS-08, 1 and 8 were tested for selectivity on hERG with the patch-clamp
assay (Figure 12B, Table 2). The selectivity of ZVS-08, 1 and 8 against the hERG channel
remains an issue similar to the purpurealidin analogues [38]. However, with structural
optimization, we increased the potency for KV 10.1, while the potency for hERG remains
the same for all three compounds ZVS-08, 1 and 8 (Table 2).
in the presence of different concentrations of Compound 8. Scale bars: 1 nA, 50 ms. (D) Dose–re-
sponse curves of compounds ZVS-08, 1 and 8 for hERG inhibition. The IC50 values from the fit
were 0.194 µ M (95% CI 0.047 µ M to 0.761 µ M, N = 2–7) for ZVS-08, 0.207 µ M (95% CI 0.113 µ M to
0.395 µ M, N = 3–8) for compound 1 and 0.156 µ M (95% CI: 0.070 µ M to 0.373 µ M, N = 3–8) for
compound 8. (E) Representative traces of hERG currents obtained in the presence of different con-
Cancers 2021, 13, 1244 centrations of Compound 1. Scale bars: 150 pA, 1 s. Only the end of the response at +20 mV and18 of 24
the tail current at −40 mV are represented. (F) Representative traces obtained in the presence of
different concentrations of Compound 8. Scale bars: 150 pA, 1 s.
3.5. Selectivity of Compounds ZVS-08 and 1 against Other Voltage-Gated Ion Channels
3.5. Selectivity of Compounds ZVS-08 and 1 against Other Voltage-Gated Ion Channels
Selected potent K 10.1 inhibitors ZVS-08 and 1 were further evaluated for their se-
Selected potent KVV10.1 inhibitors ZVS-08 and 1 were further evaluated for their se-
lectivity against other potential targets in a panel of voltage-gated ion channels using the
lectivity against other potential targets in a panel of voltage-gated ion channels using the
Xenopus laevis heterologous expression system, which allows efficient horizontal pharma-
Xenopus laevis heterologous expression system, which allows efficient horizontal pharma-
cological comparison. Several different voltage-gated ion channels relevant in cardiac
cological comparison. Several different voltage-gated ion channels relevant in cardiac
physiology, the immune system, and skeletal muscle were screened. Both KV 10.1 inhibitors
physiology, the immune system, and skeletal muscle were screened. Both KV10.1 inhibi-
ZVS-08 and 1 showed no significant inhibition and were thus considered selective for the
tors ZVS-08 and 1 showed no significant inhibition and were thus considered selective for
KV 1.1, KV 1.3, KV 1.4, KV 2.1, KV 4.2, NaV 1.4, and NaV 1.5 ion channels at a concentration
the KV1.1, KV1.3, KV1.4, KV2.1, KV4.2, NaV1.4, and NaV1.5 ion channels at a concentration
of 10 µM (Figure 13, Table S1). However, the selectivity should be further investigated to
of 10 µ M (Figure 13, Table S1). However, the selectivity should be further investigated to
check the activity of these compounds on a broader range of ion channels.
check the activity of these compounds on a broader range of ion channels.
Figure
Figure 13.
13. Selectivity
Selectivityscreening
screeningof of
ZVS-08 andand
ZVS-08 1 against voltage-gated
1 against voltage-gatedpotassium and sodium
potassium and sodium
channels
channels at
at aa concentration
concentration ofof 10
10 µµM.
M. Data
Data were
were obtained
obtained with
with the
the two-electrode
two-electrode voltage
voltage clamp
clamp on
on
Xenopus
Xenopus laevis
laevis oocytes.
oocytes. Data
Data are
are presented
presented as
as means
means ±±SEM
SEMofofnn≥≥3 3independent
independentexperiments.
experiments.
3.6. Effects
3.6. Effects on
on the
the Growth
Growth ofof Cell
Cell Lines
Lines in 2D Cell Culture
KKVV10.1
10.1 inhibitors with
withaafavourable
favourablepharmacological
pharmacological profile are are
profile promising candidates
promising candi-
for cancer therapy. To investigate the activity of the new compounds on
dates for cancer therapy. To investigate the activity of the new compounds on cancer cellscancer cells pro-
liferation, compounds ZVS-08, 1 and 8 were tested on MCF-7,
proliferation, compounds ZVS-08, 1 and 8 were tested on MCF-7, a breast cancer a breast cancer cell line
showing high
showing high KVV10.1
10.1 expression
expression and and low
low hERG
hERG expression,
expression, and
and onon Panc1,
Panc1, a pancreatic
cancer cell
cancer cell line
line with
with undetectable
undetectable K KVV10.1
10.1expression
expressionandandhigh
high level
level of
of hERG
hERG expression.
expression.
hTERT-RPE1 cells
hTERT-RPE1 cells served
served as
as aa non-cancer
non-cancer cell
cell line.
line. hTERT-RPE1
hTERT-RPE1 cells
cells show
show transient
transient physi-
phys-
ological expression of Kv10.1 that begins during the G2 phase of the cell cycle
iological expression of Kv10.1 that begins during the G2 phase of the cell cycle and extends and extends
through mitosis
through mitosis as
as present
present inin normal
normal cells. Different concentrations
cells. Different concentrations of of the
the compounds
compounds
were added to the culture medium and the growth of the cell
were added to the culture medium and the growth of the cell was monitored overwas monitored over 7272 h
h
(Figure 14).
(Figure 14).
ZVS-08 and 1 inhibited the growth of MCF-7 cells to a very similar extent and also
with similar potency, with IC50 in the range of 5 µM. Compound 8 was inactive on this cell
line, and all three compounds were significantly less efficient on Panc1; only the highest
concentrations used showed effects. hTERT-RPE1 cells, a non-transformed cell line, showed
a similar sensitivity to ZVS-08 and compound 1 as Panc1. In summary, ZVS-08 and 1 were
able to reduce the growth of cancer cells expressing KV 10.1 but were much less active on
cells expressing hERG or on normal cells, suggesting that their effect on KV 10.1 serves
more to inhibit growth in these cells. The lack of activity of compound 8 in this assay could
be caused by a higher affinity for serum proteins, depleting the compound in the medium,
as has been repeatedly observed with many compounds [39].
concentrations used showed effects. hTERT-RPE1 cells, a non-transformed cell line,
showed a similar sensitivity to ZVS-08 and compound 1 as Panc1. In summary, ZVS-08
and 1 were able to reduce the growth of cancer cells expressing KV10.1 but were much less
active on cells expressing hERG or on normal cells, suggesting that their effect on K V10.1
Cancers 2021, 13, 1244 serves more to inhibit growth in these cells. The lack of activity of compound 8 19 inofthis
24
assay could be caused by a higher affinity for serum proteins, depleting the compound in
the medium, as has been repeatedly observed with many compounds [39].
Figure 14.
Figure 14. Effects of ZVS-08 and compounds
compounds 11 and
and88on
onthe
thegrowth
growthofofMCF-7
MCF-7(A) (A)and
andPanc1
Panc1cells
cells
(B). ZVS-08
(B). ZVS-08 and compound
compound 11 inhibited
inhibitedthe
thegrowth
growthofofMCF-7
MCF-7cells
cellswith
withICIC
50 of of
50 6.8 (95%
6.8 CI:
(95% 5.14–
CI: 5.14–
9.38 µM)
9.38 µ M) and
and 5.48
5.48 µ
µMM (95%
(95% CI:
CI: 4.51–6.76
4.51–6.76 µµM).
M). Compound
Compound88was wasineffective
ineffectiveatatthe
theconcentrations
concentrations
tested. None of the compounds inhibited the growth of Panc1 cells significantly at concentration
tested. None of the compounds inhibited the growth of Panc1 cells significantly at concentration
below 10 µ M, and an IC50 could only be determined for compound 1 (over 30 µ M).
below 10 µM, and an IC50 could only be determined for compound 1 (over 30 µM).
3.7. 3D
3.7. 3D Cell
Cell Based
Based Assays
Assays
Totest
To testthe
theability
abilityofofthe
thecompounds
compounds toto inhibit
inhibit tumour
tumour progression
progression in ainmore
a more predic-
predictive
tive system,
system, we performed
we performed experiments
experiments on tumour
on tumour spheroids
spheroids of theofpancreatic
the pancreatic
cancercancer cell
cell line
line Colo357,
Colo357, which which
grows grows efficiently
efficiently in culture
in this this culture modality.
modality. The The
cellscells
werewere
seededseeded in
in the
the presence
presence of Matrigel
of Matrigel (see methods),
(see methods), allowed allowed
to formtospheroids,
form spheroids,
and the and the compounds
compounds (50 µM)
were
(50 µadded
M) were to the growth
added to themedium.
growth The controlThe
medium. contained
control the same amount
contained the same of the vehicle
amount of
(0.5% DMSO).
the vehicle Apoptosis
(0.5% DMSO).was monitored
Apoptosis wasby addition of
monitored byaaddition
fluorescent
of acaspase 2/7 activity
fluorescent caspase
reporter at the
2/7 activity same time
reporter as the
at the samecompounds.
time as the Cleavage of the reporter
compounds. Cleavagebyofcaspase results by
the reporter in
green
caspase florescence,
results in and
greentheflorescence,
intensity of and
the fluorescence
the intensitywas recorded
of the over 72was
fluorescence h. recorded
overAs depicted in Figure 15, ZVS-08 induced apoptosis efficiently starting very early,
72 h.
whileAs compound
depicted1in required
Figure longer time, induced
15, ZVS-08 but reached similar efficiently
apoptosis efficacy. Compound
starting very8 showed
early,
apoptosis statistically significant over the control starting only
while compound 1 required longer time, but reached similar efficacy. Compound 8 after 24 h and did not reach
the sameapoptosis
showed intensity statistically
as the othersignificant
compounds overinthe
thecontrol
time of the experiment.
starting only after 24 This
h andlower
did
efficacy matches the small effects detected in conventional 2D culture
not reach the same intensity as the other compounds in the time of the experiment. This with this compound.
lower efficacy matches the small effects detected in conventional 2D culture with this com-
3.8. Mutagenic Activity of ZVS-08 and 8
pound.
The association of nitro groups with mutagenicity is well known in medicinal chem-
istry. For this reason, this is not the usual functional group among approved drugs, but
recently, there have been advances in drug design incorporating the nitro group in antipara-
sitic, antitubercular, and anticancer compounds. There is a need to understand bioactivation
potential of compounds bearing aromatic nitro groups [37]. The potential mutagenic activ-
ity of compounds ZVS-08 and 8 was determined using the bacterial Salmonella/microsomal
reverse mutation assay with TA98 and TA100 Salmonella typhimurium strains in the absence
and presence of metabolic activation [40]. Compounds ZVS-08 and 8 were cytotoxic to both
bacterial strains (−/+ S9 metabolic activation) at concentrations ≥0.5 mg/plate. At non-
cytotoxic concentrations (0.0158–0.158 mg/plate), no mutagenic response was observed in
the absence (−S9) or presence (+S9) of metabolic activation (Tables S2 and S3).
Cancers 2021, 13, 1244 20 of 24
Total Green Object Integrated Intensity

8×107
(GCUxum2/Image) 6×107
4×107
ZVS-08
Compound 8
2×107 Compound 1
Control
0
0 24 48
time (h)
B Control ZVS-08 Compound 1 Compound 8
Figure
Figure 15.15.
(A)(A) Apoptosis
Apoptosis induction
induction byby ZVS-08
ZVS-08 andand compounds
compounds 1 and
1 and 8 (50
8 (50 µM)µon
M)tumour
on tumour sphe-
spheroids
roids formed by Colo 357 cells. ZVS-08 was markedly faster and compound 8 was slowest.
formed by Colo 357 cells. ZVS-08 was markedly faster and compound 8 was slowest. Data represent Data
represent mean ± standard error (N = 3). (B) Representative images of spheroids 24 h after the ad-
mean ± standard error (N = 3). (B) Representative images of spheroids 24 h after the addition of the
dition of the indicated compounds (50 µ M). Fluorescence intensity in the upper image corre-
indicated compounds (50 µM). Fluorescence intensity in the upper image corresponds to cleavage of
sponds to cleavage of the caspase 3/7 activity reporter; it has been merged with the phase contrast
theimage
caspase 3/7lower
in the activity reporter;
raw. it has
Scale bar: 400been
µ m.merged with the phase contrast image in the lower raw.
Scale bar: 400 µm.
3.8. Mutagenic Activity of ZVS-08 and 8
4. Discussion
The association
Inhibition of nitro
of KV 10.1 groups
potassium with mutagenicity
channels is well known
with small molecules in medicinal
or antibodies chem-
reduces
istry. For this reason, this is not the usual functional group among approved drugs,
cancer cell growth in vitro and in vivo. In a retrospective study in brain metastases, treat- but
recently,
ment with there
agentshave
thatbeen advances
can inhibit KVin drug
10.1 design incorporating
improved the outcome of thepatients
nitro group
who inhadan-
tiparasitic, antitubercular, and anticancer compounds. There is a need to understand
moderate expression of the channel [27]. This, together with the very high frequency of bio-
activation
abnormal potentialofofthe
expression compounds
channel inbearing
human aromatic
tumours, nitro groups
implies that K[37]. The potential mu-
V 10.1 inhibitors with
appropriate potency and selectivity may be useful in the treatment of many bacterial
tagenic activity of compounds ZVS-08 and 8 was determined using the Salmo-
tumour types.
nella/microsomal reverse mutation assay with TA98 and TA100 Salmonella
With this goal in mind, we set out to develop a new generation of K 10.1 inhibitors. typhimurium
V
Cancers 2021, 13, 1244 21 of 24
The discovery of KV 10.1-selective potassium channel inhibitors is challenging due to

the high similarity with the hERG, especially the ion-conducting pore. Most of the known
KV 10.1 inhibitors (Figure 1) also show hERG inhibition, which may lead to undesirable
QT interval prolongations in patients [28]. Moreover, the exact location of the binding
site for most of these inhibitors has not yet been experimentally determined. Structural
information about the KV 10.1 inhibitor complex is also not available, which limits the
structure-based design of new selective KV 10.1 inhibitors. Considering these limitations,
we selected purpurealidine-based KV 10.1 inhibitors that have been shown to bind to the
VSD of the channel [8]. Given the greater structural dissimilarity between KV 10.1 and
hERG in the VSD than in the pore [20], our goal was to discover a new structural class
of selective KV 10.1 inhibitors by computer-assisted ligand-based virtual screening. The
latter can overcome the limitation that the exact location of the binding site is unknown,
as it only uses information about the ligand. However, in such a case, active and inactive
ligands from the same structural class should be used in the modelling because they are
expected to bind at the same binding site and in the same orientation.
The novel structural class of diarylamine-based KV 10.1 inhibitors presented here
was identified by ligand-based virtual screening using a pharmacophore model. Phar-
macophore modelling is a powerful method that aligns active ligands based on their
pharmacophore features and then creates a ligand-based pharmacophore model consisting
of pharmacophore features common to all active ligands. Our initial model performed
well in the validation experiment as it was able to find a majority of the active ligands and
no inactive ligands from the compound library. However, it was too complex and did not
return any hits when used in the virtual screening of a library of 556,000 compounds. After
a series of simplifications, we arrived at a model with only six features (compared to 14
features in the original model) that performed excellently, finding all active compounds
and no inactive ones (Figure 7D). By using this model in the virtual screen, we found 18
potential hit compounds, nine of which were purchased and tested for KV 10.1 inhibition.
Compound ZVS-08 proved to be a hit with an IC50 value of 3.70 µM. The hit compound
ZVS-08 represents a new structural class of KV 10.1 inhibitors with diarylamine structure.
With the structure–activity relationship studies we identified structural parts impor-
tant for KV 10.1 inhibition of this new structural type of KV 10.1 inhibitors. The most potent
inhibitors from this series contain a combination of disubstituted phenyl ring with both
ortho-nitro and para-trifluoromethyl groups and basic moieties such as dimethylamino or
morpholino group. Among all virtual hits, the compound ZVS-08 was the only one potent
against the KV 10.1. Amine groups and aromatic rings are also present in all ZVS virtual
hits, but it might be possible that the substitution of the aromatic ring plays an important
role in compound binding to the active site. Hit compound ZVS-08 has the ring substituted
with two electron withdrawing groups that are not present in any other virtual hit. Based
on results, these two groups are important for KV 10.1 inhibition, as evident also in the
optimisation of ZVS-08. Structural optimization resulted in a nanomolar KV 10.1 inhibitor 1
(IC50 value of 740 nM). With structural optimization, we improved only the potency for
KV 10.1 but not for hERG, which remains the same as for the hit compound ZVS-08. New
solutions are needed for father optimization of the selectivity of KV 10.1 inhibitors against
hERG channel.
The kinetics of inhibiting by ZVS-08 and its analogues resemble those of known
com-pounds. The slow reduction in current amplitude along the stimulus is similar to
inhibition by astemizole and is interpreted as inhibition of open channels. Nevertheless, this
profile does not rule out an allosteric effect on gating, as mibefradil induces a kinetically
similar effect. ZVS-08 induces a shift in the voltage dependence of KV 10.1 currents in
the hyperpolarizing direction reminiscent of that observed with mibefradil and with
purpurealidine analogues, supporting the notion of an effect as a gating modifier rather
than a blocker of open channels.
The exact binding site for the new compounds is still unknown. It is assumed that the
parent structures bind to the VSD at a different site than mibefradil [8]. This also seems to be
Cancers 2021, 13, 1244 22 of 24
the case for the hit compound, as we observed no evidence of competition between ZVS-08
and mibefradil. We also tested competition with astemizol, due to the promiscuity of its
binding site, but again we did not observe any competition. Therefore, we hypothesize that
the new compounds bind to the VSD, probably from the inside. Mutagenesis experiments
should help to identify the binding site [6]. The similarity between the behaviour of the
ZVS-08 hit compound and astemizole would suggest that our hit shares the mechanism
of inhibition and thus possibly the binding site in the channel pore and does not bind
to the VSD like the purpurealidins from which our pharmacophore model was derived.
We performed competition studies to test for a shared binding site. We determined the
effect of different concentrations of ZVS-08 in the presence or absence of 500 nM astemizole
(Figure 10). We observed no difference in affinity between the two conditions and conclude
that ZVS-08 and astemizole do not share the same binding site.
The main interest of selective KV 10.1 inhibitors would be their use as anticancer agents.
ZVS-08 and compound 1 inhibited the growth of non-tumour and tumour cells without
KV 10.1 expression in conventional 2D culture at concentrations higher than 10 µM. At
lower concentrations, however, ZVS-08 and compound 1 derivatives were able to inhibit
efficiently the growth of MCF-7 breast cancer cells, while leaving non-transformed cells
unaffected. MCF-7 cells show significant expression levels of KV 10.1, but undetectable
expression of hERG, indicating that the effect might be related to inhibition of KV 10.1.
Importantly, the compounds did not affect the growth of Panc1 cells, which abundantly
express hERG, but not KV 10.1. There was therefore a correlation between KV 10.1 expression
and sensitivity to the compounds. In tumour spheroids, all compounds were able to induce
significant levels of apoptosis. In the case of the hit compound ZVS-08, the effect was
remarkably fast and intense. Compound 8, which was ineffective against tumour cells in
monolayer cultures showed a slower onset in the induction of apoptosis but was able to
induce cell death in longer times, which might indicate a more difficult access to the cells
in the spheroid for this compound. These results do not exclude alternative mechanisms of
action for the compounds, and further work will be required to determine to what extent
Kv10.1 block is responsible for the effects observed.
5. Conclusions
To discover a novel structural class of KV 10.1 inhibitors overexpressed in many dif-
ferent tumour types, we used a computational ligand-based approach. We identified the
novel low micromolar KV 10.1 inhibitor ZVS-08, which is based on a diarylamine scaffold.
The inhibition profile of ZVS-08 with the combination of data from the competition exper-
iments suggests that the binding site is not shared with two classical KV 10.1 inhibitors.
By synthesising the analogues, we were able to increase the potency of KV 10.1 inhibition.
Analogue compound 1 inhibited the KV 10.1 channel with an IC50 value of 740 nM and
showed a slight increase in selectivity on the hERG channel. Cancer cell proliferation was
inhibited at a concentration of 5.48 µM in KV 10.1 expressing cell line, while the cancerous
and non-cancerous cell lines without KV 10.1 expression were not inhibited. With the broad
window between mutagenic activity and inhibition of KV 10.1, the new compounds show
promising potential for further development for cancer therapy.
Supplementary Materials: The following are available online at https://www.mdpi.com/2072-6

694/13/6/1244/s1. Table S1: Selectivity screening of ZVS-08 and 1 against KV and NaV channels.
Table S2: Mutagenic activity of compound ZVS-08. Table S3: Mutagenic activity of compound 8,
chemistry: analytical procedures description and NMR spectrums.
Author Contributions: Conceptualization, Ž.T., B.Ž., J.T., T.T., L.A.P., and L.P.M.; methodology, J.T.,
T.T., L.A.P., and L.P.M.; software, Ž.T. and T.T.; formal analysis, Ž.T., L.A.H., Š.G., Š.M., A.Š., M.N.,
S.P., and L.A.P.; investigation, Ž.T., L.A.H., A.Š., M.N., X.S., T.T., and L.A.P.; data curation, Ž.T.,
L.A.H., A.Š., M.N., X.S., S.P., T.T., and L.A.P.; writing—original draft preparation, Ž.T., B.Ž., J.T., T.T.,
L.A.P., and L.P.M.; writing—review and editing, J.T., T.T., L.A.P., and L.P.M.; supervision, B.Ž., J.T.,
T.T., L.A.P., and L.P.M.; project administration, J.T., T.T., L.A.P., and L.P.M.; funding acquisition, B.Ž.,
J.T., L.A.P., and L.P.M. All authors have read and agreed to the published version of the manuscript.
Cancers 2021, 13, 1244 23 of 24
Funding: This research was funded by Slovenian Research Agency (ARRS) grant numbers J1-9192,
N1-0098, P1-0245, and CELSA project. The work has also received funding from the Max-Planck
Society and from the European Union through Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreement No. 813834-PHIONIC-H2020-MSCA-ITN-2018.
J.T. was supported by grants G0E7120N, GOC2319N and GOA4919N (FWO-Vlaanderen), and grant
CELSA/17/047 (KU Leuven). S.P. was supported by grant PDM/19/164 (KU Leuven).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Acknowledgments: We thank OpenEye Scientific Software, Santa Fe, NM, USA, for free academic
licenses for the use of their software. L.A.P. thanks the expert technical assistance of V. Díaz.
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
References
1. Lin, T.-F.; Jow, G.-M.; Fang, H.-Y.; Fu, S.-J.; Wu, H.-H.; Chiu, M.-M.; Jeng, C.-J. The Eag Domain Regulates the Voltage-Dependent
Inactivation of Rat Eag1 K+ Channels. PLoS ONE 2014, 9, e110423. [CrossRef] [PubMed]
2. Whicher, J.R.; MacKinnon, R. Structure of the Voltage-Gated K+ Channel Eag1 Reveals an Alternative Voltage Sensing Mechanism.
Science 2016, 353, 664–669. [CrossRef] [PubMed]
3. Ma, L.; Chin, Y.K.Y.; Dekan, Z.; Herzig, V.; Yuen Chow, C.; Heighway, J.; Wing Lam, S.; Guillemin, G.J.; Alewood, P.F.; King, G.F.
Novel Venom-Derived Inhibitors of the Human EAG Channel, a Putative Antiepileptic Drug Target. Biochem. Pharmacol. 2018.
[CrossRef]
4. Pardo, L.A.; Gomez-Varela, D.; Major, F.; Sansuk, K.; Leurs, R.; Downie, B.R.; Tietze, L.F.; Stuhmer, W. Approaches Targeting
KV10.1 Open a Novel Window for Cancer Diagnosis and Therapy. Curr. Med. Chem. 2012, 19, 675–682. [CrossRef]
5. Pardo, L.A.; Stühmer, W. The Roles of K+ Channels in Cancer. Nat. Rev. Cancer 2014, 14, 39–48. [CrossRef] [PubMed]
6. Movsisyan, N.; Pardo, L.A. Kv10.1 Regulates Microtubule Dynamics during Mitosis. Cancers 2020, 12, 2409. [CrossRef]
7. García-Quiroz, J.; García-Becerra, R.; Santos-Martínez, N.; Barrera, D.; Ordaz-Rosado, D.; Avila, E.; Halhali, A.; Villanueva, O.;
Ibarra-Sánchez, M.J.; Esparza-López, J.; et al. In Vivo Dual Targeting of the Oncogenic Ether-à-Go-Go-1 Potassium Channel by
Calcitriol and Astemizole Results in Enhanced Antineoplastic Effects in Breast Tumors. BMC Cancer 2014, 14, 745. [CrossRef]
8. Bai, Y.; Liao, H.; Liu, T.; Zeng, X.; Xiao, F.; Luo, L.; Guo, H.; Guo, L. MiR-296-3p Regulates Cell Growth and Multi-Drug Resistance
of Human Glioblastoma by Targeting Ether-à-Go-Go (EAG1). Eur. J. Cancer 2013, 49, 710–724. [CrossRef]
9. Wu, J.; Wu, X.; Zhong, D.; Zhai, W.; Ding, Z.; Zhou, Y. Short Hairpin Rna (ShRNA) Ether à Go-Go 1 (Eag1) Inhibition of Human
Osteosarcoma Angiogenesis via VEGF/PI3K/AKT Signaling. Int. J. Mol. Sci. 2012, 13, 12573–12583. [CrossRef]
10. Wang, X.; Chen, Y.; Li, J.; Guo, S.; Lin, X.; Zhang, H.; Zhan, Y.; An, H. Tetrandrine, a Novel Inhibitor of Ether-à-Go-Go-1 (Eag1),
Targeted to Cervical Cancer Development. J. Cell. Physiol. 2018. [CrossRef] [PubMed]
11. Gómez-Varela, D.; Zwick-Wallasch, E.; Knötgen, H.; Sánchez, A.; Hettmann, T.; Ossipov, D.; Weseloh, R.; Contreras-Jurado, C.;
Rothe, M.; Stühmer, W.; et al. Monoclonal Antibody Blockade of the Human Eag1 Potassium Channel Function Exerts Antitumor
Activity. Cancer Res. 2007, 67, 7343–7349. [CrossRef] [PubMed]
12. de Queiroz, F.M.; Suarez-Kurtz, G.; Stühmer, W.; Pardo, L.A. Ether à Go-Go Potassium Channel Expression in Soft Tissue Sarcoma
Patients. Mol. Cancer 2006, 5. [CrossRef]
13. Asher, V.; Khan, R.; Warren, A.; Shaw, R.; Schalkwyk, G.V.; Bali, A.; Sowter, H.M. The Eag Potassium Channel as a New Prognostic
Marker in Ovarian Cancer. Diagn. Pathol. 2010, 5, 78. [CrossRef]
14. Martínez, R.; Stühmer, W.; Martin, S.; Schell, J.; Reichmann, A.; Rohde, V.; Pardo, L. Analysis of the Expression of Kv10.1
Potassium Channel in Patients with Brain Metastases and Glioblastoma Multiforme: Impact on Survival. BMC Cancer 2015, 15, 839.
[CrossRef]
15. Agarwal, J.R.; Griesinger, F.; Stühmer, W.; Pardo, L.A. The Potassium Channel Ether à Go-Go Is a Novel Prognostic Factor with
Functional Relevance in Acute Myeloid Leukemia. Mol. Cancer 2010, 9. [CrossRef]
16. Ding, X.-W.; Luo, H.-S.; Jin, X.; Yan, J.-J.; Ai, Y.-W. Aberrant Expression of Eag1 Potassium Channels in Gastric Cancer Patients
and Cell Lines. Med. Oncol. 2007, 24, 345–350. [CrossRef]
17. Menéndez, S.T.; Villaronga, M.Á.; Rodrigo, J.P.; Álvarez-Teijeiro, S.; García-Carracedo, D.; Urdinguio, R.G.; Fraga, M.F.; Pardo,
L.A.; Viloria, C.G.; Suárez, C.; et al. Frequent Aberrant Expression of the Human Ether à Go-Go (HEAG1) Potassium Channel in
Head and Neck Cancer: Pathobiological Mechanisms and Clinical Implications. J. Mol. Med. 2012, 90, 1173–1184. [CrossRef]
18. Ousingsawat, J.; Spitzner, M.; Puntheeranurak, S.; Terracciano, L.; Tornillo, L.; Bubendorf, L.; Kunzelmann, K.; Schreiber, R.
Expression of Voltage-Gated Potassium Channels in Human and Mouse Colonic Carcinoma. Clin. Cancer Res. 2007, 13, 824–831.
[CrossRef]
Cancers 2021, 13, 1244 24 of 24
19. S, terbuleac, D.; Maniu, C.L. An Antiarrhythmic Agent as a Promising Lead Compound for Targeting the HEAG1 Ion Channel in
Cancer Therapy: Insights from Molecular Dynamics Simulations. Chem. Biol. Drug Des. 2016, 88, 683–689. [CrossRef]
20. Wang, W.; MacKinnon, R. Cryo-EM Structure of the Open Human Ether-à-Go-Go -Related K + Channel HERG. Cell
2017, 169, 422–430.e10. [CrossRef] [PubMed]
21. The PyMOL Molecular Graphics System, version 2.3.0; Schrodinger, LLC: New York, NY, USA, 2019.
22. Moreels, L.; Bhat, C.; Voráčová, M.; Peigneur, S.; Goovaerts, H.; Mäki-Lohiluoma, E.; Zahed, F.; Pardo, L.A.; Yli-Kauhaluoma,
J.; Kiuru, P.; et al. Synthesis of Novel Purpurealidin Analogs and Evaluation of Their Effect on the Cancer-Relevant Potassium
Channel KV10.1. PLoS ONE 2017, 12, e0188811. [CrossRef] [PubMed]
23. Gómez-Lagunas, F.; Carrillo, E.; Pardo, L.A.; Stühmer, W. Gating Modulation of the Tumor-Related Kv10.1 Channel by Mibefradil.
J. Cell. Physiol. 2017, 232, 2019–2032. [CrossRef] [PubMed]
24. Mysinger, M.M.; Carchia, M.; Irwin; John, J.; Shoichet, B.K. Directory of Useful Decoys, Enhanced (DUD-E): Better Ligands and
Decoys for Better Benchmarking. J. Med. Chem. 2012, 55, 6582–6594. [CrossRef]
25. Inte:Ligand Expert KNIME Extensions Available from Inte:Ligand GmbH. Available online: http://www.Inteligand.com/knime-
nodes (accessed on 10 January 2021).
26. Friedrich, N.-O.; de Bruyn Kops, C.; Flachsenberg, F.; Sommer, K.; Rarey, M.; Kirchmair, J. Benchmarking Commercial Conformer
Ensemble Generators. J. Chem. Inf. Model. 2017, 57, 2719–2728. [CrossRef]
27. Poli, G.; Seidel, T.; Langer, T. Conformational Sampling of Small Molecules With ICon: Performance Assessment in Comparison
With OMEGA. Front. Chem. 2018, 6, 229. [CrossRef]
28. Boydston, A.J.; Rice, J.D.; Sanderson, M.D.; Dykhno, O.L.; Bielawski, C.W. Synthesis and Study of Bidentate Benzimidazolylidene−
Group 10 Metal Complexes and Related Main-Chain Organometallic Polymers. Organometallics 2006, 25, 6087–6098. [CrossRef]
29. Surry, D.S.; Buchwald, S.L. Dialkylbiaryl Phosphines in Pd-Catalyzed Amination: A User’s Guide. Chem. Sci. 2011, 2, 27–50.
[CrossRef]
30. Marx, M.A.; Vaisburg, C.A.; Christensen, J.G.; Galemmo, J. LSD1 Inhibitors. U.S. Patent US 2017/0183308A1, 29 June 2017.
31. Sarkar, S.; Sonkar, R.; Bhatia, G.; Tadigoppula, N. Synthesis of New N-Acryl-1-Amino-2-Phenylethanol and N-Acyl-1-Amino-3-
Aryloxypropanols and Evaluation of Their Antihyperlipidemic, LDL-Oxidation and Antioxidant Activity. Eur. J. Med. Chem.
2014, 80, 135–144. [CrossRef] [PubMed]
32. Gómez-Varela, D.; Contreras-Jurado, C.; Furini, S.; García-Ferreiro, R.; Stühmer, W.; Pardo, L.A. Different Relevance of Inac-
tivation and F468 Residue in the Mechanisms of HEag1 Channel Blockage by Astemizole, Imipramine and Dofetilide. FEBS
Lett. 2006, 580, 5059–5066. [CrossRef] [PubMed]
33. García-Ferreiro, R.E.; Kerschensteiner, D.; Major, F.; Monje, F.; Stühmer, W.; Pardo, L.A. Mechanism of Block of HEag1 K +
Channels by Imipramine and Astemizole. J. Gen. Physiol. 2004, 124, 301–317. [CrossRef] [PubMed]
34. Barros, F.; de la Peña, P.; Domínguez, P.; Sierra, L.M.; Pardo, L.A. The EAG Voltage-Dependent K+ Channel Subfamily: Similarities
and Differences in Structural Organization and Gating. Front. Pharmacol. 2020, 11, 411. [CrossRef] [PubMed]
35. Wolber, G.; Langer, T. LigandScout: 3-D Pharmacophores Derived from Protein-Bound Ligands and Their Use as Virtual
Screening Filters. J. Chem. Inf. Model. 2005, 45, 160–169. [CrossRef] [PubMed]
36. LigandScout v.4.3 Available from Inte:Ligand. Available online: https://www.Inteligand.com/ligandscout (accessed on 10
January 2021).
37. Nepali, K.; Lee, H.-Y.; Liou, J.-P. Nitro Group Containing Drugs. J. Med. Chem. 2019, 62, 2851–2893. [CrossRef] [PubMed]
38. Moreels, L. The Search for Novel Inhibitors of the Cancer-Relevant Potassium Channel Kv10.1. Ph.D. Thesis, KU Leuven, Leuven,
Belgium, 2018.
39. Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P. The Extraordinary Ligand Binding
Properties of Human Serum Albumin. IUBMB Life 2005, 57, 787–796. [CrossRef] [PubMed]
40. Maron, D.M.; Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutat. Res. Mutagen. Relat. Subj.
1983, 113, 173–215. [CrossRef]

Cancers: 3D Pharmacophore-Based Discovery of Novel K 10.1 Inhibitors With Antiproliferative Activity

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cancers

1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia;

Cancers 2021, 13, 1244. https://doi.org/10.3390/cancers13061244 https://www.mdpi.com/journal/cancers

2.2. Ligand-Based Pharmacophore Modelling

2.3. Virtual Screening

Cancers 2021, 13, x FOR PEER REVIEW 5 of 25

Cancers 2021, 13, x FOR PEER REVIEW 6 of 25

Cancers 2021, 13, x FOR PEER REVIEW 6 of 25

2.6. Statistical Analysis

2.7. Cell Culture

2.8. Evaluation of Mutagenic Activity with Salmonella/Microsomal Reverse Mutation Assay

Cancers 2021, 13, x FOR PEER REVIEW 10 of 25

3.4. Analogues of the Hit Compound ZVS-08 and Structure–Activity Relationships

Steady-state current amplitude (A)

Relative current amplitude

Figure 11. Design of the hit compound ZVS-08 analogues.

Figure 11. Design of the hit compound ZVS-08 analogues.

Figure 12. Cont.

Disubstituted ortho-nitro and para-trifluoromethyl compounds with a weaker basic

Total Green Object Integrated Intensity

B Control ZVS-08 Compound 1 Compound 8

The discovery of KV 10.1-selective potassium channel inhibitors is challenging due to

Supplementary Materials: The following are available online at https://www.mdpi.com/2072-6

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