Understanding the mechanism of action of pyrrolo[3,2-: B] quinoxaline-derivatives as kinase inhibitors

Article abstract of DOI:10.1039/d0md00049c

The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors that have reached clinical development. Th

Full text of DOI:10.1039/d0md00049c

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RESEARCH ARTICLE
Understanding the mechanism of action of
pyrroloij3,2-b]quinoxaline-derivatives as kinase
inhibitors†
Cite this: DOI: 10.1039/
d0md00049c
Andrea Unzue,a Claudia Jessen-Trefzer,a Dimitrios Spiliotopoulos,b
Eugenio Gaudio,c Chiara Tarantelli,c Jing Dong,b Hongtao Zhao,b
Johanna Pachmayr,d Stefan Zahler,d Elena Bernasconi,c Giulio Sartori,c
cf
Luciano Cascione, ce Francesco Bertoni,
Paweł Śledź,b
*
a
Amedeo Caflisch b and Cristina Nevado
*
*
The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously
reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors
that have reached clinical development. These two new compounds were characterized by an array of
cell-based assays and gene expression profiling experiments. A global chemical proteomics approach was
used to generate the drug-protein interaction profile, which suggested suitable therapeutic indications.
Both inhibitors, studied in the context of angiogenesis and in vivo in a relevant lymphoma model, showed
high efficacy in the control of tumor size.
Received 12th February 2020,
Accepted 16th April 2020
DOI: 10.1039/d0md00049c
rsc.li/medchem
to obtain its global target profile, have proven efficient in this
regard. In fact, such studies have been applied to several
Introduction
Drug development is a risky and expensive process1 in which
only 8% of the candidates that enter clinical trials are
approved by the authorities.2 Late-stage failures of promising
drug candidates in clinical development have emphasized the
importance of understanding their mode of action.2 As a
result, more personalized treatments can be developed, new
clinical applications of already approved drugs can be
conceived, and a better understanding of off-target effects can
be achieved. The application of systems biology approaches,
including studies based on gene signatures that inform on
pathways and diseases that the drug of interest acts on,3 or
chemical proteomics technology, where binding of the
molecule of interest is addressed in an unbiased environment
kinase inhibitors including dasatinib, imatinib, nilotinib4
and bosutinib,5 revealing clear differences in the mode of
action of these drugs despite being all administered for the
treatment of chronic myeloid leukemia (CML).
Protein kinases are relevant targets for the treatment of
diseases including cancer, inflammation, cardiovascular
conditions and immune-related disorders.6 More than 50
small-molecule kinase inhibitors have been approved by
the FDA over the last decade.7 In our work, we have
focused on the in silico design, synthesis and
computational-aided optimization of potent and selective
receptor tyrosine kinase inhibitors.8 To date, our approach
has yielded nanomolar EphB4 inhibitors whose binding
modes, antiproliferative activities and in vivo efficacies
have been characterized.9
a Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-
8057, Zürich, Switzerland. E-mail: cristina.nevado@chem.uzh.ch;
Fax: (+41) 446353948; Tel: (+41) 446353945
Here we describe the design, synthesis and in depth
biological characterization of the mode of action of two novel
type II kinase inhibitors (8a, b) with broader scope of activity.
The two compounds showed in vivo anti-tumor activity,
especially in models derived from hematological
malignancies. The effects of these molecules on gene
expression were compared to dasatinib, an FDA approved
drug for the treatment of chronic myeloid leukemia.10 Further
characterization, including the assessment of their in vitro
selectivity, cellular angiogenesis inhibition, as well as a global
chemical proteomics approach and in vivo efficacy is reported
here.
b Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-
8057, Zürich, Switzerland
c Institute of Oncology Research, Faculty of Biomedical Sciences, USI, Bellinzona,
Switzerland
d Department of Pharmacy, University of Munich, Butenandstrasse 5-13, 81377
Munich, Germany
e SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland
f Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland
† Electronic supplementary information (ESI) available: General procedures for
the synthesis, characterization and biological evaluation of all reported
compounds can be found in the ESI. See DOI: 10.1039/d0md00049c
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Design of new type II quinoxaline
inhibitors
arthritis and other inflammatory conditions including
Crohn's disease and psoriasis that has undergone several
clinical trials.3a,12 Compound
3 is a type II kinase
Within our first generation of quinoxaline EphB4 inhibitors
(including type I, I1/2 and II),8d compound 1 proved to be the
most potent type I1/2 ligand in vitro inducing a cytostatic
effect in an in vivo cancer model. Interestingly, compound 2
represents a type II inhibitor bearing a urea linker that offers
an interesting anti-proliferative profile over a cell cancer
panel (Fig. 1).8d
inhibitor that is characterized by a pyrazole ring bearing a
tert-butyl substituent nested within the hydrophobic
pocket. Similar to our type II inhibitor 2, the urea linker
in inhibitor 3 establishes a hydrogen bond network with
Asp758 and Glu664. This structure inspired further
modifications of compound 1. In particular, the
superposition of compounds 2 and 3 reveals an overall
similar binding mode and a clear overlap of the urea
linker and its substituents (Fig. 2C), which prompted us
The type II binding mode with the Asp-Phe-Gly (DFG)-out
conformation of the kinase was confirmed by X-ray
diffraction analysis of the catalytic domain of EphA3 in
complex with 2 (Fig. 2A, PDB code 4P5Z).8d The pyrroloij3,2-
b]quinoxaline scaffold occupies the ATP binding site with the
phenyl substituent located in the hydrophobic pocket that is
generated when the kinase adopts an inactive DFG-out
conformation. Compound 2 is able to form four hydrogen
bonds within the hinge region of the kinase via the amino
and amide substituents of the pyrrole ring: the amino
substituent at position 2 is involved in a bifurcated hydrogen
bond with the side chain hydroxyl of the Thr693 gatekeeper
and the backbone carbonyl of Glu694, whereas the amide
substituent at position 3 forms two hydrogen bonds with the
backbone polar groups of Met696. In addition, the urea
linker establishes three extra hydrogen bonds acting as a
hydrogen bond acceptor from the Ser757 side chain and the
amide backbone of Asp758, and hydrogen bond donor to the
side chain of Glu664. The m-CF3-phenyl moiety is located in
the hydrophobic pocket originating from the displacement of
the Phe765 side chain of the DFG motif in the DFG-out
conformation of the kinase.
to introduce
a tert-butyl substituted pyrazole scaffold
within our quinoxaline inhibitors series. We decided to
synthesize not only the 4-methyl phenyl substituted
pyrazole ring present in compound 3, but also a quinoline
substituted one mimicking compound DCC-2036 (4),13
a
BCR-ABL1 inhibitor that underwent clinical trials for the
treatment of chronic myeloid leukemia (Fig. 2D).14
Synthesis of new type II quinoxaline
inhibitors
The synthesis of the novel type II quinoxaline derivatives
bearing the tert-butyl pyrazole moiety is shown in
Scheme 1. Compound
previously reported procedures by condensation of
commercially available 2,3-dichloroquinoxaline with
5 was prepared according to
malononitrile in the presence of sodium hydride.15 The
substitution of the chlorine at position 3 with synthetically
prepared anilines 6a, b (ref. 16) followed by cyclization
afforded intermediates 7a, b (ref. 17) (Scheme 1), which
were then hydrolyzed under strong acidic conditions
affording the final compounds 8a, b.
We have also solved the X-ray structure of the clinical
candidate BIRB796 (3) in complex with the catalytic
domain of EphA3 (Fig. 2B, PDB code 4TWN).11 Compound
3 is a selective p38 MAP kinase inhibitor developed by
Boehringer Ingelheim for the treatment of rheumatoid
Potency assessment: differential
scanning fluorimetry and EC50
determination
The binding of the two novel type II quinoxaline
inhibitors 8a, b towards the tyrosine kinases EphA3 and
EphB4 was first studied in vitro and compared to our first
generation quinoxaline inhibitors 1 and 2.8d Differential
scanning fluorimetry (DSF) was used to measure the
increase in thermal stability of EphA3 upon ligand
binding.18 Compounds
1 and 2 exhibited pronounced
thermal shifts of 11.2 and 14.3 °C respectively, while the
new inhibitors 8a, b showed even higher values (17.3 and
19.3 °C respectively, Table 1). However, when compounds
8a, b were then tested in cellular phosphorylation assays
on MEF cells transfected with Myc-tagged human EphB4,
their potency dropped compared to initial compound
series to EC50 values of 360 and 1100 nM, respectively
(Table 1).
Fig.
1 2D structures of previously developed type I1/2 and II
quinoxaline inhibitors 1 and 2.8d EC50 values were measured in a
cellular phosphorylation assay using mouse embryonic fibroblasts
(MEF) that overexpress EphB4. ΔTm values have been determined using
differential scanning fluorimetry assay.
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Fig. 2 Crystal structures of the catalytic domain of the tyrosine kinase EphA3 in complex with the low-nanomolar inhibitors 2 (A) and 3 (B) and
superposition of the two inhibitors 2 and 3 (C). The ATP binding site of the EphA3 kinase is shown in pink ribbons while the side chains mentioned
in the text and the inhibitors are shown by sticks in blue and yellow for compounds 2 and 3, respectively. (D) 2D structure of the BCR-ABL1
inhibitor DCC-2036 (4).
Scheme 1 Reagents and reaction conditions: a) 6a, b (1.2 equiv.), DMF, 80 °C, 12 h, 55–62%. b) H2SO4, 25 °C, 30 min, 89–90%.
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Table 1 EphA3/EphB4 inhibition data for the synthesized quinoxaline derivatives
b
Compound
Type of binding
R1
R2
H
R3
R4
Thermal shift (°C)a
Cellular EC50 (nM)
1
2
I1/2
II
Me
H
OH
H
H
H
11.2
14.3
5.4
89
8a
8b
II
II
H
H
H
H
H
H
17.3
19.3
360
1100
a
b
Average values of triplicate measurements. The standard deviation is smaller than 0.5 °C. Cellular EC50 values were measured in a cellular
phosphorylation assay using MEF cells overexpressing EphB4 at Proqinase.
Fig. 3 Selectivity profiles of compound 1 (left), 8a (center) and 8b (right) tested on a panel of 453 protein kinases at DiscoveRx. Measurements
were performed at a concentration of 1 μM of the inhibitor. The affinity is defined with respect to a DMSO control. The dendrogram was obtained
from KinomeScan using the KinomeTree software.19
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Selectivity profiles from biochemical
assays
developed quinoxalines (1, 2) and FDA-approved kinase
inhibitors dasatinib and imatinib (Fig. 4). The two
compounds presented a broader anti-proliferative activity
than compounds 1 and 2 (which is in line with their broader
selectivity profiles) and a significant correlation in terms of
GI50 values across the different cell lines. The leukemia K-562
cell line was particularly sensitive towards the two optimized
type II quinoxaline inhibitors, showing remarkably good GI50
values (31 and <10 nM, respectively).
In an effort to understand the mode of action of our
quinoxaline inhibitors, we compared the transcriptome of
K562 cells (one of the most sensitive cell lines towards our
compounds, Fig. 4) after 6 hours of exposure to DMSO, as
vehicle, to 1 μM of compound 8b or 1 μM of dasatinib, for
reference (ESI† – RNA-Sequencing Table). Compound 8b and
dasatinib caused very similar changes in the transcriptome of
the cells (Fig. 5A). Compound 8b downregulated MYC and
E2F targets, genes known to be involved in oxidative
phosphorylation, unfolded protein response, proteasome
pathway, PI3K/AKT/mTOR signaling, spliceasome and DNA
repair (ESI† – RNA-Seq Table compound 8b). In contrast,
genes negatively regulated by RAS were enriched among the
transcripts upregulated by compound 8b (Fig. 5B). The gene
expression signature of compound 8b overlapped with that of
dasatinib on tumor infiltrating macrophages, and with PI3K/
mTOR inhibitors in different tumor models (Fig. 5B) (ESI† –
RNA-Seq Table compound 8b). Same signatures were
observed in dasatinib treated cells (ESI† – RNA-Seq Table
dasatinib).
Given the potency loss observed towards EphB4 in the cell
based assays for compounds 8a, b (Table 1), we decided to
assess the influence of the new urea substituents on the
selectivity over a broad panel of kinases. The selectivity
profile was obtained by an in vitro competition binding assay
that reports on binding affinity with no ATP required, using
recombinant kinases (KINOMEscan at DiscoveRx).19 The
selectivity panel consisted of 453 human kinases, 58 of which
were disease related mutant kinases (mainly of ABL1, EGFR,
and PIK3CA). Single dose measurements were carried out at
1 μM concentration of the ligand.
Compounds 8a and 8b present a very similar selectivity
profile that differs from that obtained for compound 1
(Fig. 3). Quantitatively, 60 kinases (14 of which are disease-
related mutants) are more strongly inhibited by compounds
8a, b, where the difference between the affinity of 8a or 8b
and compound 1 is at least 60% (expressed as% with respect
to DMSO control). A total of 31 out of the 46 non-mutant
kinases are serine/threonine-specific protein kinases, more
than half of them belonging to the CMGC kinase family, and
several are CDK kinases, which are involved in critical
cellular processes.20 This data clearly suggests a broader
selectivity profile for compounds 8a, b, which are also able to
inhibit clinically relevant mutant kinases including ABL1,
EGFR, FLT3, KIT, and MET. Interestingly, the activity towards
EphB2, 3 and 4 kinases drops significantly in compounds 8a,
b, in line with the EC50 values obtained in cells
overexpressing EphB4 (Table 1).
Chemical proteomics
Anti-proliferative activity and gene
expression profiling
We decided to perform a chemical proteomics experiment on
our best quinoxaline inhibitor 8a aiming to obtain the global
proteomic profiles of this molecule in an unbiased cellular
setting. This approach required the synthesis of a tethered
inhibitor containing a suitable functional group that would
Compounds 8a and 8b were screened against the NCI-60
cancer cell line panel and compared to our previously
Fig. 4 The antiproliferative activity of quinoxalines 1, 2, 8a and 8b is shown together with the growth inhibition of two FDA approved anticancer
drugs (dasatinib and imatinib) evaluated on the NCI-60 cancer cell line panel. The growth inhibition is shown as a matrix with cell lines and
compounds arranged horizontally and vertically, respectively. The legend bar shows the color coding, which reflects the logIJGI50) value, with red
and green indicating high and low toxicity, respectively. The grey scale on the left hand side of the figure shows the absolute value of the
correlation of the compounds' GI50 values over for the different cell lines.
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Fig. 5 Transcriptome analysis of CML K562 cells exposed to compounds 8b or to dasatinib. (A) Scatter plots by log 2 fold gene expression
changes in compound 8b/DMSO-treated K652 vs. dasatinib/DMSO-treated K562 cells. (B) Representative GSEA plots illustrating the transcriptional
expression signature enrichment in genes downregulated (upper panel) and upregulated (lower panel) after exposure to DMSO or compound 8b in
K562 cells. Green line represents the enrichment score; bars in the middle portion of the plots show where the members of the gene set appear in
the ranked list of genes; positive or negative ranking metric indicate respectively correlation or inverse correlation with the profile; FDR, false
discovery rate; P, probability value; NES, normalized enrichment score.
allow the attachment to a resin in a covalent fashion. Based
on the X-ray structure of compound 2 (Fig. 2A) we envisioned
that the introduction of a linker at the 7 position of the
quinoxaline moiety would be tolerated due to its solvent
accessibility. Thus, the analogue of compound 8a, bearing a
primary amino group suitable for immobilization, was
synthesized, affording compound 13 (Scheme 2).
Intermediate 9 was prepared in three steps as previously
reported starting from the commercially available
4-nitrobenzene-1,2-diamine.15b,21 The substitution of the
chlorine at position 3 with synthetically prepared aniline 6a
(ref. 16) followed by cyclization in DMF at 80 °C afforded
intermediate 10.17 The nitro group was reduced in the
presence of Pd on activated charcoal and
a hydrogen
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Scheme
2
Reagents and reaction conditions: a) 6a, DMF, 80 °C, 4.5 h, 71%. b) H2, Pd/C, MeOH, rt,
4
h, 70%. c) tert-Butyl
(3-bromopropyl)carbamate, KI, DIPEA, DMF, 80 °C, 6 h, 29%. d) H2SO4, 25 °C, 30 min, quantitative yield.
atmosphere yielding the desired aniline 11 in good yield,
which was further reacted with tert-butyl (3-bromopropyl)-
carbamate to yield intermediate 12. In the final step, strong
acidic conditions were employed resulting in the hydrolysis
of the nitrile group and removal of the Boc protecting group
in quantitative yield, affording the intermediate 13.
The inhibitory activity of 13 was then compared to its
parent compound 8a by measuring the ABL2 kinase activity
in a competition binding assay (Fig. 6).19 Crucially, no activity
loss was observed for compound 13, making it a suitable
probe for our chemical proteomics study. More importantly,
the selectivity profile of compound 13 linearly correlates to
its parent compound 8a as determined at 1 μM concentration
in a panel of 453 kinases with an R2 value of 0.8 (Fig. 6C) by
a competition assay.19
loaded onto spin columns, washed, and the protein
binders were eluted with formic acid as previously
reported.5b Among the 250 proteins identified by MS
analysis as pulled down with immobilized compound 13
(Table S1†), there were 16 kinases (Table 2), five of
which turned out also as targets in the selectivity
profile analysis (LYN, BTK, YES, CSK and ABL2), plus
PRKDC, MTOR, AGK, PI4KA (Table 2).
Four of the major interactors of quinoxaline 13, LYN, BTK,
CSK and YES1, are nanomolar binders already identified in
the in vitro selectivity profile (Fig. 3). LYN, YES1, BTK and
CSK are inhibited to 0.5, 1.9, 3.3 and 0.65% control
respectively by our small molecule 13 (0.85, 9.6, 0.95 and
0.9% control, by the parent compound 8a respectively) at 1
μM concentration. The 250 human proteins belonged to
The tethered analogue 13 was covalently bound to
agarose polymer beads and incubated with the cell
lysate of K562 leukemia cells (beads treated with DMSO
were used as control). The beads were subsequently
pathways
related
to
protein
secretion,
oxidative
phosphorylation, DNA repair, MYC and E2F targets, IL6/JAK/
STAT3 signaling, glycolysis, mitotic spindle, and mTORC1
signaling.
Fig. 6 (A and B) In vitro inhibitory activity towards ABL2 kinase of compounds 8a and 13. (C) Probe compound 13 and its parent compound 8a
show a strong correlation across a 453 kinase panel at 1 μM concentration, demonstrating the validity of compound 13 as a probe for chemical
proteomic studies.
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Table 2 Kinases identified in the pull down assay with compound 13 as bait
Gene name
Description
Fold changea
PRKDC
LYN
ERLIN2
YES1
BTK
MTOR
CSK
DNA-dependent protein kinase catalytic subunit
Tyrosine-protein kinase Lyn
Erlin-2
6172
1721
1620
1020
1216
1116
1115
912
6.6
6
5.5
5
5
5
5
Tyrosine-protein kinase yes
Tyrosine-protein kinase BTK
Serine/threonine-protein kinase mTOR
Tyrosine-protein kinase CSK
Acylglycerol kinase, mitochondrial
Cyclin-dependent kinase 1
Nucleoside diphosphate kinase A
Glycerol kinase
Abelson tyrosine-protein kinase 2
Pyridoxal kinase
Nucleoside diphosphate kinase B
Phosphatidylinositol 4-kinase alpha
Sequestosome-1
AGK
CDK1
NDKA
GLPK
ABL2
PDXK
NDKB
PI4KA
SQSTM1
5
a
The peptide count average of the two duplicate measurements is given.
Fig. 7 Anti-tumor activity of compounds 8a and 8b in lymphoma models. (A) Eight B-cell lymphoma cell lines derived from mantle cell lymphoma
(MCL) (Jeko1, MAVER1), chronic lymphocytic leukemia (MEC-1, PCL-12) and diffuse large B cell lymphoma (DLBCL) of the activated B cell like
(ABC) (RI-1, SU-DHL-2) or of the germinal center B cell (GCB) (VAL, SU-DHL-4) were exposed to increased concentration of 8b or 8a for 72 h. (B)
Effects of 8b in a xenograft model of ABC-DLBCL. NOD-Scid mice subcutaneously inoculated with RI-1 cells (15 × 10 (ref. 6)) were split in three
groups respectively treated with 8a (100 mg kg−1, IP, n = 4, data not shown), 8b (100 mg kg−1, IP, n = 4) and control vehicle (n = 4). Y-Axis, tumor
volume in mm3. X-Axis, days (D) of treatment. For compound 8b vs. CTRL, D5-D15, p < 0.05. In each box-plot, the line in the middle of the box
represents the median and the box extends from the 25th to the 75th percentile (interquartile range, IQ); the whiskers extend to the upper and
lower adjacent values (i.e., 1.5 IQ); outside values have been omitted from the figure. (C) Values of the combination index (C.I.) of 8a or 8b with
birabresib in ABC-DLBCL and MCL cell lines.
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Inhibitors 8a and 8b in preclinical
Cellular angiogenesis assay
models of lymphoma
The implication of numerous kinases in angiogenesis24 led
us to examine the efficacy of our inhibitors on tube
formation via an in vitro matrigel angiogenesis assay.
Angiogenesis, the process of vascular growth by sprouting
from pre-existing vessels, is a key player during tumor growth
and metastasis in the context of several diseases including
cancer, macular degeneration, diabetic retinopathy and
arthritis.3a A remarkable effect was observed for the novel
quinoxaline 8a (65, 90, and 100% inhibition of tube length,
total branching points and total loops respectively, Fig. 8) in
comparison to our previously developed inhibitors 1 (9, 23
and 21% inhibition of tube length, total branching points
The engagement of target kinases LYN, BTK and mTOR by
quinoxaline inhibitors demonstrated by the proteomics data
prompted the investigation of their activity against
lymphomas (not included in the NCI-60 panel), for which
these three kinases are highly relevant. Anti-proliferative
activity of 8a and 8b was assessed, in parallel to dasatinib, in
eight B-cell lymphoma cell lines derived from mantle cell
lymphoma (MCL) (Jeko1, MAVER1), chronic lymphocytic
leukemia (MEC-1, PCL-12) and diffuse large B cell lymphoma
(DLBCL) of the activated B cell like (ABC) (RI-1, SU-DHL-2) or
of the germinal center
B cell (GCB) (VAL, SU-DHL-4).
and total loops respectively) and
2 (5, 9 and 13%
Compounds were active with median GI50 of 600 nM (95% C.
I. 300–1065 nM) and 250 nM (95% C.I. 100–1049 nM),
respectively and their anti-tumor activity appeared more
potent than dasatinib (median IC50 = 1325 nM, 95% C.I. 100–
16 625 nM), in particular in four of the cell lines (MAVER1,
SU-DHL-2, VAL and MEC1) (Fig. 7A).
respectively).9 We hypothesize that the promising
angiogenesis inhibition results obtained for compound 8a
could be due to its ability to inhibit several CDK kinases (in
contrast to compound 1, Fig. 3), as CDK inhibitors have been
reported to block angiogenesis in vitro and in vivo.25 Further,
these results are in line with those previously reported for
dasatinib and related TKI inhibitors.26
The anti-lymphoma activity of compounds 8a and 8b was
then assessed using a mouse xenograft model with the ABC
DLBCL RI-1 cell line. Compound 8a turned out to be very
toxic already after the first dose, due to swelling of the area
surrounding the injection and body-weight loss, and had to
be stopped although the body weight started to recover after
two weeks without treatment. Inhibitor 8b was administered
once every 5 days and was well tolerated with no general
toxicity or body-weight loss. After two weeks of treatment,
xenografts treated with 8b were three times smaller than the
control group or than the group treated with the initial single
dose of 8a at the end of the experiment (day 15) (Fig. 7B).
Dasatinib as well as inhibitors of BTK or mTOR are
synergistic when combined with BET bromodomain
inhibitors.22 Thus, we combined compounds 8a and 8b with
birabresib (MK-8628/OTX015), a BET bromodomain inhibitor
with preclinical and early clinical anti-lymphoma
activity.22b,23 Synergism, i.e., combination index (CI) values
smaller than 1.0, was observed with the combination of
compound 8a or 8b with birabresib in all the four cell lines
that were studied (Fig. 7C).
Conclusions
Two quinoxaline-containing chimeric kinase inhibitors 8a
and 8b were designed based on the available X-ray structures
of EphA3-inhibitor complexes through compound merging.
They inhibit mainly tyrosine kinases and show broader
spectrum of activity than the parent compound 1, as well as
high anti-proliferative activity against several cancer cell
lines, as demonstrated in the NCI-60 cell line panel
screening. Immortalised myelogenous leukemia cell line
K562 was shown to be particularly sensitive and was selected
for further investigations to elucidate the mode of action of
compounds 8a–b. Transcriptomic profiling revealed
orthogonal effects of compounds
1 and 8b on gene
expression, with the latter closely resembling the effect
profile of dasatinib, the SRC-family protein tyrosine kinase
inhibitor. By conducting a chemical proteomics study we
identified 17 kinases as potential binders of compounds 8a,
Fig. 8 In vitro angiogenesis assay on tube formation. Compounds were incubated with human umbilical vein endothelial cells (HUVEC) cells
seeded on matrigel for 15 h. Tube length (left), total branching points (middle) and total loops (right) were quantified. All measurements were done
at least in triplicate, and their SEM values are indicated as error bars.
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including the known dasatinib targets LYN, BTK, CSK and
YES1. Together, these studies have inspired two possible
applications of the developed chimeric compounds.
Demonstrated ability of our inhibitors to inhibit the CDKs
prompted us to investigate effect of compound 8a on
inhibiting angiogenesis in vitro, showing remarkable
improvement compared to the parent compound 1. In turn,
their engagement with the kinases LYN, BTK and mTOR,
which are relevant in lymphomas, triggered in vitro and
in vivo studies on compound 8a–b in various lymphoma
models. Both compounds have shown submicromolar IC50
values in five lymphoma cell lines. A significant decrease in
tumor size is observed in an RI-1 lymphoma xenograft mouse
model at a dose of 100 mg kg−1 of compound 8b, without any
associated mean body weight loss. These results indicate
promise for therapeutic use of developed inhibitors in vivo.
EDC
FRET
Abl
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
Fluorescence-resonance energy transfer
Abelson
murine
leukemia
viral
oncogene
homologue
EGFR
DDR
Lck
Epidermal growth factor receptor
Discoidin domain receptor
Lymphocyte-specific kinase
Cyclin dependent kinase
CDK
DMSO Dimethyl sulfoxide
MEF
VEGF
Mouse embryonic fibroblasts
Vascular endothelial growth factor
HUVEC Human umbilical vein endothelial cells
NCI
DTP
IP
National Cancer Institute
Developmental Therapeutics Program
Intraperitoneal
Further evaluation of compound 8b in a xenograft mice Conflicts of interest
model established with a more responsive cell line than the
RI-1, such as the K562 human chronic myeloid leukemia cell
There is no conflict of interest to declare.
line, might demonstrate even greater extent of tumor size
control.
Acknowledgements
We thank Dr. Erich Brunner for interesting discussions regarding
the chemical proteomic experiments. We also acknowledge and
thank the NIH/National Cancer Institute for in vitro and in vivo
evaluations of compounds under the Developmental Therapeutics
Program (DTP), and in particular Dr. Stephen L. White of the
NIH/NCI Drug Synthesis & Chemistry Branch for information and
discussions relating to the in vivo studies. The authors would like
to thank the Swiss National Science Foundation and the Swiss
Cancer League (Krebsliga) for financial support (to AC and CN)
and the Gelu Foundation (to FB). PŚ is a recipient of UZH
Entrepreneur Fellowship in Biotechnology.
Accession codes
PDB codes for EphA3 in complex with the inhibitors 2 and 3
are 4P5Z and 4TWN, respectively.
Author contributions
CN, AC, FB and SZ conceived and designed the experiments.
Experimental work was conducted by AU, CJ-T, DS, EG, CT, JD,
JL, EB, GS, LC and PS. Crystallographic and computational work
was conducted by PS, HZ and JD. The manuscript was written
and edited by CN, AC, FB, PS, AU. CJ-T and EG.
References
Ethical statement
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Mice maintenance and animal experiments were performed
under institutional guidelines established for the Animal
Facility at The Institute of Research in Biomedicine (IRB,
Bellinzona, CH). and with study protocols approved by the
local Cantonal Veterinary Authority (No. TI-20-2015).
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Abbreviations
Eph
Erythropoietin-producing human hepatocellular
carcinoma receptor
Aspartate-phenylalanine-glycine
Adenosine triphosphate
Surface plasmon resonance
Dimethylformamide
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DFG
ATP
SPR
DMF
TBTU
N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate
DIPEA Diisoproylethylamine
DCM
THF
HOBt
Dichloromethane
Tetrahydrofuran
Hydroxybenzotriazole
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