Synthesis of Indomorphan Pseudo-Natural Product Inhibitors of Glucose Transporters GLUT-1 and -3

Article abstract of DOI:10.1002/anie.201909518

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP-like chemical space and biological target space. These limitations can be overcome by combining NP-centered strategies with fragment-based compound design through combination of NP-derived fragments to afford structurally unprecedented “pseudo-natural products” (pseudo-NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo-NPs that combine biosynthetically unrelated indole- and morphan-alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT-1 and GLUT-3. Glupin suppresses glycolysis, reduces the levels of glucose-derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT-1 and GLUT-3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.

Full text of DOI:10.1002/anie.201909518

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International Edition: DOI: 10.1002/anie.201909518
German Edition: DOI: 10.1002/ange.201909518
Antitumor Agents
Synthesis of Indomorphan Pseudo-Natural Product Inhibitors of
Glucose Transporters GLUT-1 and -3
Javier Ceballos+, Melanie Schwalfenberg+, George Karageorgis, Elena S. Reckzeh, Sonja Sievers,
Claude Ostermann, Axel Pahl, Magnus Sellstedt, Jessica Nowacki, Marjorie A. Carnero Corrales,
Julian Wilke, Luca Laraia, Kirsten Tschapalda, Malte Metz, Dominik A. Sehr, Silke Brand,
Konstanze Winklhofer, Petra Janning, Slava Ziegler, and Herbert Waldmann*
Abstract: Bioactive compound design based on natural Introduction
product (NP) structure may be limited because of partial
coverage of NP-like chemical space and biological target
space. These limitations can be overcome by combining NP-
centered strategies with fragment-based compound design
through combination of NP-derived fragments to afford
structurally unprecedented “pseudo-natural products” (pseu-
do-NPs). The design, synthesis, and biological evaluation of
a collection of indomorphan pseudo-NPs that combine
biosynthetically unrelated indole- and morphan-alkaloid frag-
ments are described. Indomorphane derivative Glupin was
identified as a potent inhibitor of glucose uptake by selectively
targeting and upregulating glucose transporters GLUT-1 and
GLUT-3. Glupin suppresses glycolysis, reduces the levels of
glucose-derived metabolites, and attenuates the growth of
various cancer cell lines. Our findings underscore the impor-
tance of dual GLUT-1 and GLUT-3 inhibition to efficiently
suppress tumor cell growth and the cellular rescue mechanism,
which counteracts glucose scarcity.
Strategies for the design and discovery of novel chemical
matter endowed with bioactivity can draw from previous
insight about the biological relevance of compound classes.
This reasoning underlies, for example, biology-oriented syn-
thesis (BIOS)[1] and ring-distortion and/or -modification
approaches (“complexity to diversity”; CtD).[2] Limitations
of BIOS derive from restricted coverage of natural product
like chemical space and biological target space. These
limitations may be overcome by the combination of BIOS
with fragment-based design[3] to arrive at “pseudo-natural
products”.[4] Pseudo-natural products are obtained by the
de novo combination of natural product fragments[5] that
generate unprecedented compound classes not accessible by
known biosynthesis pathways, and, therefore, go beyond the
chemical space explored by nature. They may have new
biological targets and modes of action, such that their
bioactivity will best be evaluated in unbiased target-agnostic
cell-based assays covering a wide range of biological process-
es.[6]
Here we describe the design, synthesis, and biological
investigation of indomorphan pseudo-natural products that
[*] Dr. J. Ceballos,[+] M. Sc. M. Schwalfenberg,[+] Dr. G. Karageorgis,
Dr. E. S. Reckzeh, J. Nowacki, Dr. M. A. Carnero Corrales,
M. Sc. J. Wilke, Dr. L. Laraia, Dr. K. Tschapalda, M. Metz, Dr. S. Brand,
Dr. P. Janning, Dr. S. Ziegler, Prof. Dr. H. Waldmann
Department of Chemical Biology
Dr. J. Ceballos[+]
Current address: Laboratory of Catalysis and Organic Synthesis
EPFL SB ISIC LCSO, BCH 4221, 1015 Lausanne (Switzerland)
Dr. G. Karageorgis
Current address: School of Chemistry
University of Leeds
Max-Planck-Institute of Molecular Physiology
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Leeds, LS2 9JT (UK)
Dr. L. Laraia
Dr. E. S. Reckzeh, M. Sc. J. Wilke, Prof. Dr. H. Waldmann
Faculty of Chemistry and Chemical Biology
Technical University Dortmund
Current address: Department of Chemistry
Technical University of Denmark
Kemitorvet, Bygning 207, 2800 Kgs Lyngby (Denmark)
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
Dr. M. Sellstedt
Current address: Clinical Chemistry, Laboratory Medicine
University Hospital of Umeꢀ, 901 85 Umeꢀ (Sweden)
Dr. S. Sievers, Dr. C. Ostermann, Dr. A. Pahl
Compound Management and Screening Center, Dortmund
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
[+] These authors contributed equally to this work.
Dr. M. Sellstedt
Department of Chemistry, Umeꢀ University
901 87 Umeꢀ (Sweden)
Supporting information and the ORCID identification number for
one of the authors of this article can be found under:
https://doi.org/10.1002/anie.201909518.
M. Sc. D. A. Sehr, Prof. Dr. K. Winklhofer
Department of Molecular Cell Biology
Institute of Biochemistry and Pathobiochemistry
Ruhr University Bochum
ꢁ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
44801 Bochum (Germany)
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Figure 1. Design of the indomorphan pseudo-NP class based on the natural product fragments derived from indole (in blue) and morphan (in
red) alkaloids.
contain characteristic structural fragments of the biosynthet-
ically unrelated indole[7] and morphan[8] alkaloid classes, each
of which is endowed with diverse and different bioactivities
(Figure 1). A piperidine frag-
GLUT-1 and GLUT-3 with low nanomolar potency, inhibits
glycolysis, and efficiently suppresses the growth of various
cancer cell lines.
ment is attached in an edge-on
connection to an indole moiety
in numerous polycyclic indole
alkaloids,[9] whereas a benzene
ring is linked to a piperidine-
containing fragment through
a bridge in the scaffold of the
morphinan alkaloids.[9] In the
indomorphans, these frag-
ments are merged to a bridged,
bicyclic
indolylethylamine-
containing novel pseudo-natu-
ral product class (Figure 1).
Biological investigation of
the compound collection re-
vealed that the indomorphans
define a truly novel, structur-
ally unprecedented glucose up-
take inhibitor chemotype. The
most potent inhibitor, termed
Scheme 1. Synthesis of the indomorphan pseudo-NP class. i) MeI (5 equiv), Ag2O (5 equiv), CH2Cl2, 72 h,
rt; ii) AcCl, CH2Cl2/pyridine, 3 h, ꢀ108C; iii) allyl ethylcarbonate (2 equiv) Pd(PPh3)4 (5 mol%), THF, 808C,
Glupin, selectively blocks glu- 2.5 h; iv) ArN2H3·HCl (1 equiv), AcOH, reflux, 1.5 h; v) R3Br (3 equiv), Cs2CO3 (3 equiv), DMF, 6 h, rt;
vi) H2, Pd/C, EtOH, 5 h, rt; vii) ArCOCl (1.0 equiv), NEt3 (1.2 equiv), CH2Cl2, 12 h, rt.
cose uptake mediated by
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Results
ilarly to decreased glucose levels.[23] Thus, efficient inhibition
of glucose uptake in tumors and cancer cells may require
simultaneous targeting of both GLUT-1 and GLUT-3, and we
recently developed the first GLUT-1/GLUT-3-selective com-
pounds.[4b,24]
The glucose uptake assay monitored the inhibition of 2-
deoxyglucose (2DG) uptake as described by Yamamoto
et al.[25] Briefly, 2DG uptake is determined by quantification
of the 2DG-6-phosphate (2DG6P) that accumulates inside
the cells by using the diaphorase-resazurin/-resorufin system
(see Figure S7a). Several indomorphans inhibited 2DG
uptake with IC50 values ranging from submicromolar to low
micromolar levels (Table 1 and Table S1), with compound 5b
(Table 1, entry 2) being the most active with an IC50 value of
53 ꢁ 23 nm.
For the synthesis of the indomorphan library, bicyclic
ketone 1[10] (Scheme 1) was derivatized to yield ethers 2,
which were then transformed into different indole derivatives
3 by means of the Fisher indole synthesis. Alkylation of the
indole nitrogen atom (!4), deprotection, and acylation of the
morphan nitrogen atom yielded indomorphans 5. All the
transformations are preparatively simple and have wide
scope. The synthesis yielded 43 indomorphans in five linear
steps. In addition, 19 selected indomorphans were obtained
commercially[11] (see Table S1 in the Supporting Informa-
tion).
Calculation of the natural product score (NP-score)
distribution[12] of this pseudo-NP class, and comparison with
the scores calculated for the NP set in ChEMBL,[13] as well as
for the set of marketed and experimental drugs listed in
DrugBank,[14] revealed that indomorphans exhibit a narrow
distribution in a part of the NP-score graph that is not heavily
populated by NPs but rather by synthetically accessible
biologically relevant molecules (Figure S2a). Analysis of the
principal moments of inertia (PMI),[15] used as a crude
measure of molecular shape, revealed that these pseudo-
NPs possess a higher degree of three-dimensional character
than common synthetically accessible compound collections,
which reside typically on the rod-like to disc-like vertex of
a PMI plot (Figure S2b).[16] In addition, indomorphans
display similar shape characteristics as indole- or morphan-
containing NPs and non-naturally occurring biologically
relevant compounds (see Figure S3 and Schemes S3 and S4).
Further analysis using “Lipinski-Ro5” criteria, showed that
61% of this collection is included within the limits of drug-
like space (see Figure S2c and Table S2), thus indicating that
de novo combinations of NP-derived fragments may result in
compound collections with desirable properties.
The indomorphans were investigated in several cell-based
assays that monitor biological signaling, for example, the Wnt
and Hedgehog pathway, or metabolic processes such as
autophagy and glucose uptake. The indomorphans proved to
be inhibitors of glucose uptake in HCT116 cells, with the most
potent compounds showing IC50 values in the very low
nanomolar range (see below). Cancer cells meet their
increased demand for metabolic energy by increasing glucose
uptake, accelerated glycolysis, and conversion of pyruvate
into lactate, even in the presence of oxygen (this phenomenon
is termed the Warburg effect).[17] To this end, tumors
frequently upregulate facilitative, high-affinity glucose trans-
porters (GLUTs). Several small molecules have been report-
ed to inhibit glucose transporters (for a review, see Granchi
et al.[18]). However, many of them displayed low potency or
selectivity. Very recently BAY-876, a potent and GLUT-1-
selective inhibitor was developed by Bayer.[19] However, both
GLUT-1 and GLUT-3 are upregulated in various cancers[20]
and GLUT-3 expression can increase upon radio-chemo-
therapy or primary surgery. Therefore, cells overexpressing
GLUT-3 may possess a survival advantage and may be
clonally selected.[21] Moreover, induction of GLUT3 expres-
sion has been reported to be a protective mechanism against
hypoglycemia in neurons,[22] and cancer cells respond sim-
Investigation of the structure-activity relationship (SAR)
profile of the indomorphan scaffold revealed that potent
analogues were obtained when small groups, such as hydro-
gen, methyl, or acetyl, were used at the R1 position (Table 1,
entries 1–4). Even a PEG-based linker introduced at R1
afforded a highly potent compound (entry 5). Substitution
at the benzene ring (R2) strongly reduced the activity,
particularly with substitution at position 5, but potency was
maintained when a hydroxy group was introduced (entries 6–
9). Alkylation of the indole nitrogen atom (R3) was essential
for activity (entries 10–17). Derivatives with an ester func-
tional group embedded in the substituent yielded the most
potent compounds, whilst amides or a carboxylic acid resulted
in a loss of potency. A one-carbon linker to the ester group
was found to be optimal (entries 16 and 17). These findings
show that an ester group is best, although not absolutely
necessary, for the highest activity. For the ethyl ester and the
carboxylic acid we calculated the logP values to be 3.4 and
2.3, respectively. Moreover, calculation of the pharmacoki-
netic properties of the acid and the ethyl ester revealed
similar predicted permeability in Caco2 cells, namely, 1.12
and 1.08 (logPapp values in 10ꢀ6 cmsꢀ1) using the pkCSM
tool[41] and 38.9 and 23.0 using the preADMET tool.[42] These
data indicate that cell penetration by both the ester and the
carboxylic acid is similar, such that the activity of the acid, if at
all, represents only to a minor extent the possible hydrolysis
of the ethyl ester in cellulo. This finding is further supported
by the activity recorded for the tert-butyl ester and the amide,
both of which would be hydrolyzed only slowly or not at all.
Nevertheless, we cannot fully rule out that the ethyl ester may
represent a prodrug. To explore whether other substituents
may be tolerated, furanylmethl derivative 5o (Table 1,
entry 15) was synthesized. This analogue inhibited glucose
import with an IC50 value of 6.6 ꢁ 2.4 mm. This heterocycle is
not a full replacement of the ester, but the result indicates that
other groups are tolerated at this site. Acylation of the
morphan nitrogen atom (R4) was essential for the inhibition
of 2DG uptake (entries 18–25). Activity in the low micro-
molar range was observed for several heterocycles and more
potent analogues carried a functionalized nicotinate group.
However, over-functionalization of the pyridine ring was
disadvantageous for the activity, as exemplified by the
introduction of a CF3 group at positions 4 and 5 (entries 24
and 25).
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Table 1: Structure-activity relationship analysis for the indomorphan class (see Table S1 for further details).
Entry
Compound
R1
R2
R3
R4
IC50 [mm][a]
1
2
3
4
5
6
7
8
(ꢁ)-Glupin (5a)
Me
Ac
H
H
H
H
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
H
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
2-(methylthio)pyridin-3-yl
N-morpholino
0.055ꢁ0.017
0.053ꢁ0.023
0.13ꢁ0.05
0.087ꢁ0.015
0.083ꢁ0.026
0.10ꢁ0.06
1.4ꢁ0.2
5b
5c
5d
5e
5 f
5g
5h
5i
H
nPr
PEG*[b]
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
5-OH
5-Cl
7-Cl
5-CO2Et
H
H
H
H
H
H
H
H
H
H
H
H
H
0.21ꢁ0.09
>30
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
5j
5k
5l
5m
5n
5o
5p
5q
5r
>30
t
CH2CO2 Bu
0.19ꢁ0.06
0.093ꢁ0.035
1.45ꢁ0.6
10.0ꢁ4.0
6.7ꢁ2.4
n
CH2CO2 Pr
CH2CONHMe
CH2CO2H
furan-2-ylmethyl
CO2Et
(CH2)2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CO2Et
28ꢁ2
8.4ꢁ3.6
3.16+ ꢁ0.6
3.4ꢁ0.3
5s
5t
2-furyl
pyridine-3-yl
2-fluoropyridin-3-yl
2-methoxypyridin-3-yl
2-trifluoromethylpyridin-3-yl
2-(methylthio)-4-(trifluoromethyl)pyridin-3-yl
2-(methylthio)-5-(trifluoromethyl)pyridin-3-yl
2.3ꢁ0.8
5u
5v
5w
5x
5y
0.54ꢁ0.16
0.23ꢁ0.05
0.12ꢁ0.03
6.7ꢁ1.6
H
H
H
14ꢁ4
[a] IC50 values determined for the inhibition of 2DG uptake in HCT116 cells. Data are mean values (Nꢂ3 independent experiments, nꢂ3 independent
replicates). Error represents mean ꢁS.D. [b] PEG*: (2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamic acid.
with the same functional groups as the active compound 5a
(Table 1, entry 1). Notably, the inhibiting activity of 2-DG
uptake was not shared by the indolylethyl fragment, which
lacks the morphan scaffold, or by the bicyclic ketomorphan
fragment, which lacks the indole ring. These results suggest
that the observed biological activity of the indomorphan
scaffold is not shared by the individual NP fragments and is,
therefore, a property of the new pseudo-NP scaffold.
Based on the potency and structure, compound 5a,
termed (ꢁ)-Glupin (from glucose uptake inhibitor; Table 1,
entry 1), was selected for further analysis. (ꢁ)-Glupin showed
an IC50 value of 5 ꢁ 1 nm in separate, non-automated deter-
minations on HCT116 cells and also reduced the uptake of
Figure 2. Structure and activity of the Glupin-based fragment ana-
2DG in the highly glycolytic human breast cancer cell line
MDA-MB-231 (IC50 = 17 ꢁ 5 nm; Figure 3a).[26] This finding
was further confirmed by the inhibition of tritium-radiola-
beled 2DG uptake by (ꢁ)-Glupin with an IC50 value of 8 ꢁ
3 nm (Figure 3b). (ꢁ)-Glupin also potently suppressed 2DG
uptake in HeLa (IC50 = 22 ꢁ 2 nm) and CHO cells (IC50 = 4 ꢁ
3 nm). (ꢁ)-Glupin enantiomers were separated by prepara-
tive chiral HPLC, and the absolute configuration was assigned
logues 6 and 7. Red dashed lines show the fragments missing from
the indomorphan core, with R1 to R4 overall shown as in Glupin.
To investigate the contribution of the indole and morphan
fragments on the bioactivity and to determine whether the
novel fragment combination is required, we prepared indo-
morphan fragments 6 and 7 (Figure 2) which were decorated
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Figure 3. Inhibition of 2DG uptake by Glupin. a) (ꢁ)-Glupin inhibition of 2DG uptake in MDA-MB-231 and in HCT116. Data are mean values
3
ꢁSD (N=3, n=3). b) (ꢁ)-Glupin inhibition of 3H-2DG uptake in MDA-MB-231 cells. Cells were treated with compound or DMSO and 1 mCi H-
2DG for more than 30 min. Data are mean values ꢁSD (N=3, n=3). c) Glupin is the more potent enantiomer. 2DG uptake was measured in
MDA-MB-231 cells. Data are mean values ꢁSEM (n=3). d) Structures of (+)-Glupin (=Glupin) and the (ꢀ)-enantiomer. e) The 2DG uptake in
the presence of different amounts of Glupin was measured in a time- and concentration-dependent manner in MDA-MB-231 cells and then
analyzed by nonlinear regression using the Michaelis–Menten equation to determine the Vmax and KM,app values. f) Influence of Glupin on
glycolysis. Glucose-starved MDA-MB-231 cells were treated with Glupin and the extracellular acidification rate (ECAR) was measured over time in
a Seahorse XFp Extracellular flux analyzer. Data are mean values ꢁSD (n=3).
by means of chiral derivatization and independent synthesis
of reference compounds (see the Supporting Information for
details). The more potent (+)-(2S, 5S, 6R)-Glupin enantiomer
inhibits 2DG uptake in MDA-MB-231 cells with IC50 = 4 ꢁ
2 nm, while (ꢀ)-(2R,5R,6S)-Glupin displays an IC50 value of
335 ꢁ 47 nm (Figure 3c,d). (+)-Glupin (hereafter referred to
as “Glupin”) did not inhibit the downstream hexokinase, thus
demonstrating direct interference with glucose uptake (see
Figure S7b).
Kinetic measurements in MDA-MB-231 cells showed that
the maximum rate (Vmax) decreased, whereas the KM value
increased as the concentration of Glupin increased, thus
indicating a mixed type of inhibition (Figure 3e). Investiga-
tion of the influence of Glupin on the extracellular acidifica-
tion rate (as a measure of glycolysis) and the oxygen
consumption rate (as a measure of oxidative phosphoryla-
tion) of MDA-MB-231 cells revealed that 1 mm Glupin
inhibited glycolysis to the level of glucose-starved cells
(Figure 3 f) and increased the oxygen consumption rate
(Figure S8a). Moreover, treatment of MOLT16 cells with
50 nm (ꢁ)-Glupin reduced the levels of metabolites linked to
glycolysis (glycerol-3-phosphate, lactic acid), the tricarboxylic
acid (TCA) cycle (citric/isocitric acid), the pentose phosphate
pathway (ribitol/arabitol), and the hexosamine biosynthetic
pathway (UDP-N-acetylglucosamine), which all utilize glu-
cose-6-phosphate (Table S3). The level of aspartate, which
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Figure 4. Target engagement of GLUT-1 and GLUT-3 by Glupin. The cellular thermal shift assay was performed with lysates from SW480 cells in
the presence of 1 mm Glupin or DMSO using mass spectrometry (a) or immunoblotting (b) to detect GLUT-1 or GLUT-3 and Na+/K+-ATPase as
a control. Data of two biological replicates (a) or mean values ꢁSD and representative of n=3 (b) are shown. c) Uptake of 2DG in DLD-1 versus
DLD-1 GLUT1 (ꢀ/ꢀ) cells after incubation with the compound for 30 min. d) Uptake of 2DG in CHO cells that ectopically express GLUT-
1 compared to cells transfected with an empty vector (mock). e) Uptake of 2DG in CHO cells that ectopically express GLUT-3 compared to cells
transfected with an empty vector (mock). All data are mean values ꢁSD (n=3).
can fuel the TCA cycle, was elevated upon treatment with
Glupin, most likely as a compensatory mechanism.[27] These
results are in agreement with inhibition of glucose uptake by
Glupin and, consequently, the synthesis of glucose-6-phos-
phate.
In the absence of glucose, cells may utilize alternative
energy sources and building blocks to sustain cell growth, in
particular fatty acids, and altered lipid storage and metabo-
lism is strongly connected with cancer malignancy.[28] Treat-
ment of MDA-MB-231 cells with Glupin in the presence of
400 mm oleic acid markedly reduced the number of lipid
droplets with an IC50 value of 84.9 nm (Figure S8b). Pre-
formed lipid droplets were not affected, thus suggesting that
Glupin has no stimulating effect on lipolysis (Figure S8c).
Target engagement was demonstrated by means of
a proteome-wide cellular thermal shift assay (CETSA,
thermal proteome profiling, TPP)[29] using SW480 cells, which
revealed thermal stabilization of GLUT-3 (DTm = 7.1 ꢁ
0.48C) and GLUT-1 (DTm = 1.9 ꢁ 0.38C) in the presence of
1 mm Glupin (Figure 4a). No further members of the GLUT
family could be detected. A separate CETSA experiment
using immunoblotting (Figure 4b) confirmed this finding and
showed thermal stabilization of GLUT-1 (DTm = 6.5 ꢁ 0.68C)
and GLUT-3 (DTm > 128C).
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Figure 5. Inhibition of cancer cell growth by Glupin. a–c) The growth of MDA-MB-231 cells in 25 mm (a) or 5 mm glucose (b) was monitored in
the presence of Glupin or DMSO by means of live-cell kinetic analysis and confluence as a measure. Data are mean values (N=3) ꢁSD and
representative of n=3. c) Comparison of cell growth at 5 and 25 mm glucose. The area under the curve (AUC) was calculated for the curves in (a)
and (b). d) Glupin inhibits the growth of various cancer cell lines. 94 cell lines were treated with different concentrations of Glupin for 72 h
followed by the sulforhodamine B assay and IC50 determination.
DLD-1 GLUT1 (ꢀ/ꢀ) cells do not express GLUT-1 but
exhibit substantial expression of GLUT-3 (Figure S9).[4b] In
contrast, the isogenic parental cell line DLD-1 expresses
mainly GLUT-1 (Supporting Figure S9).[4b,30] Glupin inhibited
the uptake of 2DG in both cell lines with IC50 values of 59.6 ꢁ
8.4 nm for DLD-1 and 11.4 ꢁ 1.6 nm for DLD-1 GLUT1 (ꢀ/ꢀ;
Figure 4c). This finding confirms that Glupin targets both
GLUT-1 and GLUT-3 and indicates that Glupin inhibits
GLUT-3 more strongly than GLUT-1. Ectopic expression
and, thereby, increased abundance of GLUT-1 or GLUT-3 in
CHO cells[4b] (Figure S10a,c), led to a partial rescue of the
Glupin-mediated inhibition of glucose uptake as detected by
the increase in the IC50 values from 19.2 ꢁ 2.1 nm to 162 ꢁ
14 nm for GLUT-1 overexpression (Figure 4d) and from
19.1 ꢁ 2.9 nm to 68 ꢁ 7.2 nm for GLUT-3 (Figure 4e). In
contrast, overexpression of GLUT-2 or GLUT-4 did not have
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any influence on the activity of the compound (Figur-
syntheses of natural product hybrids, that is, NP-like com-
es S10b,d and S11 as well as Table S4). These results suggest
that Glupin interacts with GLUT-1 and GLUT-3 but not with
GLUT-2 and GLUT-4.
pounds which are composed of different NP substructures and
may be considered pseudo-NPs if they represent pseudo-NP
combinations that match our concept.[4b,5] In attempts to
generate potential alkaloid types which might not have been
identified in nature at the time, Morrison et al.[36] replaced
3,4-dihydroxyphenylalanine by tryptophan in a synthesis
following the biogenetic route leading to the morphine
scaffold. Bosch et al. (see Ref. [37] and references therein)
developed heteromorphans, that is, compounds in which the
benzene ring in 6,7-benzomorphans is replaced by a hetero-
cyclic ring, including an indomorphan analogue. “Unnatural”
polyketide natural products have been obtained by means of
synthetic biology approaches, for example, through precursor-
directed biosynthesis, mutasynthesis, or combinatorial bio-
synthesis (for reviews see Ref. [38]), and natural and non-
natural structural elements have been combined in polyketide
syntheses to yield natural product analogues not accessible by
biosynthetic pathways (see for example, Ref. [39]).
The indomorphan pseudo-NPs define a novel glucose
uptake inhibitor class with unprecedented chemotype. The
most potent compound Glupin selectively targets glucose
transporters GLUT-1 and -3 and potently inhibits the growth
of cancer cells. Our results demonstrate that inhibition of
GLUT-1 and GLUT-3 may be necessary to efficiently inhibit
the growth of cancer cells since both glucose transporters
GLUT-1 and GLUT-3 may be upregulated upon withdrawal
of glucose.[22,23,32] Dual GLUT-1- and GLUT-3-selective
compounds such as Glupin would inhibit both transporters.
Indeed, treatment with Glupin increased the expression of
both GLUT-1 and GLUT-3 in DLD-1 cells, which primarily
express GLUT-1. Upregulation of GLUT-3 upon glucose
deprivation also ensures the uptake of glucose at lower
glucose concentrations in the cell environment compared to
GLUT-1 because of its higher affinity for glucose.[40] Thus, in
particular, the overexpression of GLUT-3 may pose a hurdle
for the efficacy of GLUT-1-selective agents and emphasizes
the need to simultaneously target both proteins. These
findings may inspire new drug discovery programs aimed at
the modulation of tumor metabolism.
Real-time analysis of the growth of the MDA-MB-231 cell
line in the presence of Glupin showed a dose-dependent
suppression of cell growth in plain medium containing 25 mm
glucose (Figure 5a). This effect was much stronger at
a physiological glucose concentration (5 mm; Figure 5b,c).
Exposure of the compound to more than 90 cancer cell lines
and IMR-90 and PBMC cells as noncancerous controls (see
Figure 5d and Table S5) revealed that the growth of the
bladder cancer cell line UM-UC-3 (IC50 = 32 nm), the pan-
creas cancer cell line MIA PaCa-2 (IC50 = 61 nm), the
lymphoma cell lines WSU-NHL and SU-DHL-6 (IC50
=
62 nm and 92 nm, respectively), and the neuroblastoma cell
line SK-N-SH (IC50 = 84 nm), which depend on glucose for
cell growth,[31] was potently inhibited. IC50 values lower than
300 nm were obtained for more than 20 cell lines, whereas 24
cell lines were not affected by the compound, among them the
peripheral blood mononuclear control cells (PBMCs). Sensi-
tivity to Glupin was detected across various tissue types
(Table S6). These results indicate that targeting glucose
import by GLUT-1 and GLUT-3 may be a viable approach
to inhibit the growth of cancer cells.
Since glucose depletion may lead to increased expression
of GLUT-1 and GLUT-3,[22,23,32] we analyzed the influence of
Glupin on the expression of GLUT1 (SLC2A1) and GLUT3
(SLC2A3). The application of Glupin for 48 h induced only
a slight increase in the expression of both genes at the mRNA
level (Figure S12). However, treatment with 0.5 mm Glupin
significantly increased GLUT-1 levels after 24 h of treatment
(Figure S13a,b) and of GLUT-3 levels after 48 h of treatment
(Figure S13c,d). These results indicate that Glupin-mediated
inhibition of glucose uptake increases the levels of both
GLUT-1 and GLUT-3 in DLD-1 cells.
Discussion
Our results demonstrate that the combination of natural
product (NP) fragments in unprecedented arrangements may
yield biologically relevant pseudo-NP collections with both
novel NP-inspired structure and novel bioactivity. The
indomorphan pseudo-NPs retain the characteristic structural
elements of the guiding NPs but reside in an area of chemical
space which is inaccessible to nature, reflected by the fact that
they are not obtainable by known biosynthetic pathways. This
novel, and structurally complex scaffold may be endowed by
design with advantageous physiochemical properties, as the
pseudo-NP collection displays a NP-score distribution closer
to that of approved drugs.
The term “pseudo-natural product” and reasoning related
to the concept have inspired synthesis efforts before. Thus,
Suga and co-workers used the term to describe in vitro
synthesized cyclic peptides embodying non-natural amino
acids,[33] and Oshima and co-workers qualified alkaloid-like
compounds obtained by the interception of biosynthetic
pathways as pseudo-NPs.[34] Tietze et al.[35] reviewed the
Acknowledgements
Research at the Max Planck Institute of Molecular Physiology
was supported by the Max Planck Society and the European
Research Council under the European Unionꢀs Seventh
Framework Programme (FP7/2007–2013)/ERC Grant agree-
ment no. 268309. M.S., J.C., E.S.R., and J.W. acknowledge the
International Max-Planck Research School for a doctoral
scholarship. G.K. and L.L. received funding from the
Alexander von Humboldt Stiftung for a postdoctoral fellow-
ship; M.C.C. received funding from the Fonds der chemischen
Industrie (Kekulꢁ fellowship) and the Swiss National Science
Foundation (Doc.mobility). M. Sellstedt received funding
from the Swedish Research Council, grant number 350-2012-
6611. The compound management and screening center
(COMAS) Dortmund is acknowledged for performing the
high-throughput screening and the orthogonal and counter
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Research Articles
Chemie
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acknowledged for performing the cell panel analysis. We are
grateful to Andreas Brockmeyer (Max Planck Institute of
Molecular Physiology) for assistance during MS/MS measure-
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is acknowledged for access to, and assistance with, mass
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Conflict of interest
The development of glucose transport inhibitors is an actively
pursued drug discovery project at the Lead Discovery Center
GmbH (LDC) of the Max Planck Gesellschaft, sponsored by
H.W.
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Manuscript received: July 29, 2019
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Version of record online: && &&
,
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&&&&
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Research Articles
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Research Articles
Antitumor Agents
Combination therapy: Indomorphan
pseudo-natural products (pseudo-NPs)
have been synthesized that combine
biosynthetically unrelated indole and
morphan alkaloid fragments. This new
glucose uptake inhibitor chemotype
selectively targets and upregulates the
glucose transporters GLUT-1 and GLUT-3
and inhibits the growth of cancer cells.
J. Ceballos, M. Schwalfenberg,
G. Karageorgis, E. S. Reckzeh, S. Sievers,
C. Ostermann, A. Pahl, M. Sellstedt,
J. Nowacki, M. A. Carnero Corrales,
J. Wilke, L. Laraia, K. Tschapalda,
M. Metz, D. A. Sehr, S. Brand,
K. Winklhofer, P. Janning, S. Ziegler,
H. Waldmann*
&&&& — &&&&
Synthesis of Indomorphan Pseudo-
Natural Product Inhibitors of Glucose
Transporters GLUT-1 and -3
&&&&
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These are not the final page numbers!