Design of antitumor agents containing carbohydrate based on GLUT1, and evaluation of antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2017.03.094

A series of novel carbohydrate-modified antitumor compounds were designed based on glucose transporter 1 (GLUT1), and evaluated for their anticancer activities against four cancer cell lines. The ribose derivatives (compound 9 and 10) exhibited modest inh

Full text of DOI:10.1016/j.bmcl.2017.03.094

Accepted Manuscript
Design of Antitumor Agents Containing Carbohydrate Based on GLUT1, and
Evaluation of Antiproliferative Activity
Renshuai Zhang, Lairong Song, Bo Jiang, Lijun Wang, Ning Wu, Shuju Guo,
Dayong Shi
PII:
S0960-894X(17)30359-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.094
BMCL 24846
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
22 February 2017
14 March 2017
31 March 2017
Please cite this article as: Zhang, R., Song, L., Jiang, B., Wang, L., Wu, N., Guo, S., Shi, D., Design of Antitumor
Agents Containing Carbohydrate Based on GLUT1, and Evaluation of Antiproliferative Activity, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.094
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Design of Antitumor Agents Containing Carbohydrate Based on GLUT1, and
Evaluation of Antiproliferative Activity
§
, a, b
§, d
a, b
a, b
a, b
Renshuai Zhang,
Lairong Song, Bo Jiang, Lijun Wang, Ning Wu, Shuju
a, b
, a, b, c
Guo, Dayong Shi*
a
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese
Academy of Sciences, Qingdao, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for
b
Marine Science and Technology, Qingdao, China.
c
University of Chinese Academy of Sciences, Beijing, China.
Department of Neurosurgery, The Affiliated Hospital of Qingdao University,
d
Qingdao, China.
§
These authors contributed equally to this study
*
Corresponding author: Tel. +86-0532-8289-8719; Fax: +86-0532-8289-8741: E-Mail:
shidayong@qdio.ac.cn.
Abstract
A series of novel carbohydrate-modified antitumor compounds were designed
based on glucose transporter 1 (GLUT1), and evaluated for their anticancer activities
against four cancer cell lines. The ribose derivatives (compound 9 and 10) exhibited
modest inhibitory activity. The compound 9 significantly inhibited the migration of
A549 cell and induced A549 cell apoptosis in a concentration-dependent manner.
Moreover, compound 9 blocked A549 cells at the G0/G1 phase. The cellular uptake
studies suggested that ribose-modified compound 9 could be taken through GLUT1 in
A549 cell line.
Key word: saccharide; GLUT1; drug design; anticancer; cell uptake
Carbohydrate-based drug development is a highly promising area in contemporary
medicine. With the development of glycobiology, many biological functions of
1
-3
carbohydrates have been recognized. The studies of carbohydrate drugs have also
been paid more and more attention, and carbohydrates are also considered to be one
of the important sources of current drug-discovery leads. Carbohydrate drugs are
saccharide-containing drugs in narrow sense, including glycoside compounds (also
known as carbohydrate-modified drugs, such as Erythrimicin A and Rebeccamycin et
4
, 5
al)
and some compounds without other ingredients except carbohydrates (also
named as carbohydrate-based drugs, such as Heparin Pentasaccharide, Topiremate and
6
-8
Therafectin et al). Carbohydrate structure is essential for activity in the vast
majority carbohydrate-modified drugs.
Here, we reported series of carbohydrate-modified compounds containing a
dibromo substituted benzene ring which was derived from the red alga Rhodomela

9
confervoides (Fig 1). The conformation flexibility of carbohydrates and their polyol
1
0
structures give them many unique biological properties as we have described earlier.
More interestingly, the presence of sugar molecules may increase the ability of
compounds to enter cells through glucose transporter 1 (GLUT1) which is the most
1
1
common glucose transporter, and widely overexpress in many human cancers. The
length of the linker joining saccharide and aglycone might affect the uptake of
glycosyl-modified compounds, thus, docking was firstly performed for selecting
appropriate linker. Because of the inward open configuration of GLUT1 (PDB: 4PYP),
the design of compounds was guided by the xylose transporter XylE (PDB: 4GBZ),
which was a GLUT1 homolog with an outward open configuration that compounds
1
2, 13
would encounter when attempting to enter the cell.
The characterize function data
of compounds 1-3 and the H-bonds interaction in docking simulation were shown in
Fig 2. The total-score of compounds 1-3 suggested that the compounds 2 and 3
displayed similar affinity, which were much higher than that of compound 1. The
Gln168, Gln289, Asn294 and Typ392 were involved in the formation of H-bonds of
compound 2 complex, which was consistent with that between glucose (original
ligand) and XylE (Fig S1). In contrast, only the hydroxy groups on C2 and C4 of
D-glucose were involved in hydrogen-bonding interactions with Gln288 and Trp392
in the complex of compound 3 and XylE. Thus, the aliphatic chain containing three
carbon atoms was identified as the linker for target glycoside. Additionally,
glucosamine, xylose and ribose were also used in the design of glycoside compounds
(
Fig 1) based on GLUT1 could transport a variety of carbohydrates except for
1
4
glucose.
Figure 1. The structures of target compounds

Figure 2. Structures of compounds 1-3; the docking data including total-score, crash
and polar; docking models predict binding of 1-3 to xylose symporter XylE (PDB
4
GBZ).
The synthesis of target compounds was described in supplementary information.
All the synthesized compounds were investigated for their anticancer activity in vitro
on four human cancer cell lines (Fig 3). An interesting phenomenon was observed that
only ribose derivatives (compound 9 and 10) exhibited modest inhibitory activity
which was comparable to positive control (gefitinib), while glucose, glucosamine or
xylose modified compounds showed no activity against four cancer cell lines. The
IC values of compounds 9 and 10 in each of the four cell lines were calculated by
5
0
testing each compound in a series of concentrations (Table S1). The compounds 9 and
0 showed almost the same cytotoxicities in each cell line tested, and which were
comparable to gefitinib. Although the micromolar grade IC values were considered
1
5
0
to be undesirable in cytotoxic activity studies, compound 9 did show higher activity
2-fold) than that of gefitinib against A549. Thus, the compound 9 was used for
(
further studies of the antiproliferative mechanism.
Figure 3. Inhibitory activity of compounds (2, 4-10, 14 and gefitinib as positive
control) against four human cancer cell lines at 24 µM.
Cancer cell invasion plays a critical role during the carcinomas arising from
1
5
epithelial tissues progressed to higher pathological grades of malignancy. To

investigate the anti-migration effects of compound 9, a wound healing assay was
1
6
performed (Fig 4). Additionally, compound 9 was also examined the effects on
apoptosis and on the cell cycle in A549 cell line using flow cytometry analysis (Fig 5，
1
7
6
). The migration of A549 cell was inhibited by 48 h treatment with 9 at 8, 16 µM
concentration compared to the untreated control, and the relative mobility was
concentration dependent. Moreover, the compound 9 could induce the A549 cell
apoptosis, and the apoptosis rates increased significantly with the increase of the
concentration of the compound 9 (Fig 5). Furthermore, after incubation with 8 µM, 16
µM and 24µM compound 9 for 48h, the DNA contents of the A549 cells were 59.17%,
7
6.57% and 89.56% in G0/G1 phase, and were 40.83%, 23.44% and 10.25% in S
phase, respectively. Apparently, the newly synthesized ribose-modified compound 9
blocked A549 cell at the G0/G1 phase in a concentration-dependent manner (Fig 6).
As it is widely agreed that the synthesis of RNA occurs in G1 phase and which also
prepares for the synthesis of proteins. The ribose is one of materials for RNA
synthesis, so ribose-modified compound 9 may be erroneously identified to interfere
with RNA synthesis and block cells in G0/G1 phase, which was different from the
action of traditional antitumor drug cisplatin (inhibit DNA replication, arrests the cell
1
8, 19
cycle at the S phase).
The result (cell arrest assay) may be used to explain why
other compounds showed no activity against four cancer cell lines.
Figure 4. The relative migration rate of A549 cells in the presence of compound 9

Figure 5. The apoptosis rate induced by compound 9 in A549 cell line
Figure 6. Effects of compound 9 on A549 cell cycle arrest
Prior to cellular uptake studies of compound 9, docking simulation was performed
(Fig 7A). The result suggested this complex was capable of binding in the cavity of an

outward open XylE. The orientation of the sugar moiety in the docked complex was
similar to that of the glucose unit bound in the crystal structure, and the same residues
(Gln168, Asn294 and Trp392) were identified in the hydrogen-bonding interactions
(Fig S1, 7A). Moreover, all hydroxy groups of ribose were directly involved in the
H-bonds interaction with XylE. Next, the cellular uptake mechanism of compound 9
20
was evaluated in A549 cell line which had high level of GLUT1 expression. Cell
uptake of compound 14 (the aglycone of compound 9) in A549 was not detected by
HPLC, possibly due to the low level of uptake (Fig S11). On contrast, the
ribose-based compound 9 shown 60.3% cell uptake rate (Fig S2), suggested that the
presence of ribose increased the cellular uptake. Considering D-glucose was the main
substrate of glucose transporter, it should compete with and inhibit the glucose
transporter-mediated uptake of compound 9. Different concentrations of D-glucose
were used to detect their effect on cell uptake. The result suggested that D-glucose
exhibited a weak inhibitory effect on the uptake of compound 9, but still with
dose-dependent
(Fig
7B).
Furthermore,
the
GLUT1
inhibitor
4
,6-Oethylidene-α-D-glucose (EDG) was used to monitored its effect on cell uptake.
2
1
The result was presented in Fig 7C. A 30% reduction in cell uptake of 9 was
measured in the presence of 50 mM EDG, and with the increase of EDG
concentration (to 100 mM), a 43% decrease in the cellular uptake was observed. The
inhibition of D-glucose on cell uptake, as that of EDG showed that GLUT1 played an
important role in cell uptake of ribose-modified compound 9 in A549 cell line.
Figure 7. A: The bonding mode of compound 9 within XylE; B: Effect of externally
added D-glucose on the cellular uptake; C: Effect of GLUT1 inhibitor EDG on the
cellular uptake.
In summary, series of carbohydrate-modified antitumor compounds were designed
based on GLUT1. The ribose-modified compound could induce cell apoptosis and
arrest the cell cycle at the G0/G1 phase. The key role of GLUT1 was demonstrated in

cell uptake of antitumor agents containing ribose, which had important implications in
the research and development of carbohydrate-modified drugs.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
4
1506169), and the Key research and development project of Shandong province
(2015GSF115029, 2016ZDJS07A13, 2016GSF201193), and STIP Supported by
QNLMST (No. 2015ASKJ02), and Strategic Biological Resources Service Network
Programme of CAS (ZSTH-026), and Aoshan Talents Program (NO. 2015ASTP).
Supplementary data
Supplementary data associated with this article can be found in supporting
information.
References and Notes
1
2
3
4
5
6
. R. A. Dwek, Chem Rev 1996, 96, (2), 683-720.
. P. Sears; C. H. Wong, Proc Natl Acad Sci U S A 1996, 93, (22), 12086-93.
. M. von Itzstein; P. Colman, Curr Opin Struct Biol 1996, 6, (5), 703-9.
. A. L. Smith; K. C. Nicolaou, J Med Chem 1996, 39, (11), 2103-17.
. M. Prudhomme, Curr Med Chem 2000, 7, (12), 1189-212.
. M. Petitou; P. Duchaussoy; I. Lederman; J. Choay; P. Sinay; J. C. Jacquinet; G. Torri, Carbohydr
Res 1986, 147, (2), 221-36.
7
8
9
. R. P. Shank; J. F. Gardocki; A. J. Streeter; B. E. Maryanoff, Epilepsia 2000, 41 Suppl 1, S3-9.
. J. M. Goldsmith; J. Huprikar; S. J. Wu; J. P. Phair, J Immunopharmacol 1986, 8, (1), 1-14.
. X. Fan; N. J. Xu; J. G. Shi, Journal of Natural Products 2003, 66, (3), 455-458.
1
4
1
1
1
1
1
1
0. R. Zhang; S. Chen; X. Wang; R. Yu; M. Li; S. Ren; T. Jiang, Carbohydrate Research 2016, 429,
8-53.
1. L. Szablewski, Biochim Biophys Acta 2013, 1835, (2), 164-9.
2. L. Sun; X. Zeng; C. Yan; X. Sun; X. Gong; Y. Rao; N. Yan, Nature 2012, 490, (7420), 361-6.
3. D. Deng; C. Xu; P. Sun; J. Wu; C. Yan; M. Hu; N. Yan, Nature 2014, 510, (7503), 121-5.
4. M. Uldry; M. Ibberson; M. Hosokawa; B. Thorens, FEBS Lett 2002, 524, (1-3), 199-203.
5. D. Hanahan; R. A. Weinberg, Cell 2011, 144, (5), 646-74.
6. J. W. Astin; J. Batson; S. Kadir; J. Charlet; R. A. Persad; D. Gillatt; J. D. Oxley; C. D. Nobes, Nat
Cell Biol 2010, 12, (12), 1194-204.
1
2
1
1
2
7. K. C. Kawabata; S. Ehata; A. Komuro; K. Takeuchi; K. Miyazono, Oncogene 2013, 32, (16),
096-106.
8. Z. Wang; Z. Xu; G. Zhu, Angewandte Chemie International Edition 2016, 55, (50), 15564-15568.
9. G. I. Shapiro; J. W. Harper, J Clin Invest 1999, 104, (12), 1645-53.
0. M. Patra; T. C. Johnstone; K. Suntharalingam; S. J. Lippard, Angew Chem Int Ed Engl 2016, 55,
(
7), 2550-4.
1. J. E. Barnett; G. D. Holman; R. A. Chalkley; K. A. Munday, Biochem J 1975, 145, (3), 417-29.
2