Identification of a new bis-amino acid glycoside selectively toxic to multiple myeloma cells

Article abstract of DOI:10.1016/j.carres.2014.05.014

A bis-triazolyl phenylalaninyl galactoside was synthesized by a two-fold click reaction between an azido phenylalanine and a di-O-propynyl galactoside. By a cytotoxicity assay the compound was determined to be selectively toxic for multiple myeloma (MM) among a series of cancer cell lines with no toxicity to a control cell line. A Western blot analysis suggested that this compound could potentiate the cleavage of poly ADP-ribose polymerase in MM cells, leading to apoptosis.

Full text of DOI:10.1016/j.carres.2014.05.014

Accepted Manuscript
Identification of a new bis-amino acid glycoside selectively toxic to multiple
myeloma cells
Chao-Ying Cheng, Yan-Yan Feng, Yi Zang, Jia Li, Xiao-Peng He, Guo-Rong
Chen
PII:
S0008-6215(14)00206-7
DOI:
http://dx.doi.org/10.1016/j.carres.2014.05.014
CAR 6753
Reference:
Carbohydrate Research
To appear in:
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Revised Date:
Accepted Date:
6 December 2013
19 May 2014
19 May 2014
Please cite this article as: Cheng, C-Y., Feng, Y-Y., Zang, Y., Li, J., He, X-P., Chen, G-R., Identification of a new
bis-amino acid glycoside selectively toxic to multiple myeloma cells, Carbohydrate Research (2014), doi: http://
dx.doi.org/10.1016/j.carres.2014.05.014
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Identification of a new bis-amino acid glycoside selectively toxic to
multiple myeloma cells
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Chao-Ying Cheng,1 Yan-Yan Feng,1 Yi Zang,*2 Jia Li, Xiao-Peng He,*1 Guo-Rong Chen1
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1 Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University
of Science and Technology, 130 Meilong Rd, Shanghai 200237, PR China
2 National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
Corresponding authors. Fax: 86-21-64252758; Tel: 86-21-64253016
E-mail: yzang@mail.shcnc.ac.cn (Y. Zang), xphe@ecust.edu.cn (X.-P. He)
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Abstract
A bis-triazolyl phenylalaninyl galactoside was synthesized by a two-fold click reaction
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between an azido phenylalanine and a di-O-propynyl galactoside. By a cytotoxicity assay the
compound was determined to be selectively toxic for multiple myeloma (MM) among a
series of cancer cell lines with no toxicity to a control cell line. A western blot analysis
suggested that this compound could potentiate the cleavage of poly ADP-ribose polymerase
in MM cells, leading to apoptosis.
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Keywords: Click chemistry, Sugar, Amino acid, Cytotoxicity, Multiple Myeloma
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1. Introduction
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Sugars are key participants implicated in many physiological events such as energy storage,
cell-cell adhesion, cell growth and proliferation and intercellular signal transductions.1-6 They
also structure a number of bioactive natural products, and some pharmacological
investigations suggest that the sugar moiety is essential for both the activity and
biocompatibility of these natural products.7,8 In addition to these favorable attributes, sugars
have been used as a rigid framework upon which to display diverse pharmacophores in a
sterically defined manner.9 For example, Hirschmann et al. reported the preparation of some
sugar-based cyclic peptide (somatosatin) mimetics with sub-micromolar affinity for the
human somatostatin receptor 4.10,11 Iminosugars modified with lipid chains were synthesized
by Ye et al. as immunosuppressants.12 We also described the development of triazolyl
glycolipids that could suppress the growth of cancer cells13,14 and mono- or bidentate
glycosyl amino acids as protein tyrosine phosphatase inhibitors.15-19 The synthetic tool we
used to build these sugar-based bioactive compounds was the copper(I)-catalyzed
azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction,20,21 the most celebrated example of
“click chemistry”.22
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Despite being extensively employed in constructing bioactive compounds,23 the CuAAC
has been rarely exploited for the sugar-based drug discovery,24 a promisingly new subject in
the field of medicinal chemistry. As a continuing project as regards CuAAC-based drug
development,25-27 this research depicts the identification of a bis-triazolyl amino acid
glycoside that is selectively toxic towards multiple myeloma cells.
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2. Results and discussion
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In the course of studies on the biological activity of bis-triazolyl amino acid glycosides,28
we observed cytotoxicity of a bis-trazolyl phenylalaniny galactose (Gal23) (unpublished
observations).
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Scheme 1 Synthesis of CY01: (a) EDC, HOBt in CH2Cl2; (b) LiOH·H2O in water/MeOH (2:1,
V/V), (c) CuSO4·5H2O, sodium ascorbate in water/t-butanol (1:1, V/V).
On the basis of the observation above, here we focused on the synthesis, characterization
and biological evaluation of the hit compound. As shown in Scheme 1, CuAAC of the
di-O-propynyl Gal23 (1 equiv.)28 with the azido Phe3 (2 equiv.) in the presence of
CuSO4·5H2O and sodium ascorbate produced the desired hit compound CY01.
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Table 1
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The cytotoxicity of purified and characterized CY01 was re-evaluated using the cancerous
and normal cell lines . An MTS
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(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)2
9 assay was performed, and the viability of the cells after treatment with CY01 (50 g/mL
each) for 72 h is shown in Table 1. Interestingly, while the compound moderately suppressed
the growth of some cancerous cell lines of different nature (human MM, lung, leukemia and
cervix), it exhibited remarkable cytotoxicity against an MM cell line (RPMI 8226). In contrast,
CY01 is not toxic towards a normal cell line (mouse fibroblast).
Figure 3
As visualized by a bright-field imaging (Fig. 2a to Fig. 2c), treatment of RPMI 8266 as well
as of other two MM cell lines available (NCI-H929 and LP-1) with CY01 (100 g/mL) for 72 h
led to apparent cell death; Fig. 2d to Fig. 2f showed that cells were perfectly viable while
treated in the absence of the compound for 72 h. The half maximal inhibitory concentration
(IC50) of CY01 for RPMI 8266, NCI-H929 and LP-1 was determined to be 33.4 (± 0.2), 44.0 (±
2.8) and 71.8 (± 0.1) M, respectively.
Poly ADP-ribose polymerase (PARP) is mainly involved in DNA repair, which may serve as a
substrate of enzymes such as caspase. Cleavage of PARP produces PAR, which is a symbol of
programmed cell death (apoptosis).30 The size of cleaved PARP fragments may offer evidence
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into which enzyme participated in the cleavage, providing useful insights into the probing of
apoptosis pathways. Thus a PARP cleavage assay is commonly preliminarily conducted for
investigating apoptosis-related cell death.
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We used the western blot technique (a conventional analytical technique used to identify
specific proteins of different molecular weight based on gel electrophoresis as a key step to
separate proteins) to detect PARP cleavage in MM cells. Results indicated that CY01 could
largely potentiate the cleavage of PARP to generate PAR in 8266 cells (Fig. 2 h), suggesting
that the cell death was probably caused by an apoptotic pathway.30,31 All these data imply
that CY01 might have the potential to be employed as a lead compound for further
development of specific anti-MM drug candidates.
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3. Conclusion
In summary, this research depicted the construction of a bis-triazolyl phenylalaninyl
galactoside via a simple ‘twofold-click’ approach. The compound showed selective
cytotoxicity to MM cells among a panel of cancer cell lines, and had no influence against the
viability of a normal cell line. A western blot analysis suggested that this compound might
have the ability to induce apoptosis of MM cells. This paper would offer new insights into
the development of amino acid glycoside-based lead compounds for the MM chemotherapy.
4. Experimental section
General
All chemicals purchased were used as such. 1H NMR and 13C NMR spectra were recorded
on a Bruker AM-400 spectrometer using tetramethyl silane (TMS) as the internal standard (δ
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= 0). Chemical shifts are reported in parts per million referenced with respect to the residual
solvent (CHCl3 = 7.26 ppm and DMSO-d6 = 2.50 ppm). The following abbreviations were used
in reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd =
doublet of doublets. High resolution mass spectra (HRMS) were recorded on a Waters LCT
Premier XE spectrometer using standard conditions (ESI, 70 eV). Optical rotations were
measured using a Perkin-Elmer 241 polarimeter at room temperature and a 10 cm length
cell of a 1 mL volume. FTIR spectra were recorded on a Nicolet 380 FTIR spectrometer
(Thermo Electron Corporation, USA). The samples were mixed with KBr and then
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compressed into pellets for analysis in the spectral range of
= 4000 to 400 cm-1. All
baselines of the spectra were corrected. Analytical HPLC was measured using Agilent 1100
Series equipment with a C-18 column of a size of 0.6 mm × 250 mm × 5 μm and a flow rate
of 0.6 mL/min in 70% MeOH/30% water.
4.1. Synthesis of Phe3
The synthesis was accomplished in two sequential steps: (1) N-acetylation: To a solution of
4-azidobutanoic acid (1 equiv.) in CH2Cl2 were added EDC (1 equiv.) and HOBt (2.2 equiv.) at
0 C. After stirring for 1 h, Phe (1 equiv.) was added, and the mixture was warmed to room
temperature (rt) and stirred for another 12 h. Solvent was then removed and the resulting
residue was diluted with EtOAc and washed with water and brine. The combined organic
layer was dried over MgSO4, filtered and then concentrated in vacuum. The resulting crude
product was used directly for (2) Saponification: The azido amino acid esters (1 equiv.)
obtained was dissolved in water/MeOH (2:1, V/V), followed by addition of LiOH·H2O (3
equiv.). The mixture was stirred at 40 C for 12 h. Then solvent was removed and the
resulting residue was diluted with EtOAc and washed with water and brine. The combined
organic layer was dried over MgSO4, filtered and then concentrated in vacuum. The resulting
crude product was purified by column chromatography on silica-gel. Column
chromatography (CH2Cl2) afforded Phe3 in 2% yield. [α]D25 = +15.0 (CHCl3, concentration: 2
mg/mL); FTIR (KBr, cm-1)
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1H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.31-7.19 (m, 5H),
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4.47-4.41 (m, 1H), 3.17 (t, J = 7.0 Hz, 2H), 3.07 (dd, J = 13.8 Hz, 4.7 Hz, 1H), 2.83 (dd, J = 13.8
Hz, 10.1 Hz, 1H), 2.15-2.10 (m, 2H), 1.64 (t, J = 7.1 Hz, 2H); 13C NMR (101 MHz, MeOD) δ
175.0, 174.9, 138.7, 130.4, 129.6, 127.9, 55.0, 51.6, 38.4, 33.7, 26.1. HRMS-ESI: calc. For
C13H15N4O3 [M-H]- 275.1144, found 275.1144.
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4.2. Synthesis of CY01
To a solution of sugar alkyne Gal23 (25 mg, 0.06 mmol)28 and amino acid azide Phe3 (36
mg ,0.13 mmol) dissolved in water/t-butanol (1:1, V/V) was added CuSO4·5H2O (1 equiv.) and
sodium ascorbate (2 equiv.). The mixture was then stirred at 60 C for 12 h. Then solvent
was removed and the resulting residue was diluted with EtOAc and washed with water and
brine. The combined organic layer was dried over MgSO4, filtered and then concentrated in
vacuum. The resulting crude product was purified by column chromatography on silica-gel
(EA/ Ethanol = 3:1) to obtain CY01 as a yellow solid (12 mg, 20%). [α]D25 = +17.0 (CHCl3,
concentration: 2 mg/mL); FTIR (KBr, cm-1) 3293, 2926, 2039, 1680, 1453, 1417, 1244, 1049,
937, 700, 628. 1H NMR (400 MHz, CDCl3) δ 10.93 (s, 2H, -COOH), 8.41-6.41 (m, 24H,
benzene-H and triazole-H), 5.22-4.96 (m, 1H), 4.78 (s, 1H), 4.69-4.55 (s, 1H), 4.47-4.3 (m, 3H),
4.27-3.99 (m, 4H), 3.92-3.69 (m, 10H), 3.32-3.22 (m, 6H), 3.08-3.00 (m, 4H), 2.36 (d, J = 8.49
Hz, 1H), 2.18-2.08 (m, 1H), 2.07-1.94 (m, 2H), 1.88 (s, 2H). 13C NMR (400 MHz, DMSO-d6) δ
166.6, 135.9, 131.9, 128.6, 127.7, 123.3, 120.6, 118.3, 118.1, 111.2, 110.8, 73.0, 72.1, 69.8,
67.4, 55.6, 54.6, 50.8, 42.1, 38.0, 29.8, 28.3, 27.4, 23.2, 22.4, 13.9, 10.8. HRMS-ESI: calc. for
C53H62N8O12 [M+Na]+ 1025.4247, found 1025.4250. HPLC: tR = 3.9 min of 70% methanol/30%
water (0.6 mL/min), purity 97%.
4.3. Cell viability assay
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu
m (MTS) was purchased from Promega (Madison, WI, USA). The cell lines used were grown
in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and penicillin-Streptomycin
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from Invitrogen (Grand Island, NY, USA) at 37 C in a 5% CO2 humidified atmosphere.
For the MTS assay, cells (5,000 per well) were seeded into 96-well plates. After treatment
with compounds for 72 h, cells were added with MTS at a final concentration of 0.5 mg/mL,
followed by incubation for another 2 h. The optical density (OD) of each well was
determined at 490 nm (background subtraction at 690 nm) by a SpectraMax 340 microplate
reader (Molecular Devices, Sunnyvale, CA, USA). The growth inhibitory ratio was calculated
as follows: Growth inhibitory ratio = (Acontrol – Asample)/Acontrol (where A is the OD value per
well). IC50 values were derived from a nonlinear regression model (curvefit) based on
sigmoidal dose response curve (100, 50, 25, 12.5, 6.25 µg/mL, variable slope) and computed
using Graghpad Prism version 5.02 (Graphpad Software).
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4.4. Western blotting
Poly ADP-ribose polymerase (PARP) was purchased from Cell Signaling Technology (Boston,
MA, USA); β-actin antibody was obtained from Sigma. All antibodies were used as
recommended by the manufacturers. For the assay, 8226 cells were grown in 6-well plates
incubated with CY01 (100 g/mL) for 48 h. Cells were rinsed twice with ice-cold PBS and
lysed with 1 × SDS loading buffer. Samples were electrophoresed on 10%
SDS–polyacrylamide gels, and transferred to PVDF membranes. The membranes were
blocked for 1 h with 5% w/v milk, incubated with indicated antibodies for 2 h, washed three
times with PBS, incubated with the anti-rabbit or anti-mouse secondary antibody for 1 h,
washed 3 times with PBS, and then detected with the ECL Kit (Thermo, Pierce, USA)
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4.5. Cellular imaging
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Plates were imaged with a PerkinElmer Operetta high-content wide-field fluorescence
imaging system, coupled to Harmony software. Wells were imaged using a 40× objective lens
in a single focal plane across each plate. The bottom of each well was detected automatically
by the Operetta focusing laser.
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Acknowledgements
Prof. Yu Jin, Dr. Qing Fu, Mr. Tu Yang, Prof. Jie Tang and Prof. Fan Yang are warmly thanked
for their helpful advice during preparation of this manuscript. This research is partially
supported by the 973 project (2013CB733700), the National Science Fund for Distinguished
Young Scholars (81125023), the National Natural Science Foundation of China (21176076,
21202045), the Program of Shanghai Subject Chief Scientist (13XD1404300) and the Key
Project of Shanghai Science and Technology Commission (13NM1400900).
Supplementary data
Supplementary data associated with this article can be found in the online version.
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Figure legends
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8
9
Figure 1. The bright field image of the MM cell lines (8266, H929 and LP-1) treated with
(a-c) CY01 (100 g/mL), and (d-f) DMSO for 72 h. (g) Western blot analysis of PARP (poly
ADP-ribose polymerase) cleavage in RPMI 8226 cells treated with CY01 (100 g/mL) for 48 h;
PS341, i.e. Bortezomib and DMSO were used as positive and negative control, respectively.
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14

Table 1 Viability (%) of various cancerous or control cells treated with CY01 (50 g/mL)a
1
2
3
4
5
6
7
Cell viability (%)
K562
Hela
A549
MEF
Compound
RPMI 8226
(Myelogenous
leukemia)
(Epithelioid
(Lung
(Mouse embryonic
(Multiple myeloma)
carcinoma cervix) adenocarcinoma) fibroblast)
8
9
CY01
22.9 ± 5.0
68.4 ± 0.5
60.4 ± 3.8
64.3 ± 3.3
101.3 ± 2.8
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64
65
aValues are mean of three experiments; viability is with respect to untreated cells.
15

Figure 1
a
b
d
f
8226
c
H929
e
LP-1
g
PARP
Cleaved
PARP
-Actin




One hit is identified to be selectively toxic to multiple myeloma cells
The hit is not toxic to a control cell line
The hit induced apoptosis of multiple myeloma cells

CY01
MM cells
EnhancedPARP cleavage
induced by CY01
with CY01
without CY01