Biomolecular Interaction Assays Identified Dual Inhibitors of Glutaminase and Glutamate Dehydrogenase That Disrupt Mitochondrial Function and Prevent Growth of Cancer Cells

Article abstract of DOI:10.1021/acs.analchem.6b03849

Glutaminase (KGA/isoenzyme GAC) is an emerging and important drug target for cancer. Traditional methods for assaying glutaminase activity are coupled with several other enzymes. Such coupled assays do not permit the direct and stringent characterization of specific glutaminase inhibitors. Ebselen was identified as a potent 9 nM KGA inhibitor in the KGA/glutamate oxidase (GO)/horse radish peroxidase (HRP) coupled assay but showed very weak activity in inhibiting the growth of glutamine-dependent cancer cells. For rigorous characterization, we developed a direct kinetic binding assay for KGA using bio-layer interferometry (BLI) as the detection method; Ebselen was identified as a GDH inhibitor but not a KGA inhibitor. Furthermore, we designed and synthesized several benzo[d][1,2]selenazol-3(2H)-one dimers which were subjected to SAR analysis by several glutaminolysis specific biochemical and cell based assays. Novel glutamate dehydrogenase (GDH) or dual KGA/GDH inhibitors were discovered from the synthetic compounds; the dual inhibitors completely disrupt mitochondrial function and demonstrate potent anticancer activity with a minimum level of toxicity.

Full text of DOI:10.1021/acs.analchem.6b03849

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Article
Biomolecular interaction assays identified novel dual inhibitors
of glutaminase and glutamate dehydrogenase that disrupt
mitochondrial function and prevent growth of cancer cells
Min Zhu, Jinzhang Fang, Jingjing Zhang, Zheng Zhang, Jianhui Xie, Yan Yu, Jennifer
Jin Ruan, Zhao Chen, Wei Hou, Gensheng Yang, Wei-Ke Su, and Benfang Helen Ruan
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03849 • Publication Date (Web): 16 Jan 2017
Downloaded from http://pubs.acs.org on January 17, 2017
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Biomolecular interaction assays identified novel dual inhibitors of glutaminase and
glutamate dehydrogenase that disrupt mitochondrial function and prevent growth of
cancer cells
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Min Zhu1a, Jinzhang Fang1a, Jingjing Zhang1a, Zheng Zhang1a, Jianhui Xie1, Yan Yu1, Jennifer
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Jin Ruan2，Zhao Chen1, Wei Hou1,Gensheng Yang1, Weike Su1, Benfang Helen Ruan1*
1School of Pharmacy, Zhejiang University of Technology
2Hangzhou Jennifer Biotech. Inc.
aEqual first authors
Key words: Glutaminase, Bio-Layer Interferometry (BLI), enzyme assay, Biomolecular
interaction, cancer.
* Corresponding author
Professor: Benfang Helen Ruan
E-mail: ruanbf@yahoo.com (BR); ruanbf@zjut.edu.cn;
Tel: 86-18357023608 (BF);
Fax: (0086) 571-88871098 (BF);
¤Current address: No. 18 Chaowang Road, Xiachengqu, Hangzhou, Zhejiang, China, 310014
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Abstract:
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Glutaminase (KGA/ isoenzyme GAC) is an emerging and important drug target for
cancer. Traditional methods for assaying glutaminase activity are coupled with several other
enzymes. Such coupled assays do not permit the direct and stringent characterization of
specific glutaminase inhibitors. Ebselen was identified as a potent 9 nM KGA inhibitor in
the KGA/Glutamate Oxidase (GO)/Horse Radish Peroxidase (HRP) coupled assay, but
showed very weak activity in inhibiting the growth of glutamine - dependent cancer cells.
For rigorous characterization, we developed a direct kinetic binding assay for KGA using
Bio-Layer Interferometry (BLI) as the detection method; Ebselen was identified as a GDH
inhibitor but not a KGA inhibitor. Furthermore, we designed and synthesized several
benzo[d][1,2]selenazol -3(2H)-one dimers which were subjected to SAR analysis by several
glutaminolysis specific biochemical and cell based assays. Novel glutamate dehydrogenase
(GDH) or dual KGA/GDH inhibitors were discovered from the synthetic compounds; the dual
inhibitors completely disrupt mitochondrial function and demonstrate potent anticancer
activity with a minimum level of toxicity.
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Introduction:
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Targeting glutaminolysis is an emerging approach to address the imminent problems of
drug resistance and drug toxicity in cancer therapeutics. Glutaminolysis (Figure 1) is an
important metabolic pathway in cancer cells that converts glutamine (Gln) to α-ketoglutarate
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(α-KG), which then feeds into the TCA cycle[1,2]
. The predominant pathway in most cancer
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cells is through formation of glutamate (Glu) by the rate-limiting glutaminase (KGA/GAC)
and then to α-ketoglutarate (α-KG) by GDH or by aminotransferase in the presence of other
amino acids. Alternative pathways (Fig. 1) are (A) transamination of Glu to α-KG or (B)
first transamination of Gln to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to
a-ketoglutarate by an amidase [3]
.
The relative contribution of each pathway varies among
different cell type and tissues [4]
.
Lactate
Glutamine
Glucose
Pyruvate
Acetyl CoA
B
Citrate
GSH
GABA
Lipid
KGA/GAC
GDH
TCA
Cycle
OAA
α-KG
Glutamate
Protein
Other amino acids
A
ATP
NAD
+
NADH
Figure 1. Glucose and glutamine metabolism of cancer cells. Alternative pathways are
(A) transamination of Glu to α-KG and (B) transamination of Gln to α-ketoglutaramate
(KGM) followed by hydrolysis of KGM to a-ketoglutarate by an amidase
As the initial enzyme in glutaminolysis, glutaminase expression is increased in many
tumors from breast, brain, lung, lymph, cervix and other tissues [1]. Glutaminase inhibitors
were shown to block Gln metabolism and starve Gln-dependent cancer cells to death [5-7]; this
represents a promising therapeutic approach to develop an effective anti-cancer drug [2,8,9] with
reduced toxic side effects.
Reported glutaminase inhibitors (Figure 2) include acivicin, CK, DON, Compound 968 [10]
BPTES [11], CB-839 [12], and Ebselen [13]
Acivicin and DON are Gln analogues that bind to
the active site of KGA/GAC and show high drug toxicity due, perhaps, to the lack of
selectivity against other Gln-binding enzymes. Compound 968 was reported as an allosteric
inhibitor of GAC, but the binding mode was not validated [10]
BPTES is a known
,
.
.
glutaminase allosteric inhibitor with poor solubility (0.01 µM). CB-839 [12] is a BPTES
analog that inhibits the KGA coupled enzyme assay in a time dependent manner; it showed a
KGA inhibition with IC50 values in the range of 26-300 nM. CB-839 is in a phase I clinical
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study for “triple negative” breast cancer with an IC50 of 20 nM in cell based assays, 50%
tumor growth suppression in vivo at 200 mg/kg oral dosage, and complete tumor suppression
when used in combination with Paclitaxel.
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Figure 2. Structures of glutaminase inhibitors
Recently, Ebselen was reported as a potent KGA inhibitor with IC50 ≈ 9 nM [13] based on a
complicated KGA/GO/HRP three enzyme coupled biochemical assay using H2O2 generated
during the reaction for quantification [14]
.
This is a complicated assay involving multiple
steps: first KGA converts Gln to Glu which is then converted to α-ketoglutarate and H2O2 by
Glutamate oxidase in the presence of O2; the resulting H2O2 is then utilized by Horseradish
Peroxidase to convert Amplex red to a fluorescent compound resorufin which can be
quantified at λex=567 nm and λem=602 nm. Unfortunately as an antioxidant, Ebselen is
known to react with H2O2. In addition, even though Ebselen (IC50 ≈ 9 nM) was reported to
inhibit the KGA enzyme ten times stronger than BPTES (IC50 ≈ 100-400 nM) [13], Ebselen
showed very weak growth inhibition of Gln-dependent cancer cells such as A549 cells
(IC50>10 µM) [15], whereas the validated KGA allosteric inhibitors BPTES and CB-839 [11,12]
showed potent inhibition of A549 cancer cells (IC50 20-200 nM). Although Ebselen was
reported to induce C6 glioma cell death under oxygen and glucose depletion, the mechanism
is mediated by cellular GSH and can be reversed by addition of N-acetyl cysteine [16]. In
addition, magnetic resonance studies have shown that Ebselen administration to humans
results in slightly decreased Gln, Glu and Glx levels in the cingulated cortex [17]
.
Therefore, we suspected that the reported KGA inhibition by Ebselen might be an artifact
of the complicated KGA/GO/HRP coupled assay. As an antioxidant and the most potent
glutathione peroxidase inhibitor [18], Ebselen was reported to inhibit oxidases or reductases by
reacting with the active site cysteine or selenocysteine residue [19,20]
.
Using paper
chromatography, we demonstrated that Ebselen did not prevent KGA from hydrolyzing Gln.
For future KGA inhibitor screening, we engineered a simple KGA/GDH assay because
both are involved with glutaminolysis. Interestingly, using the GDH assay as the counter
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screen, we discovered that Ebselen is a good GDH inhibitor with an IC50 of 213 nM [15] and
could inhibit the KGA/GDH coupled assay. Therefore, the KGA/GDH coupled assay can not
be used to evaluate Ebselen specifically as a KGA inhibitor. For rigorous characterization of a
KGA binding inhibitor, the most definitive method is the biomolecular interaction analysis
which measures the direct binding of a compound to the KGA enzyme.
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Further because selenazol-3(2H)-one contains a 5-membered ring that is similar to the
thiodiazo motif of the BPTES molecules, we investigated the possibility of converting
Ebselen to an actual glutaminase inhibitor by adding a linker chain to make a series of dimers
(Figure 3). Accordingly, we developed six biochemical and cell-based
glutaminolysis-focused analyses to evaluate the synthetic compounds for their KGA and/or
GDH inhibition. Furthermore, mechanistic cell based analysis demonstrated that we have
discovered some novel GDH/ KGA dual inhibitors; these inhibitors could efficiently shut
down mitochondrial function and inhibit cancer cell growth much better than the KGA or the
GDH inhibitors.
Figure 3. Chemical synthesis of benzo[d][1,2]selenazol -3(2H)-one dimers(1B-6B) with
different linkers (methods reported [18] previously with modifications)
Material and methods
Chemicals were purchased from Aladdin (California, U.S.A). EZMTT detection reagents
were from Jennifer Biotech Inc (Hangzhou, CN). A549, T24, HL7702 cell lines, cloning
enzymes and assay kits were purchased from (Labpal, Shanghai, China). Bovine GDH from
beef liver was purchased from Rochell (5Ku). The UV absorbance was measured by
Flexstation 3 (Molecular Device, USA) or by TECAN sunrise Multimode Reader
(Switzerland). Biomolecular interaction analysis was performed using a ForteBio K2
instrument (Pall Corp., USA) with super streptavidin (SSA) biosensors. RPMI 1640
medium and others were purchased from M&C Gene Technology Inc. (Beijing, China).
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Trypsin and EDTA were purchased from Amresco (Solon, OH, USA). Fetal bovine serum
(FBS) was purchased from Zhejiang Tianhang Biological Technology Co., Ltd. (Zhejiang,
China). JC1 assay kit was purchased from Beyotime biotech. Ltd. (Shanghai, China).
Glutamine oxidase was purchased from Sigma (USA). The biotinylation kit was purchased
from ThermoScientific (USA). Compounds BPTES and CB-839 were synthesized in the lab
according to the methods reported and characterized by NMR and MS. KGA assay buffer A
contains 50 mM Hepes, 100 mM NaCl, 0.5 mM EDTA, 0.001% Tween, pH 7.5. KGA assay
buffer B contains 50 mM Hepes, 200 mM KH2PO4, 100 mM NaCl, 0.5 mM EDTA, 0.01%
BSA, 0.001% Tween 20, pH 7.5.
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1. Paper chromatography analysis of Gln hydrolysis by KGA
KGA (100 nM) was preincubated with compounds (10 µM) in buffer A for 1 hr at 25 ℃
with shaking, and then Gln (5 mM final) in buffer B was added and reacted at room
temperature overnight. An aliquot (5 µl) from each reaction was spotted on paper
chromatography which was developed using a mixture of butanol:formic acid:water (7:3:1)
and the mobility of the amino-acids was visualized by 0.5% ninhydrin.
2. HPLC analysis of the Ebselen product formed by reaction with H2O2
Ebselen (3.3 mM) and H2O2 (3.3 mM) were mixed in water (150 µl) at 25 ℃ for
overnight. Aliquots (20 µl) of Ebselen (3.3 mM), H2O2 (3.3 mM), and the reaction mixture
were injected into XB-C18 reversed phase HPLC (4.6 mm × 250 mm, 5 μm). The column
was developed with a solvent mixture of Methanol : Acetonitrile : water (62:18:20) at a flow
rate of 1.0 ml/min. The elution of each component was detected by UV absorbance at 254
nm.
3. Glutamate oxidase (GO) inhibition assay
The Glutamate oxidase inhibition assay was carried out according to the literature [20] with
modifications. Briefly, Ebselen (0-20 µM final concentration) was preincubated with 0.05
mU/ml GO in GO assay buffer (10 mM HEPES, 100 mM NaCl, pH 7.4), then a mixture of
0.3 mM Glu, 0.24 unit/ml HRP and 50 µM Amplex red were added, and the reaction rate were
followed every 30 min by fluorescence measurement at λex=540 nm and λem=590 nm.
4. Horseradish peroxidase (HRP) inhibition assay
HRP (7 ng/ml) was incubated with Ebselen (0, 0.3, 0.8 mM, respectively) for 2 hours and
then the mixture was treated with TMB stain solution (15 µl) for 30 min. Then, 1M HCl (50
µl) was added to quench the reaction, and the progress of the reaction was followed by UV
absorbance at 450 nm.
5. Chemical synthesis of 1A and 1B-7B
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Compounds (Figure 3) were synthesized using methods reported [18] previously with
modifications. The key intermediate, chlorocarbonyl phenyl hypochloroselenoite (M4), was
first prepared from the commercially available anthranilic acid following the published
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procedure [18]
.
Ebselen and selenazol-3(2H)-one dimers (1B-7B) were obtained by treating
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M4 with the corresponding amine and diamine, respectively.
Using a 50 mL round bottom flask equipped with a stir bar, we combined aniline (0.465 g,
5.0 mmol, 1.0 equiv), triethylamine (1.5 mL, 10.7 mmol, 2.1 equiv) and DCM (17.5 mL). A
solution of M4 (1.3 g, 5.1 mmol, 1.02 equiv) in DCM (10 mL) was added dropwise at 0°C.
The reaction was warmed to room temperature and stirred overnight. The precipitate was then
filtered and washed with 10mL DCM. The crude product was further purified by silica gel
column chromatography; alternatively, it can be purified by washes through water and MeOH.
Synthetic compounds were purified and characterized by LC-MS, HR-MS, 1H NMR, and/ or
13C NMR.
Ebselen (1A): Yellow solid. m.p. 173.2-174.0 ºC. 1H-NMR (500 MHz, CDCl3) δ 8.14 (d, J =
7.5 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.66 (dd, J = 8.5, 1.5 Hz, 2H), 7.64 (d, J = 1.0 Hz, 1H),
7.50-7.42 (m, 3H), 7.29 (dd, J = 12.0, 4.7 Hz, 1H). MS: ESI (MeOH), [M+H]+ m/z: 276.0.
Ethanselen (1B): Yellow solid. m.p. 295.0-295.7 ºC. 1H-NMR (500 MHz, d6-DMSO) δ 8.00
(d, J = 8.0 Hz, 2H), 7.81 (dd, J = 7.5, 1.0 Hz, 2H), 7.60-7.55 (m, 2H), 7.44-7.38 (m, 2H), 4.02
(s, 4H). MS: ESI (MeOH), [M+H] + m/z: 424.9.
Butanselen (2B): Yellow solid. m.p. 234.8-236.3 ºC. 1H-NMR (500 MHz, d6-DMSO) δ 8.04
(d, J = 8.0 Hz, 2H), 7.81 (dd, J = 7.5, 1.0 Hz, 2H), 7.64-7.57 (m, 2H), 7.45-7.39 (m, 2H), 3.77
(s, 2H), 1.66 (s, 4H).MS: ESI (MeOH), [M+H] + m/z: 453.0.
Hexanselen (3B): Yellow solid. m.p. 196.0-198.0 ºC. 1H-NMR (500 MHz, d6-DMSO) δ 8.04
(d, J = 8.0 Hz, 2H), 7.80 (dd, J = 7.5, 1.0 Hz, 2H), 7.63-7.57 (m, 2H), 7.45-7.39 (m, 2H), 3.71
(t, J = 7.0 Hz, 4H), 1.70-1.57 (m, 4H), 1.35 (t, J = 7.0 Hz, 4H). HR-MS: ESI (MeOH),
[M+H]+ m/z: calcd: 480.9855, found: 480.9928.
Octanselen (4B): Yellow solid. m.p. 176.9-178.2 ºC. 1H-NMR (500 MHz, CDCl3) δ 8.05 (d, J
= 7.5 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.59(td, J = 7.5, 1.3 Hz, 2H), 7.46-7.40 (m, 2H), 3.85
(t, J = 7.0, 4H), 1.75-1.70 (m, 4H), 1.41-1.34 (m, 4H). MS: ESI (MeOH), [M+H] + m/z: 509.0.
2,2'-(thiobis(ethane-2,1-diyl))bis(benzo[d][1,2]selenazol-3(2H)-one) (5B): Yellow solid.
m.p. 225.3-228.3 ºC. 1H-NMR (500 MHz, d6-DMSO) δ 8.05 (d, J = 8.0 Hz, 2H), 7.82 (d, J =
7.5 Hz, 2H), 7.64 – 7.58 (m, 2H), 7.46 – 7.39 (m, 2H), 3.95 (t, J = 7.0 Hz, 4H), 2.88 (t, J = 7.0
Hz, 4H). 13C-NMR (125 MHz, d6-DMSO) δ=166.5, 139.6, 131.5, 127.6, 127.3, 125.8, 125.7,
42.8, 31.1. HR-MS: ESI (MeOH), [M+Na]+ m/z: calcd: 506.9263, found: 506.9155.
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2,2'-(thiobis(propane-3,1-diyl))bis(benzo[d][1,2]selenazol-3(2H)-one) (6B) : Yellow solid.
1H NMR (500 MHz, d6-DMSO) δ 8.05 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 7.5 Hz, 2H), 7.63 –
7.57 (m, 2H), 7.44 – 7.40 (m, 2H), 3.80 (t, J = 7.0 Hz, 4H), 2.57 (t, J = 7.0 Hz, 4H), 1.93 –
1.86 (m, 4H). HR-MS: ESI (MeOH), [M+Na]+ m/z: calcd: 512.9576, found: 512.9000
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6. Cloning and purification of the human KGA enzyme
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Human KGA gene (AF327434.1, GI:12044393) encoding amino acids 128 through 669
was PCR amplified from A549 genomic DNA using primers containing NdeI and XhoI
restrictions sites at the 5’ and 3’ ends, respectively. Forward and reverse primer sequences
were as follows: AATTCATATGGAAAATAAAATAAAACAGGG and
AATTCTCGAGCAACAATCCATCAAGATTC. The purified PCR product was restriction
digested and ligated into the pET-22b vector to generate the pET-hKGA plasmid. After
DNA sequencing, the plasmid was transformed into an E. coli BL-21 strain. The
recombinant C-terminal His-tagged hKGA protein was expressed after 1 mM IPTG induction
for 3 hours and purified using nickel affinity chromatography. The purified protein was
analyzed by SDS-PAGE, which showed a single band with a molecular weight of 62 kDa, and
further was stored in buffer (25 mM HEPES, 200 mM NaCl, 5 μM BME, 10% glycerol) at
-80 ˚C.
7. Bovine GDH activity assay
The bovine GDH assay was optimized by measuring the linear dose response of the
enzymatic reaction initial velocities. To bovine GDH (0 -1.5 U/mgL final), a mixture of
NADP+ (200 μM), glutamate (16 mM) and the EZMTT detection reagent in 50 mM Tris-Cl
(pH 8.2) was added. The UV absorbance changes at 450 nm were measured every 2 min to
collect linear initial velocity.
For the bovine GDH inhibition assay in 50 mM Tris-Cl (pH 8.2) at 25︒C, 3-fold dilutions
of compounds (0 - 26 μM) were preincubated with the GDH enzyme for 0.5 hours, and a
mixture of glutamate (10 mM final), NADP+ (200 μM final) and the EZMTT detection
reagent were added and reacted for 2 hrs to measure the GDH activity.
8. KGA/GDH coupled assay
The compound was premixed with human KGA protein (1 nM) in 100 μl Hepes buffer A
for 15 min. To the 96-well plate, Gln (20 mM) in 100 μl Hepes buffer B was added and the
reaction was shaken at 25 ℃ for 3 h. The glutamate formation was then measured in a GDH
coupled assay by adding a mixture containing GDH (8 nM), NADP+ (20 μM), and the
EZMTT detection reagent [15]
.
After the solution was incubated at 25 ℃ for 5 min, the
absorbance at 450 nm was measured against the reference wavelength of 620 nm.
9. Biomolecular interaction assay using the BLI detection method
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Biomolecular interaction analysis of compounds binding to the biotinylated KGA protein
was performed using a ForteBio K2 instrument with Super Streptavidin (SSA) biosensors.
KGA was biotinylated by mixing a three-fold molar excess of Sulfo-NHS-Biotin with KGA
protein (1.0 mg/ml) at room temperature for 30 min and then at 4 ℃ for overnight. The
biotinylated KGA protein was purified on size exclusion chromatography and contained 1-2
biotins per KGA molecule, as measured by streptavidin gel-shift assay. SSA biosensors
were then loaded with the biotinylated KGA protein by dipping the sensor into sample plate
wells containing 200 µl of 50 μg/ml KGA in PBS buffer for 5 min.
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For the kinetic binding assay, PBST buffer (PBS + 0.02% tween 20) containing 1% DMSO
was used. The KGA coated sensor was first dipped into the blank buffer for 2 minutes to
obtain a baseline signal, then into a compound sample for 5 min to measure the on-rate, and
finally into the blank buffer again for 3-5 min to obtain the dissociation rate. A series
dilution of compounds was tested in duplicate to obtain a dose response. The kinetic
binding curves were obtained using double subtraction methods; the sample reference are
blank PBST solution containing 1% DMSO, and the sensor reference was immobilized with
biotinylated alanine. The KD data were analyzed using ForteBio Data analysis software;
sensorgrams (n=6) were analyzed by kinetic fit, using 1:1 binding mode and global fit
grouped by sensor. The KD values with R2 ≥ 0.5 were accepted as preliminary binding
evidence. In BLI-based assays, most small molecules showed a KD between 10-4 - 10-7 M; if
KD < 100 nM (10-7 M), the small molecule is identified as a potent and strong binder.
10. Cell proliferation assay.
Cell proliferation was measured using EZMTT assay [15], after treatment with compounds or
growth in Gln-deficient or rich media for 5 days. Briefly, cells (104/well) were plated on a
96-well plate. After setting aside the cells for 4 hours in rich media for adherence, cells
were treated with compounds or Gln-deficient media for 3-5 days. NAD(P)H production in
viable cells was measured using the EZMTT method with 1 hour incubation time [15]
.
Results are representative of at least 2 independent experiments in triplicates.
11. JC-1 assay
JC-1 assay is used to measure mitochondrial membrane potential changes in A549 cell line
after compound (0.3 and 1 μM concentration) treatment. A549 cells were treated with
compound overnight, washed once with the dilution buffer, incubated with 20 µM JC1 at
37 ℃ for 20 min, and then washed twice with dilution buffer. Rich medium (50 µl) was
added to the treated cells for immediate JC-1 fluorescence imaging and by a plate reader
measurement; the disruption of the mitochondrial membrane potential was based on the
fluorescence ratio of the Green fluorescence measured at EX 490 nm and EM 530 nm versus
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the Red fluorescence at EX 555 nm and EM 590 nm).
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12． Statistical analysis
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Statistical analysis of the samples was performed using a one-way analysis of variance
(ANOVA), and P-values < 0.05 were considered statistically significant. All data are
reported as the means ± the standard deviation (SD) unless otherwise stated.
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Results and discussion
1. Ebselen did not inhibit KGA, but inhibited GO and HRP and reacted with H2O2.
Ebselen was reported as a potent KGA inhibitor with an IC50 of 9 nM, but showed
essentially no inhibition of Gln-dependent A549 cells. To confirm the inhibition of KGA
by Ebselen, we carried out paper chromatography analysis of the KGA enzymatic reaction
products. As shown in Figure 4A, Gln and Glu were separated by paper chromatograph
with Rf values of 0.39 (Gln) and 0.42 (Glu), respectively. In the presence of KGA, > 90%
Gln was hydrolyzed to Glu. In the presence of 10 µM BPTES, no conversion of Gln to
Glu was observed, indicating that KGA activity was completely inhibited by BPTES.
However, in the presence of 10 µM Ebselen, 90% conversion of Gln to Glu was still
observed, indicating that KGA activity was not inhibited by Ebselen. Therefore, the
reported 9 nM KGA inhibition by Ebselen may be due to the inhibition of redox enzymes
(GO or HRP) or depletion of the read-out molecule H2O2. .
As shown in Figure 4B, after incubating Ebselen with H2O2 at room temperature
overnight, both the Ebselen (tR 5 min) and the H2O2 (tR 3.1 min) peaks disappeared, and a
new peak at tR 3.5 min formed; this demonstrated that indeed Ebselen can react with H2O2
as reported previously.
To test the inhibition of GO by Ebselen, we first obtained the linear dose response range
for GO as shown in Figure 4C, then used a concentration of GO within the linear range to
test the Ebselen inhibition. Under the optimal conditions, Ebselen showed an IC50 of 3
µM in inhibiting GO, as shown in Figure 4D.
In addition, we tested the inhibition of HRP by Ebselen. Figure 4E shows the linear dose
response range for HRP; and Ebselen inhibits HRP with an IC50 of 800 µM as shown in
Figure 4F.
These results demonstrated that Ebselen is not a potent inhibitor of KGA with an IC50
near 9 nM.
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Figure 4. Ebselen is not a KGA inhibitor, even though it showed inhibition in KGA/GO/
HRP coupled assay: A) Gln hydrolysis by KGA in the presence of inhibitors, demonstrating
Ebselen could not inhibit KGA; B) HPLC analysis of H2O2, Ebselen and the reaction product
of H2O2 and Ebselen, demonstrating that Ebselen could diminish H2O2 level; C) Linear dose
response curve in GO assay; D) Inhibition of GO activity by Ebselen (IC50 3 µM); E) Linear
dose response curve in HRP assay; F) Inhibition of HRP activity by Ebselen.
2. KGA biochemical assay using GDH as the coupled enzyme
We configured a human KGA/GDH coupled enzymatic assay, because both enzymes are
involved in the initial steps of glutaminolysis before the TCA cycle. Under optimized assay
conditions, the reaction initial velocities showed linear dose response to human KGA enzyme
concentration (Figure 5A), and also the assay showed excellent reproducibility with a
measured Z factor of 0.85 (Figure 5B). The dose response of Gln concentration to KGA
enzyme initial reaction velocities was curve-fitted using the Michaelus Menton equation
(Figure 5C); the obtained Gln KM of 2.78 ± 0.26 mM for the recombinant KGA is similar to
that of the native KGA enzyme [21]
.
Figure 5. KGA/GDH coupled assay showed linear dose response to KGA concentration
and high assay reproducibility.
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3. Enzyme activity and kinetic binding (BLI) assays demonstrated that Ebselen inhibits
GDH but not KGA
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In our E. coli GDH, bovine GDH and human KGA/GDH assays, the initial reaction
velocities showed linear dose response to its corresponding enzyme concentrations; under the
same optimal conditions, Ebselen showed an IC50 of 200 nM for the GDH (Figure 6A, B) and
900 nM in KGA/GDH coupled assay (Figure 6C); the IC50 values are not time-dependent and
the same IC50s were observed without or with up to 2 hrs preincubations (data not presented),
indicating Ebselen is a reversible inhibitor of GDH. In such coupled assays that involve two
enzymes, inhibition of either enzyme will give positive results. Therefore, in order to confirm
that Ebselen is a KGA inhibitor [11], we tested the compound using a direct kinetic binding
assay to the KGA enzyme alone; this direct KGA binding assay will resolve this ambiguity.
For rigorous characterization, all inhibitors from the KGA/GDH coupled assay were further
characterized based on their direct kinetic binding to a single enzyme (KGA) immobilized on
an SSA biosensor detected by BLI methods.
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The kinetic binding assay to KGA enzyme was developed and validated using its substrate
(Glutamate) and its inhibitor (BPTES). Human KGA showed dose dependent direct binding
to its substrate Gln (KD 4 µM) and its known inhibitor BPTES (KD 0.2 µM), but no binding
for Ebselen ((Figure 6D-F). Reproducible binding was observed.
Figure 6. Enzyme activity and kinetic binding (BLI) assays demonstrated that Ebselen
inhibits GDH but not KGA: Ebselen inhibited A) E. coli GDH, B) Bovine GDH, C)
Human KGA/GDH coupled assays. Kinetic binding of KGA to D) Gln (1-100 µM), E)
BPTES (0.1-10 µM) and F) Ebselen (0.1-10 µM) in PBST buffer containing 1% DMSO. (red,
low conc. 1X ; blue, middle conc. 10X ; black, high conc.100X) .
4. Chemical Synthesis of novel compound 5B and other dimers for SAR analysis
Since BPTES contains a 5-atom thiodiethyl linker, we mainly focused on the synthesis of
5B. Using Glide and DS drug design software and the GAC/BPTES co-crystal structure
[22,23], we did a preliminary autodock of the designed 5B and Ebselen into the GAC/BPTES
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pocket (Figure 7); the two Libdock scores for 5B (130, 150) are better than that for Ebselen
(102, 109), indicating that 5B is likely to fit in the allosteric site of GAC. 5B-6B are novel
compounds that are fully characterized by HPLC, 1HNMR, 13CNMR, MS, HR-MS. For
SAR studies, we also included the synthesis of dimers with shorter linker size such as 1B, 2B,
and 3B.
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The chemical synthesis of 1B has been reported and 1B was identified as the most potent
inhibitor targeting Thioredoxin reductase (TrxR)[18]
. Here, based on rigorous biological tests
using seven assays as shown in Table 1 and Figures 8,9,10, we identified the nature of the
inhibitory activity of compounds 1A, 1B-6B as glutaminolysis inhibitors.
Figure 7. Autodock of 5B into the allosteric site of BPTES/GAC co-crystal structure.
Pink and brown colors show the GAC polypeptide; the middle green is BPTES; the light blue
is 5B.
5. 3B-6B were identified as novel KGA/GDH dual inhibitors
The structure activity relationships (SAR; Table 1; Figure 8) were built by testing the
compounds in enzyme activity assays (GDH assays, KGA/GDH coupled assay), kinetic
binding assays (GDH, KGA), and cell-based assays (cancer cells and normal cells).
Table 1: Biological activity of BPTES and selenazol-3(2H)-one dimers
Enzyme Functional assay1
Kinetic KGA enzyme Binding1
IC50 (μM)
KD (μM)
Compounds
Bovine
E.coli
GDH
NO
KGA/ E.coli
Steady
state
0. 4
Curve
fitting
0.4
R2
GDH
NO
0.8
GDH
0.4
0.74
BPTES
1A
0.2
0.9
NO
NO
1.5
0.2
1.6
NO
NO
1B
1.3
0.2
0.6
NO
NO
2B
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0.8
20
0.3
0.5
0.2
0.4
0.1
1.5
1.6
3
0.2
0.4
0.1
0.2
0.64
0.51
0.89
0.80
0.86
NB
3B
4B
1.6
13
0.4
0.14
6.3
0.13
6.3
5B
2.3
6B
NO
0.7
NO
0.05
0.1
0.1
CB839
EGCG
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NB
NB
NB, no binding; NO, no inhibition; each compound was tested in triplicates and repeated for
at least 3 times. Bold is to emphasize the important data.
1The standard deviations are 10% in the enzyme activity assay (IC50) and direct binding assay
(KD).
The known KGA allosteric inhibitors BPTES and CB-839 were used to validate the
assays in Table 1 and Figure 8A, a, B, b. Both compounds did not inhibit the GDH enzyme,
but inhibited in the KGA/GDH coupled assay (IC50 400 nM). In addition, both compounds
showed binding to KGA in BLI kinetic assay (KD 200 nM).
Under the same assay conditions, compounds 3B, 4B, 5B and 6B inhibited both GDH
and KGA/GDH coupled assays with similar IC50’s as shown in Table 1 and Figure 8C, c, D, d,
E, e, F, f, and also showed direct binding to KGA in BLI assay. Therefore, compounds
3B-6B were identified as novel KGA/GDH dual specific inhibitors.
Compounds 1A, 1B, 2B inhibited the GDH activity 3-10 fold better than those of the
KGA/GDH assays (Figure 8g, i, h). However, in the direct KGA binding assays, no binding
of these compounds to KGA enzyme was observed (Figure 8G, I, H). Therefore, compounds
1A, 1B, 2B were identified as GDH inhibitors.
In summary, the kinetic binding data matches the enzyme activity assay as shown in
Table 1 and Figure 8. GDH inhibitors (1A, 1B, 2B), which do not inhibit KGA, showed
better potency in the GDH assay than in the KGA/GDH coupled assay. KGA/GDH dual
inhibitors (3B, 4B, 5B, 6B) showed better or similar potency in the KGA/GDH couple assay
than in the GDH assay; inhibition of KGA provided additional inhibitory effect in KGA/GDH
activity assay.
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Figure 8. Identification of the GDH, KGA or the novel dual KGA/GDH inhibitors. A-H)
BLI based kinetic binding assay. Binding of KGA to A) BPTES, B) CB-839, C) 3B, D) 4B,
E) 5B, F) 6B, G) 1A,H)1B, I) 2B in PBST buffer. (red 0.1 µM, blue 1 µM, black10 µM) . a-h)
IC50 of the corresponding compounds in GDH and in KGA/GDH assays.
6. A549 and T24 cell lines are strongly Glutamine-dependent.
To evaluate the Gln-dependence of the cancer cell lines, we did a viability test in both rich
medium and Gln-deficient medium for 5 days (Figure 9). Nearly complete growth inhibition
was observed for cancer cell lines (A549 and T24) in Gln-deficient media. Interestingly,
because the EZMTT cell viability detection reagents do not have inhibitory effect to the cells
[15], we treated the A549 or T24 cells grown in Gln-deficient medium for 5 days with the
EZMTT reagents for overnight, and found significant growth signals, indicating glutamine
starvation alone did not kill the cells completely, especially the T24 cells. This perhaps can
be explained by the existence of alternative or interactive metabolic pathways. For example,
under glutamine starvation conditions, the TCA cycle might be partially fueled by Acyl-CoA
from glucose or fatty acid metabolic pathways or by glutamic acid through glutamate
dehydrogenase and/or aminotransferase pathways.
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Figure 9. The Gln-dependence of A549 and T24 cell lines was measured based on the
growth difference in rich media (A, C) and in Gln-deficient media (B, D) for 5 days. E) Cell
viability (growth in the Gln-deficient versus in the rich media) was measured after incubation
with the EZMTT reagent for 1 hr and for 24 hrs. The mean and SEM of multiple
measurements are plotted. Results are representative of at least two independent experiments.
Comparisons of treated and untreated conditions were performed by unpaired t test: *,
P<0.05.
7. The KGA/GDH inhibitors demonstrated the potent growth inhibition of Gln-dependent
cancer cells but not the normal cells.
With the availability of the KGA only inhibitor (BPTES), the GDH only inhibitor (1B) and
the KGA/GDH dual specific inhibitors (3B and 5B), we measured biological action of the
glutaminolysis inhibitors using the normal cells and also the cancer cell lines A549 and T24
which showed strong Gln dependence (Figure 10).
Figure 10. Inhibition of cell proliferation by KGA inhibitor BPTES, GDH inhibitor 1A
and 1B, KGA/GDH dual inhibitor 3B and 5B.Growth inhibition of A) Gln-dependent A549; B)
Strong Gln-dependent T24; C) Normal cell HL7702. IC50 curves demonstrated the growth
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inhibition of A549 (red), T24 (blue) and HL7702 (black) cells by compounds1B (D), 3B (E)
and BPTES (F), respectively. The mean and SEM of triplicate measurements are plotted.
Results are representative of at least two independent experiments. Nonlinear 4 parameter
dose–response curve fits were used to calculate IC50 and 95% CI. Comparisons of treated
and untreated conditions were performed by unpaired t test: *, P<0.05; **, P<0.01.
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The KGA inhibitor demonstrated good correlation between potency and Gln-dependence;
BPTES showed potent inhibition of T24, but no inhibition of the normal cells.
The GDH inhibitor did not show good correlation to Gln-dependence; 1B inhibited both the
T24 cancer cells and the normal cells. Perhaps GDH inhibitors are not efficient glutaminolysis
inhibitors, but could inhibit certain cancer cells with dis-regulated GDH level [24]
.
The KGA/GDH inhibitors (e.g. 3B and 5B) demonstrated the best growth inhibition in the
most Gln-dependent cells (T24). Importantly, 3B and 5B showed essentially no inhibition
of the normal cell HL7702; at least 50 fold in selectivity was found between the cancer cells
(IC50s 200 nM) and the normal cells (IC50 > 10 µM). This is in agreement with the
observation that in normal cells, cell proliferation and NAD(P)H production are independent
of glutaminolysis. Even though the detailed mechanism for 3B remains to be discovered,
the observed high potency and low toxicity are desirable and important features for cancer
therapeutics.
8. KGA/GDH inhibitors could disrupt mitochondrial membrane potential much more
efficiently than the KGA or the GDH inhibitors
In addition, the action of the 3 types of inhibitors on mitochondrial membrane potential was
evaluated by JC1 assay in A549 cells (Figure 11). When no compound was added, red
fluorescence was the predominant color observed, which indicated normal mitochondrial
membrane potential. When A549 was treated with photonophore CCCP, green fluorescence
became the dominant color, indicating the disruption of the mitochondrial membrane potential.
Interestingly under the same conditions, the KGA/GDH inhibitor (3B, 5B) treated A549 cells
showed predominantly green fluorescence, indicating the complete disruption of
mitochondrial membrane potential. However, the KGA inhibitor (BPTES) or the GDH
inhibitors (1A or 1B) showed predominantly the red fluorescence; although small amount of
green fluorescence was detected in BPTES treated cells, indicating partial loss of membrane
potential. These results suggested that the KGA/GDH inhibitors could disrupt mitochondrial
membrane potential much more efficiently than the KGA or the GDH inhibitors.
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Figure 11. JC1 assay demonstrated that the KGA/GDH inhibitors could disrupt
mitochondrial membrane potential much more efficiently than the KGA or the GDH
inhibitors. A549 cells were treated with 1μM of A) DMSO, B) CCCP, C)5B, D)3B, E)1A,
F)BPTES, G)1B, followed by the JC1 microscopic fluorescence imaging and by a plate
reader measuring the fluorescence ratio of the Green fluorescence at EX 490 nm/ EM 530 nm
versus the Red fluorescence at EX 525 nm/EM 590 nm. Comparisons of treated and untreated
conditions were performed by unpaired t test: *, P<0.05; **, P<0.01.
Conclusion
Traditionally, the coupled KGA enzyme assays have been used to evaluate all
glutaminase inhibitors reported so far. Our results showed that without proper control, the
coupled KGA assays can easily generate false positives. Therefore, we developed the BLI
based direct binding assay that provided high throughput screening and reliable
characterization of glutaminase inhibitors. Applying the KGA biomolecular interaction
assay, we discovered that Ebselen does not bind directly to the KGA enzyme, and its reported
9 nM potent inhibition [13] of KGA activity might be due to inhibition of the second coupled
enzyme, although as an antioxidant Ebselen might react with the thio-group of the active site
cysteine and then partially inactivate KGA [16,18]
.
Also, based on the cocrystal structure of
KGA and its allosteric inhibitor BPTES, we designed and synthesized several
selenazol-3(2H)-one dimers. Using both enzyme assays and BLI kinetic binding assays, we
discovered that selenazol-3(2H)-one dimers with a 5-7 atom linkage are novel dual inhibitors
targeting both GDH and KGA allosteric sites. For Gln dependent cancer cells, the novel
dual specific glutaminolysis inhibitors efficiently disrupted the mitochondrial membrane
potential and caused cell death. Importantly, the novel dual KGA/GDH inhibitors show
essentially no inhibition of the normal cell growth, and demonstrated early promise as
effective antitumor agents with minimal toxicity.
Acknowledgement: We appreciate financial support from Hangzhou Jennifer Biotech LLC
(H1160492) and Zhejiang University of Technology (414800129).
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References
(1) Ward, P. S.; Thompson, C. B. Cancer Cell, 2012, 21, 297-308.
(2) Altman, B. J.; Stine, Z. E.; Dang, C. V. Nat. Rev. Cancer, 2016, 16, 619-634.
(3) Cooper A. J.; Shurubor Y. I.; Dorai T.; Pinto J. T.; Isakova E. P.; Deryabina Y. I.; Denton
T. T.; Krasnikov B. F. Amino Acids, 2016, 48, 1-20.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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27
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42
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46
47
48
49
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53
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55
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57
58
59
60
(4) Moreadith R. W.; Lehninger A. L. J. Biol. Chem., 1984, 259, 6215-6221.
(5) Hanahan, D.; Weinberg, R. A. Cell, 2011, 144, 646-674.
(6) Cassago, A.; Ferreira, A. P.; Ferreira, I. M.; Fornezari, C.; Gomes, E. R.; Greene, K. S.;
Pereira, H. M.; Garratt, R. C.; Dias, S. M.; Ambrosio, A. L. Proc. Natl. Acad. Sci. U. S. A.
2012, 109, 1092-1097.
(7) Ferreira, A. P.; Cassago, A.; Gonçalves, K. A.; Dias, M. M.; Adamoski, D.; Ascenção, C.
F.; Honorato, R. V.; de Oliveira, J. F.; Ferreira, I. M.; Fornezari, C.; Bettini, J.; Oliveira, P. S.;
Paes Leme, A. F.; Portugal, R. V.; Ambrosio, A. L.; Dias, S. M. J. Biol. Chem. 2013, 288,
28009-28020.
(8) Cairns, R. A.; Harris, I. S.; Mak, T. W. Nat. Rev. Cancer, 2011, 11, 85-95.
(9) Lukey, M. J.; Katt, W. P.; Cerione, R. A. Drug Discov. Today, 2016, S1359-6446,
30480-30489. Doi: 10.1016/j.drudis.2016.12.003.
(10) Katt, W. P.; Ramachandran, S.; Erickson, J. W.; Cerione, R. A. Mol. Cancer Ther. 2012,
11, 1269-1278.
(11) Elgogary, A.; Xu, Q.; Poore, B.; Alt, J.; Zimmermann, S. C.; Zhao, L.; Fu, J.; Chen, B.;
Xia, S.; Liu, Y.; Neisser, M.; Nguyen, C.; Lee, R.; Park, J. K.; Reyes, J.; Hartung, T.; Rojas,
C.; Rais, R.; Tsukamoto, T.; Semenza, G. L.; Hanes, J.; Slusher, B. S.; Le, A. Proc. Natl. Acad.
Sci. U. S. A. 2016, 113, E5328-5336.
(12) Gross, M. I.; Demo, S. D.; Dennison, J. B.; Chen, L.; Chernov-Rogan, T.; Goyal, B.;
Janes, J. R.; Laidig, G. J.; Lewis, E. R.; Li, J.; Mackinnon, A. L.; Parlati, F.; Rodriguez, M. L.;
Shwonek, P. J.; Sjogren, E. B.; Stanton, T. F.; Wang, T.; Yang, J.; Zhao, F.; Bennett, M. K. Mol.
Cancer Ther. 2014, 13, 890-901.
(13) Thomas, A. G.; Rojas, C.; Tanega, C.; Shen, M.; Simeonov, A.; Boxer, M. B.; Auld, D. S.;
Ferraris, D. V.; Tsukamoto, T.; Slusher, B. S. Biochem. Biophys. Res. Commun. 2013, 438,
243-248.
(14) McElroy K. E.; Bouchard P. J.; Harpel M. R.; Horiuchi K. Y.; Rogers K. C.; Murphy D.
J.; Chung T. D.; Copeland R. A. Anal. Biochem. 2000, 284, 382-387.
(15) Zhang, W.; Zhu, M.; Wang, F.; Cao, D.; Ruan, J. J.; Su, W.; Ruan, B. H. Anal. Biochem.
2016, 509, 33-40.
(16) Shi H.; Liu S.; Miyake M.; Liu K. J. Chem. Res. Toxicol, 2006, 19, 655-660.
(17) Masaki, C.; Sharpley, A. L.; Godlewska, B.R.; Berrington, A.; Hashimoto, T.; Singh, N.;
Vasudevan, S.R.; Emir, U.E.; Churchill, G.C.; Cowen, P. J. Psychopharmacology (Berl), 2016,
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2
3
4
233, 1097-1104.
5
6
7
(18) He, J.; Li, D.; Xiong, K.; Ge, Y.; Jin, H.; Zhang, G.; Hong, M.; Tian, Y.; Yin, J.; Zeng, H.
Bioorg. Med. Chem. 2012, 20, 3816-3827.
8
9
(19) Björnstedt M.; Kumar S.; Björkhem L.; Spyrou G.; Holmgren A. Biomed. Environ. Sci.,
1997, 10, 271-279.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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44
45
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47
48
49
50
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52
53
54
55
56
57
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59
60
(20) Fischer H.; Dereu N. Bull. Soc. Chim. Belges, 1987, 96, 757-768.
(21) Hartwick, E. W.; Curthoys, N. P. J. Enzyme Inhib. Med. Chem. 2012, 27, 861-867.
(22) Thangavelu, K.; Pan, C. Q.; Karlberg, T.; Balaji, G.; Uttamchandani, M.; Suresh, V.;
Schüler, H.; Low, B. C.; Sivaraman, J. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 7705-7710.
(23) DeLaBarre, B.; Gross, S.; Fang, C.; Gao, Y.; Jha, A.; Jiang, F.; Song, J. J.; Wei, W.;
Hurov, J. B. Biochemistry, 2011, 50, 10764-10770.
(24) Zhang, J.; Wang, G.; Mao, Q.; Li, S.; Xiong, W.; Lin, Y.; Ge, J. Oncotarget, 2016, 295,
799-800.
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Analytical Chemistry
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For TOC
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GDH
Dual GDH/KGAallosteric
KGAallosteric
O
N
Se
9
Ebselen
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GDH
(IC50)
0.2 μM
2 μM
KGA
(KD)
20 μM
A549
T24
Cell growth
(IC50)
HL7702
Green
Red
on
：
MMP (JC1)
off
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