Polar Recognition Group Study of Keap1-Nrf2 Protein-Protein Interaction Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00407

Directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as an attractive way to activate Nrf2, and Keap1-Nrf2 PPI inhibitors have been proposed as potential agents to relieve inflammatory and oxidative stress diseases. In this work, we investigated the diacetic moiety around the potent Keap1-Nrf2 PPI inhibitor DDO1018 (2), which was reported by our group previously. Exploration of bioisosteric replacements afforded the ditetrazole analog 7, which maintains the potent PPI inhibition activity (IC₅₀ = 15.8 nM) in an in vitro fluorescence polarization assay. Physicochemical property determination demonstrated that ditetrazole replacement can improve the drug-like property, including elevation of pK_a, log D, and transcellular permeability. Additionally, 7 is more efficacious than 2 on inducing the expression of Nrf2-dependent gene products in cells. This study provides an alternative way to replace the diacetic moiety and occupy the polar subpockets in Keap1, which can benefit the subsequent development of Keap1-Nrf2 PPI inhibitor.

Full text of DOI:10.1021/acsmedchemlett.5b00407

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Letter
Polar Recognition Group Study of Keap1-
Nrf2 Protein-Protein Interaction Inhibitors
Mengchen Lu, Shi-Jie Tan, Jian-Ai Ji, Zhiyun Chen, Zhenwei Yuan, Qi-Dong You, and Zhengyu Jiang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00407 • Publication Date (Web): 05 Jul 2016
Downloaded from http://pubs.acs.org on July 6, 2016
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ACS Medicinal Chemistry Letters
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Polar Recognition Group Study of Keap1ꢀNrf2 ProteinꢀProtein Inꢀ
teraction Inhibitors
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a
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a
a,b *
MengꢀChen Lu, ShiꢀJie Tan, JianꢀAi Ji, ZhiꢀYun Chen, ZhenꢀWei Yuan, QiꢀDong You
ZhengꢀYu Jiang
and
a,b *
a
State Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design and Optimiꢀ
b
zation, China Pharmaceutical University, Nanjing 210009, China; Department of Medicinal Chemistry,
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
KEYWORDS: Keap1 Nrf2 Protein-Protein Interaction
ABSTRACT: Directly disrupting the Keap1ꢀNrf2 proteinꢀprotein interaction (PPI) has emerged as an attractive way to activate
Nrf2, and Keap1ꢀNrf2 PPI inhibitors have been proposed as potential agents to relieve inflammatory and oxidative stress diseases.
In this work, we investigated the diacetic moiety around the potent Keap1ꢀNrf2 PPI inhibitor DDO1018 (2), which was reported by
our group previously. Exploration of bioisosteric replacements afforded the diꢀtetrazole analog 7, which maintains the potent PPI
inhibition activity (IC50 = 15.8 nM) in an in vitro fluorescence polarization assay. Physicochemical property determination demonꢀ
strated that diꢀtetrazole replacement can improve the drugꢀlike property, including elevation of pKa, logD and transcellular permeaꢀ
bility. Additionally, 7 is more efficacious than 2 on inducing the expression of Nrf2ꢀdependent gene products in cells. This study
provides an alternative way to replace diacetic moiety and occupy the polar subꢀpockets in Keap1, which can benefit the subsequent
development of Keap1ꢀNrf2 PPI inhibitor.
Oxidative stresses exacerbate many chronic diseases, inꢀ
cluding cancer, cardiovascular diseases, chronic inflammation,
and neurodegenerative diseases. The constant stresses imꢀ
have therapeutic potential in a range of disease types including
inflammatory conditions, chronic kidney diseases and cancer
chemoprevention.
1
9ꢀ11
posed by electrophiles and oxidants can increase the levels of
oxidized proteins, phospholipids and DNA, which finally conꢀ
tribute to the neoplastic and chronic degenerative diseases.
A wide variety of dietary and synthetic compounds have
been identified as potent activators of AREꢀregulated genes,
2
12
e.g. sulforaphane, chalcone and curcumin. These traditional
Nrf2ꢀARE activators are all electrophiles that form a covalent
The transcription factor nuclear factor erythroid 2ꢀrelated facꢀ
tor 2 (Nrf2), member of the Cap’N’collar family of proteins, is
the primary regulator of cell defensive mechanisms against
oxidative stresses. Nrf2 binds to the antioxidant response eleꢀ
ment (ARE) and regulates the transcription of a battery of cell
protective genes (about 200), including phase 1 and 2 enꢀ
zymes, glutathioneꢀbased and thioredoxinꢀbased antioxidant
1
3
bond to a cysteine residue in the target Keap1 protein. The
electrophilic nature of these activators could increase the unꢀ
certainty in further clinical development. Thus, directly disꢀ
rupting the Keap1ꢀNrf2 proteinꢀprotein interaction (PPI) has
been regarded as an innovative strategy to activate Nrf2. Owꢀ
ing to the competitive and reversible nature, the Keap1ꢀNrf2
PPI inhibitors may show some advantages towards the elecꢀ
3
systems.
14
Kelchꢀlike ECHꢀassociated proteinꢀ1(Keap1), a Bricꢀaꢀ
brac/Tramꢀtrack/Broad (BTB) protein, is responsible for the
regulation of Nrf2 activity. Under normal conditions, Keap1
acts as a substrate adaptor component in the assembly of the
Cullin3 (Cul3)ꢀbased ubiquitin E3 ligase complex. This E3
ligase complex shepherds Nrf2 toward the polyubiquitination
and degradation by the 26S proteasome, which limits the Nrf2
trophilic Nrf2 activators.
Recently, a series of Keap1ꢀNrf2 PPI inhibitors, including
15ꢀ17
18ꢀ23
peptides
and small molecules
have been identified.
Among these agents, the diacetate compound series showed
the most predominant PPI inhibition activity. The discovery of
DDO1002 (1), the first nanomolar Keap1ꢀNrf2 PPI inhibitor,
confirmed that necessity of fulfilling the five subꢀpockets in
Keap1 and making multiple polar interactions with key arꢀ
ginines in Keap1 binding cavity should be stressed in the deꢀ
4
ꢀ6
activity at a basal level. Under stress conditions, the cysteine
residues in the Keap1 protein are covalently modified by oxiꢀ
dative and/or electrophilic agents, which cause the conformaꢀ
24
sign of potent Keap1ꢀNrf2 PPI inhibitors. Further optimizaꢀ
tion of 1 resulted in DDO1018 (2) with higher solubility and
better Nrf2 inducing activity. The in vivo experiments also
7
, 8
tional changes of the complex. These changes relieve Nrf2
from Keap1ꢀmeditated depression and activate the Nrf2ꢀARE
pathway to induce the transcription of downstream genes,
which finally activate cell defensive system to protect cells
from endogenous and exogenous stresses. Thus, compounds
that upꢀregulate the expression of ARE gene products may
24, 25
confirmed the antiꢀinflammatory effects of 2.
These proꢀ
gress provided useful tools to further investigate the pharmaꢀ
cological effects of the Keap1ꢀNrf2 PPI inhibitors. However,
the diacetate moiety of these compounds may have some poꢀ
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Page 2 of 12
tential risks for further development. Thus, it is quite necesꢀ sized based upon our previously identified compound 2 (as
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sary to investigate whether the diacetate moiety can be reꢀ
placed without sacrificing the activity.
shown in Table 1). These compounds were synthesized via the
derivation of 3. The different side chains were introduced by
the electrophilic substitution of the sulfonamides, which have
been used to construct the diacetic compound series (Scheme
24, 25
1).
Diꢀtetrazole analog can maintain the potent PPI inhibition
activity
These compounds were evaluated for their Keap1ꢀNrf2 PPI
inhibition activity in a previously described fluorescence poꢀ
24, 28
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Figure 1. Discovery and optimization process of diꢀacetic comꢀ
pound series.
larization (FP) assay.
As shown in Figure 2, compound 7
bearing the tetrazole groups maintained the PPI inhibition
activity with an IC50 of 15.8 nM, which is comparable to 2
(IC = 13.4 nM). While the introduction of other bioisosteric
Design and Synthesis of compounds with various bioisoꢀ
steric substituents of diacetic groups
50
substituents led to the dramatic decrease in activity, only 8
with 1,2,3ꢀtriazole groups displayed the nanomole activity
Our previous studies have identified that the P1 and P2 poꢀ
lar subꢀpockets of the Keap1 cavity are the determinants for
(
IC = 609 nM). The prominent performance of the tetrazole
50
26, 27
the potent Keap1ꢀNrf2 PPI inhibitors.
Accordingly, we
and 1,2,3ꢀtriazole groups indicated that electronꢀrich aromatic
ring may be an alternative pattern for occupying the P1 and P2
subꢀpockets. The carboxamide group, which can form similar
hydrogen bond but has significant high pKa value, resulted in
the inactive compound. It is consistent with the results providꢀ
successfully developed the diacetate compound series, which
utilized the two carboxylic acid groups to occupy the P1 and
2
4, 25
P2 subꢀpockets (as shown in Figure 1).
The diacetate
moiety can form multiple polar interactions, including hydroꢀ
gen bonds and salt bridges, with key polar residues, especially
arginines in the P1 and P2 subꢀpockets. However, the diacetic
acid moiety is highly polar and easily ionized, which may
possess potential hurdles in druggability issues.
29
ed by Jnoff, E. et al.. However, the carboxamide group can
30
also be active in certain cases. The research of Jain et al.
showed that when the substituents on the sideꢀchain phenyl
ring was the pꢀmethoxy group, the diꢀacetamide moiety can
insert into the P1 and P2 subꢀpockets and good inhibition acꢀ
tivity can be retained. The hydroxamic acid analog also
showed moderate activity. Considering the similar properties
of the two functional groups in forming hydrogen bonds, the
difference in acidity may be the main reason for the loss of
activity. The methyl ether of 2 did not show activity, providꢀ
ing a possible choice for the prodrug design of Keap1ꢀNrf2
PPI inhibitor.
Figure 2. Doseꢀresponse inhibition curves of 2 and 7 deterꢀ
mined by the FPꢀbased binding assay.
Binding mode investigation indicated diꢀtetrazole moiety
has novel ‘sandwich’ꢀlike binding pattern
In order to compare the role of diacetic moiety and tetrazole
groups in Keap1 binding, we carried out molecular docking
studies between the Keap1 Kelch domain and the two active
compounds 2 and 7. The LigandFit tool, which has been provꢀ
en suitable for Keap1 target and has been applied in our previꢀ
Scheme 1. Reagents and conditions: (a) NH OH·HCl, 95% ethaꢀ
2
20, 24
ous work,
was used to propose the binding pose. As shown
nol, MeOH, 60 °C, 2 h; (b) Pd/C, H , THF, rt, 4 h; (c) 4ꢀ
2
acetamido benzenesulfonyl chloride, toluene, pyridine, 100 °C, 2
h, 71%; (d) DMF, K CO , bromoacetonitrile, rt, 3 h, 53%; (e)
in Figure 3A, the diacetic moiety of 2 can insert into the P1
and P2 subꢀpockets and interact with key arginines by hydroꢀ
gen bonds and salt bridges. Although the tetrazole groups of 7
can occupy the P1 and P2 subꢀpockets, the binding pattern
showed some differences. In the case of 7, most of the hydroꢀ
gen bonds and salt bridges interactions were retained. Besides,
the diꢀtetrazole moiety can make extra piꢀcharge interactions
with guanidyl group of arginines. As shown in Figure 3B, the
diꢀtetrazole moiety packed between Arg380 and Arg415 and
formed multiple electrostatic interactions including chargeꢀ
charge and piꢀcharge interactions, which seems like a ‘sandꢀ
2
3
DMF, K CO , 3ꢀbromopropꢀ1ꢀyne, rt, 3 h, 59%; (f) DMF, K CO ,
2
3
2
3
methyl bromoacetate, rt, 3 h, 54%; (g) DMF, K CO , 2ꢀ
2
3
bromoacetamide, rt, 3 h, 64%; (h) DMF, NH Cl, NaN , N , 100
4
3
2
°
C, 6 h, 67%; (i) DMF, NH Cl, NaN , N , 100 °C, 6 h, 63%; (j)
4 3 2
NH OH·HCl, NaOH (aq) , MeOH, rt, 6 h, 64%; (k) LiAlH , THF,
2
4
NaOH (aq), rt, 3 h, 51%; (l) LiOH, CH OH/H O, rt, 6 h, 68%.
3
2
To investigate whether the diacetate moiety can be replaced
without sacrificing the activity, a series of compounds with
various bioisosteric substituents were designed and syntheꢀ
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Overall, the docking results indicated that the tetrazole moiety
wich’ binding mode, further enhancing the binding in the P1
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and P2 subꢀpockets. In order to match up this binding pose,
the core naphthalene ring also had a little bit of deflection, but
the hydrophobic interactions with Arg415 and Ala556 were
retained. The movement of the naphthalene ring also induced
the displacement of two phenyl rings. But the overall binding
pattern was similar, and the key piꢀpi interactions were kept.
not only interacted with key arginines by hydrogen bonds and
salt bridges, but also formed favorable piꢀcharge interactions.
The ‘sandwich’ꢀlike binding pattern taken by the diꢀtetrazole
moiety provided an alternative for the design of potent Keap1ꢀ
Nrf2
PPI
inhibitors.
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Figure 3. Binding mode investigation of 2 and 7 using molecular docking. Figure A and B represent the binding modes of 2 and
, respectively. The surface of Keap1 is colored as the property of hydrogen bonds. The ligand is represented as sticks. Green
7
dashed lines represent hydrogen bonds, yellow solid lines represent electrostatic interactions, the pink dashed lines represent PiꢀPi
interactions and the violet dashed lines represent hydrophobic interactions.
Table 1. IC , Physicochemical Properties and ARE Induction Fold Results of the Compounds Containing Various Substituꢀ
5
0
a
ents.
Induction Fold
0.1 ꢁM 1 ꢁM
IC50 (nM)
95% CI
Solubility
(pH = 7.4)
Log D
No.
Rꢀgroup
pKa
4.79
(pH = 7.4)
0.01 ꢁM
5 ꢁM
1
4.4
5.0
mg/mL
2
1.02
1.33 + 0.38
3.10 + 0.44
5.67 + 0.67 10.77 + 0.90
1
2.4ꢀ16.7
1
ꢁ
1
ꢁ
4.1
g/mL
1.2
g/mL
4
5
>50 ꢁM
> 50 ꢁM
10.51
10.73
4.65
4.75
1.11 + 0.17
0.94 + 0.12
1.18 + 0.16
1.03 + 0.06
2.00 + 0.37
1.24 + 0.23
3.50 + 0.72
4.10 + 0.86
36.4
ꢁg/mL
6
7
8
> 50 ꢁM
10.46
5.12
9.67
3.91
2.31
3.01
1.11 + 0.12
2.11 + 0.34
1.01 + 0.10
1.22 + 0.06
1.35 + 0.23
2.75 + 0.86
15.8
13.7ꢀ18.3
3.5
mg/mL
4.37 + 1.36 12.39 + 0.89 15.17 + 0.89
609
342.7ꢀ1084
697.9
ꢁg/mL
1.33 + 0.01
1.67 + 0.37
2.43 + 0.57
1.71 + 0.13
4.01 + 0.54
4.54 + 0.30
2
600
941.8
ꢁg/mL
9
8.12
2.87
1.19 + 0.14
2
150ꢀ3189
6
55.1
1
0
1
> 50 ꢁM
> 50 ꢁM
10.77
9.8
2.99
3.21
1.00 + 0.08
1.07 + 0.08
1.12 + 0.09
1.27 + 0.24
1.17 + 0.01
2.46 + 0.18
1.32 + 0.14
3.56 + 0.30
ꢁg/mL
3
88.5
1
ꢁg/mL
Physicochemical property determination confirms diꢀ
tetrazole replacement can elevate pKa and log DpH=7.4
spectively, which further confirmed 2 as an easily ionizable
and high polar compound. All of the compounds showed
higher pKa and logDpH=7.4 values than those of 2, which reꢀ
flected that the replacement of the diacetic moiety can imꢀ
We further evaluated the impact of bioisosteric substituents
on the physicochemical properties of 2 and 4ꢀ11. As shown in
Table 1, the pKa and logDpH=7.4 of 2 were 4.79 and 1.02, reꢀ
prove the physicochemical properties. The pKa and logDpH=7.4
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of the most potent analogue in the PPI inhibition experiment,
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compound 7, were 5.12 and 2.31, respectively. They were
both higher than those of 2, but lower than the other comꢀ
pounds, especially for the pKa value. It indicated that the P1
and P2 subꢀpockets favor the easily negatively charged groups
and the electrostatic interactions are important for Keap1ꢀ
ligand binding. Although the pKa and logDpH=7.4 changed, 7
retained the advantages in solubility, similar with 2. Taken
together, the higher pKa and logDpH=7.4 of 7 could enhance the
passive cell membrane permeability, which may be beneficial
for the cellular activity.
2
0.265±0.086
Diꢀtetrazole analog 7 is more potent in cellular ARE ꢀ luꢀ
ciferase reporter assay
In view of the promising character of 7, we investigated the
cellular activities of these compounds using AREꢀluciferase
reporter assay in HepG2ꢀAREꢀC8 cells, which has been apꢀ
plied extensively to determine the Nrf2 inducing ability of
small molecules. As shown in Table 1, 7, which is optimal in
both PPI inhibition activity and physicochemical properties,
showed significant improvement in the Nrf2ꢀARE inducing
activity. The level of ARE to about 15 fold can be achieved by
7 at 5 ꢁM, while 2 can only get about 11 fold at the same conꢀ
centration. These results showed that 7 has obvious adꢀ
vantages compared with 2 in the cellular AREꢀluciferase reꢀ
porter assay.
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Diꢀtetrazole replacement resulted in better cell membrane
permeability
Considering the significant change of the physicochemical
property induced by the polar recognition group, we then exꢀ
amined whether diꢀtetrazole replacement could affect the cell
membrane permeability. Permeability coefficients (Pe) were
determined using a standard parallel artificial membrane perꢀ
meability assay (PAMPA) on a PAMPA Explorer instrument
qRTꢀPCR analysis confirms 7 can more significantly inꢀ
duce the expression of Nrf2ꢀdriven genes
(pION). PAMPA is used as an in vitro model of passive,
transcellular permeability.
We then investigated whether 7 is more potent in elevating
the transcription of Nrf2ꢀdriven genes. Quantitative realꢀtime
PCR (qRTꢀPCR) analysis was performed to monitor the
mRNA level of three selected target genes, GCLM (glutamateꢀ
cysteine ligase, modifier subunit), NQO1 (NAD(P)H): Quiꢀ
none Oxidoreductase 1) and HOꢀ1 (Heme oxygenase 1). Both
of the two compounds concentrationꢀdependently increased
the transcription of Nrf2ꢀregulated genes. In the case of 2 at
the highest concentration (10 ꢁM), the levels of HOꢀ1 and
GCLM were about 21ꢀ and 14ꢀfold, respectively (Figure 4A
and B). Remarkably, for the optimized compound 7, there was
approximately 30ꢀ and 20ꢀ fold increase in the mRNA level of
HOꢀ1 and GCLM, respectively (Figure 4A and B). 7 also
showed the superiority in the elevation of NQO1 mRNA.
Treatment with 7 can induce about 5ꢀfold at the concentration
of 5 ꢁM, while similar elevation effects was achieved by 2 at
10 ꢁM (Figure 4C). Taken together, these results indicated
that 7 can obviously activate the transcriptional activity of
Surprisingly, 7 with the diꢀtetrazole showed a Pe value of
−6
4
2.3 × 10 cm/s, while 2 with the diacetic group only gave a
−6
Pe value of 0.3 × 10 cm/s. The results clearly showed that the
diꢀtetrazole analog can permeate the cell membrane through
the passive diffusion mechanism, while the diacetic analog can
hardly accomplish the transmembrane transport by this mechꢀ
anism. It confirmed that the diꢀtetrazole replacement can imꢀ
prove the cell membrane permeability which is beneficial for
the cellular activity.
Table 2. Membrane permeability of the selected comꢀ
pounds
−6
Compound
7
Rꢀgroup
Pe, pH = 7.4 (10 cm/s)
42.266 ± 4.476
Nrf2
at
a
lower
concentration
than
2.
Figure 4. Expression of Nrf2ꢀregulated genes after treatment with 2 and 7. HCT116 cells were treated with these two comꢀ
pounds respectively at the indicated concentrations for 12 h. The histogram showed qRTꢀPCR analysis of (A) HOꢀ1, (B) GCLM
and (C) NQO1 expression in HCT116 cells after treatment with compound 2 and 7, respectively. The expression of Nrf2 targeted
genes used βꢀactin for normalization (n=3).
Compound 7 is more efficacious on upꢀregulating the proꢀ
tein level of Nrf2 targeted genes
comparison. As shown in Figure 5, the upꢀregulation of
downstream protein NQO1 was not obvious at 8 h, which may
3
1, 32
result from the high basal level of NQO1 in HCT116 cells.
Then we examined the concentrationꢀdependent effects of 7
on the protein level of Nrf2 targeted genes including HOꢀ1,
NQO1 and γꢀGCS. The effects of 2 were also evaluated as
Compared to NQO1, the elevation of HOꢀ1 and γꢀGCS was
much more obvious. Compound 7 doubled the expression of
HOꢀ1 and γꢀGCS at the concentration of 10 ꢁM at 8 h, while 2
4
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semiꢀquantitative data). These data further substantiated the
only increased the expression of these two genes at 1.5ꢀ and
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1.6ꢀfold, respectively. While at 16h, compound 7 showed sigꢀ
nificant superiority in the elevation of NQO1, HOꢀ1 and γꢀ
GCS (Figure 5 for the protein band and Figure S1 for the
higher cellular Nrf2 inducing activity of 7 bearing tetrazole
groups.
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Figure 5. Doseꢀdependent increase of Nrf2ꢀregulated genes NQO1, HOꢀ1 and γꢀGCS in HCT116 cells after treatment with 2 and 7,
respectively.
In summary, with the aim to improve the physicochemical
properties and cell membrane permeability, we used the biꢀ
oisosteric groups to replace the diacetate groups of 2, which is
a potent Keap1ꢀNrf2 PPI inhibitor with lessꢀthanꢀideal drugꢀ
like properties. Among the compounds with various bioisoꢀ
steric substituents, 7 with tetrazole groups retains the Keap1ꢀ
Nrf2 PPI inhibition potency with an IC of 15.8 nM in the FP
State Key Laboratory of Natural Medicines, China Pharmaceutiꢀ
cal University (No.SKLNMZZCX201611).
The authors declare no competing financial interest.
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AUTHOR INFORMATION
Corresponding Author
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9) Suzuki, T.; Motohashi, H.; Yamamoto, M. Toward clinical
application of the Keap1–Nrf2 pathway. Trends Pharmacol. Sci.
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013, 34, 340ꢀ346.
*
*
Eꢀmail: youqidong@gmail.com
Eꢀmail: jiangzhengyucpu@163.com
(10) Lu, M.ꢀC.; Ji, J.ꢀA.; Jiang, Z.ꢀY.; You, Q.ꢀD. The Keap1–
Nrf2–ARE Pathway As a Potential Preventive and Therapeutic
Target: An Update. Med. Res. Rev, published online May 18, 2016;
DOI: 10.1002/med.21396.
Author Contributions
All authors have given approval to the final version of the manuꢀ
script.
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11) Lu, M.ꢀC.; Ji, J.ꢀA.; Jiang, Y.ꢀL.; Chen, Z.ꢀY.; Yuan, Z.ꢀW.;
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This work is supported by the projects 81230078 (key program)
and 81173087 of National Natural Science Foundation of China;
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(SRFDP, 20130096110002); the Fundamental Research Funds for
the Central Universities 2016ZPY016; and the Project Program of
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Figure 1. Discovery and optimization process of di-acetic compound series.
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Figure 2. Dose-response inhibition curves of 2 and 7 de-termined by the FP-based binding assay.
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Figure 3. Binding mode investigation of 2 and 7 using molecular docking. Figure A and B represent the
binding modes of 2 and 7, respectively. The surface of Keap1 is colored as the property of hydrogen bonds.
The ligand is represented as sticks. Green dashed lines represent hydrogen bonds, yellow solid lines
represent electrostatic interactions, the pink dashed lines represent Pi-Pi interactions and the violet dashed
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Figure 4. Expression of Nrf2ꢀregulated genes after treatment with 2 and 7. HCT116 cells were treated with
these two compounds reꢀspectively at the indicated concentrations for 12 h. The histogram showed qRTꢀPCR
analysis of (A) HOꢀ1, (B) GCLM and (C) NQO1 exꢀpression in HCT116 cells after treatment with compound 2
and 7, respectively. The expression of Nrf2 targeted genes used βꢀactin for normaliꢀzation (n=3).
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Figure 5. Doseꢀdependent increase of Nrf2ꢀregulated genes NQO1, HOꢀ1 and γꢀGCS in HCT116 cells after
treatment with 2 and 7, respectively.
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