Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction

Article abstract of DOI:10.1016/j.bmcl.2013.03.013

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

Full text of DOI:10.1016/j.bmcl.2013.03.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3039–3043
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a small-molecule inhibitor and cellular probe
of Keap1–Nrf2 protein–protein interaction
a
a
c
c
a
Longqin Hu a,b, , Sadagopan Magesh , Lin Chen , Lili Wang , Timothy A. Lewis , Yu Chen ,
Carol Khodier c, Daigo Inoyama a, Lesa J. Beamer d, Thomas J. Emge e, Jian Shen a, John E. Kerrigan b,
Ah-Ng Tony Kong b,f, Sivaraman Dandapani c, Michelle Palmer c, Stuart L. Schreiber c, Benito Munoz c
⇑
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
c Chemical Biology Platform and Probe Development Center, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
d Department of Biochemistry, University of Missouri, Columbia, MO, USA
e Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
f Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization
assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein inter-
action. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation
demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated
as ML334 (LH601A), which is at least 100Â more potent than the other stereoisomers. The stereochem-
istry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific
synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translo-
cation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary
but tractable structure–activity relationships support its use as a lead for our ongoing optimization
Ó 2013 Elsevier Ltd. All rights reserved.
Received 27 January 2013
Revised 24 February 2013
Accepted 4 March 2013
Available online 14 March 2013
Keywords:
Keap1
Nrf2
Inhibitors of protein–protein interaction
Nrf2 activation
Three major cellular components are involved in the regulation
of cellular defense mechanisms that protect cells from oxidative
stress; they are Kelch-like ECH-associated protein 1 (Keap1), nu-
clear factor erythroid 2-related factor 2 (Nrf2), and antioxidant re-
sponse elements (ARE). The Keap1–Nrf2–ARE system is the main
signaling pathway that regulates transcription of a series of cyto-
protective proteins including glutathione S-transferases (GSTs),
NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1
(HO-1), and Nrf2 itself (Fig. 1). This system plays a key role in
oxidative stress response, inflammation, and carcinogenesis.1–4
Targeting the Keap1–Nrf2–ARE signaling pathway represents an
attractive strategy to discover chemopreventive agents for cancer
as well as preventive and therapeutic agents for a variety of other
diseases and conditions, including diabetes, Alzheimer’s, and Par-
kinson’s, that involve oxidative stress and inflammation.5–13
Keap1–Nrf2 protein–protein interaction is considered a critical
point of the pathway that can be targeted for intervention.1 Some
of the known Nrf2-activating/ARE-inducing agents are already in
human clinical trials as chemopreventive agents for cancer or as
therapeutic agents for conditions involving inflammation. These
include sulforaphane for the treatment and prevention of prostate
cancer and for the treatment of chronic obstructive pulmonary
disease (COPD) and bardoxolone methyl for the treatment of ad-
vanced chronic kidney disease (CKD) in patients with type 2 diabe-
tes mellitus.14–18 However, all currently known small-molecule
Nrf2 activators/ARE inducers, including many natural products
(e.g., sulforaphane, curcumin, and epigallocatechin gallate from
natural sources such as fruits, vegetables, and tea products) and
synthetic compounds (e.g., oltipraz, anethole dithiolethione, and
bardoxolone methyl), function as eletrophiles and indirectly inhi-
bit Keap1–Nrf2 protein–protein interaction through covalent mod-
ification of the sensitive cysteine residues found in Keap1.1,19
To discover novel non-reactive small molecules as direct
inhibitors of the protein–protein interaction between Keap1 and
Nrf2, we developed a fluorescence polarization (FP) assay with
fluorescein-labeled 9mer Nrf2 peptide amide as the fluorescent
probe and Keap1 Kelch domain as the target protein20 and used
it to screen the NIH MLPCN library of small molecules. Herein,
we report the discovery of a small molecule hit as a first-in-class
direct small-molecule inhibitor of Keap1–Nrf2 protein–protein
interaction. Because of the presence of three chiral centers and
the uncertain stereochemical composition of the hit sample, we
synthesized the hit and a series of analogs with defined composition
⇑
Corresponding author. Tel.: +1 848 445 5291; fax: +1 732 445 6312.
E-mail address: LongHu@rutgers.edu (L. Hu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.013

3040
L. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3039–3043
Figure 1. A model depicting Nrf2 activation by a direct inhibitor of Keap1–Nrf2 interaction. Nrf2 is sequestered in the cytosol by its protein inhibitor, Keap1, and is
transcriptionally inactive. A direct inhibitor binds to the Kelch domain of Keap1 and suppresses the ubiquitination of Nrf2, which decreases degradation of Nrf2 and allows
more Nrf2 to translocate to the nucleus and form a transcriptionally active complex with Maf, leading to the induced expression of Nrf2 itself and oxidative stress response
enzymes that deactivate reactive oxygen species and electrophiles.
for activity confirmation, determination of stereochemical require-
ment for Keap1 binding, and preliminary structure–activity rela-
tionship analysis.
predominantly composed of one set of enantiomers as later con-
firmed by chiral HPLC analysis. It became clear that we needed
to determine which of the stereoisomer is responsible for binding
to Keap1 Kelch domain.
Hit identification and confirmation. We developed an FP assay,20
which was successfully used to screen the MLPCN library of
337,116 compounds (PubChem Assay ID: 504523, 504540). Using
inhibition >3Â standard deviation of DMSO wells (corresponding
Resynthesis of hit 1 for activity confirmation and stereochemistry
assignment. When we synthesized hit 1 according to Scheme 1 by
reacting 1-phthalimidomethyl tetrahydroisoquinoline (( )-4) with
cis-cyclohexanedicarboxylic anhydride, we obtained an expected
mixture of four stereoisomers (5, LH601) based on our chiral HPLC
analyses. The sample of 5 was shown to be two fold less active than
the hit 1 sample obtained from the NIH MLPCN library in our SPR
solution competition assay. Further analysis of the hit 1 sample
from the NIH MLPCN library, we found that the hit 1 sample con-
tained ꢀ90% of one set of enantiomers, probably as a result of
recrystallization used for purification in the commercial process.
We then separated the sample of 5 into diastereomers (A/B and
C/D) using flash silica gel chromatography. A/B was shown to con-
tain the active isomer in hit 1 and was further separated into the
two enantiomers A and B by preparative normal phase chiral sep-
aration on a Chiralcel OD column. The activity of isomer A, B and C/
D were then compared to that of hit 1 in our FP and SPR assays. As
shown in the SPR dose–response curves inFigure 2D, we have iden-
tified the most active stereoisomer in hit 1 being the isomer A
to 12% inhibition) for hit calling, the primary screen at 10 lM gen-
erated a list of 489 hits. After excluding fluorescent compounds,
460 of the initial hits were cherry-picked and retested in the FP as-
say for eight-point dose–response curves and in a thermal shift
secondary assay. Among the eight confirmed hits, hit 1 (Fig. 1A)
was the most promising with an IC50 of 3
lM (Fig. 2B). The binding
constant (Kd) of hit 1 to Keap1 Kelch domain was confirmed to be
1.9 lM (Fig. 2C and D) using an SPR solution competition assay we
recently developed.21 Hit 1 has no chemically reactive functional
groups present and is not expected to modify sulfhydryl groups
of cysteine residues in proteins.
Since there are three stereogenic elements in hit 1 and the two
substituents on the cyclohexyl ring are known to be of cis configu-
ration, we were expecting four stereoisomers that could be pres-
ent; one of the four stereoisomers is likely to be a direct
inhibitor of the Keap1–Nrf2 interaction since ligand–target inter-
actions are often stereospecific. However, the composition of hit
1 sample in the MLPCN library was assigned with uncertainty as
a pair of diastereomers. Only after our synthesis and determination
of Keap1 binding of an equal mixture of all four stereoisomers did
we realize that the hit 1 sample in the MLPCN library was
(LH601A) with a Kd of 1
lM while its enantiomer B has a Kd of only
100 M and the other diastereomers C/D are inactive. The four
l
trans-stereoisomers resulting from the reaction of 1-phthalimi-
domethyl tetrahydroisoquinoline (( )-4) with trans-cyclohexan-
edicarboxylic anhydride were also inactive.

L. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3039–3043
3041
X-ray crystallography was used to assign the stereochemistry of
the four isomers. We first attempted but failed to grow single crys-
tals of pure isomer A. Interestingly, the A isomer could only be
crystallized as a pair with its enantiomer B even when using 96%
ee of A. Fortunately, isomer D readily crystallized as single crystals
of the pure D (Fig. 2E), which was used to assign the absolute ste-
reochemistry of all four isomers as indicated in Scheme 1.
A
C
B
O
N
O
OH
O
O
N
Hit 1
We then undertook a stereospecific synthesis of (SRS)-5 as fur-
ther confirmation of its absolute stereochemistry as shown in
Scheme 2. Starting from the commercially available (S)-tetrahydro-
isoquinoline-1-carboxylic acid ((S)-6), we protected the secondary
amine with Cbz and reduced the carboxylic acid to give Cbz-pro-
tected (S)-(tetrahydroisoquinolin-1-yl)methanol ((S)-7). Mitsun-
obu reaction, using phthalimide in the presence of DIAD and PPh3,
followed by hydrogenolysis afforded stereospecifically the (S)-4.
Alternatively, (S)-4 was prepared from the racemic amine by reso-
lution through crystallization of their diastereomeric salts with (À)-
dibenzoyl
L-tartaric acid ((À)DBTA). A known alkaloid-mediated
desymmetrization procedure of cis-cyclohexanedicarboxylic anhy-
dride with benzyl alcohol in the presence of quinidine22,23 was used
to control the chiral centers on the cyclohexyl ring to obtain
(1R,2S)-2-(benzyloxycarbonyl)cyclohexane carboxylic acid ((R,S)-
8). Simple amide bond coupling between the optically active (S)-4
and (R,S)-8 produced the benzyl protected (SRS)-9, which upon
hydrogenolysis gave the desired enantiomer (SRS)-5.
After confirming the Keap1-binding activity of (SRS)-5 in our FP
and SPR assays, we determined its cellular activity in two cell-
based functional assays. Figure 3A illustrates the ARE-inducing
D
E
activity of (SRS)-5 (isomer A) with an EC50 of 18 lM as compared
to >100 lM for its enantiomer (RSR)-5 (isomer B) and its diastereo-
mers C/D in the CellSensorÒ ARE-bla HepG2 cell line, obtained from
Invitrogen, where ARE controls the expression of b-lactamase.24
Figure. 3B demonstrates that (SRS)-5 promotes the nuclear translo-
cation of Nrf2 with a similar EC50 of 12 l
M in the PathHunterÒ
U2OS Keap1–Nrf2 functional assay, obtained from DiscoveRx,
which uses b-galactosidase-based enzyme fragment complemen-
tation technology and luminescence for the detection of Nrf2 nu-
clear translocation.25 All these data indicate that (SRS)-5 is cell
permeable and is capable of inhibiting the Keap1–Nrf2 interaction,
leading to the dissociation of Nrf2 from Keap1 in the cytosol, its
subsequent translocation to the nucleus, and the upregulation of
ARE-controlled genes.
Figure 2. (A) The structure of hit 1 identified through HTS, (B) dose–response curve
of the hit 1 in the confirmative FP assay, (C) sensorgrams of the hit 1 in the SPR
assay, (D) comparison of Kd of different stereoisomers of 5 (LH601) to hit 1, and (E)
X-ray crystal structures of A/B as a pair and D as a single enantiomer.
We also synthesized several analogs of (SRS)-5 to derive preli-
minary structure–activity relationships suggesting that (SRS)-5 is
O
O
OH
N
N
O
O
H2N
NH2
O
NaBH(OAc)3
O
O
N
O
N
NH
POCl3
OH
O
O
O
1
2
3
4
O
O
A
B
C
(SRS)-5
(RSR)-5
(SSR)-5
O
A/B
N
O
OH
Flash silica gel
chromatography
O
O
Chiral separation
on Chiralcel OD
O
N
p-Xylene
C/D
Diastereomers
Scheme 1. Synthesis and separation of hit 1 stereoisomers.
(RRS)-5
Enantiomers
D
5
(LH601)

3042
L. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3039–3043
O
O
HO
COOH
NH
N
N
1) Phthalimide
DIAD, PPh3
Cbz
1) NaBH(OAc)3
O
O
1) CbzCl
2) BH3
N
S
NH
N
2) H2, Pd/C
2) (-) DBTA resolution
(S)-6
(S)-7
4
3
(S)-
O
O
O
HOOC
BnOH/Toluene
COOBn
Quinidine
EDC/HOBt
TEA
(R,S)-8
O
O
N
N
O
OBn
H2, Pd/C
O
OH
O
O
O
O
N
S
N
S
S
S
R
R
(SRS)-9
(SRS)-5
Scheme 2. Stereospecific synthesis of (SRS)-5.
A
B
100
80
60
40
20
0
Isomer A EC50 = 18 μM
Isomer B EC50 > 100 μM
Isomer C/D EC50 > 100 mM
100
80
60
40
20
0
Isomer A EC50 = 12 μM
Isomer B EC50 > 100 μM
Isomer C/D EC50 > 100 μM
-20
-20
10-7
10-6
10-5
10-4
10-7
10-6
10-5
10-4
[Compound] (M)
[Compound] (M)
Figure 3. ARE-inducing activity of (SRS)-5 (isomer A) in CellSensorÒ b-lactamase reporter assay (A) and its effect on Nrf2 nuclear translocation in the PathHunterÒ U2OS
Keap1–Nrf2 functional assay (B) in comparison to other stereoisomers.
viable lead for further structure optimization into more potent di-
rect inhibitors of Keap1–Nrf2 interaction. The following is a sum-
mary of some of the more important SARs (Fig. 4): (1) Keap1
binding activity resides primarily in one stereoisomer. As discussed
earlier, (SRS)-5 (isomer A) is at least 100 times more active than
other stereoisomers (B/C/D and trans isomers). (2) Acidic function-
ality is preferred on the cycloalkane ring. The corresponding
methyl ester was completely inactive and the corresponding amide
was about 20-fold less active than (SRS)-5, suggesting that the free
carboxylic acid group in (SRS)-5 is optimal for binding to Keap1 Kelch
domain. (3) A one-carbon linker between tetrahydroisoquinoline
(THIQ) and phthalimido group is optimal. Analogs with
one-carbon extension, that is, an ethylene linker instead of a meth-
ylene, between THIQ and phthalimido group, are inactive. (4) One
of the carbonyls in phthalimido group can be removed to give a
lactam with only slight decrease in Keap1 binding. Additional
analogs are being synthesized in our continuing lead optimization
efforts to explore the chemical spaces around the various points of
the scaffold to improve binding affinity, membrane permeability,
and other physico-chemical and pharmaceutical properties.
We docked (SRS)-5 to the Nrf2 peptide binding site in Keap1
Kelch domain starting from the cocrystal structure of Keap1 Kelch
domain with the 16mer Nrf2 peptide (PDB code: 2FLU); Figure. 5A
shows a representative binding pose of (SRS)-5 in the Nrf2 peptide
binding site of Keap1 Kelch domain and Figure. 5B depicting the
overlay of (SRS)-5 to Nrf2 peptide as bound to Keap1 and major
interactions between Keap1 and the ligands. Based on this model,
reduction = ~2× less active
aminolysis = ~3-4× less active
O
homologation = inactive
N
the binding of (SRS)-5 to Keap1 is strengthened by two
p–cation
O
OH
ester = inactive
O
interactions between THIQ and Arg415 and between phthalimido
and Arg380 in addition to the hydrogen-bonding interactions that
are also observed between Keap1 and Nrf2 peptide.
O
amide = 20× less active
N
inactivating molecular dissections
The solubility of (SRS)-5 is >100 lM in phosphate buffered sal-
ine and (SRS)-5 is highly stable over a period of 24 h. Even though
Figure 4. Preliminary structure–activity relationships. The activity noted was
based on the solution competition SPR assay.
it does not contain electrophilic groups, distinguishing it from

L. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3039–3043
3043
Figure 5. Docking of (SRS)-5 to the Nrf2 binding site of Keap1 Kelch domain. (A) Top view with (SRS)-5 (shown in stick model) bound to Keap1 Kelch domain shown in ribbon
graphics and interacting Keap1 residues in VDW surface. (B) Overlay of the (SRS)-5 colored by element type with Nrf2 peptide in green as docked onto the Nrf2 binding site of
Keap1 Kelch domain (rotated ꢀ90° around the X-axis in A/B for a better view of the interactions with Keap1 residues shown at the bottom.
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that we used to mimic cysteine residues. No thiol addition or
decomposition could be detected over 48 h, indicating that (SRS)-
5 inhibits the Keap1–Nrf2 protein–protein interaction by non-
covalent binding and thus is a first-in-class direct inhibitor of
Keap1–Nrf2 interaction. (SRS)-5 was designated as a probe with
the NIH Molecular Libraries Program (ML334) due to these proper-
ties and its potency as an inhibitor of Keap1–Nrf2 interaction.
In summary, we have discovered, through high-throughput
screening of the NIH MLPCN library of small molecules, a first-
in-class direct inhibitor of the Keap1–Nrf2 protein–protein interac-
tion. It was found that only one stereoisomer among the possible
eight isomers is active. The different binding activity between
the stereoisomers is expected of structurally specific ligand–target
interactions. The strong binding affinity of (SRS)-5 (ML334,
LH601A) over its stereoisomers and the preliminary, yet tractable,
structure–activity relationships provide further evidence of its
direct binding to Keap1 and support its use as a lead for structure
optimization.
Acknowledgments
We thank Dr. Suzanne Forry-Schaudies from the National Can-
cer Institute for her advice and continuing efforts in overseeing the
success of this project. We gratefully acknowledge the financial
support of grants CA133791 and MH093197 (to L.H.) from the Na-
tional Institutes of Health. This work was funded in part by the
NIH-MLPCN program (1 U54 HG005032-1 awarded to S.L.S.).
22. Bolm, C.; Schiffers, I.; Atodiresei, I.; Hackenberger, C. P. R. Tetrahedron:
Asymmetry 2003, 14, 3455.
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24. Invitrogen. Cell Sensor™ ARE-bla Hep G2 Cell-based Assay Protocol, 2006; p 8.
25. DiscoveRx. PathHunterÒ Keap1–Nrf2 Functional Assay for chemiluminescent
detection of activated Nrf2. Product Booklet: 93-0821C3, 2011; p 12.
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