Effects of 3-Bromo-4,5-dihydroisoxazole Derivatives on Nrf2 Activation and Heme Oxygenase-1 Expression

Article abstract of DOI:10.1002/open.201800185

Natural and synthetic electrophilic compounds have been shown to activate the antioxidant protective Nrf2 (nuclear factor erythroid 2-related factor 2)/heme oxygenase-1 (HO-1) axis in cells and tissues. Here, we tested the ability of different isoxazoline-based electrophiles to up-regulate Nrf2/HO-1. The potency of activation is dependent on the leaving group at the 3-position of the isoxazoline nucleus, and an additional ring on the molecule limits the Nrf2/HO-1 activating properties. Among the synthetized compounds, we identified 3-bromo-5-phenyl-4,5-dihydroisoxazole 1 as the derivative with best activating properties in THP-1 human monocytic cells. We have confirmed that the target of our compounds is the Cys151 of the BTB domain of Keap1 by using mass spectrometry analyses and X-ray crystallography. Our findings demonstrate that these compounds affect the Nrf2/HO-1 axis and highlight a positive activity that can be of relevance from a therapeutic perspective in inflammation and infection.

Full text of DOI:10.1002/open.201800185

DOI: 10.1002/open.201800185
Effects of 3-Bromo-4,5-dihydroisoxazole Derivatives on
Nrf2 Activation and Heme Oxygenase-1 Expression
[
a]
[b, c]
[d]
[e]
Andrea Pinto, Zeina El Ali,
Sꢀbastien Moniot, Lucia Tamborini,*
[d]
[b, c]
[e]
Clemens Steegborn, Roberta Foresti,
and Carlo De Micheli
Natural and synthetic electrophilic compounds have been
shown to activate the antioxidant protective Nrf2 (nuclear
factor erythroid 2-related factor 2)/heme oxygenase-1 (HO-1)
axis in cells and tissues. Here, we tested the ability of different
isoxazoline-based electrophiles to up-regulate Nrf2/HO-1. The
potency of activation is dependent on the leaving group at
the 3-position of the isoxazoline nucleus, and an additional
ring on the molecule limits the Nrf2/HO-1 activating proper-
ties. Among the synthetized compounds, we identified 3-
bromo-5-phenyl-4,5-dihydroisoxazole 1 as the derivative with
best activating properties in THP-1 human monocytic cells. We
have confirmed that the target of our compounds is the
Cys151 of the BTB domain of Keap1 by using mass spectrome-
try analyses and X-ray crystallography. Our findings demon-
strate that these compounds affect the Nrf2/HO-1 axis and
highlight a positive activity that can be of relevance from a
therapeutic perspective in inflammation and infection.
1
. Introduction
The Nrf2 (nuclear factor erythroid 2-related factor 2) transcrip-
tion factor plays a key role in cellular stress response mecha-
nisms, controlling the transcription of more than 1000 genes
involved in detoxification, anti-oxidant, metabolic and anti-in-
the nucleus where it binds to the antioxidant responsive ele-
ment (ARE) located in the promoter region of detoxifying and
protective genes. Keap-1 also controls Nrf2 by mediating its
ubiquitination. Among Nrf2 dependent genes, Heme oxygen-
ase-1 (HO-1) is one of the enzymes controlled by Nrf2 that
converts the heme to biliverdin, iron and carbon monoxide
(CO), important antioxidant and signaling molecules active
[
1–3]
flammatory activities.
Under unstressed conditions, Nrf2 is
retained in the cytoplasm in a silent form by its repressor pro-
tein, Keap-1 (Kelch-like ECH-associated protein 1), which con-
tains a subset of 27 highly reactive cysteine (Cys) residues in
Human. Oxidative and environmental stimuli modify the cys-
teine residues of Keap-1, thus enabling translocation of Nrf2 to
[4]
during stress conditions. Therefore, the Nrf2/HO-1 axis has
been shown to be very important for tissue protection and is
currently the target of drug discovery approaches.
A successful strategy used to disrupt the Keap1-Nrf2 interac-
tion consists in using electrophiles from synthetic or natural
sources. Bardoxolone methyl (CDDO-Me, Figure 1) is a potent
[
a] Prof. A. Pinto
Department of Food, Environmental and Nutritional Sciences (DeFENS)
University of Milan
viaCeloria 2, 20133 Milan (Italy)
[
b] Dr. Z. El Ali, Prof. R. Foresti
Inserm U955, Equipe 12
Crꢀteil, 94000 (France)
[
c] Dr. Z. El Ali, Prof. R. Foresti
Universitꢀ Paris Est, Facultꢀ de Mꢀdecine
Crꢀteil, 94000 (France)
[d] Dr. S. Moniot, Prof. C. Steegborn
Department of Biochemistry, University of Bayreuth
Universitaetsstr. 30, 95447 Bayreuth (Germany)
[
e] Dr. L. Tamborini, Prof. C. De Micheli
Department of Pharmaceutical Sciences, University of Milan
viaMangiagalli 25, 20133 Milan (Italy)
E-mail: lucia.tamborini@uimi.it
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/open.201800185.
ꢁ
2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Figure 1. Electrophilic modulators of the Keap1-Nrf2 pathway.
ChemistryOpen 2018, 00, 0 – 0
1
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

activator of Nrf2 and like the fumarates is thought to act via
2
. Results and Discussion
[
5,6]
reaction with Cys151 of the Keap-1 protein.
In addition to
these compounds, other electrophilic modulators of the
Keap1-Nrf2 pathway are known including curcumin, 4-hydroxy-
2.1. Chemistry
2
-nonenal, and ebselen (Figure 1). In this context, one of the
The synthesis of derivatives 1, 2 and 4–8 was based on the
1,3-dipolar cycloaddition of bromonitrile oxide, generated in
situ by dehydrohalogenation of the stable precursor dibromo-
most studied compounds is sulforaphane, a naturally occurring
[
7–1]
isothiocyanate isolated from cruciferous vegetables.
[23]
Numerous electrophiles (e.g., acrylamides and other a,b-un-
saturated groups, boronic acids, and a-halogen-substituted ke-
tones) have been widely employed to design enzymatic cova-
lent inhibitors. Some functional groups such as epoxides, aziri-
dines, halomethyl- and acyloxymethyl-ketones are often re-
garded as too reactive for potential drug applications. None-
theless, examples of selective and relatively safe inhibitors
formaldoxime (DBF) to the suitable dipolarophile, affording
in all cases the 5-substituted 3-bromo-isoxazoline derivative in
very high yield. Derivatives 1, 2, 4 and 7 were previously de-
[
24–26]
scribed by us,
compound 3 was obtained following a liter-
[27]
ature procedure whereas derivatives 5, 6 and 8 were ob-
tained according to Scheme 1.
[
11–13]
bearing those functionalities exist (e.g., Fosfomycin).
The
3-halo-4,5-dihydroisoxazole warhead can be considered a rare
electrophile in therapeutics since it reacts solely with cysteine
residues activated by surrounding amino acid residues present
in the catalytic site of a number of enzymes. Such a peculiarity
has already been used to design efficacious inhibitors of differ-
[
14–17]
ent enzymatic targets, ranging from parasitic
and bacterial
to human targets involved in the modulation
of neuronal metabolic pathways or in tumor cell metabo-
[
18,19]
[20]
enzymes
[
21]
lism. In this context, we tested different isoxazoline-based
electrophiles (Figure 2) to examine their effect on the Nrf2/HO-
1
axis.
2 3
Scheme 1. Reagents and conditions: a) NaOAc, Ac O, AcOH; b) DBF, NaHCO ,
EtOAc, room temperature.
2.2. Effects on Nrf2 Activation and HO-1 Expression
Electrophilic compounds can exert cytotoxic activity and we
first examined the viability of human monocytic THP-1 cells ex-
posed to increasing concentrations of the different com-
pounds. As shown in Figure 3, compounds 1, 2, 4 and 5 did
not cause evident cytotoxicity up to 100 mm. Only compound
Figure 2. Isoxazoline-based electrophiles.
3
promoted cell toxicity (34%) at the highest concentration of
100 mm; such a profile could be due to the presence of the
Here the influence of different leaving groups in position 3,
i.e., bromine, chlorine and nitro (compounds 1–3, Figure 2)
and of the substituents in position 4 and 5 of the isoxazoline
ring have been investigated (compounds 4–5, Figure 2). The
more interesting derivatives in terms of cytotoxic activity, i.e.,
compounds 1 and 5, and other three para-phenyl substituted
analogues, i.e., compounds 6–8, have been submitted to a
mass spectrometry analysis to get the proof of concept of the
covalent addition to Keap1-BTB domain (Broad complex, Tram-
track, and Bric-ꢂ-Brac domain). Subsequently, in order to deter-
mine the covalent addition of the ligands to Cys151 of Keap1-
BTB domain, the structure of the complex between Keap1-BTB
and 5 was solved by X-ray crystallography. Cysteine-151, in
fact, represents one of the major sensors in KEAP1 and its
nitro group which can be reduced to a toxic hydroxylamine
derivative. It is noted that there are many compounds that will
induce Nrf2-HO-1 at concentrations lower than those used in
[3,4]
the present study but there are also examples of already ap-
proved drugs, such as dimethylfumarate (DMF), that can be
used in the higher micromolar range (50–100) to affect Nrf2
[5]
due to their low cytotoxicity profile. On the contrary, CDDO is
very powerful at nanomolar range but it is also very toxic at
low micromolar concentrations. Thus, the ability of compounds
to induce the Nrf2 system needs to be balanced with their in-
trinsic cytotoxicity. When tested in Western blot experiments,
we observed that compounds 1, 3 and 5 increased Nrf2 and
HO-1 protein expression at different extents with compound 1
being the most efficient inducer of both proteins (Figure 4a).
In contrast, compound 2 increased Nrf2 but not HO-1 protein
[22]
modification is sufficient for robust activation of NRF2.
(
Figure 4b) while compound 4 did not affect Nrf2 or HO-1 ex-
&
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
2
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Figure 3. Cytotoxicity data of compounds 1–5. Human monocytic THP-1 cells were exposed for 24 h to increasing concentrations of the different compounds
and assessed for cell viability using the LDH assay, as described in the Experimental Section. Results are mean Æ SEM of three independent experiments.
Figure 4. Compound 1 is the most efficient Nrf2 and HO-1 activator in THP-1 cells. A) Western blot results of THP-1 cells treated for 6 h with the different
compounds. B) Densitometric analysis of Western blots. C+ is the positive control obtained from THP-1 cells incubated for 6 h with 100 mm DMF. Results are
mean Æ SEM of three independent experiments; * p<0.05 vs. control.
pression (data not shown). The fact that compound 2 activated
Nrf2 but did not induce HO-1 expression is not understood at
present. The activation of Nrf2 by derivatives 1, 2, 3 and 5 is
strictly related to the nature of the leaving group appended at
the 3 position of the isoxazoline nucleus. As previously ob-
prises three of the 27 cysteines of Keap1, including Cys151 as
[22]
the critical stress sensor. The mass for the protein incubated
with DMSO was determined to be 15,190.9 Da in good
agreement (within <30 ppm) to its calculated mass of
15,190.4645 Da (Figure 5A). Incubation of Keap1 BTB with our
compounds resulted in the emergence of a peak with a higher
mass confirming covalent modification of the protein by addi-
tion of a single compound molecule. For all compounds but 1,
the most abundant species corresponds to the derivatized pro-
tein with the expected mass (Figure 5B). For all compounds,
however, an additional peak is observed with a mass that fits
to a degradation product in which the phenyl substituent in
position 5 of the isoxazoline ring was eliminated (Figure 5C).
Variable amounts of the unmodified protein, intact protein
adduct and protein modified with degradation product are ob-
served with the different compounds (Figure 5B).
[
16,17,25]
served on different targets,
the 3-bromo-isoxazoline war-
head is more active than the 3-chloro counterparts. Finally, the
additional ring present in derivative 4 seems to limit the Nrf2/
HO-1 activating properties.
2.3. Mass Spectrometry Analysis
To confirm that the target of our compounds is the BTB
domain of Keap1, we purified recombinant protein and investi-
gated by mass spectrometry its covalent modification in the
absence or presence of 1, 5, 6, 7 or 8. The BTB domain com-
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
3
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

[
a]
Table 1. Data collection and refinement statistics. Keap1 BTB domain in
complex with 5.
Wavelength [ꢃ]
Resolution range [ꢃ]
Space group
0.91814
36.8–2.2 (2.28–2.20)
P6 22
5
Unit cell
a=42.491, b=42.491, c=265.213
Total reflections
Unique reflections
Multiplicity
47296 (4795)
7968 (752)
5.9 (6.4)
98.73 (99.08)
9.33 (0.57)
50.25
Completeness [%]
Mean I/s(I)
2
Wilson B-factor [ꢃ ]
R-meas
0.149 (3.161)
1.00 (0.33)
7946 (750)
0.2506 (0.4153)
0.2919 (0.3979)
1085
1052
15
18
131
CC1/2
Reflections used in refinement
R-work
R-free
No. non-hydrogen atoms
No. macromolecule atoms
No. ligand atoms
No. solvent molecules
No. protein residues
RMS(bonds) [ꢃ]
Figure 5. The isoxazoline covalently modifies a single cysteine in the BTB
domain of Keap1. A) Deconvoluted intact protein mass spectrum of the
native protein (top) and after treatment with 5 (bottom). The masses deter-
mined by MS are in good agreement with the theoretical mass for the
native protein (15190.46 Da) and with that of the protein after covalent
modification of a cysteine residue of the protein by compounds. B) Popula-
tion distribution between native unmodified protein, protein modified by
the intact compound, and modified protein after elimination of the aryl sub-
stituent at position-5 of the isoxazoline moiety. C) The theoretical and calcu-
lated masses are given for the example of the reaction of BTB with 5 and for
the degradation product common to all compounds (Figure 5B).
0.002
RMS(angles) [8]
0.40
96.90
0.00
0.00
71.21
71.63
59.33
Ramachandran favored [%]
Ramachandran outliers [%]
Rotamer outliers [%]
Average B-factor [ꢃ ]
Average macromolecules
Average ligands
2
Average solvent molecules
56.54
2.4. Crystallographic Studies
[
a] Values for the highest-resolution shell are shown in parentheses.
Kelch-like ECH-associated protein 1 (Keap1) controls Nrf2 acti-
vation by binding Nrf2 in the cytoplasm and inducing its deg-
radation by the proteasome. Keap 1 contains a number of cys-
teins that react with oxidants and electrophiles, thus inducing
modification in the Keap1 protein that prevents the binding to
and degradation of Nrf2. As a consequence, Nrf2 accumulates
[28]
and enters the nucleus to up-regulate its target genes. The
Cys151 residue in the Keap1 protein has been shown to be es-
[
29]
sential for Nrf2 activation by several electrophiles. To confirm
Cys151 modification and to identify further interactions and
mechanistic consequences, we solved the structure of the
Keap1-BTB domain bound to 5 by X-ray crystallography
(
Table 1).
Figure 6. Crystal structure of the Keap1/5 complex (PDB ID 6FFM): Stereo-
scopic view of the region of Cys151 covalently bound to 5 (purple). The
backbone and residue 151 of the BTB domain are shown in white with two
types of electron density maps. A feature-enhanced map of type 2mFo-DFc
in blue is contoured at 1sigma and a Fourier difference omit map (mFo–
DFc) of the ligand is presented in green contoured at 3sigma. Both maps
support the presence of the covalently bound inhibitor and suggest the ex-
istence of 2 to 3 alternative conformations of the cysteine side chain.
The structure comprises residues 48 to 180, which adopt a
conformation very similar to the previously reported Keap1-
BTB structures (root mean square deviations on the position of
[
6,30]
1
21 to 131 Ca range from 0.38 to 0.53 ꢃ). C The presence of
continuous electron density extending from the Sg atom of
Cys 151 shows the presence of covalently bound ligand
(Figure 6). Residual difference density extending from Cys151
further suggests that the cysteine side-chain points in at least
two different directions, which could either, indicate that the
compound induces a change in orientation or that the ligand
induces extreme flexibility and is present in multiple conforma-
tions rather than adopting a unique one.
tain flexibility of this part and/or by the presence of multiple
conformations. This is consistent both with the absence of in-
teraction with the protein and with the coexistence of two
enantiomers of 5. This limited and rather unspecific interac-
tions between Keap1-BTB and a covalent inhibitor of Cys151 is,
however, not uncommon since it was observed in most other
The electron density, however, does not cover the ligand
completely and the aryl substituent in position 5 of the isoxa-
zoline moiety is only partially defined, the signal fading away
from the chiral centre onward. This can be explained by a cer-
[6,30]
published complex structures.
The major conformation of 5
that is observed here is consistent with previous Keap1 Cys151
&
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
4
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

and are uncorrected. Microanalyses (C, H, N) of new compounds
were within Æ0.4% of theoretical values.
General Procedure for the Cycloaddition Reaction
Solid NaHCO (417 mg, 4.96 mmol) and DBF (402 mg, 1.98 mmol)
3
were added to a solution of the appropriate alkene (0.99 mmol) in
CH Cl (4.2 mL). The reaction mixture was stirred at room tempera-
2
2
ture for 48 h and the progress of the reaction was monitored by
TLC. Water was added and the organic layer was separated, dried
over anhydrous Na SO and evaporated under reduced pressure.
2
4
Figure 7. Superposition of the BTB/5 complex to the complex with CDDO.
The structure of the BTB/5 complex (white/purple) (PDB ID 6FFM) was super-
posed to the BTB/CDDO complex structure (light green, PDB ID 4CXT) based
Purification of the crude material was performed by column chro-
matography.
[
6]
a
on 101 C positions (rmsd 0.221 ꢃ).
N-(4-(3-Bromo-4,5-dihydroisoxazol-5-yl)phenyl)acetamide (5)
a) 4-Vinylaniline (200 mL, 1.71 mmol) was dissolved in AcOH
modifier conformations (Figure 7) and, since the isoxazoline
substituent is flexible and not significantly involved in binding,
is representative of complexes between Keap1-BTB and our
other isooxazoline compounds, including the most potent in-
hibitor 1 (Sup. Figure 1).
(
0.5 mL) and NaOAc (34 mg, 0.51 mmol) and Ac O (157 mL,
2
1
.71 mmol) were added. The reaction was stirred at room tempera-
ture for 30 min. Water (5 mL) was added and the solution was ex-
tracted with EtOAc (3ꢅ3 mL). The organic phase was dried over
anhydrous Na SO and evaporated under reduced pressure. Purifi-
2
4
cation of the crude material by column chromatography (cyclohex-
ane/EtOAc 7:3) afforded the protected aniline (160 mg, 58% yield).
b) Compound 5 was synthesized following the general procedure
for the cycloaddition reaction reported above using the acetamide
prepared in the previous step.
3
. Conclusions
Our work demonstrates that certain isoxazoline-based electro-
philes, and especially bromo-based derivatives such as com-
pound 1, are good activators of the Nrf2/HO-1 protective
system. It appears that the potency of activation is dependent
on leaving group appended at the 3 position of the isoxazoline
nucleus and that an additional ring on the molecule, such as
in the case of compound 4, limits the Nrf2/HO-1 activating
properties. The ability of compound 5 to covalently bind the
Keap1-BTB domain at Cys151 indicates the most likely cysteine
target that is critical for allowing the modulation of Nrf2 ex-
pression in cells. However, we cannot exclude that other cys-
teine residues of Keap1 are modified by our compound and
contribute to functional effects. It is interesting to note that
isoxazoline derivatives have been already shown to inactivate
glyceraldehydes-3-phosphate dehydrogenase from Plasmodium
1
Yield: 82%; H NMR (300 MHz, CDCl ): 2.18 (s, 3H), 3.18 (dd, J=9.2,
3
17.3, 1H), 3.59 (dd, J=10.7, 17.3, 1H), 5.63 (dd, J=9.2, 10.7, 1H),
1
3
7.20 (bs, 1H), 7.30 (d, J=8.5, 2H), 7.52 (d, J=8.5, 2H); C NMR
(75 MHz, CDCl ): 24.8, 42.2, 83.2, 120.4, 127.0, 135.0, 137.1, 138.7,
68.8; Anal. calcd for C H BrN O : C, 46.66; H, 3.92; N, 9.89;
3
1
11 11
2
2
found: C, 47.00; H, 3.99; N, 9.80.
3
-Bromo-5-(4-fluorophenyl)-4,5-dihydroisoxazole (6)
Compound 6 was synthesized following the general procedure for
the cycloaddition reaction reported above.
Yield: 93%; white solid; crystallized as white needles from n-
hexane; mp=59.2–59.98C; Rf =0.37 (cyclohexane/EtOAc, 9:1);
1
H NMR (300 MHz, CDCl ): d=3.18 (dd, J=9.1, 17.3, 1H) 3.61 (dd,
3
[
25,26]
falciparum.
·Therefore, our findings that these compounds
J=10.7, 17.3, 1H), 5.66 (dd, J=9.1, 10.7, 1H), 7.05–7.15 (m, 2H),
1
3
7
2
.30–7.40 (m, 2H); C NMR (75 MHz, CDCl ): d=163.1 (d, JC-F=
also affect Nrf2/HO-1 highlight an additional positive activity
for these derivatives that can be of relevance from a therapeu-
tic perspective in inflammation and infection.
3
47.9), 136.9, 135.3 (d, JC-F=3.1), 128.1 (d, JC-F=8.3), 116.1 (d, JC-
F=21.7), 82.8, 49.4; MS(ESI): 243.9 [M+H] . Anal. calcd for
+
C H BrFNO: C, 44.29; H, 2.89; N, 5.74; found: C, 44.50; H, 2.99; N,
9
7
5
.60.
Experimental Section
3
-Bromo-5-(4-nitrophenyl)-4,5-dihydroisoxazole (8)
Materials and Methods
Compound 8 was synthesized following the general procedure for
the cycloaddition reaction reported above.
1
13
All reagents were purchased from Sigma. H NMR and C NMR
spectra were recorded with a Varian Mercury 300 (300 MHz) spec-
trometer. Chemical shifts (d) are expressed in ppm, and coupling
constants (J) are expressed in Hz. TLC analyses were performed on
commercial silica gel 60 F254 aluminium sheets; spots were further
evidenced by spraying with a dilute alkaline potassium permanga-
nate solution or ninhydrin. Flash chromatography separations were
performed on Bꢄchi Pump Manager C-615 and C-601 instruments.
Melting points were determined on a model B 540 Bꢄchi apparatus
Yield: 80%; solid; crystallized as white needles; mp=101.1
1
À101.58C; R =0.41 (cyclohexane/EtOAc, 8:2); H NMR (300 MHz,
f
CDCl ): d=3.17 (dd, J=8.2, 17.3, 1H), 3.74 (dd, J=11.1, 17.3, 1H),
3
5.78 (dd, J=8.2, 11.1, 1H), 7.50–7.58 (m, 2H), 8.22–8.30 (m, 2H);
1
3
C NMR (75 MHz, CDCl ): d=148.2, 146.8, 136.9, 126.9, 124.4, 81.9,
3
+
49.6; MS(ESI): 270.8 [M+H] . Anal. calcd for C H BrN O : C, 39.88;
9
7
2
3
H, 2.60; N, 10.33; found: C, 40.00; H, 2.71; N, 9.98.
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
5
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Cell culture and Determination of Cell Viability
Crystallization was carried out by hanging drop vapour diffusion
by mixing equal amounts of protein solution with a crystallization
solution composed of 25–28% PEG 4000, 0.1m Tris-HCl pH 8.0 and
The human monocytic cell line THP-1 was cultured under standard
conditions in RPMI medium (Gibco) supplemented with 10% fetal
bovine serum, 1% sodium pyruvate (Gibco) and 1% penicillin/
streptomycin (Gibco). One million cells/well were used for viability
assay studies. THP-1 cells were incubated for 24 h with increasing
concentrations (1–100 mm) of the compounds 1, 2, 3, 4 and 5 and
solvent control (0.1% DMSO). At the end of the incubation the cell
supernatant was collected and lactate dehydrogenase (LDH) activi-
ty was measured as an index of cell toxicity according to the man-
ufacturer’s instructions (Roche). Only damaged cells release this
enzyme in the supernatant and the level of LDH activity correlates
with the extent of cellular damage. Triton 2% was used as a posi-
tive control. Results were expressed as the percentage of cell via-
bility.
0
.2m lithium sulfate. For cryoprotection, the crystals were briefly
soaked in a solution composed of the original crystallization
mother liquor supplemented with 25% glycerol. Diffraction data
have been collected on BL14.1 operated by the Helmholtz-Zen-
trum Berlin (HZB) at the BESSY II electron storage ring (Berlin-
[31]
[32]
Adlershof, Germany) and processed using the XDS package.
[33]
The structure was solved by molecular replacement using Phaser
and the previously published structure of the BTB domain of
Keap1 as a model (PDB code 4CXI). Refinement of the structure
[34]
was performed using phenix.refine until convergence of the R
factors. The statistics of the diffraction data as well as of the final
model are presented in Table 1. The coordinates of the model as
well as the structure factors were deposited to the PDB under ac-
cession code 6FFM.
Western Blot Analysis for Determination of Nrf2 and HO-1
Protein Expression
Mass Spectrometry Measurement of the Intact Protein Masses
Based on the toxicity results, the ability of 1, 2, 4 and 5 to induce
Nrf2/HO-1 was assessed at concentrations up to 100 or 200 mm.
Compound 3, which exhibited significant toxicity at 100 mm, was
tested only up to 50 mm. THP-1 cells were incubated with the com-
pounds for 6 h after which induction of Nrf2 and HO-1 proteins by
Western blot was assessed using specific antibodies. Briefly, cells
were washed in cold PBS before lysis in lysis buffer (20 mm Tris
pH 7.4, 137 mm NaCl, 2 mm EDTA pH 7.4, 1% Triton, 25 mm b-glyc-
erophophate, 1 mm Na3VO4, 2 mm sodium pyrophosphate, 10%
glycerol, 1 mm PMSF, 1% mammalian protease inhibitor). The ho-
mogenates were centrifugated at 15,000 rpm for 20 min at 48C.
Equal amounts of denaturated protein were loaded onto 10%
SDS-PAGE gel and transferred on PVDF membrane (Amersham Bio-
sciences, Les Ulis, France). Membranes were then incubated with
antibodies directed against Nrf2 (H-300, Santa Cruz biotechnology,
Santa Cruz, USA, 1:1000 dilution in Tris buffer saline Tween 0.2%,
overnight incubation at 48C) or HO-1 (Abcam, UK, 1:1000 dilution
in 3% bovine serum albumin solution prepared with Tris buffer
saline-Tween 0.2%, 2 h incubation at room temperature). Secon-
dary antibodies were anti-rabbit-HRP (7074S, Cell Signaling, 1:2500
dilution in 3% bovine serum albumin solution prepared with Tris
buffer saline-Tween 0.2%) for Nrf2 detection and anti-mouse-HRP
To analyze covalent modification(s) of the BTB domain, 10 mm BTB
4
8–180 (S172A) were incubated with 3 mm 1, 5, 6, 7 or 8 (or 3%
DMSO final concentration for control) for 1 h on ice in 20 mm Tris-
HCl pH 8.0 and 150 mm NaCl. Intact protein masses were deter-
mined through HPLC-coupled electrospray ionization (ESI)-MS on
an AB sciex TripleTOF 5600+ mass spectrometer. Intact proteins
were first concentrated and washed on a Piccolo Proto 200 C4 5-
mm 2.5ꢅ0.5-mm trap column (Higgins Analytical) and subsequently
switched in line with, and separated on, a Jupiter C4 5-mm 300-ꢃ
1
50ꢅ1-mm analytical column (Phenomenex) mounted onto a Shi-
À1
madzu Prominence UFLC (Shimadzu) at a 70 mLmin flow rate
with the following buffers: AÀ5% ACN, 5% DMSO, and 0.1% FA;
BÀ90% ACN, 5% DMSO, and 0.1% FA. Proteins were then eluted
over with a gradient of 3 min of 1% B to 55% B followed by 1 min
of 55% B to 90% B. Mass analysis was performed by ESI-TOF-MS
on an AB Sciex TripleTOF 5600+ mass spectrometer (Sciex) with a
DuoSpray Ion Source with the following settings: floating voltage
of 5,500 V, temperature of 3508C, declustering potential of 120
with four separate TOF experiments, each, respectively with 4, 12,
2
0, and 40 time bins summed. Data analysis was performed as fol-
lows: spectra were integrated over a retention time period, and
the summed TOF experiment with the greatest resolution selected.
The raw data were then converted and deconvoluted using the
MaxEnt I algorithm (Waters) at a resolution of 0.1 Da.
(
7076S, Cell Signaling, 1:5000 dilution in 3% bovine serum albumin
solution prepared with Tris buffer saline-Tween 0.2%) for HO-1, in-
cubated for 1 h at room temperature. The wash buffer was Tris
buffer saline-Tween 0.2%. Immunoreactive bands were detected
by chemiluminescence (ECL solution, Amersham Biosciences, Les
Ulis, France). b-actin (Santa Cruz biotechnology, Santa Cruz, USA) Conflict of Interest
was used as a loading control. Images were captured using a
G:Box F3 imagery station (Syngene, Cambridge, UK).
The authors declare no conflict of interest.
Recombinant Expression, Purification and Crystal Structure
Determination of the BTB Domain of Keap1
Keywords: 3-bromo-4,5-dihydroisoxazole · crystal structure ·
cycloaddition · Keap1 · mass spectrometry
The gene encoding residues 48–180 of human Keap1 (Uniprot
Q14145) were subcloned in a modified pET19 vector to incorpo-
rate a TEV cleavable N-terminal hexahistidine tag. As previously re-
ported, a single point mutation was also introduced at position
[
1] K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N.
Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, Y. Nabeshima, Biochem.
Biophys. Res. Commun. 1997, 236, 313–322.
2] H. Motohashi, M. Yamamoto, Trends Mol. Med. 2004, 10, 549–557.
3] R. Foresti, S. K. Bains, T. S. Pitchumony, E. L. de Castro Brꢆs, F. Drago, J.
Dubois-Randꢀ, C. Bucolo, R. Motterlini, Pharm. Res. 2013, 76, 132–148.
4] A. Nikam, A. Olliver, M. Rivard, J. L. Wilson, K. Mebarki, T. Martens, J. L.
Dubois-Randꢀ, R. Motterlini, R. Foresti, J. Med. Chem. 2016, 59, 756–
762.
[
[
[6]
1
72 (S172A), which enhanced protein stability and crystallizability.
The protein was expressed in E. coli BL21 (DE3) and purified to ho-
[6]
mogeneity as previously described. Prior to crystallization, the
[
À1
BTB domain at 10 mgmL concentration was incubated for 1 h on
ice in the presence of 3 mm 5 (10% final DMSO concentration).
&
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
6
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

[
[
5] B. Haas, S. Chrusciel, S. Fayad-Kobeissi, J. L. Dubois-Randꢀ, F. Azuaje, J.
Boczkowski, R. Motterlini, R. Foresti, J. Cell. Physiol. 2015, 230, 1128–
[22] a) L. Quinti, S. D. Naidu, U. Trꢇger, X. Chen, K. Kegel-Gleason, D. Llꢈres,
C. Connolly, V. Chopra, C. Low, S. Moniot, E. Sapp, A. R. Tousley, P. Vo-
dicka, M. J. Van Kanegan, L. S. Kaltenbach, L. A. Crawford, M. Fuszard, M.
Higgins, J. R. C. Miller, R. E. Farmer, V. Potluri, S. Samajdar, L. Meisel, N.
Zhang, A. Snyder, R. Stein, S. M. Hersch, L. M. Ellerby, E. Weerapana,
M. A. Schwarzschild, C. Steegborn, B. R. Leavitt, A. Degterev, S. J. Tabrizi,
D. C. Lo, M. DiFiglia, L. M. Thompson, A. T. Dinkova-Kostova, A. G. Ka-
zantsev, Proc. Natl. Acad. Sci. USA 2017, E4676–E4685; b) M. McMahon,
D. J. Lamont, K. A. Beattie, J. D. Hayes, Proc. Natl. Acad. Sci. USA 2010,
107, 18838–18843.
1138.
6] A. Cleasby, J. Yon, P. J. Day, C. Richardson, I. J. Tickle, P. A. Williams, J. F.
Callahan, R. Carr, N. Concha, J. K. Kerns, H. Qi, T. Sweitzer, P. Ward, T. G.
Davies, PLOS 2014, 9, e98896.
[
[
7] A. J. Wilson, J. K. Kerns, J. F. Callahan, C. J. Moody, J. Med. Chem. 2013,
56, 7463–7476.
8] S. Zheng, Y. R. Santosh Laxmi, E. David, A. T. Dinkova-Kostova, K. H. Shia-
voni, Y. Ren, Y. Zheng, I. Trevino, R. Bumeister, I. Ojima, W. C. Wigley, J. B.
Bliska, D. F. Mierke, T. Honda, J. Med. Chem. 2012, 55, 4837–4846.
[23] A. Pinto, P. Conti, M. De Amici, L. Tamborini, U. Madsen, B. Nielsen, T.
Christesen, H. Brauner-Osborne, C. De Micheli, J. Med. Chem. 2008, 51,
2311–2315.
[9] T. Satoh, S. R. McKercher, S. A. Lipton, Free Radical Biol. Med. 2014, 66,
45–57.
[
10] W. Li, S. Zheng, M. Higgins, R. P. Morra, Jr., A. T. Mendis, C.-W. Chien, I.
[24] P. Conti, M. De Amici, A. Pinto, L. Tamborini, G. Grazioso, B. Frølund, B.
Nielsen, C. Thomsen, B. Ebert, C. De Micheli, Eur. J. Org. Chem. 2006, 24,
5533–5542.
[25] S. Bruno, A. Pinto, G. Paredi, L. Tamborini, C. De Micheli, V. La Pietra, L.
Marinelli, E. Novellino, P. Conti, A. Mozzarelli, J. Med. Chem. 2014, 57,
7465–7471.
[26] S. Bruno, M. Margiotta, A. Pinto, G. Cullia, P. Conti, C. De Micheli, A. Moz-
zarelli, Bioorg. Med. Chem. 2016, 24, 2654–2659.
[27] O. A. Ivanova, E. M. Budynina, E. B. Averina, T. S. Kuznetsova, N. S. Zefir-
ov, Synthesis 2006, 4, 706–710.
[28] T. Suzuki, M. Yamamoto, J. Biol. Chem. 2017, 292, 16817.
[29] D. V. Chartoumpekis, N. Wakabayashi, T. W. Kensler, Biochem. Soc. Trans.
2015, 43, 639–644.
[30] C. Huerta, X. Jiang, I. Trevino, C. F. Bender, D. A. Ferguson, B. Probst,
K. K. Swinger, V. S. Stoll, P. J. Thomas, I. Dulubova, M. Visnick, W. C.
Wigley, Biochimica et Biophysica Acta 2016, 1860, 2537–2552.
[31] U. Mueller, R. Fçrster, M. Hellmig, F. U. Huschmann, A. Kastner, P. Male-
cki, S. Pꢄhringer, M. Rçwer, K. Sparta, M. Steffien, M. ꢉhlein, P. Wilk,
M. S. Weiss, Eur. Phys. J. Plus 2015, 130, 1–10.
Ojima, D. F. Mierke, A. T. Dinkova-Kostova, T. Honda, J. Med. Chem. 2015,
5
8, 4738–4748.
11] J. D. Hayes, A. T. Dinkova-Kostova, Trends Biochem. Sci. 2014, 39, 199–
18.
12] S. De Cesco, J. Kurian, C. Dufresne, A. K. Mittermaier, N. Moitessier, Eur.
J. Med. Chem. 2017, 138, 96e114.
13] E. L. Kwak, R. Sordella, D. W. Bell, N. Godin-Heymann, R. A. Okimoto,
B. W. Brannigan, P. L. Harris, D. R. Driscoll, P. Fidias, T. J. Lynch, S. K. Ra-
bindran, J. P. McGinnis, A. Wissner, S. V. Sharma, K. J. Isselbacher, J. Set-
tleman, D. A. Haber, Proc. Natl. Acad. Sci. USA 2005, 102, 7665–7670.
14] R. Ettari, A. Pinto, S. Previti, L. Tamborini, I. C. Angelo, V. La Pietra, L. Ma-
rinelli, E. Novellino, T. Schirmeister, M. Zappalꢂ, S. Grasso, C. De Micheli,
P. Conti, Bioorg. Med. Chem. 2015, 23, 7053–7060.
[
[
[
2
[
[
15] R. Ettari, L. Tamborini, I. C. Angelo, S. Grasso, T. Schirmeister, L. Lo Presti,
C. De Micheli, A. Pinto, P. Conti, ChemMedChem 2013, 8, 2070–2076.
16] L. Tamborini, A. Pinto, T. K. Smith, L. L. Major, M. C. Iannuzzi, S. Cosco-
nati, L. Marinelli, E. Novellino, L. Lo Presti, P. E. Wong, M. P. Barrett, C.
De Micheli, P. Conti, ChemMedChem 2012, 7, 1623–1634.
[
[
17] P. Conti, A. Pinto, P. E. Wong, L. L. Major, L. Tamborini, M. C. Iannuzzi, C.
De Micheli, M. P. Barrett, T. K. Smith, ChemMedChem 2011, 6, 329–333.
18] C. JÅst, C. Nitsche, T. Scholz, L. Roux, C. D. Klein, J. Med. Chem. 2014, 57,
[32] W. Kabsch, Acta Crystallogr. 2010, D66, 133–144.
[33] G. Bunkoczi, N. Echols, A. J. McCoy, R. D. Oeffner, P. D. Adams, R. J. Read,
Acta Crystallogr. 2013, D69, 2276–2286.
[
7
590–7599.
[34] P. V. Afonine, R. W. Grosse-Kunstleve, N. Echols, J. J. Headd, N. W. Moriar-
ty, M. Mustyakimov, T. C. Terwilliger, A. Urzhumtsev, P. H. Zwart, P. D.
Adams, Acta Crystallogr. 2012, D68, 352–367.
[
[
19] R. Orth, T. BÅttcher, S. A. Sieber, Chem. Commun. 2010, 46, 8475–8477.
20] A. Pinto, L. Tamborini, E. Pennacchietti, A. Coluccia, R. Silvestri, G. Cullia,
C. De Micheli, P. Conti, D. De Biase, J. Enzyme Inhib. Med. Chem. 2016,
3
1, 295–301.
21] A. Pinto, L. Tamborini, G. Cullia, P. Conti, C. De Micheli, ChemMedChem
016, 11, 10–14.
[
2
Received: September 5, 2018
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
7
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
A. Pinto, Z. El Ali, S. Moniot,
L. Tamborini,* C. Steegborn, R. Foresti,
C. De Micheli
An eye on the target: The ability of dif-
ferent isoxazoline-based electrophiles to
up-regulate the antioxidant protective
Nrf2/heme oxygenase-1 (HO-1) axis is
evaluated and a 3-bromo-isoxazoline
derivative endowed with activating
properties in THP-1 human monocytic
cells is identified. Through mass spec-
trometry analyses and X-ray crystallog-
raphy, it is possible to confirm that the
target of the synthesized compounds is
the Cys151 of BTB domain of Keap1.
&& – &&
Effects of 3-Bromo-4,5-
dihydroisoxazole Derivatives on Nrf2
Activation and Heme Oxygenase-1
Expression
&
ChemistryOpen 2018, 00, 0 – 0
www.chemistryopen.org
8
ꢁ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!