Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain

Article abstract of DOI:10.1021/acs.jmedchem.9b00445

The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

Full text of DOI:10.1021/acs.jmedchem.9b00445

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Article
Discovery of first in vivo active inhibitors of soluble
epoxide hydrolase (sEH) phosphatase domain
Jan S Kramer, Stefano Woltersdorf, Thomas Duflot, Kerstin Hiesinger, Felix F Lillich, Felix
Knöll, Sandra K Wittmann, Franca M Klingler, Steffen Brunst, Apirat Chaikuad, Christophe
Morisseau, Bruce D. Hammock, Carola Buccellati, Angelo Sala, G. Enrico Rovati, Matthieu
Leuillier, Sylvain Fraineau, Julie Rondeaux, Victor Hernandez Olmos, Jan Heering, Daniel Merk,
Denys Pogoryelov, Dieter Steinhilber, Stefan Knapp, Jeremy Bellien, and Ewgenij Proschak
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00445 • Publication Date (Web): 22 Aug 2019
Downloaded from pubs.acs.org on August 24, 2019
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Chemistry
Bellien, Jeremy; University Hospital Centre Rouen
Proschak, Ewgenij; Goethe-Universitat Frankfurt am Main, Institute of
Pharmaceutical Chemistry
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Discovery of first in vivo active inhibitors of soluble
epoxide hydrolase (sEH) phosphatase domain
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1‡
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Jan S. Kramer , Stefano Woltersdorf , Thomas Duflot , Kerstin Hiesinger , Felix F. Lillich ,
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1,3
Felix Knöll , Sandra K. Wittmann , Franca-M. Klingler , Steffen Brunst , Apirat Chaikuad ,
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Christophe Morisseau , Bruce D. Hammock , Carola Buccellati , Angelo Sala , G. Enrico
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1,7
Rovati , Matthieu Leuillier , Sylvain Fraineau , Julie Rondeaux , Victor Hernandez Olmos ,
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,7
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1,2
Jan Heering , Daniel Merk , Denys Pogoryelov , Dieter Steinhilber , Stefan Knapp , Jeremy
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1,*
Bellien , and Ewgenij Proschak
1
Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9,
0438 Frankfurt a. M., Germany.
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2
Laboratory of Pharmacokinetics, Toxicology and Pharmacogenetics, Rouen University Hospital,
76000 Rouen, France
3
Structural Genomics Consortium, Buchmann Institute for Life Sciences, Goethe-University
Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt a. M., Germany.
4
Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center,
University of California Davis, 95616 Davis, USA.
5
Department of Pharmacological and Biomolecular Sciences, University of Milan, Via
Balzaretti, 9, 20133, Milan, Italy
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Normandie Univ, UNIROUEN, INSERM U1096, 1 rue de Germont, 76000 Rouen, France
Fraunhofer IME-TMP, Max-von-Laue-Strase 9, 60438 Frankfurt a. M., Germany
Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, 60438
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Frankfurt a. M., Germany.
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Department of Clinical Pharmacology, Rouen University Hospital, 76000 Rouen, France
KEYWORDS
soluble epoxide hydrolase, phosphatase, oxazoles, crystal structure, chemical probe,
lysophosphatidic acid
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ABSTRACT
The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme
exhibiting two different catalytic activities, which are located in two distinct domains. Although
the physiological role of the C-terminal hydrolase domain is well-investigated, little is known
about its phosphatase activity located in the N-terminal domain of the sEH (sEH-P). Herein, we
report the discovery and optimization of the first inhibitor of human and rat sEH-P, applicable in
vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor
provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to
rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-
yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile
in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in
vivo.
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Introduction
The homodimeric soluble epoxide hydrolase (sEH) consists of two functionally different
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domains which are connected by a flexible proline-rich linker . The C-terminal domain catalyzes
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the conversion of epoxyeicosatrienoic acids (EETs) and related epoxy fatty acids (EpFAs)
towards the corresponding α,β-diols. The role of this hydrolase activity of sEH is well studied,
and inhibitors of the C-terminal domain (sEHI) hold promising therapeutical potential in various
diseases including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative
2
diseases . The very broad therapeutical scope of sEHI is based on the hypothesis that while an
accumulation of EpFAs has no or little effect under physiological conditions, they can prevent
mitochondrial dysfunction and reduce subsequent reactive oxygen species (ROS) formation and
3
the endoplasmic reticulum stress conditions . Accordingly, sEHI targeting the hydrolase domain
are in the focus of drug discovery and two candidates have already reached clinical
2
,4
development .
In contrast to the well-understood function of the C-terminal hydrolase domain, the role of the N-
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terminal phosphatase domain remains unclear. In 2003, Cronin et al. and Newman et al.
independently reported a phosphatase activity of the N-terminal domain of the sEH (sEH-P). In
vitro, sEH-P is able to hydrolyze diverse lipid phosphates including farnesyl pyrophosphate7,
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sphingosine-1-phosphate, and lysophosphatidic acid . The mechanism of lipid phosphate
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0
hydrolysis is well-investigated . However, neither the endogenous substrate nor its
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physiological and possibly pathophysiological role have been identified so far . Several
molecules with sEH-P inhibitory activity have been identified up to date. Sodium dodecyl sulfate
7
12,13
(
1, SDS) and N-acetyl-S-farnesyl-L-cysteine (2)
are lipid-like inhibitors of sEH-P. SMTP-7
(3), an investigational thrombolytic drug for treatment of ischemic stroke that simultaneously
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inhibits both, the hydrolase and phosphatase activities of sEH . The development of high-
1
5
16
throughput screening assays for sEH-P led to discovery of ebselen (4) and oxaprozin (5) as
novel sEH-P inhibitors. While 4 also affects sEH hydrolase activity, 5 is a selective but weak
inhibitor of sEH-P (IC = 5 µM). In this study, we investigate the structure-activity relationships
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(
SAR) of oxazole-based sEH-P inhibitors using the non-steroidal anti-rheumatic drug 5 as a
starting point.
O
OH
O
S
O
O
N
H
Na
S
O
O
1
(sodium dodecyl sulfate)
2 (N-acetyl-S-farnesyl-L-cysteine)
O
O
HO
OH
N
N
O
OH
O
O
HO
OH
OH
3
(SMTP-7)
O
O
O
N
N
Se
4
(ebselen)
5 (oxaprozin)
Figure 1. Structures of previously identified inhibitors of sEH-P activity 1-5
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Results and Discussion
Chemistry
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The 2,4,5-trisubstituted oxazole scaffold of compound 5 is accessible via Blümelein-Lewy
synthesis or starting from -acyloxy ketones. In both methods, -bromo-deoxybenzoins or
benzoin derivatives can be employed as starting materials to generate the heterocycle. However,
crossed benzoin derivatives with two different aryl moieties are not easily accessible whereas,
asymmetric deoxybenzoins can be obtained from O-protected cyanohydrins as reported by Stork
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et al. which are subsequently converted into -bromo-ketones. This approach allows a very
flexible alteration of the oxazole scaffold in all three substituent positions and enables a
comprehensive structure-activity relationship analysis.
To investigate the importance of the carbon in the alkyl linker of 5, thioglycolic acid derivative 9
was synthesized in two steps from 4,5-diphenyloxazole-2-thiol 6 and methyl 2-bromoacetate 7
resulting in methyl ester 8 which was subsequently hydrolysed (Scheme 1).
O
N
O
O
N
O
N
i)
ii)
SH
S
O
S
O
Br
CH3
8
8%
65%
O
O CH3
OH
6
7
8
9
Scheme 1: i) Et N, THF, rt, 24 h; ii) KOH, MeOH/H O/THF, 70 °C, 15 min µw.
3
2
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Amide derivative 12 was obtained from commercially available -bromo-deoxybenzoin 10 and
succinic amide 11 reacting under basic conditions to an -acyloxy ketone which under heating in
acetic acid in presence of excess of ammonium acetate cyclized to the oxazole 12 (Scheme 2).
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O
Br
O
O
N
i)
NH2
NH2
HO
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8%
O
O
10
11
12
Scheme 2: i) 1. Et N, acetonitrile, 45°C, 6 h; 2. NH OAc, AcOH, reflux, 3 h.
3
4
Synthesis of oxaprozin derivatives 20a-m, with alterations in the 4-phenyl moiety of the oxazole
core structure, started from benzaldehydes 13a-m as shown in Scheme 3. The substitution
18
strategy followed the Topliss optimization scheme . Benzaldehydes 13a-m were treated with
trimethylsilyl cyanide (TMSCN, 14) in the presence of catalytic amounts of triethylamine under
1
7
neat conditions to yield silyl protected cyanohydrins 15a-m. As described by Stork et al. O-
protected cyanohydrins are easily deprotonated in α-position by LDA in THF, obtaining a stable
nucleophile at low temperatures, which can react with an electrophile, in this case benzyl
bromide 16. The desired deoxybenzoins 17a-m were subsequently obtained by cleaving the
remaining silyl ether group of the intermediate with tetrabutylammonium fluoride solution
(TBAF). Deoxybenzoins derivatives 17a-m were then transformed to the appropriate -bromo-
deoxybenzoins 18a-m under heating with bromine in dichloromethane. The respective -bromo-
deoxybenzoins 18a-m were subsequently reacted with succinic acid 19 under basic conditions to

-acyloxy ketones which under heating in acetic acid and in presence of excess of ammonium
acetate yielded oxaprozin derivatives 20a-m.
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TMS
O
O
i)
CN
Br
ii)
TMS CN
R1
88%-99%
23%-93%
O
R1
R1
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1
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3a-m
14
15a-m
16
17a-m
Br
O
O
O
iii)
iv)
OH
OH
O
N
HO
11%-34%
over 2 steps
O
R1
R1
18a-m
19
20a-m
Scheme 3: i) Et N, 24 h; ii) 1. LDA, THF, -78°C→rt, 24 h; 2. TBAF, 30 min; iii) Br , CHCl ,
3
2
3
reflux, 2 h; iv) 1. Et N, MeCN, 45°C, 6 h; 2. NH OAc, AcOH, reflux, 3 h.
3
4
Synthesis of oxaprozin derivatives 22a-e, with alterations in the oxazole 2-substituent is depicted
in Scheme 4. Starting from intermediate 18l, 22a-e were obtained by the same strategy as
described above for oxaprozin derivatives 20a-m using dicarboxylic acids 21a-e with different
length and rigidity.
Br
O
O
O
N
i)
R2
R2
HO
13%-16%
Cl
Cl
Cl
22a-e
Cl
18l
21a-e
O
OH
O
OH
O
2
R =
,
,
,
OH ,
OH
O
OH
O
a
b
c
d
e
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Scheme 4: i) 1. dicarboxylic acid 21a-e, Et N, MeCN, 45°C, 6h; 2. NH OAc, AcOH, reflux 3h.
3
4
For alteration of the 5-phenyl moiety of the oxazole ring, a different synthetic approach was
chosen to reduce the synthetic efforts. The adjusted strategy (Scheme 5) employed 3,4-
dichlorophenylacylbromide 23 and glutaric acid 21b to obtain -acyloxy ketone 24 under basic
conditions which reacted under heating in BF *Et O and in presence of excess acetamide to
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oxazole 25. The oxazole 5-position was then activated for following Suzuki-cross-coupling by
introduction of bromine with NBS in acetonitrile yielding key intermediate 26 for final
modification. Coupling of 26 to oxaprozin derivatives 28a-k with the appropriate boronic acid
derivatives 27a-k succeeded in DMF/water with tetrakis-(triphenylphosphine)-palladium(0) as
catalyst and potassium phosphate as base.
O
O
O
Br
O
OH
N
i)
ii)
OH
7
9%
O
O
58%
Cl
Cl
O
Cl
Cl
23
Cl
Cl
24
25
OH
B
R3
Br
OH
O
N
iv)
O
N
R3
iii)
67%
27a-k
OH
OH
3%-50%
O
Cl
O
Cl
Cl
Cl
26
28a-k
Scheme 5: i) glutaric acid 21b, Et N, acetone, TLC-control; ii) acetamide, BF *Et O, 140 °C,
3
3
2
3
0 h; iii) NH OAc, NBS, acetonitrile, rt, 1.5 h; iv) boronic acid derivative 27a-k, K PO ,
4 3 4
DMF/H O 2/1, Pd(PPh ) , 55-80 °C, 30-48 h.
2
3 4
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Structure-activity relationships
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The exploration of structure-activity relationships of substituted oxazoles was performed using a
16
fluorescence-based enzyme activity assay as reported previously . In this kinetic assay, the
conversion of the fluorogenic substrate fluorescein diphosphate by the recombinant sEH-P
domain is monitored to assess the inhibitory potency of the test compounds. Bioisosteric
replacement of the β-methylene bridge by sulfur (9) slightly diminished potency while
replacement of the carboxylate by a primary amide (12) resulted in complete loss of activity.
Replacement of the oxazole core in pyrazole 29 was also not tolerated (Figure 2).
OH
O
OH
O
NH2
O
OH
O
N
O
N
O
N
O
N
N
S
5
9
12
inactive
29
IC50 = 5 µM
IC50 = 17 µM
IC50 > 50 µM
Figure 2. Initial diversification of 5.
The next optimization round addressed the 4-position of the oxazole ring (Table 1). Ortho-
substituted phenyl moieties as they were present in compounds 20a and 20b were poorly
tolerated, while meta substituted phenyl residues (20c and 20d) retained similar potency as 5.
The SAR of para substituted phenyl residues in the 4-position of the oxazole core was more
sophisticated. While fluorine (20e) only slightly diminished the potency, larger electron-
withdrawing moieties such as trifluoromethyl (20f) or nitrile (20g) led to a loss of activity. A
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chlorine substituent at this position (20h) slightly enhanced inhibitory potency while the
electron-releasing methoxy moiety (20i) decreased activity. Following the Topliss optimization
1
8
scheme , a second chlorine substituent was introduced in the meta-position of the phenyl ring
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(20k) which led to a more than tenfold increase in sEH-P inhibition. Other variants of the
chlorine substitution pattern (20l and 20m) did not lead to potency improvement.
Table 1. Optimization of the 4-position of the oxazole core.
OH
R
N
O
O
Cpd
5
R
IC [µM]
Cpd
20g
R
IC [µM]
5
0
50
5 ± 1
NC
44 ± 5
3 ± 1
F
Cl
2
2
2
0a
0b
0c
40 ± 12
33 ± 7
10 ± 1
20h
20i
20j
Cl
O
18 ± 3
2 ± 1
Cl
NC
F3C
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2
2
2
0d
0e
0f
7 ± 1
9 ± 1
33 ± 7
20k
20l
Cl
Cl
0.12 ± 0.06
17 ± 2
F
F
Cl
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20m
Cl
9 ± 2
F3C
Cl
We then addressed optimization of the oxazole 2-position to adjust the interactions of the
important carboxylate moiety (Table 2). While the rigidization of the flexible ethyl linker by a
double bond resulted in a twofold loss of potency (22a), introduction of an additional methylene
(
22b) further improved the inhibitory activity. Replacement of the propyl bridge by a rigid
phenyl linker (22d) demonstrated that optimal interactions of the carboxylate are achieved at a
distance of 4 bonds from the oxazole core. The conformationally restrained ortho and para
substituted benzoic acids did not retain this high potency (22c and 22e).
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Optimization of the linker in 2-position of the oxazole core.
Cl
Cl
N
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
Cpd
R
IC50 [µM]
0.12 ± 0.06
2
2
2
0k
2a
2b
COOH
COOH
0.51 ± 0.08
0.058 ± 0.005
15 ± 1
COOH
22c
COOH
2
2d
0.15 ± 0.04
12 ± 2
COOH
COOH
22e
Finally, the impact of the phenyl substituent in 5-position of the oxazole was investigated, while
3
,4-dichlorophenyl substituent in the 4-position and the propionic acid in the 2-position were
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maintained (Table 3). When the phenyl substituent was omitted, the potency dropped
dramatically (25) but was partially restored by introduction of a bromine substituent (26).
Pyridine moieties were not favored (28a and 28b) while almost every substituent in ortho or
meta position was well-tolerated, with a slight preference for fluorine (compound 28e) or linear
substituents such as nitrile (28g) or ethinyl (28j). However, no further potency improvement over
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2b and 22d was achieved rendering these oxaprozin derivatives as most active sEH-P inhibitors
for further characterization.
Table 3. Optimization of the 5-position of the oxazole core.
Cl
Cl
O
OH
N
O
R
Cpd
R
IC50 [µM]
>50
Cpd
R
IC50 [µM]
0.12 ± 0.02
25
28e
F
H
2
6
3 ± 1
28f
O
0.31 ± 0.05
0.12 ± 0.01
Br
22b
0.058 ± 0.005
28g
NC
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8a
20 ± 1
4 ± 1
28h
28i
F3C
0.41 ± 0.19
0.31 ± 0.04
N
28b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
2
2
8c
0.31 ± 0.04
0.28 ± 0.01
28j
0.14 ± 0.01
0.23 ± 0.11
F
8d
F
28k
Cl
F
In vitro characterization of 22b and 22d
To verify binding of the most potent compound 22b to sEH-P, orthogonal biophysical assays
were performed. Differential scanning fluorimetry (DSF), also known as thermal shift assay, was
conducted with the N-terminal domain and the C-terminal domain of sEH. The N-terminal domain
was stabilized by 22b, while the reference inhibitor of the sEH hydrolase activity CIU (N-
19
cyclohexyl-N'-(4-iodophenyl)urea) had no stabilizing effect on the phosphatase domain (Figure
2
A). A full list of the DSF experiments with additional reference inhibitors is included in the
Supporting Information. In contrast, 22b did not stabilize the C-terminal domain which melting
point was instead significantly increased by CIU (Figure 2B). Isothermal titration calorimetry
revealed tight binding of 22b to the N-terminal domain of sEH with a K value of 0.3 µM (Figure
d
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2
C). The analysis of the thermodynamic properties of binding revealed balanced enthalpic and
entropic contributions to binding enthalpy (Figure 2D).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
B
N-terminal domain
C-terminal domain
P=0.0161
7
0
P<0.0001
58
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
56
65
54
60
52
55
50
50
48
o
U
b
o
U
I
b
/
I
/
2
2
w
C
2
w
C
2
C
-
500
0
500
1000 1500
2000
2500 3000
3500 4000
4500
0
0
.2
.0
-
-
-
-
-
-
0.2
0.4
0.6
0.8
1.0
1.2
-1.4
-1.6
-1.8
5
∆G
∆H
-T∆S
0
0
-10
-
5
-
20
-30
-40
-
10
15
20
-
K = 0.3 µM
d
-
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Mole Ratio
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Figure 2. Biophysical characterization of 22b. A. 22b, but not sEH hydrolase inhibitor CIU
stabilized the N-terminal domain of sEH in differential scanning fluorimetry (DSF) assay. B. 22b
did not stabilize the C-terminal domain of sEH, in contrast to CIU. The concentration of the
inhibitors in the DSF experiments was 50 µM. Statistical significance of the measured melting
points compared to the DMSO control was determined by a two-tailored T-test using GraphPad
Prism (version 7.05; GraphPad Software, Inc.). C. ITC measurement of the complex formation of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2b and N-terminal sEH domain. Shown are raw binding heats of each injection (top panel) as
well as normalized binding heats fitted to a single binding site model (solid line). The
thermodynamic parameters of this titration experiment are shown in the insert.
Compound 5 (oxaprozin) is an approved non-selective inhibitor of cyclooxygenases 1 and 2.
Therefore, the inhibitory potency of 22b towards cyclooxygenases was investigated. 22b
exhibited slightly lower potency towards both cyclooxygenase isoforms compared to 5, which
inhibits COX-1 with an IC of 0.74 µM (29%CV) and COX-2 with an IC of 0.11 µM
5
0
50
(38%CV). Furthermore, agonistic potency towards peroxisome proliferator-activated receptor
gamma (PPARγ) and retinoid X receptor alpha (RXRα) was observed, which needs to be
considered when using 22b in cellular assays.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Selectivity screen of 22b and 5 (oxaprozin) towards diverse targets of fatty acid
mimetics. All values are measures at least as duplicate (n ≥ 2). Values for nuclear receptors are
mean ± SD% transactivation of reference agonist; inactive - no statistically significant reporter
transactivation at the indicated concentration
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Target
Effect
22b
5
Cyclooxygenase 1 (COX-1)
IC = 1.2 µM (20% CV)
IC = 0.74 µM (29%CV)
5
0
50
Cyclooxygenase 2 (COX-2)
IC = 0.42 µM (9.6% CV)
IC = 0.11 µM (38%CV)
5
0
50
PPARα
PPARβ/δ
PPARγ
FXR
inactive at 10 µM
inactive at 10 µM
29±1% at 10 µM
inactive at 10 µM
inactive at 10 µM
inactive at 10 µM
77±2% at 10 µM
inactive at 10 µM
inactive at 10 µM
inactive at 10 µM
inactive at 10 µM
inactive at 10 µM
inactive at 10 µM
14.4±0.4% at 10 µM
LXRα
LXRβ
RXRα
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In order to understand the structural basis of sEH-P inhibition by oxazole-based compounds, co-
crystallization experiments of sEH-P domain with oxazole inhibitors 22b and 22d were
performed. While the most affine inhibitor 22b did not yield crystals, 22d was co-crystallized
with the sEH-P. Figure 3A shows the N-terminal domain, with the bound compound 22d, while
Figure 3B and Figure 3C show details of the inhibitor interaction. The main interaction of 22d
with the protein was mediated by its carboxyl group that formed hydrogen bonds with the
residues Asn124 and Asn189 as well as three water molecules. Two of these water molecules
were tightly coordinated by a magnesium ion which was present in the active site. In addition,
the nitrogen of the oxazole ring formed a water-mediated hydrogen bond with the backbone NH
of Val19. The aromatic oxazole system exhibited aromatic edge-to-face interactions with the side
chain of Phe41. The phenyl substituents fitted tightly into a deeply buried hydrophobic pockets.
Figure 3C shows that the phenyl substituent in the 5-position of the oxazole core of the inhibitor
is in close proximity to the amino acids Trp126, Phe92, Leu60, Trp63, Leu53, and Phe41 that
constitute the most hydrophobic surface area of the binding site. Furthermore, these residues
belong to a region in the structure where the most prominent structural changes upon inhibitor
binding were observed. The environment around the 3,5-dichloro phenyl group in the 4-position
of the oxazole core comprises three hydrophobic amino acids Ile96, Val19 and Trp126 in close
proximity to this ring system. Here, the importance of the chlorine substituents can be explained
by contacts towards backbone carbonyl oxygens of Phe92 and Ile 96, comparable to ones
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0
21
observed by Heroven et al. and Falke et al. (Figure 3D). The phenyl ring in the 2-position of
the oxazole core carrying the carboxyl group was positioned in close proximity to the entrance of
the active site. The amino acids Phe20, Phe41, Val19 and Thr50 were shielding one side of the
phenyl ring while the other side was mainly solvent exposed.
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2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Phe41
A
Phe92
0
1
2
3
4
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7
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9
0
1
2
3
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9
0
1
2
3
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8
9
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
Lys160
Arg99
Trp63
Asp11
Ile186
Asn189
Phe20
B
C
Phe92
Phe41
Asn124
Ile96
Asn189
Asp11
Val19
D
Phe92
Ile96
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Figure 3. X-ray structure of 22d in complex with the N-terminal domain of sEH (PDB code
5
MWA). A. 2F -F electron density map at 1σ of 22d in the binding pocket (blue mesh) as well
o c
as the polder omit map at 3σ (green mesh). B. The interactions of the carboxylate head group
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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0
2+
with the Mg ion are mediated by water molecules. C. Hydrophobic interaction of the phenyl
residues. D. Interactions of the chlorine substituents with backbone carbonyl atoms.
In order to investigate the applicability of 22b in typical model organisms in vivo, the inhibitory
potency against full length (fl) human, mouse, and rat sEH was compared (Table 5). 22b
inhibited the phosphatase activity of the human full length enzyme, however, its potency was
significantly decreased compared to the isolated N-terminal domain. Furthermore, the sEH-H
activity was also affected showing an IC of about 9 µM. Most strikingly, 22b did not inhibit
5
0
neither hydrolase nor phosphatase activity of the mouse sEH, while rat sEH-P was selectively
impaired.
Table 5. Inhibitory activity of 5 (oxaprozin) and 22b against sEH enzymes from different
species
Human fl sEH
Mouse fl sEH
sEH-H
Rat fl sEH
sEH-H
>300
sEH-H
>300
sEH-P
sEH-P
sEH-P
5
IC50
70.0 ± 3.0
>300
35.5 ± 5.8
62.5 ± 1.9
(M)
22b IC50
M)
9.5 ± 0.5
4 ± 1
>30
>30
>30
2.8 ± 2.2
(
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In order to explain the lack of activity against mouse sEH, residues in close proximity to the
inhibitor in the enzyme were investigated. Five amino acids which differed between the human
and the mouse enzymes were identified including Val19Ile, Phe20Ala, Phe41Tyr, Leu60Phe,
Ile96Met. The rat protein features the same sequence changes as mouse protein except Phe41
which remains unchanged in the rat protein compared to human sEH. To investigate the influence
of these five sequence changes to inhibitor binding, in silico mutagenesis of the sEH-P complex
structure was performed using MOE software suite. Figure 4 shows the influence of the mutations
on the shape of the binding site. We identified Phe41Tyr as the most significant difference between
the human and mouse enzymes, while Leu60Phe might also have a slight influence on the inhibitor
binding. The reason for the prominent effect of Phe41Tyr mutation on inhibitor binding is the
addition of the hydroxyl group which collides with the oxazole core of 22d, efficiently blocking
the inhibitor binding. Phe41 is not altered in rat sEH, which supports the model shown in Figure
4.
0
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0
Ala20
Ile19
Tyr41
Met96
Phe60
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Figure 4. Analysis of sequence changes in different species and its implication in inhibitor
potency. The crystal structure of the human sEH-P was used to calculate the surface of the
receptor binding site (shown in green for hydrophobic and pink for hydrophilic areas), with the
bound inhibitor 22d. In addition, the 5 amino acids that differ between the human and mouse
sEH in the binding pocket were mutated in silico and are shown in the picture, where the two
most significant changes Tyr41 and Phe60 are shown as space filling representations.
1
1
1
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9
0
Structural analysis of 22d binding to sEH-P suggest possible explanation of the pronounced
differences between the inhibition of the full length enzyme and the isolated sEH-P domain.
Although the structure was refined at high resolution (1.55 Å), the position of the amino acids 74-
84 and 131-138 could not be determined due to disorder resulting in the lack of interpretable
electron density in these regions. A comparison of the crystal structure with the crystallized full
length protein revealed that the areas that were not modeled in our structure have quite high B-
2
2
factors in full length sEH (pdb: 5alu) . Additionally, the protein sequences of the human and the
mouse enzyme show high sequence variability in the areas 82- 88 and 89 -100 as well as 130-146.
2
2
Comparison to one of the published full length sEH crystal structures (pdb: 5alu) showed that
the binding of the inhibitor led to a prominent induced fit in the binding site (Figure 5A). The
phenyl ring in the 5-position of the oxazole core pushes Leu60 and Trp126 towards the interface
between the N-terminal and the C-terminal domain. This leads to a movement of the interacting
residues Asp128 and Arg130. These structural changes lead to shift in the residues Met145,
His146, and Glu142, which are in contact with the interface between the N-terminal and the C-
terminal domain (Figure 5B and 5C).
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A
S61
L60
D129
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0
D128
W126
R130
B
C
9
0°
E236
M145
H146
T230
N283
E142
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Figure 5. Induced fit binding of 22d to the N-terminal domain leads to structural distortion of
the interface to the C-terminal domain. A. Binding site of 22d; B. global view on the superposed
proteins (PDB codes 5mwa and 5alu); C. Interface between the N-terminal and the C-terminal
domain.
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0
The structural data suggests that there is a probability that the C-terminal domain hinders the
binding of 22d to the N-terminal domain in the context of the full length enzyme. Therefore, we
performed kinetic experiments to validate the mode of inhibition and determine the K of 22b
i
towards the isolated N-terminal domain and the full length sEH (Figure 6). We used DiFMUP, an
sEH-P substrate with only one phosphate moiety, as a substrate and determined a K value of 219.9
m
µM for the isolated N-terminal domain and a K value of 628.5 µM for the full length sEH. We
m
were able to demonstrate that 22b is a competitive inhibitor under both conditions tested. We
determined a K value of 0.05 µM for the isolated N-terminal domain and a K value of 4.8 µM for
i
i
the full length sEH, which is in agreement with the discrepancy we observe for the IC values
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using FDP as a substrate.
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A
3
2
1
0
1
nM 22b
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0 nM 22b
30 nM 22b
00 nM 22b
300 nM 22b
1
1
000 nM 22b
000 nM 22b
3
0
10000 nM 22b
0.0000
0.0002
0.0004
0.0006
c(DiFMUP) [M]
B
1.0
0
.8
0.6
0
0
.4
.2
0.0
.0000
0
0.0002
0.0004
0.0006
c(DiFMUP) [M]
Figure 6. K determination of 22b on the isolated sEH-P domain (A) as well as on the sEH-FL
i
(B) by a mixed model inhibition fit in GraphPad (version 7.05; GraphPad Software, Inc.).
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Due to the decreased activity of 22b against full length sEH-P compared to the isolated N-
terminal domain as well as its inhibitory activity against sEH-H in the full length enzyme (Table
5), we also evaluated and compared the inhibitory behavior of previously published sEH-P
inhibitors. Initial DSF experiments showed that only compounds 2 and 22b are able to stabilize
the N-terminal domain of sEH (Table 6 and Supporting Information). 1 (SDS) displays inhibitory
potency against N-terminal and full length sEH-P, while leaving sEH-H almost unaffected. 2 (N-
acetyl-S-farnesyl-L-cysteine) potently inhibited the phosphatase activity of the single N-terminal
domain and the full length sEH without affecting hydrolase activity. In contrast, 4 (ebselen)
potently and unselectively inhibited both activities of separated sEH domains and the full length
enzyme. 22b turned out as most potent inhibitor of the phosphatase activity of the N-terminal
domain. However, as mentioned above, the activity towards the full length sEH-P was markedly
reduced and in the full length protein, both domains were inhibited with similar IC values (Table
1
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1
1
1
1
1
1
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0
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9
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9
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9
0
5
0
6). These results were surprising due to the fact that the initial screening hit 5 (oxaprozin) inhibited
16
the N-terminal domain, while leaving the sEH-H unaffected . Taken together, the weak activity
of current inhibitors limits their applications as chemical probes in cellular assay systems and
interspecies differences in the sEH-P active site need to be considered in murine model systems.
2
3
12
Furthermore, reported posttranslational modifications of sEH by nitration or phosphorylation
might also influence inhibitor binding.
Table 6. DSF experiments with the isolated N-terminal sEH domain and inhibitory activity of
sEH-P inhibitors against isolated human sEH domains and full length human sEH enzyme.
sEH-P DSF sEH-H
°C] IC [µM]
sEH-P
fl sEH-H
fl sEH-P
Compound
[
IC [µM]
IC [µM]
IC [µM]
5
0
50
50
50
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