Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency towards fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2018.01.003

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.

Full text of DOI:10.1016/j.bmcl.2018.01.003

Accepted Manuscript
Identification and optimization of soluble epoxide hydrolase inhibitors with dual
potency towards fatty acid amide hydrolase
Sean D. Kodani, Saavan Bhakta, Sung Hee Hwang, Svetlana Pakhomova,
Marcia E. Newcomer, Christophe Morisseau, Bruce D. Hammock
PII:
S0960-894X(18)30003-9
https://doi.org/10.1016/j.bmcl.2018.01.003
BMCL 25529
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 November 2017
31 December 2017
2 January 2018
Please cite this article as: Kodani, S.D., Bhakta, S., Hee Hwang, S., Pakhomova, S., Newcomer, M.E., Morisseau,
C., Hammock, B.D., Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency
towards fatty acid amide hydrolase, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.01.003
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Title: Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency
towards fatty acid amide hydrolase
Authors: Sean D. Kodania, Saavan Bhaktaa, Sung Hee Hwanga, Svetlana Pakhomovab, Marcia
E. Newcomerb, Christophe Morisseaua, Bruce D. Hammocka
Affiliations:
aDepartment of Entomology and Nematology, and UC Davis Comprehensive Cancer Center,
University of California, Davis, Davis, CA 95616
bDepartment of Biological Sciences, Louisiana State University, Baton Rouge, LA 70809
Corresponding Author:
Bruce D. Hammock, Ph.D.
Department of Entomology and Nematology
University of California, Davis, Davis, CA 95616
FAX: 1 530 752 1537
bdhammock@ucdavis.edu

ABSTRACT
Multi-target inhibitors have become increasing popular as a means to leverage the advantages of
poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for
soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to
synergize when treating inflammatory and neuropathic pain. The structure activity relationship
(SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-
trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was
previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency
while maintaining high sEH potency were developed by optimization. Interestingly, compared to
most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and
related compounds appear to inhibit FAAH through a time-independent, competitive mechanism.
These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH
inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new
dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be
useful for future studies in understanding the therapeutic application of dual sEH/FAAH
inhibition.
Keywords: Soluble Epoxide Hydrolase, Fatty Acid Amide Hydrolase, Urea Inhibitors,
Neuropathic Pain
Abbreviations and definitions: AADAC, arylacetamide deacetylase; AEA, arachidonoyl
ethanolamide; EETs, epoxyeicosatrienoic acids; FAAH, fatty acid amide hydrolase; hCE, human
carboxylesterase; MAP, methoxy arachidonoyl phosphonate; NSAIDS, non-steroidal anti-
inflammatory drugs; PF-3845, N-(pyridin-3-yl)-4-(3-((5-(trifluoromethyl)pyridin-2-
yl)oxy)benzyl)piperidine-1-carboxamide; PON, paraoxonoase; sEH, soluble epoxide hydrolase;
t-TUCB, trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid; URB597,
3'-carbamoyl-[1,1'-biphenyl]-3-yl cyclohexylcarbamate

TEXT
Chronic pain is poorly managed by current treatment options. The available therapies,
including non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, are not effective on all
types of pain, can be debilitating or have a high potential for abuse.1 Furthermore, few new
therapies have come to the market in the past decade. One recent approach towards designing
analgesics with high efficacy and reduced side effects has been the combination of inhibitors for
two or more targets known to regulate pain, known as poly-pharmacology.2 These multi-target
inhibitors have the potential for higher efficacy and reduced drawbacks arising from the use of a
single-target drug or a combination of multiple drugs.3 In particular, enzymes involved in the
regulation of signaling lipids, including soluble epoxide hydrolase (sEH) and fatty acid amide
hydrolase (FAAH), have been proposed as suitable targets for the application of poly-
pharmacology for pain treatment.4,5
The sEH is responsible for the regulation of lipid epoxides acting as potent chemical
mediators such as epoxyeicosatrienoic acids (EETs).6 These signaling lipids are responsible for
mediating a number of biological processes including nociception,7,8 inflammation9 and
hypertension.10,11 By converting the biologically active epoxides to their respective largely
inactive diols, sEH negatively regulates the activity of the EETs. The in vivo stability of EETs
and other chemically stable epoxy-fatty acids is low due to the high catalytic efficiency of sEH.12
Thus, sEH inhibition has been the major approach for studying the biological role of lipid
epoxides in numerous disease states including neuropathic and inflammatory pain.13,14 Treating
with sEH inhibitors reduces both forms of pain in a manner that may be dependent, in part, on
cannabinoid signaling,15 endoplasmic reticulum stress16 and/or other mechanisms. Several of
these inhibitors have been developed as IND candidates that have reached Phase I

(GSK2256294A) and Phase II (AR9281) clinical trials for COPD and hypertension, respectively
(Figure 1).17-19 GSK2256294A has not progressed to further stages of clinical trials and AR9281
was unable to demonstrate efficacy in human patients.
FAAH is a separate enzyme that is studied as a potential therapeutic target for
neuropathic and inflammatory pain.20-22 This enzyme hydrolyzes arachidonoyl ethanolamide
(AEA), an endocannabinoid that regulates nociception and other physiologies through activation
of the cannabinoid receptors.22,23 Like EETs, AEA is quickly metabolized in vivo by FAAH and
therefore in vivo studies investigating AEA require FAAH inhibitors. Although activation of
cannabinoid receptors has numerous undesired effects including hypothermia, catalepsy and
hyperphagia, treatment with FAAH inhibitors or AEA alone is not sufficient for producing these
effects.24,25 Several FAAH inhibitors have been developed. Among them, PF-04457845 reached
Phase II clinical trials without success due to lack of efficacy despite its excellent target
engagement.26 Recently, BIA 10-2474 was also pulled from a Phase I clinical trial after the death
of a study subject,27 which was independent of FAAH inhibition.28
Concurrent inhibition of both sEH and FAAH synergistically reduces both inflammatory
and neuropathic pain.29 Interestingly, the sEH inhibitor trans-4-[4-(3-trifluoromethoxyphenyl-l-
ureido)-cyclohexyloxy]-benzoic acid (t-TUCB), which was thought to be a selective potent sEH
inhibitor (IC50 = 0.4 nM), was recently identified as a weak FAAH inhibitor (human FAAH IC50
= 260 nM). t-TUCB demonstrates excellent efficacy with multiple indications including
neuropathic pain, but it was not clear whether its high efficacy is derived from its poly-
pharmacology. This excellent efficacy has led to its use as a tool to treat various diseases30-32 in
several animal species.33,34 Despite the extensive use of this compound for studying sEH biology,
the contribution of FAAH inhibition to results in the experimental or clinical disease models has

Figure 1. A. Structures of several sEH inhibitors (t-TUCB, TPPU, GSK2256294A, AR9281) and
FAAH inhibitors (URB597, PF-3845, PF-04457845) B. Modifications of t-TUCB skeleton were
tested at the trifluoromethoxyphenyl group (“Ring A”), the trans cyclohexyl group (“Ring B”) or
the benzoic acid (“Ring C”).

not been explored. Thus, our primary goal was to produce novel inhibitors with improved
potency towards sEH and FAAH. Our secondary goal was to test the plausibility of FAAH
inhibition contributing to the observed beneficial effects of t-TUCB and related compounds by
defining the potency for dual inhibition in other species.
Synthesis of all inhibitors was done according to established procedures (described in
detail in the Supplementary Material). Recombinant enzyme preparations were used with
fluorescent-based substrates to quantify potency of inhibitors on sEH and FAAH (described in
the Supplementary Material). All of the newly synthesized inhibitors are relatively potent
towards sEH (IC50 < 50 nM) as expected. Thus, we primarily focused on determining the
chemical structures essential for optimizing potency on FAAH. Compared to the known FAAH
inhibitors PF-3845 and URB597, t-TUCB is 233-fold and 6-fold less potent, respectively (Table
1).20,35 Rings “A” and “B” and substituents on the 4-position of “C” were modified on t-TUCB to
determine the portion of the structure that primarily confers potency on FAAH (Figure 1B).
Urea-based FAAH inhibitors described previously have an aromatic substitution on one side of
the urea, similar to ring “A” on t-TUCB. Since these compounds had higher potency for the 4-
fluoro or unsubstituted rings than the 4-trifluoromethoxy substituent,36 the 4-trifluoromethoxy
group on t-TUCB was replaced by a hydrogen (2), fluoride (3) or chloride (4). Potency on FAAH
decreased as the size and hydrophobicity of the para position substituent increased, with 4-
trifluoromethoxy (1) being the most potent. Substituting the aromatic ring for a cyclohexane (5)
or adamantane (6) resulted in a complete loss in activity against FAAH. Switching the
cyclohexane linker of ring “B” to a cis conformation (c-TUCB) resulted in a 20-fold loss of
potency while replacing it with a butane chain (9) resulted in a completely inactive compound.
Modification of the cyclohexane to an aromatic linker (10) had essentially no effect on potency

Table 1. Potency of inhibitors with modifications on Ring A (R1) and Ring B (R2) against both
human sEH and human FAAH.
IC50 (nM)
R1
R2
hsEH
1100
hFAAH
23
URB597
PF-3845
>10,000
0.6
t-TUCB
0.8
30
18
7
140
9,200
4,600
680
2
3
4
5
6
>10,000
>10,000
2,800
>10,000
170
6
c-TUCB
9
3
2
15
7
10
13
8
1,800

for FAAH relative to t-TUCB. Although many potent urea-based FAAH inhibitors use a
piperidine as the carbamoylating nitrogen,21,37,38 the modification to piperidine-incorporated tri-
substituted urea reduced potency 13-fold (13). Together, these changes on ring “B” indicate the
trans-cyclohexyl ring provides the exact fit in the active site of FAAH essential for t-TUCB’s
inhibitory potency.
To further explore the relationship between structure and function on the FAAH enzyme,
we focused on the substitutions on the 4-position of ring “C” (Table 2). The importance of the
terminal carboxylic acid group was explored by testing the potency of the corresponding
aldehyde (15) and alcohol (16) in addition to the amide (21) and nitrile (14). Generally, the
higher oxidation state of the terminal portion correlates with higher potency towards FAAH. t-
TUCB was 10-times more potent than 15 and 50-times more potent than 16. Similarly, the amide
(21) was over 100-times more potent than the nitrile (14). Converting the benzoic acid to the
phenol (18) had a minor effect on potency. Interestingly, modifying the phenol to the anisole (19)
completely removed activity while creating an acetate ester (20) is equipotent to the phenol.
Since the substrates for FAAH tend to be relatively hydrophobic lipids, we speculated conversion
of the acid and primary amide to the corresponding esters or substituted amides, respectively,
would result in improved potency by introducing hydrophobic groups. As expected, the methyl
ester (22) had 4-fold improved potency relative to the corresponding acid (t-TUCB). However,
incorporating a bulkier substitution than the methyl group such as isopropyl ester (23) showed
11-fold less potency compared to the methyl ester (22). Interestingly, the benzyl ester (24) gives
approximately the same potency as the methyl ester, suggesting that π-π stacking between
benzylic group and a residue at the active site may be important. Relative to t-TUCB, the methyl
(25), ethyl (26) and glycinyl (27) amides all had essentially the same potency; however, the

Table 2. Potency of inhibitors with modifications on Ring C (R3) against both human sEH and
human FAAH.
IC50 (nM)
R3
hsEH
0.8
hFAAH
t-TUCB
140
14
5
>10,000
15
16
17
4
3
4
1,100
5,800
5,300
18
19
2
3
120
>10,000
20
21
4
2
120
70
22
23
7
5
35
400
24
3
24
25
26
2
2
170
100
27
2
130

28
29
3
5
30
1,100

benzyl amide (29) was substantially less potent (16-fold). Between the methyl- (22 and 25) and
benzyl-substituted (24 and 29) compounds, the esters generally appear to be more potent than the
amides. This difference in the potency may be due to the specific angle of the substituted groups
or the increased electron density of the ester carbonyl compared to the amide carbonyl.
Generating the methyl ester of the glycinyl amide (28) increased the potency 4-fold compared to
the corresponding free acid. Since the amide and esters appeared to be active, the amide
bioisostere oxadiazole (17) was tested and had 38-fold less potency than the initial compound.
Many compounds in this series could act as prodrugs following esterase or amidase catalyzed
hydrolysis.39
Many FAAH inhibitors work through carbamoylation of the catalytic serine
residue.20,36,40 Inhibition through this mechanism is time-dependent because the inhibitory
potency depends on the rate of carbamoylation. However, to our surprise, the potency of t-TUCB
and two new inhibitors (18 and 24) does not change with time (Figure 2A), suggesting that these
inhibitors are unlikely to inhibit through the formation of a covalent intermediate. URB597, an
inhibitor known to carbamoylate FAAH, has a 3-fold increase in potency over the same period of
time. To further test the mechanism of action of these inhibitors we determined the effect of
varying substrate concentrations on the inhibitory potency of t-TUCB (Figure 2B). Results
app
app
showed an increase in KM with only a minor change in vmax indicating that inhibition
primarily occurs through a competitive mechanism. Assuming a simple competitive model, the
calculated Ki for t-TUCB is 156 ± 85 nM. Using the same analysis, the Ki for 18 and 24 are 9.1 ±
2.5 nM and 43 ± 25 nM, respectively.
Since the endogenous substrates, EETs and AEA, for sEH and FAAH, respectively, share
the arachidonic acid backbone, it is reasonable to speculate t-TUCB may competitively inhibit

Figure 2. A. t-TUCB, 18, 24 and URB597 were pre-incubated with FAAH enzyme at various
time intervals followed by addition of substrate ([S]final = 5 μM). Potency of t-TUCB, 18 or 24
inhibition is independent on pre-incubation time while inhibition by URB597 is time dependent.
B. Reaction kinetics of OMP hydrolysis by FAAH was measured at various [OMP] and [t-
TUCB] to determine inhibition mechanism. As [t-TUCB] increases the KMapp substantially
increases (from KMapp = 30 μM to KMapp >200 μM) and the vmax marginally increases (4.0 to 5.4
RFU/sec), consistent with a primarily competitive mechanism of inhibition.

FAAH by mimicking and displacing the substrate. Although these lipids are linear carbon chains,
a folded orientation in the substrate tunnel may resemble the linked-ring structure of t-TUCB.
The crystal structure of methoxy arachidonoyl phosphonate (MAP) bound to FAAH
demonstrates the bound substrate is not linear in the active site but bends around the unsaturated
bonds.41 To investigate this possibility, we docked t-TUCB, 18 and 24 into the FAAH active site
using AutoDock Vina (Figure 3A and Supplementary Figure 1). Docking in the FAAH active
site, the polar aromatic groups on t-TUCB, 18 and 24 form hydrogen-bond interactions with the
catalytic serine and orient in a manner similar to its lipid substrates when docked in the FAAH
active site. In the case of t-TUCB and 18, the A ring occupies the acyl-binding pocket of the
active site; while 24 stays in the membrane access channel. The binding orientation from docking
t-TUCB and 18 is similar to the co-crystal structure described for covalently bound PF-384520
and is consistent with the SAR described in this manuscript. If the 4-trifluoromethoxyphenyl
group on ring A occupies the acyl-binding pocket, then modification of this group to bulky
lipophilic groups such as an adamantyl group will be not suitable for this site and modification to
smaller groups such as an unsubstituted phenyl will decrease the ability to stably occupy that
pocket. Further, modification of ring B will either decrease the stability of that conformation in
the case of the butane chain or force the molecule to unfavorable conformation in the case of the
cis form of TUCB (c-TUCB) and the tri-substituted urea (13). Finally, modifications to the 4-
substituent on ring C determine potency based on the interactions required to generate an acyl-
intermediate; thus, we would predict
a
highly electrophilic organophosphate or
trifluoromethylketone analogues that mimic these acyl-intermediates may improve the potency
of future inhibitors.42, 43
By comparison, the disubstituted urea on t-TUCB is known to form strong hydrogen

Figure 3. A. Docking of t-TUCB (yellow) in the active site of FAAH (PDB: 1MT5) using
AutoDock Vina and B. Co-crystal structure of t-TUCB (yellow) in the active site of sEH. The
key catalytic residues for FAAH (Ser241) and sEH (Asp335) are represented in addition to the
proton-donating residue on sEH (Tyr 383 and Tyr466). Hydrogen bonds are represented on the
co-crystal structure as yellow dashed lines.

bonds with the catalytic aspartate residue and the H-bond donating tyrosine residues in the active
site of the sEH enzyme.44 In the co-crystallization of t-TUCB in the active site of sEH (Figure
3B) the urea fits between the aspartate and the two tyrosine residues. The flanking pockets on
either side of the catalytic site are large and can accommodate a variety of shapes. Thus,
consistent with the SAR, a large variety of shapes may be accommodated by sEH on either side
of the urea and potency will remain relatively high (IC50 < 50 nM). The atomic coordinates and
structure factors (code 6AUM) have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
To test the selectivity of these inhibitors toward FAAH, we investigated their ability to
inhibit a series of related enzymes, especially serine hydrolases (Table 3). t-TUCB, 18, or 24 did
not inhibit the tested carboxylesterases (hCE1 and hCE2), hydrolases involved in xenobiotic
detoxification, or paraoxonases (PON1, PON2 and PON3), esterases involved in the regulation
of artherosclerosis,45 but 18 did partially inhibit arylacetamide deacetylase (AADAC), a poorly
characterized enzyme that is known to metabolize several xenobiotics.46 These results indicate
that, unlike most known FAAH inhibitors that have poor selectivity against other serine
hydrolases,47, 48 broad off-target inhibition of other serine hydrolases by t-TUCB, 18 and 24 is
unlikely.
t-TUCB has been used extensively for studying the role of sEH in multiple animal
models of pain.33, 34, 49 To test whether potency towards FAAH may account for the efficacy in
these animal models, the effectiveness of t-TUCB, 18 and 24 to inhibit FAAH was measured on
other species than human using brain microsome preparations. As positive controls, the IC50

Table 3. IC50 values of compounds t-TUCB, 18 and 24 on other human serine hydrolases.
IC50 (nM)
Enzyme
FAAH
sEH
t-TUCB
140
18
120
2
24
24
3
0.8
mEH
>10,000 >10,000 >10,000
>10,000 >10,000 >10,000
>10,000 >10,000 >10,000
>10,000 >10,000 >10,000
>10,000 >10,000 >10,000
>10,000 >10,000 >10,000
hCE1
hCE2
PON1
PON2
PON3
AADAC
>10,000
5,400
>10,000

values of two well characterized FAAH inhibitors, PF-3845 and URB-597 were measured also
(Table 4, Supplementary Data). These two inhibitors are structurally distinct and have excellent
selectivity in rat brain microsomes for the FAAH enzyme over other hydrolases.20,38 As expected,
both inhibitors blocked >80% of the activity in the microsomes of the different species tested
(Supplementary Figure 2). Interestingly, both t-TUCB and 24 are less potent against the FAAH
of all other species tested than human FAAH. 18 had comparable IC50s for FAAH from all
species tested except from rat, where 18 is 10–fold less potent. The hypothesized mechanism of
inhibition based on docking relies on a relatively specific fit in the enzyme active site. Thus, we
speculate small modifications to this acyl-binding pocket across species accounts for the
substantial species selectivity observed for these inhibitors. By comparison, sEH inhibition relies
primarily on strong hydrogen-bonding interactions and less on enzyme fit. t-TUCB, 18 and 24
also had species differences on sEH but all three inhibitors were still relatively potent in all
species tested (IC50 < 100 nM). Due to the low potency towards FAAH relative to sEH (Table 4),
it is unlikely that FAAH inhibition contributes to the previously observed effects of t-TUCB on
pain in a mouse model49.
In conclusion, we have designed dual sEH/FAAH inhibitors with nanomolar potency
towards both enzymes. These inhibitors have potency independent of incubation time and a
mechanism consistent with competitive inhibition. Furthermore, these inhibitors are selective for
FAAH over other serine hydrolases and have relatively low species selectivity. Use of these
inhibitors will support future biological studies investigating the importance of dual sEH/FAAH
inhibition.
Acknowledgements: This work was supported by the National Institute of Environmental
Health Sciences grant R01 ES002710 and the National Institute of Environmental Health

Table 4. IC50 values of compounds t-TUCB, 18 and 24 on sEH and FAAH in multiple species.
IC50 (nM)
Enzyme
FAAH
Species
t-TUCB
18
24
PF-3845 URB597
Human
Mouse
Rat
140
120
350
24
0.6
23
11
12
12
22
7
6,300
510
12
>10,000 1,700 >10,000
4.4
Cat
1,200
3,900
3,300
0.8
92
280
270
2
240
280
87
<0.1
Dog
0.4
Horse
Human
Mouse
Rat
0.3
sEH
3
-
-
-
-
-
-
-
5.7
0.4
1.0
4.3
11
2.1
1.6
42
-
54
-
Cat
2.0
-
Dog
5.6
28
-
Horse
19
21
77
-

Sciences Superfund Research Program grant P42 ES004699. SDK was supported by a NIGMS-
funded Pharmacology Training Program (T32GM099608). MEN and SP acknowledge support
from NIH HL107877. Structural data used in this publication were collected at the Gulf Coast
Protein Crystallography Beamline at the Center for Advanced Microstructures and Devices. This
beamline is supported by the National Science Foundation grant DBI-9871464 with co-funding
from the National Institute for General Medical Sciences. We would also like to thank Drs.
Patricia Pesavento and Carrie Fino for providing samples of animal tissue used in these studies.
Competing financial interests declaration: SDK and BDH have filed a patent through the
University of California for compounds described in this text for use as dual sEH/FAAH
inhibitors. BDH is founder and SHH is an employee of Eicosis Animal Health, a company
developing t-TUCB for use in companion animals.

References
1.
2.
3.
4.
Allerton CMN, Ali Z, Smith DM. CHAPTER 1. The Disease of Pain and Current
Market Trends. 2013: 1-36.
Reddy AS, Zhang S. Polypharmacology: drug discovery for the future. Expert Rev
Clin Pharmacol. 2013;6(1): 41-47.
Anighoro A, Bajorath J, Rastelli G. Polypharmacology: challenges and opportunities
in drug discovery. J Med Chem. 2014;57(19): 7874-7887.
Hwang SH, Wagner KM, Morisseau C, et al. Synthesis and structure-activity
relationship studies of urea-containing pyrazoles as dual inhibitors of
cyclooxygenase-2 and soluble epoxide hydrolase. J Med Chem. 2011;54(8): 3037-
3050.
5.
Migliore M, Habrant D, Sasso O, et al. Potent multitarget FAAH-COX inhibitors:
Design and structure-activity relationship studies. Eur J Med Chem. 2016;109: 216-
237.
6.
7.
Morisseau C, Hammock BD. Impact of soluble epoxide hydrolase and
epoxyeicosanoids on human health. Annu Rev Pharmacol Toxicol. 2013;53: 37-58.
Inceoglu B, Jinks SL, Schmelzer KR, Waite T, Kim IH, Hammock BD. Inhibition of
soluble epoxide hydrolase reduces LPS-induced thermal hyperalgesia and mechanical
allodynia in a rat model of inflammatory pain. Life Sci. 2006;79(24): 2311-2319.
Wagner KM, McReynolds CB, Schmidt WK, Hammock BD. Soluble epoxide
hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative
diseases. Pharmacol Ther. 2017.
8.
9.
Schmelzer KR, Kubala L, Newman JW, Kim IH, Eiserich JP, Hammock BD. Soluble
epoxide hydrolase is a therapeutic target for acute inflammation. Proc Natl Acad Sci
U S A. 2005;102(28): 9772-9777.
10.
11.
Yu Z, Xu F, Huse LM, et al. Soluble Epoxide Hydrolase Regulates Hydrolysis of
Vasoactive Epoxyeicosatrienoic Acids. Circ Res. 2000;87(11): 992-998.
Ulu A, Lee KSS, Miyabe C, et al. An Omega-3 Epoxide of Docosahexaenoic Acid
Lowers Blood Pressure in Angiotensin-II-Dependent Hypertension. J Cardiovasc
Pharmacol. 2014;64: 87-99.
12.
13.
14.
15.
16.
Morisseau C, Inceoglu B, Schmelzer K, et al. Naturally occurring monoepoxides of
eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids.
J Lipid Res. 2010;51(12): 3481-3490.
Inceoglu B, Jinks SL, Ulu A, et al. Soluble epoxide hydrolase and
epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc Natl Acad
Sci U S A. 2008;105(48): 18901-18906.
Inceoglu B, Wagner K, Schebb NH, et al. Analgesia mediated by soluble epoxide
hydrolase inhibitors is dependent on cAMP. Proc Natl Acad Sci U S A. 2011;108(12):
5093-5097.
Wagner K, Inceoglu B, Hammock BD. Soluble epoxide hydrolase inhibition,
epoxygenated fatty acids and nociception. Prostaglandins Other Lipid Mediat.
2011;96(1-4): 76-83.
Inceoglu B, Bettaieb A, Trindade da Silva C, Lee KSS, Haj FG, Hammock BD.
Endoplasmic reticulum stress in the peripheral nervous system is a signiicant driver of
neuropathic pain. Proc Natl Acad Sci USA. 2015;112: 9082-9087.

17.
18.
19.
20.
21.
Yang L, Cheriyan J, Gutterman DD, et al. Mechanisms of Vascular Dysfunction in
COPD and Effects of a Novel Soluble Epoxide Hydrolase Inhibitor in Smokers.
Chest. 2017;151(3): 555-563.
Lazaar AL, Yang L, Boardley RL, et al. Pharmacokinetics, pharmacodynamics and
adverse event profile of GSK2256294, a novel soluble epoxide hydrolase inhibitor. Br
J Clin Pharmacol. 2016;81(5): 971-979.
Shen HC, Hammock BD. Discovery of inhibitors of soluble epoxide hydrolase: a
target with multiple potential therapeutic indications. J Med Chem. 2012;55(5): 1789-
1808.
Ahn K, Johnson DS, Mileni M, et al. Discovery and characterization of a highly
selective FAAH inhibitor that reduces inflammatory pain. Chem Biol. 2009;16(4):
411-420.
Johnson DS, Stiff C, Lazerwith SE, et al. Discovery of PF-04457845: A Highly
Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor. ACS Med Chem
Lett. 2011;2(2): 91-96.
22.
23.
24.
Piomelli D, Sasso O. Peripheral gating of pain signals by endogenous lipid mediators.
Nat Neurosci. 2014;17(2): 164-174.
Blankman JL, Cravatt BF. Chemical probes of endocannabinoid metabolism.
Pharmacol Rev. 2013;65(2): 849-871.
Cravatt BF, Demarest K, Patricelli MP, et al. Supersensitivity to anandamide and
enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide
hydrolase. Proc Natl Acad Sci U S A. 2001;98(16): 9371-9376.
Fegley D, Gaetani S, Duranti A, et al. Characterization of the fatty acid amide
hydrolase inhibitor cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester
(URB597): effects on anandamide and oleoylethanolamide deactivation. J Pharmacol
Exp Ther. 2005;313(1): 352-358.
Huggins JP, Smart TS, Langman S, Taylor L, Young T. An efficient randomised,
placebo-controlled clinical trial with the irreversible fatty acid amide hydrolase-1
inhibitor PF-04457845, which modulates endocannabinoids but fails to induce
effective analgesia in patients with pain due to osteoarthritis of the knee. Pain.
2012;153(9): 1837-1846.
25.
26.
27.
28.
29.
von Schaper E. Bial incident raises FAAH suspicions. Nat Biotechnol. 2016;34(3):
223.
Kerbrat A, Ferre JC, Fillatre P, et al. Acute Neurologic Disorder from an Inhibitor of
Fatty Acid Amide Hydrolase. N Engl J Med. 2016;375(18): 1717-1725.
Sasso O, Wagner K, Morisseau C, Inceoglu B, Hammock BD, Piomelli D. Peripheral
FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.
Pharmacol Res. 2015;97: 7-15.
30.
31.
32.
Wang L, Yang J, Guo L, et al. Use of a soluble epoxide hydrolase inhibitor in smoke-
induced chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol.
2012;46(5): 614-622.
Harris TR, Bettaieb A, Kodani S, et al. Inhibition of soluble epoxide hydrolase
attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon
tetrachloride in mice. Toxicol Appl Pharmacol. 2015;286(2): 102-111.
Sun D, Cuevas AJ, Gotlinger K, et al. Soluble epoxide hydrolase-dependent
regulation of myogenic response and blood pressure. Am J Physiol Heart Circ

Physiol. 2014;306(8): H1146-1153.
33.
34.
35.
Guedes AG, Morisseau C, Sole A, et al. Use of a soluble epoxide hydrolase inhibitor
as an adjunctive analgesic in a horse with laminitis. Vet Anaesth Analg. 2013;40(4):
440-448.
Ulu A, Appt S, Morisseau C, et al. Pharmacokinetics and in vivo potency of soluble
epoxide hydrolase inhibitors in cynomolgus monkeys. Br J Pharmacol. 2012;165(5):
1401-1412.
Mor M, Rivara S, Lodola A, et al. Cyclohexylcarbamic Acid 3'- or 4'-Substituted
Biphenyl-3-yl Esters as Fatty Acid Amide Hydrolase Inhibitors: Synthesis,
Quantitative Structure-Activity Relationships, and Molecular Modeling Studies. J
Med Chem. 2004;47: 4998-5008.
36.
37.
Keith JM, Apodaca R, Xiao W, et al. Thiadiazolopiperazinyl ureas as inhibitors of
fatty acid amide hydrolase. Bioorg Med Chem Lett. 2008;18(17): 4838-4843.
Johnson DS, Ahn K, Kesten S, et al. Benzothiophene piperazine and piperidine urea
inhibitors of fatty acid amide hydrolase (FAAH). Bioorg Med Chem Lett.
2009;19(10): 2865-2869.
38.
39.
40.
41.
42.
43.
Ahn K, Johnson DS, Fitzgerald LR, et al. Novel Mechanistic Class of Fatty Acid
Amide Hydrolase Inhibitors with Remarkable Selectivity. Biochemistry. 2007;46:
13019-13030.
Kim IH, Nishi K, Tsai HJ, et al. Design of bioavailable derivatives of 12-(3-
adamantan-1-yl-ureido)dodecanoic acid, a potent inhibitor of the soluble epoxide
hydrolase. Bioorg Med Chem. 2007;15(1): 312-323.
Moreno-Sanz G, Duranti A, Melzig L, et al. Synthesis and structure-activity
relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid
amide hydrolase inhibitors. J Med Chem. 2013;56(14): 5917-5930.
Bracey MH, Hanson MA, Masuda KR, Stevens RC, Cravatt BF. Structural
adaptations in a membrane enzyme that terminates endocannabinoid signaling.
Science. 2002;298(5599): 1793-1796.
Wheelock CE, Nishi K, Ying A, et al. Influence of sulfur oxidation state and steric
bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and
fatty acid amide hydrolase (FAAH). Bioorg Med Chem. 2008;16(4): 2114-2130.
Quistad GB, Sparks SE, Casida JE. Fatty acid amide hydrolase inhibition by
neurotoxic organophosphorus pesticides. Toxicol Appl Pharmacol. 2001;173(1): 48-
55.
44.
45.
46.
47.
Morisseau C, Hammock BD. Epoxide hydrolases: mechanisms, inhibitor designs, and
biological roles. Annu Rev Pharmacol Toxicol. 2005;45: 311-333.
Chistiakov DA, Melnichenko AA, Orekhov AN, Bobryshev YV. Paraoxonase and
atherosclerosis-related cardiovascular diseases. Biochimie. 2017;132: 19-27.
Fukami T, Yokoi T. The Emerging Role of Human Esterases. Drug Metab
Pharmacokinet. 2012;27(5): 466-477.
Zhang D, Saraf A, Kolasa T, et al. Fatty acid amide hydrolase inhibitors display broad
selectivity and inhibit multiple carboxylesterases as off-targets. Neuropharmacology.
2007;52(4): 1095-1105.
48.
Crow JA, Bittles V, Borazjani A, Potter PM, Ross MK. Covalent inhibition of
recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the
carbamates JZL184 and URB597. Biochem pharmacol. 2012;84(9): 1215-1222.

49.
Wagner K, Inceoglu B, Dong H, et al. Comparative efficacy of 3 soluble epoxide
hydrolase inhibitors in rat neuropathic and inflammatory pain models. Eur J
Pharmacol. 2013;700(1-3): 93-101.

Graphical Abstract