Rational design of fatty acid amide hydrolase inhibitors that act by covalently bonding to two active site residues

Article abstract of DOI:10.1021/ja4014997

The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.

Full text of DOI:10.1021/ja4014997

Article
pubs.acs.org/JACS
Rational Design of Fatty Acid Amide Hydrolase Inhibitors That Act by
Covalently Bonding to Two Active Site Residues
Katerina Otrubova,†,∥ Monica Brown,§ Michael S. McCormick,§ Gye W. Han,§ Scott T. O’Neal,⊥
Benjamin F. Cravatt,‡,∥ Raymond C. Stevens,§,∥ Aron H. Lichtman,⊥ and Dale L. Boger*,†,∥
†Department of Chemistry, ‡Department of Chemical Physiology, §Department of Molecular Biology, and ∥The Skaggs Institute for
Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
⊥Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States
S
* Supporting Information
ABSTRACT: The design and characterization of α-ketohe-
terocycle fatty acid amide hydrolase (FAAH) inhibitors are
disclosed that additionally and irreversibly target a cysteine
(Cys269) found in the enzyme cytosolic port while
maintaining the reversible covalent Ser241 attachment
responsible for their rapid and initially reversible enzyme
inhibition. Two α-ketooxazoles (3 and 4) containing
strategically placed electrophiles at the C5 position of the
pyridyl substituent of 2 (OL-135) were prepared and
examined as inhibitors of FAAH. Consistent with the observed
time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed
that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to
Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site.
In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH
substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors
accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the
targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in
mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged
over the 1−6 h time course monitored.
INTRODUCTION
■
Inhibitors that react sequentially with two nucleophilic residues
in enzyme active sites are rare.1,2 Representative of the
examples, a recent inhibitor discovered by pursuing a high-
throughput screening lead for O-linked N-acetylglucosamine
transferase was shown to function by cross-linking two active-
site catalytic residues.3 Building on these rare examples and
complementary to their serendipitous discovery, we report
herein the design and characterization of α-ketoheterocycle-
based fatty acid amide hydrolase (FAAH) inhibitors that
additionally and irreversibly target a cysteine (Cys269) found in
the enzyme’s cytosolic port, while maintaining the reversible
covalent Ser241 attachment responsible for their rapid and
initially reversible enzyme inhibition. In addition to their
discovery by structure-guided design rather than serendipity, an
Figure 1. Endogenous FAAH substrates and corresponding hydrolysis
products.
in vivo efficacious but short acting FAAH inhibitor is converted
into an enzyme inhibitor with a long-acting in vivo duration of
action.
growing class of signaling fatty acid amides9 at their sites of
action.6 Although FAAH is a member of the amidase signature
FAAH4,5 inactivates several endogenous signaling lipid
amides6−9 including the endogenous cannabinoid (endocann-
binoid) anandamide (1a)10−12 and the sleep-inducing sub-
stance oleamide (1b, Figure 1).13−16 FAAH’s cellular and
subcellular distribution is consistent with its role in regulating a
Received: February 10, 2013
Published: April 12, 2013
© 2013 American Chemical Society
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family of serine hydrolases for which there are a number of
prokaryotic enzymes, it is the only well-characterized
mammalian enzyme bearing the family’s unusual Ser-Ser-Lys
catalytic triad.17−19
Herein, we report the examination of two prototypical
inhibitors containing strategically placed electrophiles at the
pyridyl C5-position of 2 (Figure 2). The modifications were
designed to subsequently react with and trap Cys269 found in
the enzyme cytosolic port following hemiketal formation of the
electrophilic carbonyl of 3 and 4 with the active site Ser241,
thus providing dual-binding inhibitors. Time-dependent
inhibition, Lineweaver−Burk kinetic analysis, and irreversibility
studies indicate that the inhibitors ultimately function by a
noncompetitive mechanism rather than the reversible, com-
petitive inhibition observed for 2 and related α-ketoheterocycle
inhibitors. X-ray crystallographic characterization of 3 in
complex with r/hFAAH confirms that the inhibitor is covalently
bound in the two distinct positions as designed. In vivo
characterization of 3 and 4 demonstrates that such inhibitors
raise endogenous FAAH substrates levels both to a greater
extent and for an extended duration relative to the reversible
inhibitor 2, and that 3 exhibits a sustained longer acting in vivo
analgesic effect relative to 2.
Because of the therapeutic potential20 of FAAH inhibitors for
the treatment of pain,21 inflammatory,22 and sleep disor-
ders,15,23 there has been widespread interest in the develop-
ment of selective inhibitors of the enzyme.24 Because FAAH
inhibition only potentiates an activated signaling pathway,
increasing the endogenous levels of the released lipid signaling
molecules only at their sites of action, it provides a temporal
and spatial pharmacological control not available to a classical
receptor agonist (e.g., cannabinoid receptor agonists). Follow-
ing early studies with substrate-inspired inhibitors that served to
characterize FAAH as a serine hydrolase,25−33 a series of potent
and selective inhibitors that display excellent in vivo activity
have been disclosed, serving to support the use of FAAH as a
target for therapeutic intervention.34 The earliest of such
inhibitors were α-ketoheterocycles35−46 that bind to FAAH by
reversible hemiketal formation with an active site serine. Many
of these competitive inhibitors were found to be potent and
selective for FAAH relative to other mammalian serine
hydrolases, and members of this class have been shown to
exhibit analgesic activity in vivo.46,47 Of these, 2 (OL-135)
emerged as a potent (Ki = 4.7 nM) and selective (>60−300-
fold) FAAH inhibitor that induces analgesia and increases
endogenous anandamide levels (Figure 2).47 It exhibits
RESULTS AND DISCUSSION
■
Chemistry. The core of the inhibitors was accessed by Stille
coupling48 of the stannylated oxazole intermediate 546 with the
appropriately functionalized 2-chloropyridines (Scheme 1).
Scheme 1
Figure 2. Design of the active-site cross-linking inhibitors 3 and 4.
Thiophilic electrophiles were placed at the pyridine C5 position of the
α-ketoheterocycle inhibitor 2, proximal to residue Cys269 found in the
FAAH cytosolic port.
Subsequent TBS ether deprotection (Bu4NF) and oxidation
of the liberated alcohols with Dess−Martin periodinane49
(DMP) provided the corresponding α-ketoheterocycles 4 and
10. The candidate inhibitor 3 was accessed by further
modification of the pyridyl C5-substituent of 10, entailing
methoxymethyl (MOM) ether deprotection and subsequent
conversion of the resulting primary alcohol 11 to the
corresponding bromide 3.
analgesic or anti-inflammatory activity at doses that approach
or are lower than those of common pain or anti-inflammatory
medications.47 It lacks significant offsite target activity, does not
bind cannabinoid (CB1 or CB2) or vanilloid (TRP) receptors,
and the in vivo effects are observed without the respiratory
depression and dosing desensitization characteristic of opioid
administration or the increased feeding and decreased motor
control characteristic of cannabinoid agonists.
Enzyme Inhibition. X-ray crystallographic structures of r/
hFAAH in complex with 2 and related α-ketoheterocycles and
their analyses were previously reported.50−52 Along with
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verification of the hemiketal linkage of the active site
nucleophilic Ser241 to the electrophilic carbonyl and capturing
the active site catalytic residues in an in-action state, Cys269
was observed adjacent to the pyridine ring. The cysteine,
located proximal to the active site in the cytosolic port near the
pyridine of 2, is spatially oriented to react with a second
electrophile placed on C5 of the pyridyl group of the inhibitor.
In principle, this might be achieved by either a second slower
and irreversible alkylation as envisioned from the benzylic
bromide 3 or a second, potentially reversible addition as
envisioned for the otherwise benign nitrile 4. In both instances
and characteristic of an irreversible enzyme inhibitor, the
second covalent attachment would be expected to extend the in
vivo duration of action of these inhibitors relative to the
reversible inhibitor 2.
4 was examined alongside that of 2. As expected, the potency of
2 did not change with inhibitor−enzyme preincubation times of
0−6 h, whereas the apparent Ki values of 3 and 4 improved
more than 3−15-fold, consistent with slow irreversible
inhibition of FAAH (Figure 3).
Lineweaver−Burk Kinetic Analysis. Inhibitor 2 exhibited
simple competitive reversible FAAH inhibition by Lineweaver−
Burk kinetic analysis38 (Figure 4A). In contrast and after 3 h of
The initial characterization of 3 and 4 and their comparison
with 2 were conducted using purified recombinant rat FAAH
(rFAAH) expressed in Escherichia coli53 at 20−23 °C as
previously disclosed.38 The initial rates of hydrolysis (≤10−
20% reaction) were monitored using enzyme concentrations at
least 3 times below an initially measured Ki by following the
breakdown of 14C-oleamide, and Ki values were established as
previously described (Dixon plot).38 Without preincubation
with the enzyme, the inhibitors 3 (Ki = 3.1 nM) and 4 (Ki = 1.5
nM) exhibited apparent Ki values similar to that observed with
2 (Ki = 4.7 nM) (Figure 3).
Time-Dependent Enzyme Inhibition. Because we
expected the second, subsequent Cys269 alkylation to proceed
more slowly than the rapid hemiketal formation of the
inhibitors, the time-dependent inhibition of FAAH by 3 and
Figure 4. Lineweaver−Burk analysis demonstrates reversible, com-
petitive inhibition by 2 (A) and the noncompetitive inhibition by 3
(B) and 4 (C) after inhibitor−enzyme preincubation for 3 h.
preincubation with FAAH, 3 and 4 exhibited noncompetitive
FAAH inhibition by Lineweaver−Burk analysis (Figure 4B and
C), indicative of irreversible enzyme inhibition and consistent
with Cys269 alkylation of the benzylic bromide in the case of 3
and thioimidate formation in the case of 4.
Irreversible Enzyme Inhibition. Dialysis dilution (4 °C,
18 h, 370-fold) of the FAAH−inhibitor mixture following 3 h of
preincubation with 2 restored nearly full enzyme activity
consistent with its reversible enzyme inhibition, whereas the
analogous dialysis dilution of the mixtures containing 3 and 4
remained unchanged, failing to restore FAAH activity,
Figure 3. Time-dependent FAAH inhibition. Apparent Ki values were
measured after 0−6 h preincubation of inhibitor with rFAAH.
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indicative of irreversible enzyme inhibition (Figure 5). It is
especially notable that 4, presumably forming a Cys269
thioimidate adduct with the nitrile, does not appear to be
even slowly reversible under these conditions.
bromide Cys269 alkylation. In effort to better understand the
structural details of 3 binding in the FAAH active site, the
complex was crystallographically characterized. The X-ray
structure of bromide 3 bound to h/rFAAH54 was solved to
2.3 Å resolution, with the data processing and refinement
statistics reported in Table 1.
Table 1. X-ray Data Collection and Refinement Statistics for
a
r/hFAAH in Complex with 3
r/hFAAH−3
crystal data
space group
P3221
cell dimensions
a, b, c (Å)
104.1 × 104.1 × 261.0+
α = β, γ (deg)
data collection
processing software
wavelength (Å)
resolution (Å)
Rmerge (%)
90, 120
HKL2000
0.979
Figure 5. Dialysis dilution of inhibitor−FAAH mixtures confirms
reversible inhibition by 2 and establishes irreversible FAAH inhibition
by 3 and 4. After 3 h preincubation of FAAH with compounds at
concentrations that result in inhibition of ca. 80% enzyme activity (22
°C, 3 h; 100 nM 2, 80 nM 3 and 4) and following measurement of
residual enzyme activity, dialysis dilution (4 °C, 18 h, 370-fold
dilution) of the mixtures resulted in nearly full recovery of enzyme
activity for 2, but little or no recovery of enzyme activity for 3 and 4.
50.00−2.30
12.7 (90.9)
11.8 (1.2)
97.4 (88.0)
71, 819
mean I/σ(I)
completeness (%)
no. of unique reflns
redundancy
6.4 (2.8)
refinement
resolution (Å)
33.38−2.30 (2.36−2.30)
Role of the Electrophilic Carbonyl. While the kinetic
behavior and irreversible nature of the inhibitors 3 and 4 are
consistent with the targeted irreversible Cys269 alkylation,
these analyses alone do not confirm the observation of the
designed alkylation or whether it might indicate the
involvement of other active site nucleophiles including those
of the Ser-Ser-Lys catalytic triad. In addition, the results would
not distinguish between a delivered Cys269 active site
alkylation with or without continued Ser241 hemiketal
participation that would result in cross-linking two nucleophilic
active site residues. The hemiketal participation is reversible
and conceivably could participate in delivering the inhibitor to
Cys269 proximal to the FAAH active site, but not remain
engaged during or subsequent to the eventual covalent
alkylation. To establish the role of the electrophilic carbonyl,
the corresponding alcohols 7 and 12, as well as the methylene
derivative 13, were also examined (Figure 3). These studies
establish that the electrophilic carbonyl of 3 and 4, and its
hemiketal formation with Ser241, is central to their relatively
rapid (≤1 h) and potent (apparent Ki = 500−200 pM)
irreversible inhibition of FAAH. Although the two alcohols 7
and 12 also exhibited a slow time-dependent increase in FAAH
inhibition indicative of an active-site binding and delivery of
Cys269 alkylation, they remained at least 100-fold less effective
than the corresponding ketones at each time point examined.
The corresponding methylene derivative 13 was a further 100-
fold less effective, being 10 000-fold less effective than 4.
X-ray Cocrystal Structure of 3 Bound to FAAH.
Although these studies provide convincing, albeit indirect,
evidence that the benzylic bromide of 3 and nitrile of 4 are
effectively delivered to the FAAH active site and alkylate
Cys269, they do not establish whether 3 and 4 simply benefit
from the electrophilic hemiketal formation or whether it
remains intact in the bound inhibitor. That is, it was still not
clear whether the reversible hemiketal formation simply
participates in delivering the inhibitor to Cys269, or whether
it remains intact during or subsequent to the putative benzylic
Rwork/Rfree (%)
22.0/26.5
8787
8405
52
no. of atoms
protein
ligand
PEG/ion
36
water
294
average total B factor (Å2)
A chain
61.5
B chain
protein
62.2
61.4
80.2
54.0
ligand
66.3
PEG/ion
69.1
water
52.4
rmsd bond length (Å)
rmsd bond angle (deg)
0.016
1.764
b
Ramachandran plot
preferred (%)
allowed (%)
outliers (%)
97.2
2.7
0.1
a
b
Highest resolution shell is shown in parentheses. As defined in
Molprobity.
Beautifully, inhibitor 3 was found covalently attached to the
catalytic Ser241 residue through its electrophilic carbonyl
bound as a deprotonated hemiketal mimicking the enzymatic
tetrahedral intermediate, a hallmark of the α-ketoheterocycle
class of FAAH inhibitors. More importantly, a second covalent
attachment between the methylene at C5 of the pyridine and
Cys269 is also clearly observed in the electron density (Figure
6). This presumably occurs through SN2 displacement of the
primary bromide following Ser241 hemiketal formation.
The overlays of the cocrystal structures of 3 and 250 are
essentially identical, indicating that capture of the second,
irreversible Cys269 alkylation of the benzylic bromide does not
distort the inhibitor binding from that observed with 2 (Figure
7). Even the precise positioning of Cys269 is unperturbed in
the covalent complex with 3 in comparison to both its position
and its orientation in complex with 2, which lacks the covalent
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Of note, Gly268−Cys269 in the FAAH cytosolic port define
an anion binding site that represents a key interaction for
specific classes of emerging FAAH substrates (N-acyl
taurines)55 and perhaps others yet to be discovered. As
illustrated herein, it represents a site unique to FAAH that can
be exploited to enhance the potency or selectivity of FAAH
inhibitors. In addition to the hemiketal formation observed with
3 and its exquisite interaction with the oxyanion hole defined
by Ile238, Gly239, Gly240, and Ser241 and the unusual Ser217-
mediated OH−π H-bond to the activating oxazole of 3, the key
anchoring terminal phenyl group of the C2 acyl chain overlays
nicely with that of 2 benefiting from the same CH−π
interactions.50−52 The oxazole and pyridyl rings are quasi-
coplanar with the pyridyl nitrogen directed toward the oxazole
aryl CH rather than oxygen (anti vs syn). This strongly
preferred orientation avoids a destabilizing electrostatic
interaction between the pyridyl nitrogen and oxazole oxygen
lone pairs while maintaining the stabilizing conjugation
between the two aromatic rings.56 The Thr236 side-chain
hydroxyl group is oriented toward the pyridyl nitrogen atom of
3 at a distance consistent with a bridging hydrogen-bonded
water molecule, although it was not observed in the electron
density. Thr236 is H-bonded to Lys142, which is an integral
member of the Ser241-Ser217-Lys142 catalytic triad. This
intricate H-bonding network, which locks the pyridyl ring into a
fixed location and orientation, serves to enhance inhibitor
affinity 20-fold.38 In FAAH complexes with 3 and 4, it also
serves to preposition the pyridyl C5-electrophile for reaction
with Cys269.
Figure 6. Inhibitor 3 in the active site of r/hFAAH from the cocrystal
structure. The protein backbone is shown as ribbons in green. Select
residue side-chains and bound 3 are shown as sticks in light blue
(carbon), red (oxygen), blue (nitrogen), and yellow (sulfur). 2Fo − Fc
electron density contoured to 1 σ about the ligand and select side-
chains is shown as a gray mesh.
Inhibitor Selectivity. Activity-based protein profiling
(ABPP), which uses active site-directed chemical probes to
evaluate the activity of enzymes in native biological systems, has
emerged as a powerful tool for evaluating candidate serine
hydrolase inhibitors directly in complex proteomes.57 ABPP
methods offer the advantage of testing enzymes in their native
state and eliminate the need for their recombinant expression,
purification, and the development of specific substrate assays.
Because inhibitors are screened against many enzymes in the
proteome in parallel, both potency and selectivity can be
simultaneously evaluated. Previous studies29 have shown that
the α-ketoheterocycle class of inhibitors are exquisitely selective
for FAAH, although two enzymes did emerge as potential
competitive targets: triacylglycerol hydrolase (TGH) and the
membrane-associated hydrolase, KIAA1363. Each inhibitor (3
and 4) was tested for its effects on the fluorophosphonate
(FP)-rhodamine probe labeling of serine hydrolases in the
mouse brain (Figure 8) and heart membrane proteome
(Supporting Information) at concentrations ranging from 10
nM to 100 μM. As was observed in previous studies, inhibitors
3 and 4 showed excellent selectivity for FAAH over KIAA1363
(>103-fold) and good selectivity over TGH (Supporting
Information Figure S2) with a 20 min inhibitor preincubation,
where FAAH inhibition may still be reversible. As expected, the
activity against KIAA1363 did not change with the 6 h
incubation consistent with a reversible interaction with the
inhibitors. Similarly, competitive inhibition of monoacylglycerol
lipase (MAGL) and α,β-hydrolase containing domain 6
(ABHD6) was observed only at concentrations 102−103
times higher than that required to inhibit labeling of FAAH.
This selectivity (>104−105 fold vs KIAA1363, >10 fold vs
TGH, >102 fold vs MAGL, >104 fold vs ABHD6), as well as
FAAH potency (>10-fold), increased with the more prolonged
6 h preincubation with inhibitor where FAAH inhibition is
Figure 7. Overlay of key active site regions of the X-ray cocrystal
structures of 3 and 2 bound to r/hFAAH. r/hFAAH residues and
bound ligands 2 and 3 are shown as sticks. Carbon atoms
corresponding to the cocrystal structure with 2 are shown in green
(r/hFAAH) and cyan (2), and those corresponding to the cocrystal
structure with 3 are shown in pink. Nitrogen, oxygen, and sulfur atoms
from both crystal structures are shown in blue, red, and yellow,
respectively.
thioether bond. Consequently, the reversible Ser241 hemiketal
covalent attachment to the electrophilic carbonyl inhibitor
remains unperturbed. This results in an exquisite cross-linking
at the FAAH active site, wherein covalent, reversible hemiketal
formation drives a subsequent slower irreversible Cys269
covalent reaction. That we also observed time-dependent
inhibition with 4 further indicates that covalent modification of
Cys269 can even occur with electrophiles as benign as a nitrile
without disturbing the reversible covalent hemiketal.
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reactive benzylic bromide as well as the inhibitor 4 containing a
relatively unreactive nitrile exhibit good FAAH selectivity even
after 6 h preincubation with mouse brain membrane proteome.
In this assay, both inhibitors exhibit a slow time-dependent
increase in potency (6 h > 20 min) reflective of their slow
irreversible enzyme inhibition, displaying IC50 values <10 nM
(4 > 3) after the 6 h preincubation. Notably, the addition of a
second and slow covalently modifying functionality did not
erode, and may have additionally enhanced, the inhibitor
selectivity, especially upon prolonged proteome exposure.
Preliminary in Vivo Characterization. In initial efforts to
establish in vivo inhibition of FAAH and its subsequent
pharmacological effects, the inhibitors 3 and 4 were examined
alongside 2 for their ability to increase the endogenous levels of
a series of lipid amide signaling molecules in both the brain
(CNS effect) and the liver (peripheral effect). This entailed
monitoring the effects of the inhibitors on the endogenous
levels of the FAAH substrates anandamide (AEA), oleoyl
ethanolamide (OEA), and palmitoyl ethanolamide (PEA). It is
the increase in endogenous levels of anandamide (AEA) in the
brain and its subsequent action at cannabinoid (CB1 and CB2)
receptors that are thought to be responsible for the analgesic
effects of FAAH inhibitors. The effects were established at two
time points (1 and 3 h) following intraperitoneal (i.p.)
administration of 30 mg/kg inhibitor in a single mouse per
time point for the initial screen. Both inhibitors 3 and 4
increased the endogenous levels of the key lipid amides in both
the brain and the liver (not shown). In addition, these effects
appeared to not only persist over the 3 h time course, but they
were also greater in magnitude relative to 2.
As a result, a dose- and time-dependent study of the effect of
4 on the endogenous brain levels of AEA, OEA, and PEA was
conducted. Compound 4 was administered to mice (10, 30, and
50 mg/kg, i.p., two mice at each dose), and the animals were
sacrificed at various time points up to 6 h post administration.
Brains from these mice were analyzed for FAAH substrate
levels. Compound 4 increased the levels of AEA (2−4-fold at
30 and 50 mg/kg) in a dose-dependent manner with maximum
increases still observed 6 h after administration (Figure 9A).
Significant in these observations is the fact that partial blockage
of FAAH in vivo can cause elevations in PEA and OEA, but
>90% FAAH inhibition is required to elevate AEA levels in
vivo.59c Brain levels of palmitoyl ethanolamide and oleoyl
Figure 8. ABPP screen of 3 and 4 in mouse brain membrane
proteome (1 mg/mL) with FP-rhodamine (100 nM). Inhibitor
preincubation with the proteome was conducted at both 20 min and 6
h.
irreversible (Figure 8). Only MAGL, which acts on a similar
substrate and is known to possess at least one reactive active
site cysteine,58 appears to exhibit a weaker (>100-fold) but
time-dependent increase in potency at 6 h with inhibitor 4.
Other significant off target inhibition was only observed at 20
min or 6 h at inhibitor concentrations of 100 μM, a
concentration that is >104-fold over the concentration at
which 3 and 4 inhibit FAAH.
A subtle impact of the nature of the pyridyl C5 substituent is
also apparent in this proteome-wide assay with 4 displaying a
greater potency than 3 at both time points. This may be
reflective of some nonproductive consumption of the more
reactive benzylic bromide 3 as compared to the otherwise
benign nitrile 4 in the proteome millue. Nonetheless and even
under such conditions, both the inhibitor 3 containing a
Figure 9. Lipid levels in the brain 1−6 h post administration of 4, i.p. at 10, 30, and 50 mg/kg: (A) AEA, (B) OEA, (C) PEA.
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Figure 10. Comparison of lipid levels in the brain 1−6 h post administration of 2 and 4 at 30 mg/kg, i.p.: (A) AEA, (B) OEA, (C) PEA.
ethanolamide also increased in a dose-dependent manner. After
1 h, levels of PEA increased 5.9-fold, and levels of OEA
increased 4.5-fold (Figure 9B and C). Moreover, these levels
continued to rise 3 h post administration of 4 (PEA increases
12-fold). These elevations exceeded the effects observed with 2
in earlier studies with administration at 50 mg/kg46 where both
the maximum lipid amide increase is lower and the duration of
the increase is much shorter (1 h max). Consequently, a side-
by-side comparison of 4 with 2 was also conducted, measuring
the impact 2 had on the same signaling amides when also
administered at 30 mg/kg (i.p.). Because the effects of 2 had
been previously established to dissipate by 6 h, measurements
of the effects of 2 were recorded at 1 and 3 h. As shown in
Figure 10, the maximum effect of 2 on all three lipid amides is
observed at 1 h, is already attenuated at 3 h, and can be
projected from the trends to have dissipated at 6 h as previously
observed.46 Because 2 has been shown to exhibit good brain
exposure effectively crossing the blood brain barrier such that
plasma and brain concentrations are equivalent,46 the
distinctions in the in vivo activity of 2 versus 4 (or 3) are
not due to poor brain exposure to 2 (Supporting Information
Figure S3). Both the duration (>6 h for 4) and the magnitude
of the lipid amide elevations are much more robust with 4
versus 2. Thus, administration of 4 caused substantial
accumulations of all three measured FAAH substrates in
brain, with peak levels of AEA achieved within 1.5−3 h (Figure
11A and B), and elevations in these lipids were maintained over
the 6 h time course, similar to the behavior of irreversible
carbamate inhibitors of FAAH such as URB597 and PF-
3845.59−61 The impact of the benzylic bromide 3 at 30 mg/kg
(i.p.) in increasing brain levels of AEA was also examined and
found to be indistinguishable from that of the nitrile 4 (data in
Supporting Information Figure S1). These sustained and long-
lasting increases in the in vivo lipid amide levels indicate that
inhibitors 4 and 3 achieve sufficiently high brain levels after its
initial dosing to produce durable (>6 h) and near-complete
inhibition of FAAH (>90%), consistent with their slow
irreversible inhibition of FAAH.
Inhibitor 3 Exhibits a Sustained in Vivo Duration of
Action in a Model of Neuropathic Pain. In an important
extension of the studies and following observation of the long-
acting in vivo effects on endogenous AEA levels following i.p.
administration, mice were subjected to chronic constriction
injury (CCI) and examined 14 days later for signs of
neuropathic pain. Inhibitor 3, administered i.p. (30 mg/kg),
Figure 11. (A) Levels of anandamide (AEA) in the brain 0−6 h post
administration of 4 at 30 and 50 mg/kg administered i.p., and (B)
levels of PEA and OEA in the brain 0−6 h post administration of 4
given at 50 mg/kg, i.p.
significantly attenuated cold allodynia in paws ipsilateral to CCI
surgery, exhibiting a maximal efficacy that matches that
observed with the reversible inhibitor 2 (OL-135) but with a
much longer duration of action (Figure 12). In the control
paws of the same mice, 3 had no effect on cold allodynia,
indicating a lack of effects (e.g., sedative) on the uninjured
paws. Significantly, the effects of 3 following its administration
were sustained, remaining unchanged over the 1−6 h time
course monitored and indicating a pronounced in vivo efficacy
lasting >6 h. This contrasts the shorter duration of action of the
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recombinant rFAAH was used in the inhibition assays. The inhibition
assays were performed as described.15 The enzyme reaction was
initiated by mixing 1 nM of rFAAH (800, 500, or 200 pM rFAAH for
inhibitors with Ki ≤ 1−2 nM) with 20 μM of 14C-labeled oleamide in
500 μL of reaction buffer (125 mM TrisCl, 1 mM EDTA, 0.2%
glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9.0) at room
temperature in the presence of three different concentrations of
inhibitor. The enzyme reaction was terminated by transferring 20 μL
of the reaction mixture to 500 μL of 0.1 N HCl at three different time
points. The 14C-labeled oleamide (substrate) and oleic acid (product)
were extracted with EtOAc and analyzed by TLC as detailed. The Ki of
the inhibitor was calculated using a Dixon plot as described (standard
deviations are provided in the Supporting Information tables).
Lineweaver−Burk analysis was performed as described confirming
competitive, reversible inhibition for 2, and noncompetitive inhibitions
for 3 and 4 (Figure 4A−C).
Reversibility of FAAH Inhibition (Dialysis). The reversibility of
FAAH inhibition by 2, 3, and 4 was assessed by dialysis dilution using
recombinant rFAAH. Cell pellets were homogenized in 15 mL of
FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02%
Triton X-100, 0.4 mM Hepes, pH 9.0). A 3 mL aliquot of membrane
homogenate was used for each sample dialyzed. The dialysis
experiment was performed in the predialysis mix at or near the
apparent IC80. The final assay inhibitor concentrations used were 100
nM 2, 80 nM 3, and 80 nM 4. Samples were preincubated with the
enzyme for 3 h at room temperature (22 °C) before 300 μL was
removed and assayed in triplicate in a FAAH activity assay. The
remaining sample (2.7 mL) was injected into a dialysis cassette
employing a 10 000 MW cutoff membrane. The mixture was dialyzed
against 1 L of PBS at 4 °C on a stir plate for 18 h. The post dialysis
FAAH activity was assessed by assaying 300 μL samples taken from
the dialysis cassettes in triplicate. FAAH activity is expressed as a
percentage of vehicle treated FAAH (DMSO alone).
Competitive ABPP of FAAH Inhibitors with FP-Rhodamine.
Mouse tissues were Dounce-homogenized in PBS buffer (pH 8.0) and
membrane proteomes isolated by centrifugation at 4 °C (100 000g, 45
min), washed, resuspended in PBS buffer, and adjusted to a protein
concentration of 1 mg/mL. Proteomes were preincubated with
inhibitors (10−100 000 nM; DMSO stocks) for 20 min and 6 h and
then treated with FP-rhodamine (100 nM, DMSO stock) at room
temperature for 10 min. Reactions were quenched with SDS-PAGE
loading buffer, subjected to SDS-PAGE, and visualized in-gel using
flatbed fluorescence scanner (MiraBio). Labeled proteins were
quantified by measuring integrated band intensities (normalized for
volume); control samples (DMSO alone) were considered 100%
activity. IC50 values were determined from dose−response curves
using Prism software.
In Vivo Pharmacodynamic Studies with Inhibitors. Inhibitors
were prepared as a saline-emulphor emulsion for intraperitoneal (i.p.)
administration by vortexing, sonicating, and gently heating neat
compound directly into an 18:1:1 v/v/v solution of saline:ethanol:e-
mulphor. Male C57Bl/6J mice (<6 months old, 20−28 g) were
administered inhibitors in saline-emulphor emulsion or an 18:1:1 v/v/
v saline:emulphor:ethanol vehicle i.p. at a volume of 10 μL/g weight.
After the indicated amount of time (1, 3, and 6 h), mice were
anesthetized with isofluorane and decapitated. Total brains (∼400 mg)
and a portion of the liver (∼100 mg) were removed and flash frozen in
liquid N2. Animal experiments were conducted in accordance with the
guidelines of the Institutional Animal Care and Use Committee of The
Scripps Research Institute.
Measurement of Brain Lipids. Tissue was weighed and
subsequently Dounce homogenized in 2:1:1 v/v/v CHCl3:MeOH:Tris
pH 8.0 (8 mL) containing standards for lipids (50 pmol of d4-PEA, 2
pmol of d4-AEA, and 10 nmol of pentadecanoic acid). The mixture was
vortexed and then centrifuged (1400g, 10 min). The organic layer was
removed, evaporated under a stream of N2, resolubilized in 2:1 v/v
CHCl3:MeOH (120 μL), and 10 μL of this resolubilized lipid solution
was injected onto an Agilent G6410B QQQ instrument. LC separation
was achieved with a Gemini reverse-phase C18 column (5 μm, 4.6 mm
× 50 mm, Phenomonex) together with a precolumn (C18, 3.5 μm, 2
Figure 12. FAAH inhibition in vivo by 3 significantly attenuated
neuropathic pain for >6 h with a potency matching that of the
reversible inhibitor 2, but for a sustained duration. Male C57BL/6
mice were subjected to chronic constriction injury (CCI) of the sciatic
nerve and tested 10 days later for mechanical allodynia, as measured
with acetone-induced cold allodynia. Inhibitors 2 and 3 (30 mg/kg,
i.p.) significantly attenuated CCI-induced cold allodynia in paws
ipsilateral to CCI surgery, but had no effect in paws contralateral to
□
○
▼
CCI surgery. , vehicle treatment; , 2 treatment; , 3 treatment.
Data expressed as mean SEM (n = 10).
reversible inhibitor 2 whose in vivo effects dissipated over this
time course.
CONCLUSIONS
■
The design of α-ketohetorocycles additionally targeting the
remote Cys269 found in the cytosolic port of FAAH provided
inhibitors that covalently react with two active site nucleophiles.
The cross-link design was sufficiently accurate that the first of
the two covalent attachments, the hemiketal, while being itself
reversible, remains intact after the second, slower irreversible
reaction with Cys269. The second electrophile targeting
Cys269 was incorporated as a C5 substituent on the pyridyl
group of 5-(pyrid-2-yl)oxazoles including 2, and ranged from
the irreversibly reactive benzylic bromide 3 to the covalent
reversible and typically benign nitrile 4. Consistent with
expectations, the inhibitors exhibited time-dependent, non-
competitive enzyme inhibition, produced sustained accumu-
lation of the endogenous substrates for FAAH in vivo for >6 h,
and exhibited efficacious and long-acting antinociceptive
activity (>6 h) in an in vivo model of neuropathic pain. The
exquisite FAAH active site cross-linking by 3 was confirmed by
X-ray crystallography, unambiguously establishing it as a rare
example of sequential reaction of an inhibitor with two
nucleophiles in the active site of an enzyme,1−3 and the only
characterized example, to our knowledge, both where the cross-
linking was achieved through rational, structure-guided design
and where one of the covalent attachments is reversible. Our
studies thus serve as a prototype for an approach to convert
selective short-acting reversible, competitive serine hydrolase
inhibitors into in vivo efficacious compounds that produce a
robust long-acting pharmacological response suitable for target
validation or drug development by targeting peripheral active
site nucleophiles.
EXPERIMENTAL SECTION
■
FAAH Inhibition. 14C-labeled oleamide was prepared from 14C-
labeled oleic acid as described.15 The truncated rat FAAH (rFAAH)
was expressed in E. coli and purified as described.53 The purified
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mm × 20 mm). Mobile phase A was composed of a 95:5 v/v
H2O:MeOH, and mobile phase B was composed of a 65:35:5 v/v/v i-
PrOH:MeOH:H2O. 0.1% formic acid or 0.1% ammonium hydroxide
was included to assist in ion formation in positive and negative
ionization mode, respectively. The flow rate for each run started at 0.1
mL/min with 0% B. At 5 min, the solvent was immediately changed to
60% B with a flow rate of 0.4 mL/min and increased linearly to 100%
B over 10 min. This was followed by an isocratic gradient of 100% B
for 5 min at 0.5 mL/min before equilibrating for 3 min at 0% B at 0.5
mL/min (23 min total per sample). MS analysis was performed with
an electrospray ionization (ESI) source. The following MS parameters
were used to measure the indicated metabolites in positive mode
(precursor ion, product ion, collision energy in V): AEA (348, 62, 11),
OEA (326, 62, 11), PEA (300, 62, 11), d4-AEA (352, 66, 11), d4-PEA
(304, 62, 11). For negative polarity, the analysis was performed in
MS2 scan mode from 100−1000 m/z. The capillary was set to 4 kV,
the ionization source was set to 100 V, and the delta EMV was set to 0.
Lipids were quantified by measuring the area under the peak in
comparison to the standards.
Chronic Constriction Injury (CCI) Allodynia Assay. Surgery
was performed as described previously.62 Allodynia was initially tested
a minimum of 14 days after surgery. Male C57BL/6 mice were
habituated to the test apparatus for 2 h on the 2 days prior to testing.
On test days, the mice were brought into the test room, weighed, and
allowed to acclimate for at least 1 h before the start of the experiment.
Mice were administered inhibitors 2 or 3 (30 mg/kg, n = 10) or
vehicle (i.p.) as detailed above, then placed in ventilated polycarbonate
cylinders on a mesh table. A within subject design was used in which
the mice were administered the different regimens in a counter-
balanced fashion, with at least 1 week between test days. Cold
(acetone-induced) allodynia were tested at 1, 3, and 6 h post drug
administration. Testing was carried out by an observer who was
blinded to treatment conditions. Cold allodynia was tested by
propelling 10 μL of acetone (Fisher Bioscience) via air burst, from a
200 μL pipet (Rainin Instruments, Oakland, CA) onto the plantar
surface of each hind paw. Total time lifting or clutching each paw was
recorded, with a maximum time of 20 s.63
structure have been deposited in the RCSB Protein Data Bank with
accession code 4J5P.
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental details for the synthesis and characterization
of the candidate inhibitors and the dose- and time-dependent in
vivo effects of 3 on lipid amide levels. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
boger@scripps.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support of the National
Institutes of Health (DA015648, D.L.B.; DA017259 and
DA033760, B.F.C.; DA017259, R.C.S.; DA009789 and
DA017259, A.H.L.).
REFERENCES
■
(1) Vijayalakshmi, J.; Meyer, E. F., Jr.; Kam, C. M.; Powers, J. C.
Biochemistry 1991, 30, 2175−2183.
(2) Drawz, S. M.; Bonomo, R. A. Clin. Microbiol. Rev. 2010, 23, 160−
201.
(3) Jiang, J.; Lazarus, M. B.; Pasquina, L.; Sliz, P.; Walker, S. Nat.
Chem. Biol. 2012, 8, 72−77.
(4) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner,
R. A.; Gilula, N. B. Nature 1996, 384, 83−87.
(5) Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
2238−2242.
(6) Patricelli, M. P.; Cravatt, B. F. Vitam. Horm. 2001, 62, 95−131.
(7) Egertova, M.; Cravatt, B. F.; Elphick, M. R. Neuroscience 2003,
119, 481−496.
(8) Boger, D. L.; Fecik, R. A.; Patterson, J. E.; Miyauchi, H.; Patricelli,
M. P.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 2000, 10, 2613−2616.
(9) Ezzili, C.; Otrubova, K.; Boger, D. L. Bioorg. Med. Chem. Lett.
2010, 20, 5959−5968.
For data analyses, behavioral data were analyzed using a two-way
mixed factorial analysis of variance (ANOVA) for each paw, with drug
treatment as the between subjects measure, and time as the within
subjects measure. Follow-up comparisons were made using the
Bonferroni test. All animals (n = 10 per treatment group) were
included in the analyses. Differences between groups were considered
statistically significant at p < 0.05.
(10) Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson,
L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;
Mechoulam, R. Science 1992, 258, 1946−1949.
(11) Martin, B. R.; Mechoulam, R.; Razdan, R. K. Life Sci. 1999, 65,
573−595.
FAAH Production, Crystallization, and Crystal Structure
Determination. The N-terminal transmembrane-deleted humanized
version of FAAH (amino acids 32−579) was expressed in E. coli and
purified as previously described,54 using 0.08% n-undecyl-β-D-malto-
side in the ion exchange and size exclusion chromatography steps of
the purification. Purified protein was crystallized as previously
described,52 with the modifications described below. Precipitant
solution contained 50 mM MES pH 5.5, 0.02% UM-LA, 15% PEG
400, 4% polypropylene glycol P400, 13% xylitol, 1 mM DTT, 50 mM
KCl, and 50 mM NaF. Crystals were grown by the sitting drop vapor
diffusion method at 14 °C in 96-well plates (Innovaplate SD-2;
Innovadyne Technologies), and frozen in liquid nitrogen immediately
after harvesting. Crystallographic data were collected at 100 K using
the Blu-Ice data collection suite64 at the Stanford Synchrotron
Radiation Laboratory on beamline 11-1, and processed using
HKL2000.65 The structure was determined to 2.30 Å resolution in
the space group P3221 by molecular replacement using FAAH
coordinates from PDB code 3K84. Molecular replacement and
structure refinement were conducted using Phaser66 and REFMAC,67
respectively, from the CCP4 software suite.68 The Dundee PRODRG
Web server69 was used to calculate restraint parameters for the
covalently bound inhibitor 3. Crystallographic model building was
conducted using Coot,70 and images of the structure were prepared in
PyMOL (DeLano Scientific, LLC). Results from data processing and
structure refinement are provided in Table 1. Coordinates for the
(12) Schmid, H. H. O.; Schmid, P. C.; Natarajan, V. Prog. Lipid Res.
1990, 29, 1−43.
(13) Boger, D. L.; Henriksen, S. J.; Cravatt, B. F. Curr. Pharm. Des.
1998, 4, 303−314.
(14) Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996,
118, 580−590.
(15) Cravatt, B. F.; Prospero-Garcia, O.; Suizdak, G.; Gilula, N. B.;
Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506−
1509.
(16) Lerner, R. A.; Siuzdak, G.; Prospero-Garcia, O.; Henriksen, S. J.;
Boger, D. L.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
9505−9508.
(17) (a) Patricelli, M. P.; Cravatt, B. F. Biochemistry 1999, 38,
14125−14130. (b) Patricelli, M. P.; Cravatt, B. F. J. Biol. Chem. 2000,
275, 19177−19184. (c) Patricelli, M. P.; Lovato, M. A.; Cravatt, B. F.
Biochemistry 1999, 38, 9804−9812. (d) McKinney, M. K.; Cravatt, B.
F. J. Biol. Chem. 2003, 278, 37393−37399.
(18) McKinney, M. K.; Cravatt, B. F. Annu. Rev. Biochem. 2005, 74,
411−432.
(19) Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.;
Cravatt, B. F. Science 2002, 298, 1793−1796.
6297
dx.doi.org/10.1021/ja4014997 | J. Am. Chem. Soc. 2013, 135, 6289−6299

Journal of the American Chemical Society
Article
(20) (a) Cravatt, B. F.; Lichtman, A. H. Curr. Opin. Chem. Biol. 2003,
7, 469−475. (b) Ahn, K.; McKinney, M. K.; Cravatt, B. F. Chem. Rev.
2008, 108, 1687−1707. (c) Ahn, K.; Johnson, D. S.; Cravatt, B. F. Exp.
Opin. Drug Discovery 2009, 4, 763−784.
(43) Kimball, F. S.; Romero, F. A.; Ezzili, C.; Garfunkle, J.; Rayl, T. J.;
Hochstatter, D. G.; Hwang, I.; Boger, D. L. J. Med. Chem. 2008, 51,
937−947.
(44) Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang,
I.; Boger, D. L. J. Med. Chem. 2008, 51, 4393−4403.
(21) (a) Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.;
Giang, D. K.; Martin, B. R.; Lichtman, A. H. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 9371−9376. (b) Lichtman, A. H.; Shelton, C. C.;
Advani, T.; Cravatt, B. F. Pain 2004, 109, 319−327. (c) Cravatt, B. F.;
Saghatelian, A.; Hawkins, E. G.; Clement, A. B.; Bracey, M. H.;
Lichtman, A. H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10821−10826.
(22) Karsak, M.; Gaffal, E.; Date, R.; Wang-Eckhardt, L.; Rehnelt, J.;
Petrosino, S.; Starowicz, K.; Steuder, R.; Schlicker, E.; Cravatt, B. F.;
Mechoulam, R.; Buettner, R.; Werner, S.; Di Marzo, V.; Tueting, T.;
Zimmer, A. Science 2007, 316, 1494−1497.
(45) DeMartino, J. K.; Garfunkle, J.; Hochstatter, D. G.; Cravatt, B.
F.; Boger, D. L. Bioorg. Med. Chem. Lett. 2008, 18, 5842−5846.
(46) Ezzili, C.; Mileni, M.; McGlinchey, N.; Long, J. Z.; Kinsey, S. G.;
Hochstatter, D. G.; Stevens, R. C.; Lichtman, A. H.; Cravatt, B. F.;
Bilsky, E. J.; Boger, D. L. J. Med. Chem. 2011, 113, 7277−7287.
(47) (a) Lichtman, A. H.; Leung, D.; Shelton, C. C.; Saghatelian, A.;
Hardouin, C.; Boger, D. L.; Cravatt, B. F. J. Pharmacol. Exp. Ther.
2004, 311, 441−448. (b) Chang, L.; Luo, L.; Palmer, J. A.; Sutton, S.;
Wilson, S. J.; Barbier, A. J.; Breitenbucher, J. G.; Chaplan, S. R.; Webb,
M. Br. J. Pharmacol. 2006, 148, 102−113. (c) Schlosburg, J. E.; Boger,
D. L.; Cravatt, B. F.; Lichtman, A. H. J. Pharmacol. Exp. Ther. 2009,
329, 314−323. (d) Kinsey, S. G.; Long, J. Z.; O’Neal, S. T.; Abdulla, R.
A.; Poklis, J. L.; Boger, D. L.; Cravatt, B. F.; Lichtman, A. H. J.
Pharmacol. Exp. Ther. 2009, 330, 902−910. (e) Booker, L.; Kinsey, S.
G.; Abdullah, R. A.; Blankman, J. L.; Long, J. Z.; Boger, D. L.; Cravatt,
B. F.; Lichtman, A. H. Br. J. Pharmacol. 2012, 165, 2485−2496.
(48) Farina, V.; Krishnamurthy, V.; Scott, W. J. Org. React. 1997, 50,
1−652.
(23) (a) Huitro
Henriksen, S. J. Exp. Neurol. 2001, 172, 235−243. (b) Huitron
Resendiz, S.; Sanchez-Alavez, M.; Wills, D. N.; Cravatt, B. F.;
Henriksen, S. J. Sleep 2004, 27, 857−865.
́ ́
n-Resendiz, S.; Gombart, L.; Cravatt, B. F.;
́
-
́
(24) Clement, A. B.; Hawkins, E. G.; Lichtman, A. H.; Cravatt, B. F. J.
Neurosci. 2003, 23, 3916−3923.
(25) Patricelli, M. P.; Patterson, J. P.; Boger, D. L.; Cravatt, B. F.
Bioorg. Med. Chem. Lett. 1998, 8, 613−618.
(26) Koutek, B.; Prestwich, G. D.; Howlett, A. C.; Chin, S. A.;
Salehani, D.; Akhavan, N.; Deutsch, D. G. J. Biol. Chem. 1994, 269,
22937−22940.
(49) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277−
7287.
(50) Mileni, M.; Garfunkle, J.; DeMartino, J. K.; Cravatt, B. F.; Boger,
D. L.; Stevens, R. C. J. Am. Chem. Soc. 2009, 131, 10497−10506.
(51) Mileni, M.; Garfunkle, J.; Kimball, F. S.; Cravatt, B. F.; Stevens,
R. C.; Boger, D. L. J. Med. Chem. 2010, 53, 230−240.
(52) Mileni, M.; Garfunkle, J.; Ezzili, C.; Cravatt, B. F.; Stevens, R.
C.; Boger, D. L. J. Am. Chem. Soc. 2011, 133, 4092−4100.
(53) Patricelli, M. P.; Lashuel, H. A.; Giang, D. K.; Kelly, J. W.;
Cravatt, B. F. Biochemistry 1998, 37, 15177−15187.
(54) Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.; Liimatta,
M.; Pabst, B.; Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt,
B. F.; Ahn, K.; Stevens, R. C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
12820−12824.
(27) Patterson, J. E.; Ollmann, I. R.; Cravatt, B. F.; Boger, D. L.;
Wong, C.-H.; Lerner, R. A. J. Am. Chem. Soc. 1996, 118, 5938−5945.
(28) Boger, D. L.; Sato, H.; Lerner, A. E.; Austin, B. J.; Patterson, J.
E.; Patricelli, M. P.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 1999, 9,
265−270.
(29) Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat.
Biotechnol. 2003, 21, 687−691.
(30) Deutsch, D. G.; Omeir, R.; Arreaza, G.; Salehani, D.; Prestwich,
G. D.; Huang, Z.; Howlett, A. Biochem. Pharmacol. 1997, 53, 255−260.
(31) Deutsch, D. G.; Lin, S.; Hill, W. A. G.; Morse, K. L.; Salehani,
D.; Arreaza, G.; Omeir, R. L.; Makriyannis, A. Biochem. Biophys. Res.
Commun. 1997, 231, 217−221.
(32) Edgemond, W. S.; Greenberg, M. J.; McGinley, P. J.; Muthians,
S.; Campbell, W. B.; Hillard, C. J. J. Pharmacol. Exp. Ther. 1998, 286,
184−190.
(33) Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Boger, D. L.
Bioorg. Med. Chem. Lett. 2005, 15, 103−106.
(55) (a) McKinney, M. K.; Cravatt, B. F. Biochemistry 2006, 45,
9016−9022. (b) Saghatelian, A.; McKinney, M. K.; Bandell, M.;
Patapoutian, A.; Cravatt, B. F. Biochemistry 2006, 45, 9007−9015.
(c) Saghatelian, A.; Trauger, S. A.; Want, E. J.; Hawkins, E. G.;
Suizdak, G.; Cravatt, B. F. Biochemistry 2004, 43, 14332−14339.
́
(56) Guimaraes, C. R. W.; Boger, D. L.; Jorgensen, W. L. J. Am.
(34) (a) Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem.
Lett. 2011, 21, 4674−4685. (b) Deng, H. F. Exp. Opin. Drug Discovery
2010, 5, 961−993. (c) Seierstad, M.; Breitenbucher, J. G. J. Med. Chem.
2008, 51, 7327−7343. (d) Vandevoorde, S. Curr. Top. Med. Chem.
2008, 8, 247−267.
Chem. Soc. 2005, 127, 17377−17384.
(57) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279−3301.
(58) (a) Saario, S. M.; Salo, O. M. H.; Nevalainen, T.; Poso, A.;
Laitinen, J. T.; Jarvinen, T.; Niemi, R. Chem. Biol. 2005, 12, 649−656.
̈
(b) Labar, G.; Bauvois, C.; Borel, F.; Ferrer, J.-L.; Wouters, J.;
Lambert, D. M. ChemBioChem 2010, 11, 218−227. (c) Zvonok, N.;
Pandarinathan, L.; Williams, J.; Johnston, M.; Karageorgos, I.; Janero,
D. R.; Krishnan, S. C.; Makriyannis, A. Chem. Biol. 2008, 15, 854−862.
(d) King, A. R.; Lodola, A.; Carmi, C.; Fu, J.; Mor, M.; Piomelli, D. Br.
J. Pharmacol. 2009, 157, 974−983.
(35) Otrubova, K.; Boger, D. L. ACS Chem. Neurosci. 2012, 3, 340−
348.
(36) Boger, D. L.; Sato, H.; Lerner, A. E.; Hedrick, M. P.; Fecik, R.
A.; Miyauchi, H.; Wilkie, G. D.; Austin, B. J.; Patricelli, M. P.; Cravatt,
B. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5044−5049.
(37) Boger, D. L.; Miyauchi, H.; Hedrick, M. P. Bioorg. Med. Chem.
Lett. 2001, 11, 1517−1520.
(59) (a) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.;
Arendse, A.; Stevenson, T.; Lund, E. T.; Nugent, R. A.; Normanbhoy,
T.; Alexander, J. P.; Cravatt, B. F. Biochemistry 2007, 46, 13019−
13030. (b) Johnson, D. S.; Ahn, K.; Kesten, S.; Lazerwith, S. E.; Song,
Y.; Morris, M.; Fay, L.; Gregory, T.; Stiff, C.; Dunbar, J. B., Jr.;
Liimatta, M.; Beidler, D.; Smith, S.; Nomanbhoy, T. K.; Cravatt, B. F.
Bioorg. Med. Chem. Lett. 2009, 19, 2865−2869. (c) Ahn, K.; Johnson,
D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.;
Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith,
S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Van
Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Chem. Biol. 2009, 16, 411−
420. (d) Johnson, D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L.
K.; Morris, M.; Beidler, D.; Liimatta, M. B.; Smith, S. E.; Dudley, D.
T.; Sadagopan, N.; Bhattachar, S. N.; Kesten, S. J.; Nomanbhoy, T. K.;
Cravatt, B. F.; Ahn, K. ACS Med. Chem. Lett. 2011, 2, 91−96.
(60) Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179−1187.
(38) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.;
Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.;
́
Guimaraes, C. R. W.; Jorgensen, W. L.; Cravatt, B. F. J. Med. Chem.
2005, 48, 1849−1856.
(39) Leung, D.; Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.;
Cravatt, B. F.; Boger, D. L. Bioorg. Med. Chem. Lett. 2005, 15, 1423−
1428.
(40) Romero, F. A.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006,
68, 14004−14005.
(41) Romero, F. A.; Du, W.; Hwang, I.; Rayl, T. J.; Kimball, F. S.;
Leung, D.; Hoover, H. S.; Apodaca, R. L.; Breitenbucher, B. J.; Cravatt,
B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 1058−1068.
(42) Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.;
Hwang, I.; Cravatt, B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 3359−
3368.
6298
dx.doi.org/10.1021/ja4014997 | J. Am. Chem. Soc. 2013, 135, 6289−6299

Journal of the American Chemical Society
Article
(61) (a) Jayamanne, A.; Greenwood, R.; Mitchell, V. A.; Aslan, S.;
Piomelli, D.; Vaughan, C. W. Br. J. Pharmacol. 2006, 147, 281−288.
(b) Keith, J. M.; Apodaca, R.; Xiao, W.; Seierstand, M.; Pattabiraman,
K.; Wu, J.; Webb, M.; Karbarz, M. J.; Brown, S.; Wilson, S.; Scott, B.;
Tham, C.-S.; Luo, L.; Palmer, J.; Wennerholm, M.; Chaplan, S.;
Breitenbucher, J. G. Bioorg. Med. Chem. Lett. 2008, 18, 4838−4843.
(c) Karbarz, M. J.; Luo, L.; Chang, L.; Tham, C.-S.; Balmer, J. A.;
Wilson, S. J.; Wennerholm, M. L.; Brown, S. M.; Scott, B. P.; Apodaca,
R. L.; Keith, J. M.; Wu, J.; Breitenbucher, J. G.; Chaplan, S. R.; Webb,
M. Anesth. Analg. 2009, 108, 316−329. (d) Keith, J. M.; Apodaca, R.;
Tichenor, M.; Xiao, W.; Jones, W.; Pierce, J.; Seierstad, M.; Palmer, J.;
Webb, M.; Karbarz, M.; Scott, B.; Wilson, S.; Luo, L.; Wennerholm,
M.; Chang, L.; Brown, S.; Rizzolio, M.; Rynberg, R.; Chaplan, S.;
Breitenbucher, J. G. ACS Med. Chem. Lett. 2012, 3, 823−827.
(62) Kinsey, S. G.; Long, J. Z.; Cravatt, B. F.; Lichtman, A. H. J. Pain
2010, 11, 1420−1428.
(63) Decosterd, I.; Woolf, C. J. Pain 2000, 87, 149−158.
(64) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.;
Deacon, A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.;
Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. J. Synchrotron Radiat. 2002,
9, 401−406.
(65) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307−
326.
(66) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658−674.
(67) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr.
1997, D53, 240−255.
(68) Collaborative Computational Project, Number 4. Acta
Crystallogr. 1994, D50, 760−763.
(69) Schuttelkopf, A. W.; van Aalten, D. M. F. Acta Crystallogr. 2004,
̈
D60, 1355−1363.
(70) Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126−2132.
6299
dx.doi.org/10.1021/ja4014997 | J. Am. Chem. Soc. 2013, 135, 6289−6299