Design, synthesis, and characterization of α-ketoheterocycles that additionally target the cytosolic port Cys269 of fatty acid amide hydrolase

Article abstract of DOI:10.1021/jm401820q

A series of α-ketooxazoles incorporating electrophiles at the C5 position of the pyridyl ring of 2 (OL-135) and related compounds were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH) that additionally target the cytosolic port Cys269. From this series, a subset of the candidate inhibitors exhibited time-dependent FAAH inhibition and noncompetitive irreversible inactivation of the enzyme, consistent with the targeted Cys269 covalent alkylation or addition, and maintained or enhanced the intrinsic selectivity for FAAH versus other serine hydrolases. A preliminary in vivo assessment demonstrates that these inhibitors raise endogenous brain levels of anandamide and other FAAH substrates upon intraperitoneal (i.p.) administration to mice, with peak levels achieved within 1.5-3 h, and that the elevations of the signaling lipids were maintained >6 h, indicating that the inhibitors effectively reach and remain active in the brain, inhibiting FAAH for a sustained period.

Full text of DOI:10.1021/jm401820q

Article
pubs.acs.org/jmc
Open Access on 01/23/2015
Design, Synthesis, and Characterization of α‑Ketoheterocycles That
Additionally Target the Cytosolic Port Cys269 of Fatty Acid Amide
Hydrolase
Katerina Otrubova,†,§ Benjamin F. Cravatt,‡,§ and Dale L. Boger*,†,§
‡
§
†Department of Chemistry, Chemical Physiology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: A series of α-ketooxazoles incorporating electrophiles at the
C5 position of the pyridyl ring of 2 (OL-135) and related compounds
were prepared and examined as inhibitors of fatty acid amide hydrolase
(FAAH) that additionally target the cytosolic port Cys269. From this
series, a subset of the candidate inhibitors exhibited time-dependent
FAAH inhibition and noncompetitive irreversible inactivation of the
enzyme, consistent with the targeted Cys269 covalent alkylation or addition, and maintained or enhanced the intrinsic selectivity
for FAAH versus other serine hydrolases. A preliminary in vivo assessment demonstrates that these inhibitors raise endogenous
brain levels of anandamide and other FAAH substrates upon intraperitoneal (i.p.) administration to mice, with peak levels
achieved within 1.5−3 h, and that the elevations of the signaling lipids were maintained >6 h, indicating that the inhibitors
effectively reach and remain active in the brain, inhibiting FAAH for a sustained period.
Early studies following the initial identification of the enzyme
led to the disclosure of a series of substrate-inspired inhibitors
that were used to characterize the enzyme as a serine
hydrolase.16−22 Subsequent studies disclosed several classes of
inhibitors that provide opportunities for the development of
inhibitors with therapeutic potential. These include the reactive
aryl carbamates and ureas23−31 that irreversibly carbamylate the
FAAH active site catalytic serine.32 A second, and one of the
earliest classes, is the α-ketoheterocycle-based inhibitors33−44
that bind to FAAH by reversible hemiketal formation with the
active site catalytic serine. Many of these reversible, competitive
inhibitors have been shown to be selective for FAAH versus other
mammalian serine hydrolases as well as efficacious analgesics in
vivo.44,45 In these studies, 2 (OL-135)36 emerged as a potent (Ki
= 4.7 nM)36 and selective (>60−300 fold)19 prototypical FAAH
inhibitor that induces analgesia and increases endogenous
anandamide levels.45 It lacks significant off-site target activity,
does not bind cannabinoid (CB1 or CB2) or vanilloid (TRP)
receptors, and does not significantly inhibit common P450
metabolism enzymes or the human ether-a-go-go related gene
product (hERG). The analgesic effects of 2 are observed without
the respiratory depression or chronic dosing desensitization
characteristic of opioid administration or the increased feeding
and decreased motor control characteristic of cannabinoid (CB)
agonist administration.45 It possesses a relatively short duration
of in vivo activity relative to irreversible inhibitors, although
further conformational constraints in the C2 acyl chain of 2 have
provided inhibitors that are not only orally active but also exhibit
extended durations of in vivo activity.44
INTRODUCTION
■
Because of the therapeutic potential of inhibiting fatty acid amide
hydrolase (FAAH)1,2 for the treatment of pain,3,4 inflammatory,5
or sleep disorders,6 there is a continuing interest in the
development of selective inhibitors of the enzyme.7 The
distribution of FAAH is consistent with its role in regulating
signaling fatty acid amides8−10 including anandamide (1a)11 and
oleamide (1b)12,13 at their sites of action (Figure 1). Although
FAAH is a member of the amidase signature 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.14,15
Received: November 25, 2013
Published: January 23, 2014
Figure 1. Substrates of fatty acid amide hydrolase.
© 2014 American Chemical Society
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Complementary to a series of systematic structure−activity
relationship (SAR) studies on 2 exploring substitution of the
central oxazole, the C2 acyl side chain, and the central
heterocycle,33−46 the X-ray characterization of inhibitor-bound
complexes defined key features that impact inhibitor affinity and
selectivity.47−50 These include not only the Ser241 hemiketal
formation with the inhibitor electrophilic carbonyl and its
interaction with the enzyme oxyanion hole but also an unusual
Ser217-mediated OH−π H-bond to the activating heterocycle
and the key anchoring interaction of the terminal phenyl group of
the C2 acyl chain. The structural studies also revealed that
Cys269 is located adjacent to C5 of the inhibitor pyridine
substituent, which in turn is engaged in a series of intricate
interactions in the enzyme cytosolic port.51 Herein, we report
results of a systematic study of candidate inhibitors containing
modifications at the pyridyl C5-position of 2 and related
inhibitors that in principle could covalently trap this proximal
Cys269 to provide inhibitors that alkylate or cross-link the FAAH
active site. In turn, this could be expected to enhance their
potency, potentially enhance their selectivity, and extend their in
vivo duration of action (Figure 2). Herein, we detail the
systematic inhibitor modifications that led to the discovery and
characterization of such inhibitors52 and the unexpected trends
that the additional strategically placed electrophiles display.
RESULTS AND DISCUSSION
■
Chemistry. The series 1 analogues (3−22) were accessed
from 5-(tributylstannyl)oxazole 1e36 by Stille coupling53 with the
appropriate 2-chloro- or 2-bromopyridine (Scheme 1). This was
followed typically by TBS ether deprotection (Bu4NF) and
oxidation of the liberated alcohol with Dess−Martin periodinane
(DMP)54 to provide the corresponding α-ketoheterocycles: 3, 7,
9, 14, and 18−22. The remaining inhibitors were accessed by
further modification of the pyridyl C5 substituent (Scheme 1).
The second series, in which the pyridine of 2 is replaced with
an alkyl linker to the pendant electrophile, was accessed by
Sonogashira coupling of 5-bromooxazole 1f43 with the
appropriate alkyne (Scheme 2). The alkyne intermediate was
reduced to the corresponding alkane with H2 and palladium on
carbon or palladium hydroxide. This was followed by TBS ether
deprotection (Bu4NF) and oxidation of the liberated alcohol
with Dess−Martin periodinane (DMP) to yield the series 2 C5-
substituted oxazoles: 23 and 28. Further elaboration of the
terminal electrophile (R group) yielded the remaining
compounds: 24−27 and 29−32.
Enzyme Inhibition. The initial characterization of the
candidate inhibitors and their comparison with 2 was conducted
using purified recombinant rat FAAH (rFAAH) expressed in
Escherichia coli55 at 20−23 °C as previously disclosed.38 The
initial rates of hydrolysis (>10−20% reaction) were monitored
using enzyme concentrations below the initially measured Ki
values by following the breakdown of 14C-oleamide, and Ki values
were established as previously described (Dixon plot).
Series 1 was developed directly on the basis of 2 (Ki = 4.7 nM),
placing a potential thiol-capturing electrophile at the 5-position
of the pyridine ring (5−8, 11, 12, 14, and 16−22). Thioesters 5
and 16 were expected to be the most straightforward traps for the
Cys269 thiol by thioester exchange. Without preincubation of
the inhibitors with the enzyme, these inhibitors along with their
precursors (3−22) were tested for binding and inhibition of
rFAAH (Figure 3). All display potencies similar to 2, exhibiting
Ki values in the low nanomolar range. In series 2, the pyridine
ring was replaced by an alkyl chain of appropriate length capped
with the thiol-engaging moiety. As modeled, this flexible linker is
able to reach through the cytosolic pocket and place the
potentially reactive electrophile proximal to Cys269. Like the
series 1 inhibitors and without preincubation with the enzyme, all
series 2 inhibitors exhibited effective FAAH inhibition with
potencies that approach or match that of 2 (Figure 3).
Time-Dependent Enzyme Inhibition. Because the Cys269
alkylation was expected to be slow relative to the rapid hemiketal
formation, the time-dependent inhibition of FAAH was
examined. This was accomplished by preincubation of the
inhibitors with recombinant rFAAH for a period of 1−6 h. As
previously observed, reversible, competitive inhibitor 2 does not
display time-dependent inhibition of FAAH, and its Ki value
remains unchanged with the enzyme−inhibitor preincubation
times of 0−6 h (Figure 4). In contrast, a select subset of inhibitors
(11, 14, 17, and 20−22) in series 1 exhibited significant increases
in potency, displaying 2−20-fold improvements in Ki over the
same time period, consistent with slow irreversible inhibition of
FAAH. Surprisingly, thioesters 5 or 16 did not exhibit this time-
dependent increase in enzyme inhibition potency. Similarly,
chloride 12 was found to be relatively nonpotent and insensitive
to preincubation with the enzyme, whereas the corresponding
Figure 2. Inhibitor series examined.
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Scheme 1
Scheme 2
dependent increases in potency, whereas both the homologated
nitrile 14 and its imidate 17, where a methylene spacer separates
the electrophile and pyridyl ring, did exhibit increases in potency
with the enzyme−inhibitor preincubation. Of the series of
inhibitors that might be expected to serve as Michael acceptors
for a thiol conjugate addition (18−22), including the α,β-
unsaturated ester 18 and nitrile 19, only those bearing the weaker
activating substituents (20−22 vs 18 and 19) that would be
expected to react slower and to be intrinsically less reversible
displayed the exceptionally potent and time-dependent FAAH
inhibition improvements. Notably and throughout this series, it
was not the anticipated electrophiles that exhibited the time-
dependent inhibition of FAAH characteristic of a slow
irreversible inhibitor, but rather it was a less-well-recognized
alternative (14 and 17 vs 16, 11 vs 12, and 20−22 vs 18−19).
Finally, no inhibitor in series 2 that bears the flexible linker to the
second electrophile displayed the time-dependent increases in
potency, indicating that the conformationally restricted place-
bromide 11 was initially more potent and exhibited the most
pronounced time-dependent increase in potency of all inhibitors.
Both nitrile 7 and its imidate 8, where the candidate electrophile
is attached directly to the pyridyl ring, did not display time-
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Figure 3. Enzyme inhibition.
ment of the second electrophile is important to observation of
the targeted alkylation. For the inhibitors that displayed time-
dependent increases in inhibitor potency, enzyme activity did not
recover after this time period and is indicative of irreversible
enzyme inhibition.
Lineweaver−Burk Kinetic Analysis. The compounds that
demonstrated time-dependent improvements in potency were
further investigated by Lineweaver−Burk kinetic analysis. In
previous studies, α-ketoheterocycle inhibitors including 2 were
shown to display well-behaved competitive, reversible inhibition
kinetics. Despite expectations but consistent with the lack of
time-dependent FAAH inhibition, Lineweaver−Burk kinetic
analysis of thioesters 5, 16, and 25 after 3 h preincubation with
the enzyme confirmed that they also behave as reversible,
competitive inhibitors, analogous to 2 and related α-ketohetero-
cycle inhibitors (Figure 5). Thus, despite the expectations of a
Figure 4. Time-dependent inhibition. Ki values were measured after 0−
6 h inhibitor preincubation with rFAAH.
facile transthioesterification with Cys269, the thioesters exhibit
enzyme inhibition characteristic of reversible inhibitors,
suggesting that reaction with Cys269 does not occur.56
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inhibition presumably entails thiol nucleophilic addition to the
electrophile to provide the Cys269-linked thioimidate for 14 and
17, and it presumably involves an apparent irreversible thiol
conjugate addition to 20−22. Interestingly, both the α,β-
unsaturated nitrile 19 and ester 18, which do not exhibit time-
dependent increases in inhibitor activity or the potent Ki values
consistent with irreversible inhibition, displayed mixed kinetics,
exhibiting competitive inhibition at low inhibitor concentrations
and noncompetitive inhibition at high concentrations. Presum-
ably, this indicates that the thiol conjugate addition products
derived from 18 and 19 are either formed less effectively or, more
likely, that they may be sufficiently reversible at 23 °C to less
effectively trap Cys269 as an apparent irreversible inhibitor of the
enzyme.
Figure 5. Lineweaver−Burk kinetic analysis of 5, 16, and 25
demonstrate reversible, competitive inhibition.
Irreversible Enzyme Inhibition. Dialysis dilution (4 °C, 18
h, 370-fold) of the FAAH-inhibitor mixture following 3 h of
preincubation with 2 restored full enzyme activity, consistent
with its reversible enzyme inhibition, whereas the mixtures
containing 11, 14, and 17−22 remained relatively unchanged,
failing to restore FAAH activity, indicative of irreversible enzyme
inhibition under the conditions monitored (4 °C, pH 9, Figure
7). It is notable that 14 and 17 (not shown), which presumably
form a Cys269 thioimidate adduct, do not appear to be even
slowly reversible under these conditions. Similarly, 20−22
displayed irreversible inhibition of FAAH, consistent with their
time-dependent, noncompetitive enzyme inhibition. Interest-
ingly, dialysis dilution at 4 °C also did not restore enzyme activity
with both the α,β-unsaturated nitrile 19 and ester 18, which do
not exhibit time-dependent FAAH inhibition and displayed
concentration-dependent mixed competitive/noncompetitive
Significantly, thioesters 5 and 25 were recovered unchanged
from the assay buffer (6 h) and from enzymatic assays (5),
indicating that they are not undergoing chemical hydrolysis or
transient enzyme adduct formation and subsequent hydrolysis
under the conditions of the assay.
In contrast, the inhibitors that demonstrated a time-dependent
increase in inhibitor potency also exhibited noncompetitive
inhibition of FAAH when preincubated with the enzyme for 3 h
prior to Lineweaver−Burk kinetic analysis (Figure 6). This is
expected of irreversible enzyme inhibition and consistent with
Cys269 alkylation or addition to the pendant electrophile. In the
case of 11, this entails Cys269 thiol nucleophilic displacement of
the benzylic bromide to provide the corresponding thioether,
and its structure has been confirmed by X-ray analysis of the
inhibitor bound to FAAH.52 The noncompetitive enzyme
Figure 6. Lineweaver−Burk analysis demonstrates noncompetitive FAAH inhibition for (A) 11, (B) 14, (C) 17, (D) 20, (E) 21, and (F) 22.
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Figure 8. ABPP selectivity screen in mouse brain membrane proteome
(1 mg/mL) with FP-rhodamine (100 nM), n = 2−4. Inhibitor
preincubation with the proteome was conducted for 6 h.
Figure 7. Dialysis dilution of inhibitor−FAAH mixtures illustrates
reversible inhibition by 2 and establishes irreversible FAAH inhibition
by 11, 14, and 17−22. After 3 h preincubation of purified recombinant
rat FAAH with compounds at concentrations that result in inhibition of
ca. 80% enzyme activity (22 °C; 3 h; 100 nM, 2; 80 nM, 11 and 14; 100
nM, 18 and 19; 150 nM, 20; and 80 nM, 21 and 22), 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 11,
14, and 17−22 under the conditions monitored (4 °C, pH 9);
conducted in triplicate and reported as the percent enzyme inhibition
SD.
inhibitors on the endogenous levels of the FAAH substrates
anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl
ethanolamide (PEA) were measured. Notably, it is the increase in
endogenous levels of anandamide and its subsequent action at
cannabinoid (CB1 and CB2) receptors that are thought to be
responsible for the analgesic and anti-inflammatory effects of
FAAH inhibitors. The effects were established 3 h following
intraperitoneal (i.p.) administration of inhibitor in three mice per
time point for an initial screen (30 mg/kg). Significantly,
increases in endogenous levels of anandamide in the brain
requires >90% inhibition of FAAH for in vivo enzyme
inhibition.58 With the exception of imidate 17, which matched
the increased anandamide levels observed with 2 after 3 h, each of
the additional inhibitors proved to be roughly equivalent (11, 14,
and 20 > 21 and 22), increasing anandamide levels
approximately 2-fold over that of 2 and approximately 3-fold
over vehicle treatment (Figure 9).
kinetics in the Lineweaver−Burk analysis at 23 °C. This suggests
that their inhibition of FAAH following the 3 h incubation (22
°C) is not reversible at 0 °C. Unfortunately, the reversibility of 18
and 19 at 23 °C could not be established because of the instability
of FAAH at 23 °C over the dialysis time frame.
Inhibitor Selectivity. The selectivity of the time-dependent,
irreversible FAAH inhibitors 17 and 20−22 were examined along
with 11 and 14 that were recently disclosed52 using activity-based
protein profiling (ABPP) of the serine hydrolases.57 ABPP
methods permit the testing of serine hydrolases 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 relative potency and selectivity can be
simultaneously evaluated. Previous studies19,37,52 have shown
that the α-ketoheterocycle class of inhibitors are selective for
FAAH, although four enzymes have emerged as potential
competitive targets: triacylglycerol hydrolase (TGH), αβ
hydrolase containing domain 6 (ABHD6), monoacylglycerol
lipase (MAGL), and the membrane-associated hydrolase
KIAA1363. Each inhibitor was tested for its effects on the
fluorophosphonate (FP)-rhodamine probe labeling of serine
hydrolases in the mouse brain (contains KIAA1363, MAGL, and
ABHD6) and heart membrane (contains TGH) proteome at
concentrations ranging from 10 nM to 100 μM. The selectivity
assessments were conducted following 6 h inhibitor incubation
with the proteomes and all inhibitors showed superb selectivity
for FAAH over KIAA1363 and ABHD6 (>104-fold), excellent
selectivity over MAGL (>200-fold), and good selectivity over
TGH (Figure 8).
With PEA and OEA, which show significant enhancements in
endogenous levels with partial enzyme inhibition and are less
sensitive to the extent of FAAH inhibition, all of the inhibitors
that displayed time-dependent, irreversible FAAH inhibition
matched or exceeded the activity of 2, producing elevations of 3−
12-fold over vehicle. Of these, both bromide 11 and nitrile 14
exhibited the largest increases. As a result, more detailed dose-
and time-dependent studies of 11 and 14 were conducted as
reported elesewhere.52 The results of these studies revealed that
they cause accumulation of all three lipid amides in the brain with
peak levels achieved within 1.5−3 h, that these elevations exceed
those achieved with the reversible inhibitor 2, that these
elevations are maintained >6 h (vs 2−3 h for 2), consistent
with irreversible enzyme inhibition, and that they exhibit long
acting in vivo activity in a mouse model of neuropathic pain.52
CONCLUSIONS
■
The design, synthesis, and characterization of α-ketoheterocycles
that additionally target the remote Cys269 nucleophile found in
the cytosolic port of FAAH59 provided inhibitors that slowly
react with the enzyme nucleophile, effectively providing time-
dependent, irreversible inhibitors of the enzyme that maintain or
enhance their selectivity for FAAH over other serine hydrolases.
The electrophiles capable of targeting Cys269 were incorporated
as a C5 substituent on the pyridyl group of the 5-(pyrid-2-yl)
oxazole of 2 and ranged from the reactive benzylic bromide 11 to
the otherwise benign nitrile 14. The irreversible inhibitors of
FAAH displayed an expected sensitivity to the position of the
electrophile introduction, but those that were successful
exhibited surprising trends in apparent reactivity toward
Cys269 that would not be easily predicted. A preliminary in
vivo characterization of the identified irreversible FAAH
Preliminary in Vivo Characterization. In initial efforts to
screen for in vivo inhibition of FAAH and its subsequent
pharmacological effects, the set of inhibitors displaying the time-
dependent, irreversible FAAH inhibition (11, 14, 17, and 20−
22) were examined alongside of 2 for their ability to increase the
endogenous levels of a series of lipid amide signaling molecules
that are substrates for FAAH in both the brain (CNS effect) and
liver (peripheral effect, not shown). Thus, the effects of the
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recombinant rFAAH was used in the inhibition and reversibility assays
unless otherwise indicated. The purity of each tested compound (>95%)
was determined on an Agilent 1100 LC/MS instrument using a
ZORBAX SB-C18 column (3.5 mm, 4.6 mm × 50 mm, with a flow rate
of 0.75 mL/min and detection at 220 and 253 nm) with a 10−98%
acetonitrile/water/0.1% formic acid gradient (two different gradients).
The inhibition assays were performed as described.36 The enzyme
reaction was initiated by mixing 1 nM rFAAH (800, 500, or 200 pM
rFAAH for inhibitors with Ki ≤ 1−2 nM) with 20 μM 14C-labeled
oleamide in 500 μL of reaction buffer (125 mM TrisCl, 1 mM EDTA,
0.2% glycerol, 0.02% Triton X-100, and 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.13 The Ki of
the inhibitor was calculated using a Dixon plot. Lineweaver−Burk
kinetic analysis was performed as described,36 confirming competitive,
reversible inhibition for 5, 16, and 25 and noncompetitive inhibition for
11, 14, 17, and 20−22 (Figures 5 and 6).
Reversibility of FAAH Inhibition (Dialysis). The reversibility of
FAAH inhibition by 2, 11, 14, and 17−22 was assessed by dialysis
dilution using purified recombinant rFAAH. The enzyme was placed in
15 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol,
0.02% Triton X-100, and 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, 18,
and 19; 80 nM, 11, 14, 21, and 22; and 150 nM, 20. 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
postdialysis 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) and is shown in
Figure 7.
Competitive ABPP of FAAH Inhibitors with FP-Rhodamine.
Mouse tissues were Dounce-homogenized in PBS buffer (pH 8.0), and
membrane proteomes were 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 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 a flatbed
fluorescence scanner (MiraBio). Labeled proteins were quantified by
measuring integrated band intensities (normalized for volume); control
samples (DMSO alone) were considered to have 100% activity. IC50
values (n = 2−4) were determined from dose−response curves using
Prism software and are reported in Figure 8.
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 in an 18:1:1 v/v/v solution of saline/ethanol/
emulphor. 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, or 6 h), mice (n = 3 for each
compound at each time point) were anesthetized with isofluorane and
killed by decapitation. 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.
Figure 9. Lipid levels in the brain 3 h post inhibitor administration (i.p.,
30 mg/kg, n = 3).
inhibitors confirmed their ability to raise endogenous brain levels
of the enzyme substrates, including anandamide, in mice to a
greater extent (>2-fold) and for a longer duration (>6 h) than the
reversible α-ketoheterocycles on which they are based. Two of
these (11 and 14) were characterized in greater detail, as
reported elsewhere, along with their long acting in vivo efficacy in
a mouse model of neuropathic pain.52
EXPERIMENTAL SECTION
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
■
FAAH Inhibition. 14C-labeled oleamide was prepared from 14C-
labeled oleic acid as described.13 The truncated rat FAAH (rFAAH) was
expressed in E. coli and purified as described,55 and the purified
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vortexed and then centrifuged (1400g, 10 min). The organic layer was
removed, dried under a stream of N2, and resolubilized in 2:1 v/v
CHCl3/MeOH (120 μL), and 10 μL of this resolubilized lipid 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 mm ×
20 mm). Mobile phase A was composed of 95:5 v/v H2O/MeOH, and
mobile phase B was composed of 65:35:5 v/v/v i-PrOH/MeOH/H2O.
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), and d4-PEA (304, 62, 11). 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 (n = 3 for each inhibitor at each
time point).
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́ ́
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ASSOCIATED CONTENT
* Supporting Information
Full experimental details and characterization of the candidate
inhibitors, inhibitor purities, and enzyme inhibition measure-
ment standard deviations for Figures 3, 4, and 8. This material is
available free of charge via the Internet at http://pubs.acs.org.
N.; Cravatt, B. F.; Henriksen, S. J. Characterization of the Sleep-Wake
Patterns in Mice Lacking Fatty Acid Amide Hydrolase. Sleep 2004, 27,
857−865.
■
S
(7) (a) Otrubova, K.; Ezzili, C.; Boger, D. L. The Discovery and
Development of Inhibitors of Fatty Acid Amide Hydrolase (FAAH).
Bioorg. Med. Chem. Lett. 2011, 21, 4674−4685. (b) Deng, H. F. Recent
Advances in the Discovery and Evaluation of Fatty Acid Amide
Hydrolase Inhibitors. Expert Opin. Drug Discovery 2010, 5, 961−993.
(c) Seierstad, M.; Breitenbucher, J. G. Discovery and Development of
Fatty Acid Amide Hydrolase (FAAH) Inhibitors. J. Med. Chem. 2008,
51, 7327−7343. (d) Vandevoorde, S. Overview of the Chemical
Families of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase
Inhibitors. Curr. Top. Med. Chem. 2008, 8, 247−267.
AUTHOR INFORMATION
Corresponding Author
*Phone: 858-784-7522. Fax: 858-784-7550. E-mail: boger@
scripps.edu.
■
Notes
(8) Ezzili, C.; Otrubova, K.; Boger, D. L. Fatty Acid Amide Signaling
Molecules. Bioorg. Med. Chem. Lett. 2010, 20, 5959−5968.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We gratefully acknowledge the financial support of the National
Institutes of Health (DA015648, D.L.B.; DA017259, B.F.C.).
■
ABBREVIATIONS USED
■
AA, arachidonic acid; ABHD6, αβ hydrolase containing domain
6; ABPP, activity-based protein profiling; AEA, anandamide; CB,
cannabinoid; DMP, Dess−Martin periodinane; FAAH, fatty acid
amide hydrolase; i.p, intraperitoneal; MAGL, monoacylglycerol
lipase; OEA, oleoyl ethanolamide; PEA, palmitoyl ethanolamide;
TBS, tert-butyldimethylsilyl; TGH, triacylglycerol hydrolase
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