Assay and inhibition of diacylglycerol lipase activity

Article abstract of DOI:10.1016/j.bmcl.2012.05.101

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″- ¹⁴C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-¹⁴C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-¹⁴C]arachidonic acid.

Full text of DOI:10.1016/j.bmcl.2012.05.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4585–4592
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Assay and inhibition of diacylglycerol lipase activity
Meghan Johnston a, Shachi R. Bhatt b, , Surina Sikka b,à, Richard W. Mercier a, Jay M. West a,
Alexandros Makriyannis a, S. John Gatley b, Richard I. Duclos Jr. b,
⇑
a Center for Drug Discovery, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA
b Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-formyl-a-amino acid esters of b-lactone derivatives structurally related to tetrahydrolipst-
Received 13 April 2012
Revised 25 May 2012
Accepted 29 May 2012
Available online 6 June 2012
atin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL)
activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen
inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance
energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol
lipase activity utilizing the labeled endogenous substrate [100-14C]1-stearoyl-2-arachidonoyl-sn-glycerol
with phosphorimaging detection was used to quantify inhibition by following formation of the initial
product [100-14C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-14C]ara-
chidonic acid.
Keywords:
Diacylglycerol lipase
2-Arachidonoylglycerol
Endocannabinoid
Labeled
Ó 2012 Elsevier Ltd. All rights reserved.
Assay
Fluorescence resonance energy transfer
The catalytic sites of diacylglycerol lipases (DAGLs) are unique
among the lipases and hydrolases in effecting good selectivity for
the hydrolysis of the 1-acyl group of 1,2-diacyl-sn-glycerol sub-
molecules that inhibit DAGL would have major effects on lipid
metabolism. For example, less DAGL-catalyzed biosynthesis of
endocannabinoid 2-arachidonoylglycerol (2-AG) may reduce acti-
vation of cannabinoid receptors. The resulting reduction of recep-
tor signaling would be distinct from the actions of cannabinoid
receptor inverse agonists and may be of pharmacological utility.
The three lead inhibitors of DAGL activity include bis-oximino-
carbamate RHC80267 1,1,25 fluorophosphonate O-3841 2,26 and
strates.1 The DAGL
a
(120 kDa) and DAGLb (70 kDa) isoforms1,2
are both present during brain development3 where their presynap-
tic axonal location suggests an important role in this period of neu-
ritogenesis, rapid cell growth, and plasticity.4,5 However, the
ultimate postsynaptic dendritic location of the
a-isoform in the
adult brain is consistent with the subsequent role of DAGL
a
in
tetrahydrolipstatin (3, THL)1,27 (Fig. 1) that inhibit hDAGL
a with
endocannabinoid paracrine retrograde signaling with a lesser role
for the DAGLb isoform.1,5–13 Thus, DAGLs have an important role
in the endocannabinoid system as they are primarily responsible
for releasing endocannabinoid 2-arachidonoylglycerol (2-AG) from
diacylglycerols, including 1-stearoyl-2-arachidonoyl-sn-glycerol
that is the principal 1,2-diacyl-sn-glycerol component of brain
and nerves,14–16 for signaling at cannabinoid receptors.17–24 Small
apparent IC50 values of 65,000 nM, 160 nM, and 60 nM, respec-
tively. The transition-state-mimicking fluorophosphonate group
(RP=OOEtF) of FP-fluorescein (see Supplementary data for struc-
ture) utilized for the identification of serine hydrolases does not re-
act appreciably with DAGL,27 unlike fluorophosphonate O-3841 2
(ROP=OMeF)26 and its t-butyl analog O-5596.28 Also, DAGL activity
is not affected by phenylmethylsulfonylfluoride (PhCH2SO2F).1
There have been several previously reported analytical methodol-
ogies for measuring DAGL activities including radio-TLC,1,26,29,30
LC–MS,2,27 and the use of general esterase reporter molecules.2
We now report our studies of new inhibitors of DAGL activity
that generally resemble diglycerides. We will also discuss the
DAGL proteins utilized in these studies. Our work on assay condi-
tions using a radiolabeled endogenous diglyceride substrate will be
detailed. Finally, the development of novel fluorescent resonance
energy transfer (FRET) reporter substrates for the in vitro assay
of DAGL activity will be discussed including their utilizations for
the assays of related lipases.
Abbreviations: AA, arachidonic acid; 2-AG, 2-arachidonoylglycerol; DAGL,
diacylglycerol lipase; FAAH, fatty acid amide hydrolase; FRET, fluorescence reso-
nance energy transfer; MAGL, monoacylglycerol lipase; NBD, nitrobenzoxadiazole;
[
14C]SAG, [100-14C]1-stearoyl-2-arachidonoyl-sn-glycerol; THL, tetrahydrolipstatin.
⇑
Corresponding author. Tel.: +1 617 373 3163; fax: +1 617 373 8886.
E-mail addresses: meghanryan.johnston@gmail.com (M. Johnston), shachi.r.
bhatt@gmail.com (S.R. Bhatt), surinasikka4u@gmail.com (S. Sikka), r.mercier@
neu.edu (R.W. Mercier), j.west@neu.edu (J.M. West), a.makriyannis@neu.edu
(A. Makriyannis), s.gatley@neu.edu (S.J. Gatley), duclos@neu.edu (R.I. Duclos).
Present address: Organogenesis Inc., 150 Dan Road, Canton MA 02021, USA.
à
Present address: Courtagen Life Sciences, Inc., Suite 3700, 12 Gill Street, Woburn
MA 01801, USA.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.101

4586
M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
O
H
N
O
P
O
O
N
CH3
1
N
N
H
O
O
F
2
O
O
1
2
OCH3
3
RHC80267
O-3841
O
O
O
O
R
O
O
O
O
O
C6H13
C6H13
C6H13
H23C11
H23C11
H
H15C7
H15C7
9
3 - 8
10
11
(racemic)
(racemic)
(racemic)
R-
S
NHCHO
O
3 (THL)
tetrahydrolipstatin (orlistat, Xenical®
)
N-formyl-L-leucyl ester
S
R
S
R
S
S
R
R
NHCHO
NHCHO
NHCHO
NHCHO
O
O
O
O
4
5
6
7
(OMDM-188)
N-formyl-
-allo-isoleucyl ester
N-formyl-
N-formyl-
N-formyl-
L
D
D
L-isoleucyl ester
-isoleucyl ester
-allo-isoleucyl ester
S
NHCHO
O
8
(S)-N-formyl-
α-aminobutyryl ester
Figure 1. Diacylglycerol lipase inhibitors.
We first prepared new analogs of THL 3 and OMDM-188 4.31
THL 3 has an N-formyl- -leucyl ester and OMDM-188 4 is the cor-
responding N-formyl- -isoleucyl ester. The three other novel iso-
leucine diastereomers (5, 6, 7) as well as the (S)- -aminobutyryl
ester 8 were prepared via the reported method31 from the corre-
sponding benzyloxycarbonyl protected -amino acids 13 (see
Scheme 1). The b-lactone analog 9 completely lacking both the
N-formyl- -aminoacyloxy and the 2-hexyl groups was prepared
by treating racemic 3-hydroxypalmitic acid with N-phenyl-bis(tri-
fluoromethanesulfonimide). The shorter chain separable trans-10
and cis-11 b-lactones were prepared according to the reported
method.32 Also, a new series of racemic ether lipids derived from
lead compounds RHC80267 1 and O-3841 2 was prepared (see
Supplementary data).
or at least 10 lg of total protein from membrane preparations
L
was required per well. The proteins were utilized such that the
substrate hydrolysis would proceed to the extent of about 5% in
20 min. The more readily expressed mDAGLb isoform (that has a
L
a
79% homology1 with the human isoform) or mDAGL
a isoform (that
a
has a 97% homology1 with the human isoform) were also used to
confirm inhibition of DAGL activities.
a
It was of particular importance for biological relevance and
inhibitor evaluation that assays of DAGL activities utilize pure
endogenous substrate such as radiolabeled [100-14C]1-stearoyl-2-
arachidonoyl-sn-glycerol ([14C]SAG).29,34 Any radiolabeled 1-stea-
royl-3-arachidonoyl-sn-glycerol present in the substrate, due to
rearrangement of [14C]SAG to labeled 1,3-diglyceride, would be
readily hydrolyzed by other enzymes in the relatively crude en-
zyme preparations and the result misinterpreted to be due to DAGL
activity. The 1,2-diglyceride substrate [14C]SAG that we used con-
tained less than 0.5% of the 1,3-diglyceride isomer (see Supple-
mentary data).
We used diacylglycerol lipase (DAGL) protein from either cell
lysates or membrane fractions that were prepared according to
the previously reported method.1,33 The Cravatt group at Scripps
provided hDAGLa, mDAGLa, and mDAGLb from overexpression
by transient infection of HEK293T cultures in addition to cell lysate
of the empty vector HEK293T control. Some experiments used a
second commercially prepared plasmid to provide additional hu-
Although the [14C]SAG substrate has previously been used for
TLC-based assays of DAGL activity,1,26,30 details of these TLC assays
were not fully described. Also, the apparent IC50 of THL 3 for hDA-
man
ology. All attempts to overexpress human DAGL
disappointing. The lipase activity of hDAGL expressed in the hu-
man cell line was still sufficient for assay of newly synthesized
inhibitors. At least 100 g of total protein from crude cell lysates
a
-isoform overexpressed in HEK293T using the same method-
GLa has been reported by the Di Marzo group to range from
a
were
60 nM1,25,26 to 1000 nM,31 postulated to be due to the effect of
DAGL protein concentration. Therefore, we developed a standard-
ized assay that contained 10% DMSO.35,37 Higher DMSO concentra-
tions did not increase rates of substrate conversion to fluorescent
a
l

M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
4587
R
NHCbz
Z2O
O
R
O
NH2
OH
EDCI, DMAP
DCM
R
O
NHCbz
OH
NaOH
O
OH
O
O
O
O
+
dioxane
C6H13
H
23C11
C6H13
H23C11
12
13
14
15
R
NH2
O
R
O
NHCHO
H2, Pd/C
THF
formic anhydride
DCM
O
O
O
O
O
O
C6H13
H23C11
C6H13
H23C11
16
4 - 8
R
O
NHCHO
4 - 8
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
O
O
O
O
O
5
6
7
8
4
L-allo-Ile
D-Ile
D-allo-Ile
α-aminobutyryl
L-Ile
(OMDM-188)
Scheme 1. Synthesis of analogs of THL 3 and OMDM-188 4.
products in any of the in vitro assays. Non-denaturing detergents
n-heptyl-b- -thioglucopyranoside and Triton X-100 at concentra-
JZL184 interference with hDAGL
fluorescent assays.
a
activities in radioassays and
D
tions up to 0.2% were also used in some experiments (see Tables
1 and 3). Detergent use dramatically increases apparent DAGL spe-
cific activities and also decreases the apparent IC50 for DAGL inhib-
itory compounds. The DAGL enzyme suspensions had a 15 min
preincubation period of covalent quasi-irreversible inactivation
by THL 3 or other potential inhibitors. The substrate [14C]SAG
The use of 10
man) hDAGL
lM THL 3 resulted in complete inhibition of (hu-
a
activity for all protein preparations (Table 1). Using
TLC under basic conditions1,26 and phosphorimaging analysis,34,41
the radioassays consistently showed the apparent IC50 of THL 3
to be in the range of 10–100 nM. Analogs 4–8 were also all extre-
mely potent inhibitors of hDAGL
tones 9–11 and other compounds including JZL184, PMSF, and
RHC80267 1 were poor inhibitors of hDAGL . The b-lactone SD41
and ether lipid analogs of O-3841 we synthesized (see Supplemen-
tary data) did not inhibit hDAGL
concentrations.
a in radio-TLC assays. The b-lac-
was then added (20 lM final concentration) and residual DAGL
activity was quantified after 20 min by quenching the reaction
with 2:1 chloroform/methanol and vortexing to denature the pro-
tein and move all lipids out of the aqueous phase. Very little rear-
a
a or mDAGLb at 10 lM screening
rangement of
[
14C]SAG (1,2-diglyceride) to 1,3-diglyceride
occurred under the reaction and workup conditions as assessed
by TLC using boric acid treated silica gel plates.
The b-lactones THL 3, OMDM-188 4, and new analogs 5–9, were
assayed for the inhibition of (murine) mDAGL
a
D
at 10 nM to 10
-allo-isoleucyl-ana-
-amino
acids. Several analogs (THL 3, OMDM-188 4, and the new -allo
lM
TLC DAGL assays with [14C]SAG substrate (or LC–MS assays with
pure unlabeled SAG substrate) could result in significant errors if
subsequent hydrolysis of 2-AG was not considered. It was critical
that evaluations of enzymatic hydrolysis of [14C]SAG substrate in-
clude the sums of radiolabeled 2-AG and free radiolabeled arachi-
donic acid released. The crude cell preparations that were used had
considerable monoacylglycerol lipase (MAGL), fatty acid amide
hydrolase (FAAH), and other lipase activities which further de-
graded the radiolabeled 2-AG as it was formed under the assay
conditions. It was very clear from the controls and from experi-
ments with poor inhibitors that the release of labeled 2-AG was
followed by further hydrolysis to labeled arachidonic acid. Thus
DAGL activity was calculated via the sum of [14C]2-AG plus
concentrations (Table 2). The -isoleucyl- and
D
logs (6 and 7) were clearly less potent than analogs of (S)-
a
L
analog 5) showed good selectivity for diacylglycerol lipase over
monoacylglycerol lipase and fatty acid amide hydrolase enzymes.
Future studies can include shorter alkyl chain analogs similar to
the fatty acid synthase inhibitors from the Romo group42 that
might have better solubility and membrane penetration properties.
Ether lipid substrates 17–22 (Fig. 2) for in vitro fluorescence
resonance energy transfer (FRET) assay of DAGL and related lipase
activities were developed from related lipase activity reporter mol-
ecules.43–47 This series of novel ether lipid molecules was synthe-
sized (Scheme 2) with the biomimetic stereochemistry at sn-2
and incorporated terminally functionalized sn-1 and sn-2 fatty acyl
groups. The epoxide of (R)-(ꢀ)-glycidyl methyl ether (23) was
opened with benzyl alcohol and sodium hydride followed by silyl
protection of the secondary alcohol 24, and hydrogenolysis of the
benzyl ether 25 in ethanol/ethyl acetate/acetic acid (1:1:0.2). Sub-
sequent acylation of the primary hydroxyl group of 26, deprotec-
tion of the secondary alcohol t-butyldimethylsilyl group,
followed immediately by acylation of the secondary hydroxyl of
28 gave ether lipid products 17–22 with only 5% impurity from
acyl rearrangement.
[
14C]AA released divided by the sum of [14C]2-AG, [14C]AA, and fi-
nal [14C]SAG concentrations for each lane. Also, DAGL activity in
this human cell line (HEK293T) was not adjusted for hDAGL
a
and hDAGLb activities demonstrated to be present in cell lysates
following the mock infections.
We observed that the conversion of [14C]2-AG to [14C ]AA was
reduced in modified radio-TLC assays by pre-treatment of cell ly-
sate with the highly selective MAGL inhibitor JZL184 at 10
for 15 min. Then, 10-fold dilution to 1 M for screening assay use
in some experiments as noted in the data tables gave little
lM
l

4588
M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
Table 1
Assays of the inhibition of hDAGL
a
(radio-TLC assay with [14C]SAG substrate),37 rFAAH,57 and hMAGL63 enzyme activities
Compound
number
Detail of N-formyl-
ester
a
-amino hDAGL
a
inhibition at 10
l
M detergent free or (with Triton rFAAH inhibition at
hMAGL inhibition at
10 M (%)
X-100) (%)
10 lM (%)
l
3 (THL)
100, (100a)
(98a)
(95a)
(78a)
(86a)
6
7
3
47
16
1
L
L
L
-Leucyl
4 (OMDM-188)
-Isoleucyl
5
6
7
-allo-Isoleucyl
-Isoleucylc
28
0
39
42
D
D
-allo-Isoleucyl
8
9
(S)-
a
-aminobutyryl
(99a)
(25a, 45b)
(37b)
(25b)
(37b)
ND
12
10
79
45
4
55
None
None
none
NA
NA
NA
NA
NA
NA
trans-10
cis-11
JZL184
URB597
n-C16H33SO2F
PMSF
94d
97e
100e
100e
100e
95e
10
100d
100f
18
ND
19
82
5
22
13
RHC80267 (1)
SD41
30
<0g
NA, not applicable; ND, not done.
a
0.05% Triton X-100 present.
0.015% Triton X-100 present.
b
c
5% impurity in
D
-isoleucyl analog due to
cis-11 inhibits rFAAH only 13% at 1
8 pt rFAAH IC50 (95% confidence). JZL184, 974 nM (784–1210); URB597, 4.9 nM (4.1–6.0); n-C16H33SO2F, 6.3 nM (4.5–8.7); PMSF, 833 nM (746–931); RHC80267 (1),
D
-allo-isoleucyl analog.
d
e
l
M, but inhibits hMAGL 66% at 100 nM.
2240 nM (2010–2500).
f
8 pt hMAGL IC50 (95% confidence). JZL184, 57 nM (53–62).
Protein was pretreated with JZL184 to completely inhibit MAGL activity.
g
Table 2
Radio-TLC assay with [14C]SAG substrate of the inhibition of mDAGL
a
activity
Compound
number
Detail of N-formyl-
amino ester
a
-
mDAGL
10 nM (%)
a
inhibition at
mDAGL
100 nM (%)
a
inhibition at
mDAGL
1000 nM (%)
a
inhibition at
mDAGLa inhibition at
10,000 nM (%)
3 (THL)
55
69
60
33
19
72
80
61
29
51
92
96
88
69
75
98
96
L
L
L
-Leucyl
4 (OMDM-188)
-Isoleucyl
5
6
7
100
85
-allo-Isoleucyl
-Isoleucyla
D
D
90
-allo-Isoleucyl
8
9
(S)-
NA
NA
NA
NA
NA
a-Aminobutyryl
50
68
96
100
<0
3
<0
64
10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18
JZL184
URB597
n-C16H33SO2F
JZL195
ND
NA, not applicable; ND, not done.
a
5% impurity in D-isoleucyl analog due to D-allo-isoleucyl analog.
The FRET pairs initially studied were the pyrene and nitro-
benzoxadiazole (NBD) fluorophors with either the dinitrophenyl
or nitroxyl group quenchers. Excitation of the pyrene or NBD results
in radiationless energy transfer to the quenchers when close en-
ough and sufficiently well oriented. The fully extended distances
between the fluorophors and quenchers were estimated (Schrö-
dinger Suite 2010, in an aqueous environment with a dielectric con-
stant of 80) to be 18 Å for 17, 18, and the NBD analog 20; and, to be
24 Å for the pyrenedecanoyl analog 19. The assays with NBD analog
20 had too much baseline instability as this fluorescent group is
quite sensitive to the polarity of its environment. The pyrene was
estimated to be 15 Å and 24 Å from the nitroxyl stable free radical
quenching groups48 of the 5-doxylstearoyl analog 21 and the 16-
doxylstearoyl analog 22, respectively, and both compounds were
poor substrates for enzymatic hydrolysis. The pyrene-dinitrophenyl
FRET pairings 17 and 18 were stable to uncatalyzed hydrolysis at
neutral pH, were the best enzyme substrates, and were readily
utilized in a 96-well format. A convenient in vitro fluorometric
esterase assay utilizing the BioTek Instruments Synergy™ HT
Multi-Mode Microplate 96-well reader was developed that mea-
sured nanomolar concentrations of fluorescent reaction product.49
Cell lysate or membrane preparations containing overexpressed
DAGL catalyzed the hydrolysis of the reporter substrate, and a fluo-
rescence response increased at a nearly linear rate for over two
hours. Wells that had a 15 min pre-incubation with DAGL inhibitors
and that showed a concentration dependent attenuation of fluores-
cence response were identified as ‘hits.’ At a screening concentra-
tion of 10
lM, compounds 3–8 were identified as potent
inhibitors of hDAGL
a
(Table 3). However, any potential DAGL inhib-
itor identified from the in vitro FRET assay should then be submit-
ted to the TLC assay with the radiolabeled endogenous diglyceride
substrate to identify any false positives for DAGL inhibition. Fluo-
rescence results could reflect inhibition of other hydrolytic en-
zymes in the crude cell preparations that hydrolyze the ether
lipid substrates 17–22 to a much greater extent than the endoge-
nous 1,2-diacyl-sn-glycerol substrate. Using highly selective en-
zyme inhibitors JZL184, URB597, and others, the enzymes
responsible for false positives include monoacylglycerol lipase

M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
4589
Table 3
Results of in vitro FRET-based screening using reporter compound 17 of the inhibition of lipase activities
Compound number
Detail of N-formyl-
a-
hDAGL
a
inhibition at 10
lM detergent free or
Lipoprotein lipase (bacterial)
Pancreatic lipase (porcine)
amino ester
(0.05% Triton X-100) (%)
inhibition at 10
lM (%)
inhibition at 10 lM (%)
3 (THL)
92, 86b (99)
92 (99)
92
84
61
86
68
98
99
98
99
95
L
L
L
-Leucyl
4 (OMDM-188)
-Isoleucyl
5
6
7
92 (99)
-allo-Isoleucyl
-Isoleucyla
90 (98)
D
D
90 (98)
-allo-Isoleucyl
8
9
(S)-
a
-Aminobutyryl
93 (100)
14 (72)
69b
80
<0
67
33
36
21
8
<0
<0
<0
<0
97
38
96
94
36
68
29
17
78
<0
43
None
None
None
NA
NA
NA
NA
NA
NA
NA
trans-10
cis-11
JZL184
URB597
n-C16H33SO2F
PMSF
RHC80267 (1)
SD41
JZL195
65b
59, 44b
22, 7b
40, 94b
8b
70, 40b
17, 7b
84, 55b
NA, not applicable.
a
5% impurity in D-isoleucyl analog due to D-allo-isoleucyl analog.
Protein was pretreated with JZL184 to completely inhibit MAGL activity.
b
O
H
N
O
NO2
1
O
H
OCH3
2
O
O2N
3
17
O
O
H
O
O2N
O2N
N
O
O
O
H
OCH3
NO2
18
O
O
H
N
O
H
OCH3
NO2
19
O
H
N
O
NO2
H
N
O
H
OCH3
O2N
O2N
N
N
20
O
.
O
O
O
N
O
O
O
H
OCH3
21
.
O
N
O
O
O
O
O
H
OCH3
22
Figure 2. Newly synthesized reporter substrates for in vitro FRET-based DAGL and related lipase assays.
(MGL) and fatty acid amide hydrolase (FAAH). The ether lipid FRET-
substrate 17 will be most useful for assays with DAGLs purified to
homogeneity.
Additional assays were performed to establish the selectivity of
DAGL inhibitors. Compounds 3–11 were assayed for binding to the
CB1 (rat brain preparation) and CB2 (mouse or human receptor

4590
M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
TBS-Cl
BnOH
NaH
1
OCH3
OBn
H
OCH3
OBn
H
OCH3
H2,Pd/C
imidazole
HO 2
TBSO
O
EtOH
EtOAc
CH3COOH
(1:1:0.2)
DCM
THF
3
23
24
25
(R)-(-)-glycidyl
methyl ether
O
O
O
R1
OH
O
H
R1
OH
O
H
R1
TEA(HF)3
DCM
EDCI, DMAP
TBSO
TBSO
H
HO
OCH3
OCH3
DCM
OCH3
26
28
27
O
O
R2
OH
O
O
H
R1
EDCI, DMAP
R2
O
DCM
OCH3
17 - 22
O
4-pyrenebutyryl, 10-pyrenedecanoyl, 5-doxylstearoyl,
16-doxylstearoyl, dinitrophenyl-ε-amino-n-caproyl
R1
O
4-pyrenebutyryl, dinitrophenyl-ε-amino-n-caproyl,
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl
R2
Scheme 2. Synthesis of new ether lipid reporter compounds 17–22 for in vitro assays of DAGL and related lipase activities.
expressed in HEK293),50 and none had a Ki below 1
lM in these
used, the assay will be suitable for the preliminary screening of
compound libraries. An improved and detailed radio-TLC assay of
DAGL activity with the labeled endogenous substrate [100-14C]1-
stearoyl-2-arachidonoyl-sn-glycerol was utilized to unambigu-
ously distinguish DAGL activity from the activities of MAGL and
competition binding experiments. Compounds 3–11 were also as-
sayed for inhibition of endocannabinoid hydrolytic enzymes in
fluorescence-based assays (Table 1). Assays of the inhibition of
fatty acid amide hydrolase (rFAAH) used the reported coumarin
amide reporter compound,56,57 and assays of the inhibition of mon-
oacylglycerol lipase (hMGL) used the 7-hydroxy-6-methoxy
analog62,63 of the reported coumarin ester.66 The assays were vali-
dated with standard compounds including the selective FAAH
inhibitor URB59759 and the selective MAGL inhibitor JZL184.36 Pre-
liminary studies with [100-14C ]SAG using the purified rFAAH and
hMAGL endocannabinoid hydrolytic enzymes in these fluorescent
assays showed low activities of these enzymes for the 1,2-diglycer-
ide substrate. Also, preliminary studies with ether lipid substrate
17 in the in vitro FRET-based assay confirmed the potent inhibition
by THL 367,68 of commercially available homogeneous bacterial
lipoprotein lipase and porcine triacylglycerol lipase enzymes (Ta-
ble 3).69 This new FRET-based methodology should be suitable
for the assay of new inhibitors of human recombinant proteins
including lipoprotein lipase, triacylglycerol lipase, and other re-
lated hydrolases to determine DAGL selectivity in vitro.
FAAH. Thus, methodologies were established to determine
a/b-
subtype selectivity as well as selective inhibition of DAGL over
esterases, amidases, and other lipases.
Acknowledgments
This work was supported with NIH research Grant R03-DA-
24842 (R.I.D., Jr.) and by the Office of Science (BER) of the U.S.
Department of Energy with research Grant DE-SC0005251 (S.J.G.).
The first author (M.J.) was supported with NIH training Grant
T32-DA-07312 (A.M.). We are extremely grateful to Jacqueline
Blankman, Ku-Lung (Ken) Hsu, and Benjamin Cravatt at The Scripps
Research Institute for generously providing rFAAH, hDAGL
a,
mDAGLb, and mDAGL proteins. We wish to thank the office staffs
a
of NU Environmental Health and Safety, the Department of Phar-
maceutical Sciences, and the Center for Drug Discovery. We are
also grateful to JodiAnne Wood, Aneetha Halikhedkar, Kirin Vemu-
ri, Jianxin Guo, David Budil, Vidyanand Shukla, Sonyuan Lin, Frank
Desarnaud, Christopher Kearns, Yan Peng, and Haotian Wang for
their consultations and assistance here at NU.
In summary, our structure–activity relationship (SAR) studies
have demonstrated molecular features of inhibitors that result in
inactivation of human and murine DAGLs at nanomolar inhibitor
concentrations. The importance of a small (S)-N-formyl-a-amino
group as a structural feature in targeting DAGLs was clearly dem-
onstrated. The b-lactone was the most active of the covalently
reactive quasi-irreversible inactivating functional groups that we
tested for DAGL inhibition. In pilot experiments, we have looked
at many factors that may affect assay results including protein
concentration, substrate structure and concentration, length of
the incubation period for enzyme inactivation, and the presence
of co-solvent and detergent. An in vitro FRET-based screen was
established for rapidly identifying inhibitors of DAGL activity.
Although false positives can occur due to inhibition of other hydro-
lytic enzymes present in the cell lysate or membrane preparations
Supplementary data
Supplementary data (the structures and names of the standard
chemicals used, structures and synthetic schemes for newly syn-
thesized ether lipid analogs of O-3841, western blot of hDAGL
a
and mDAGLb proteins, TLC of [14C]SAG substrate for radioassays,
radio-TLC assay data for inhibitors 3–8 (at 100 nM and 10 nM) of
mDAGL
hDAGL
compound substrates 17–22 at 10% DMSO, the inhibition of
a
, the comparison of the effect of DMSO concentration on
a
activity, the comparison of hDAGL activities on reporter
a

M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
4591
reported from the literature are actually apparent IC50 as all inhibitors in this
investigation undergo covalent reactions with the hydrolytic enzymes.
36. Long, J. Z.; Li, W.; Booker, L.; Burston, J. J.; Kinsey, S. G.; Schlosburg, J. E.; Pavon,
F. J.; Serrano, A. M.; Selley, D. E.; Parsons, L. H.; Lichtman, A. H.; Cravatt, B. F.
Nat. Chem. Biol. 2009, 5, 37.
hDAGL
a activity by THL 3 using reporter compound 17 with 10%
DMSO conditions, and the characterizations including 1H NMR
spectra of 3–11, 17, and 18) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.05.101.
37. Radio-TLC assay of DAGL inhibition: All cells, lysates (fresh or stored), and
membrane preparations were probe sonicated for 5 periods of 3 s with ice bath
cooling immediately prior to use. To the protein (100
containing DAGL in 90 L of buffer (50 mM Tris, pH 7.4, 10 mM CaCl2) in
screwtop eppendorfs (with O-rings) was added 5 L of pure DMSO (for the
control, 0% inhibition) or the inhibitors in 5 L of DMSO to be assayed at their
appropriate concentrations. As a positive control, 5 L of a stable solution of
lipoprotein lipase (0.23
g, Sigma, from Pseudomonas sp.,30 0.2% n-heptyl-b-
thioglucopyranoside, 10 mM CaCl2, 100 mM NaCl, 50 mM Tris, pH 7.4) was
always used. Each vial was hand mixed briefly then incubated for 15 min in a
sand bath at 37 °C. Then, [14C]SAG substrate (304,000 dpm, specific activity
lg) suspensions
References and notes
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l
l
D-
55 Ci/mol) in 5
sample of [14C]SAG in DMSO was always checked by scintillation counting to
evaluate substrate concentration. After brief hand mixing and 20 min
incubation at 37 °C, the reaction was terminated by adding 200 L of 2:1
CHCl3/MeOH and vortexing for 1 min. Centrifugation for 2 min at 10,000ꢁg
gave a 150 L bottom phase that was predominantly chloroform, a small
lL of DMSO was added by microcap to all vials. An extra 5 lL
a
l
l
protein interphase, and an upper phase that was predominantly water. The
upper phase and protein interface contained negligible (twice background)
radioactive material by scintillation counting. Using
approximately 100 L of each bottom phase was transferred to new eppendorf
vials. Then, 5 L samples of the bottom phases (approximately 10,000 dpm)
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a 200 lL pipette tip,
l
l
l
counting. The silica gel 60 TLC plates were eluted with chloroform/methanol/
aqueous ammonium hydroxide. Though literature reports range from
85:15:0.11,26,28 to 85:15:1,31 an optimized ratio of 86:14:0.75 elutes
substrate [14C]SAG (Rf 0.88), [14C]2-AG (Rf 0.59), and [14C]arachidonic acid (Rf
0.11). In addition to the characteristic decompositions under the basic
conditions of radiolabeled arachidonic acid and diglyceride substrate [14C]2-
AG, there generally appeared to be more degradation of radiolabeled substrate
if it did not contain carrier lipids from cell extraction. The air dried TLC plates
were opposed to Perkin Elmer multisensitive screens for 12 h. Raw data as
gross digital light units (DLU) were obtained from the Perkin Elmer Cyclone
phosphorimaging system for quantitative analysis (OptiQuant software version
5.0).38,39 Percent inhibition was calculated following background subtraction.
The protein specific activities were obtained from the controls. The specific
activities of the hDAGLa were in the range of 0.003–0.01 nmol/mg-min for the
cell lysates (specific activity was 0.06 in the presence of 0.05% Triton X-100)
and up to 0.1 nmol/min-mg for membrane (10,000ꢁg fraction) preparations.
DAGL activities of protein from empty plasmid transfections were 0.003–
0.01 nmol/min-mg. Thus, the specific activities of hDAGL
times the activities of mock infections with empty plasmid. The radio-TLC
analyses of mDAGLb inhibition (data not shown) used 20 g of protein from
cell lysate with a specific activity above 0.1 nmol/min-mg. The radio-TLC
analyses of mDAGL inhibition (Table 2) used 8.8 of protein from
a were only 1.5–3
l
a
l
g
a
19. Piomelli, D. Nat. Rev. Neurosci. 2003, 4, 873.
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conditions was >400 nmol/mg-min. Other standard compounds from prior
literature that were used, JZL184,36 PMSF,1 and RHC80267,1,25,40 have been
reported to be poor inhibitors of DAGL activity.
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pyrene donor and dinitrophenyl acceptor 17 and 18 were satisfactory, though
the 2-pyrenyl analog 17 was used for all in vitro FRET assays. The fluorescent
assays were run in the same Tris buffer with calcium used for the
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35. General: THL 3, JZL184, URB597, and other inhibitors used (see Supplementary
data for structures) were commercially available and used as freshly prepared
DMSO solutions. All arachidonates were maintained under argon or in argon-
degassed solvents. Glass-backed Silica Gel 60 TLC plates were used, and long
delays between spotting and elution were avoided. All solvent ratios are by
volume. All data are reported as the mean of triplicate experiments except
n = 1 or 2 for radio-TLC assays and FRET screenings with DAGLs, lipoprotein
lipase, and pancreatic lipase. Increased enzymatic activity (rather than
inhibition) is indicated by <0% inhibition, and is likely due to detergent or
radiochemical assays except that 200
efficient reading. The lipoprotein lipase standard (0.23
positive control. The freshly sonicated HEK cell lysate (100
l
L
final volumes were needed for
g) was again used for a
g total) protein
l
l
containing DAGL was used as a suspension for each assay. A 15 min period of
gentle shaking at ambient temperature was used following the addition of
10
The ether lipid substrate (25
(10 L) to all wells, and after 2 min of shaking at 37 °C, an initial reading was
taken with excitation at 320 nm and emission observed at 400 nm. Every
l
L of pure DMSO (for the control) or the 10
lL DMSO solutions of inhibitors.
lM final concentration) was then added in DMSO
l
other effects at higher compound (>10
lM) assay concentrations as has
previously been observed in DAGL assays.36 All IC50 data discussed and

4592
M. Johnston et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4585–4592
14 min, another 1 min of shaking would precede the fluorescence readings. The
below) which correlates well with the IC50 of 7 nM using the radiolabeled N-
arachidonoylethanolamine (anandamide) substrate.53 All other standards THL
3,25,27,31 JZL184,36 PMSF,53,61 and RHC8026727 have been reported to be poor
inhibitors of FAAH activity.
readings were followed over 2 h, but the timepoint of 1.5 h was used to
calculate percent inhibitions. The inhibition of hydrolysis of the ether lipid
reporter compound 17 by the DAGL-containing protein preparations with the
compounds under investigation screened at 10
l
M was then compared with
58. Patricelli, M. P.; Lashuel, H. A.; Giang, D. K.; Kelly, J. W.; Cravatt, B. F.
Biochemistry 1998, 37, 15177.
10 lM THL control, which gives complete inhibition of all human and murine
DAGLs tested with the radiolabeled endogenous substrate [14C]SAG. Using the
59. Mor, M.; Rivara, S.; Lodola, A.; Plazzi, P. V.; Tarzia, G.; Duranti, A.; Tontini, A.;
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63. Inhibition of human MAGL: The N-terminal his-tagged full length human
monoacylglycerol lipase used was expressed in E. coli.62 The coumarin
substrate fluorescence assay demonstrated JZL184 to have an apparent IC50
of 57 nM that is comparable to human recombinant MAGL expressed in COS7
cells where the IC50 of JZL184 was reported to be 2–6 nM with the endogenous
substrate 2-AG.36,64 All other standards, THL 3,25–27,31 JZL184,36 URB597,54,65
palmitylsulfonyl fluoride (n-C16H33SO2F),65 PMSF,65 and RHC80267,27 were
reported to be poor inhibitors of MAGL activity.
pyrene-dinitrophenyl reporter compound 17, the apparent IC50 of THL was
always approximately 10 nM with hDAGL
assay.
a using this in vitro FRET-based
50. Cannabinoid receptor binding: Assays were performed by the previously
reported methods.51,52 Palmitylsulfonyl fluoride (n-C16H33SO2F) had a Ki 440
nM, which correlates well with
a
literature report.53 All other standards
(including RHC80267 and SD41) did not bind to the cannabinoid receptors,
analogous to the previous reports for THL 3,25,31 JZL184,36 URB597,54,55 and
PMSF.53
51. Khanolkar, A. D.; Lu, D.; Ibrahim, M.; Duclos, R. I., Jr.; Thakur, G. A.; Malan, T. P.,
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57. Inhibition of rat FAAH: The N-terminal his-tagged rFAAH deletion sequence
used was expressed in an E. coli cell line provided by the Cravatt group.58 The
rFAAH coumarin ester substrate fluorescence assay demonstrated URB597 to
have an apparent IC50 of 4.9 nM. This is comparable to inhibition of rat
membrane preparations used for the hydrolysis of tritiated anandamide.54,59,60
Palmitylsulfonyl fluoride (n-C16H33SO2F) had an apparent IC50 of 6.3 nM in the
67. Lookene, A.; Skottova, N.; Olivecrona, G. Eur. J. Biochem. 1994, 222, 395.
68. Hadvary, P.; Sidler, W.; Meister, W.; Vetter, W.; Wolfer, H. J. Biol. Chem. 1991,
266, 2021.
69. Other novel in vitro FRET-based assays: The bacterial lipoprotein lipase assays
(0.46
pancreatic lipase type II assays (1.0
L3126, labeled 100–400 units/mg with olive oil substrate) also used pyrene-
dinitrophenol FRET reporter compound 17 (25 M in 200 L volumes), which
l
g of high specific activity lipase as described above) and the porcine
lg suspended in 1:9 DMSO/water, Sigma
l
l
was a better substrate than the isomeric 18. The apparent IC50 of THL was
approximately 100 nM with lipoprotein lipase, but less than 1 nM with the
porcine pancreatic lipase using this FRET assay.
fluorescent rFAAH assay (approximately 2 lM in the hMAGL assay detailed