Académique Documents
Professionnel Documents
Culture Documents
Original article
CNRS e Aix-Marseille Universit e Enzymologie Interfaciale et Physiologie de la Lipolyse e UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
CNRS e UMR 7313, Ecole Centrale Marseille e Aix-Marseille Universit, Equipe Chirosciences, Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2012
Received in revised form
5 September 2012
Accepted 21 October 2012
Available online 29 October 2012
We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e. MmPPOX and
MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human
(HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipaserelated proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts
(PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the
FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared
however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities
obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of
DGL, we demonstrated that the phenoxy group plays a key role in specic molecular interactions within
the lipases active site. The absence of this group in the case of MPOX, as well as its connectivity to the
neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory
efciency of these oxadiazolones and leads to a signicant gain in selectivity towards the lipases tested.
The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone
derivatives could also provide useful leads for the development of novel and more discriminative
inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual
lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal
lipolysis process.
2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Enzyme
Enzyme inhibition
Lipases
Oxadiazolone
In silico molecular docking
1. Introduction
Lipase inhibitors have a wide range of applications in the elds
of pharmaceutics and medicine. During the last decade, the use of
digestive lipase inhibitors to reduce the adsorption of dietary fat
453
Fig. 1. Structure of Orlistat and preparation of MmPPOX (1c), MpPPOX (2c) and MPOX (3c). Reagents and conditions: i) NaNO2, HCl, 0 C; ii) SnCl2, HCl, 0 C, 73e81%; iii)
MeOCOCl, pyridine, 0 C to RT, 68e78%; iv) ClCO2CCl3, CHCl2, pyridine, 0 C to RT, 70e80%.
providing this compound with potential applications in the treatment of type II diabetes [6] and Alzheimers disease [7], as well as in
anti-tumoral [5,8] and anti-mycobacterial [9,10] activities. Similar
b-lactone metabolites exhibiting inhibitory properties towards
lipase and other esterases have also been identied from various
microbial sources [11]. However, these compounds never reached
similar levels of inhibition to those exerted by Orlistat [12,13].
Among the wide range of lipolytic enzyme inhibitors, 3-aryl-5methoxy-1,3,4-oxadiazol-2(3H)-one compounds have been reported to show tunable inhibitory properties, depending on the
nature of the N-3 substituent used. Oxadiazolones with aliphatic
substituents at position N-3 were rst reported by Huang et al. [14] to
be efcient inhibitors of housey head acetylcholinesterase (AchE)
as well as potent insecticides. In the early 2000s, high-throughput
screening strategies were developed at Sano-Aventis Pharma for
identifying various classes of hormone sensitive lipase (HSL) inhibitors [15]. This research led to the identication of 3-phenyl oxadiazolone derivatives (e.g. CAY10499 from Cayman Chemical, and
MmPPOX formerly known as compound 7600) due to their high
in vitro inhibitory activity on partially puried HSL from rat epididymal adipose tissue [16]. Further tests showed that the process of
HSL inhibition by MmPPOX was partially reversible, and the mechanism possibly underlying the reaction between this inhibitor and
the enzymes catalytic serine residue has been described [17,18]. It
was also noted that apart from the members of the HSL family, no
other carboxylester hydrolases (including esterases such as AchE and
pig liver esterase; and lipases such as HPL, Thermomyces lanuginosus
lipase and Bacillus subtilis LipA lipase) were inhibited by this
compound under the experimental conditions tested [17]. Contradictory results were however reported at the same time in a SanoAventis Pharmas patent, showing that the same oxadiazolone
molecules could inhibit the lipase activity from porcine pancreas
crude extracts [19]. It was assumed in this work that porcine
pancreatic lipase (PPL) could be inhibited by oxadiazolones. More
recently, similar N-3 substituted oxadiazolones developed for
HSL inhibition have been described as reversible inhibitors of
endocannabinoid-hydrolyzing enzymes, namely fatty acid amide
hydrolase (FAAH) and recombinant human monoacylglycerol lipase
(rhMGL) [20,21], both involved in several physiological processes of
pharmaceutical interest such as pain and inammation [22].
In this context, designing and synthesizing more selective
inhibitors of the various animal and microbial lipases is not only of
fundamental value for understanding the function and catalytic
mechanism of these lipases, but could also provide better drug
454
Table 2
Inhibition levels (%) of HPL, HPLRP2, GPLRP2, DGL, hCEH, PPL and PPE after a 30-min
incubation period with each inhibitor.a
Lipolytic enzymes
xI
MpPPOX
MPOX
Orlistat
HPL
100
400
100
400
2
20
2
20
2
20
2
20
400
26.3
42.7
85.1
87.7
63.7
81.2
80.1
90.4
10.9
81.9
54.5
84.7
89.7
NI
20.0
72.9
87.1
66.0
88.8
13.3
23.6
12.4
92.7
77.1
78.6
90.6
NI
NI
10.7
35.8
49.9
92.7
77.6
86.0
1.4
6.6
71.6
81.3
34.1
77.2
85.0
85.5
90.9
83.6
86.3
82.7
87.3
51.5
56.3
80.1
83.6
93.1
PPL
HPLRP2
GPLRP2
DGL
hCEH
PPE
a
Results are expressed as mean values of at least two independent assays (CV
% < 5.0%). NI no inhibition.
to 50% lipase inhibition were then determined from these inhibition curves (Table 1). The t values were found to increase
concomitantly with the decrease in the inhibitory power, as shown
in the case of PPL: MmPPOX (t 1.69 min) > MpPPOX
(t 1.84 min); HPLRP2: MmPPOX (t 0.78 min) > MpPPOX
(t 2.06 min) > MPOX (t 14.6 min) and hCEH: MPOX
(t 1.00 min) > MpPPOX (t 1.43 min) > MmPPOX
(t 1.55 min). With GPLRP2, although MmPPOX and MPOX
displayed almost the same xI50 value, the inhibition rate with
MmPPOX (t 0.46 min) was nearly 22 times faster than that
observed with MPOX (t 9.97 min) (Fig. 3C). A similar pattern of
behaviour was also observed with MmPPOX (t 1.88 min) and
MpPPOX (t 9.31 min) acting on DGL (Fig. 3D).
In order to compare the inhibitory activity of these oxadiazolones with that of Orlistat, the same kinetic parameters were also
measured with this inhibitor and each of the lipases tested. The xI50
and t values obtained with PPL (2.76; 1.66 min), HPLRP2 (0.77;
0.55 min), GPLRP2 (0.53; 0.40 min), DGL (5.60; 0.59 min) and hCEH
(0.78; 0.52 min) as well as the lipase inhibition rates, which are
presented in Tables 1 and 2, were found to be of the same order
of magnitude as those reached with the three oxadiazolone
compounds.
2.3. Mass spectrometry analysis
To investigate whether these oxadiazolones inhibit HPLRP2,
GPLRP2, DGL and hCEH by forming a covalent bond with the
catalytic site, mass spectrometry analyses were conducted on
inhibited enzymes. MmPPOX was used in these experiments
because it efciently inhibits all these four lipases. After preincubating each lipolytic enzyme for 30 min at 25 C with
Table 1
Half-inactivation time (t) and xI50 values of MmPPOX, MpPPOX, MPOX and Orlistat measured on digestive lipases.a
Lipolytic enzymes
HPL
PPL
HPLRP2
GPLRP2
DGL
hCEH
xI50
t (min)
MmPPOX
MpPPOX
MPOX
Orlistat
MmPPOX
MpPPOX
MPOX
Orlistat
>400
26.5
0.62
0.77
8.33
1.48
>400
57.6
1.04
>40
7.12
0.93
NI
>400
2.27
0.74
>40
0.77
18.4
2.76
0.77
0.53
5.60
0.78
>120
1.69
0.78
0.46
1.88
0.90
>120
1.84
2.06
>60
9.31
1.43
NI
>60
14.6
9.97
>60
1.00
1.64
1.66
0.55
0.40
0.59
0.52
a
The half-inactivation time (t) and the xI50 values were determined as described in Experimental section. The inhibitor molar excess (xI) used for the kinetic determinations of t was: 100, 5, 2, 20 and 4 for HPL & PPL, HPLRP2, GPLRP2, DGL and hCEH, respectively. Results are expressed as mean values of at least two independent assays (CV
% < 5.0%). NI no inhibition.
Fig. 2. Effects of increasing molar excess (xI) of MmPPOX on the rates of hydrolysis of
TC4 emulsions by PPL (-) and HPL (,). Each enzyme was pre-incubated at various
inhibitor molar excess for 30 min at 25 C. Kinetic assays were performed as described
in Experimental section. Results are expressed as mean values of at least two independent assays (CV% < 5.0%).
455
456
Fig. 3. Residual activities of GPLRP2 (upper panels) and DGL (lower panels) measured on TC4 emulsion using the pH-stat technique. Effects of increasing molar excess (xI) of
compounds MmPPOX (-), MpPPOX (,), MPOX (;) and Orlistat (A) on the rates of hydrolysis of TC4 by (A) GPLRP2 and (B) DGL. Each enzyme was pre-incubated at various
inhibitor molar excess for 30 min at 25 C. (C) GPLRP2 and (D) DGL residual activities measured as a function of the incubation time at a constant inhibitor molar excess (xI 2 with
GPLRP2; xI 20 with DGL). Kinetic assays were performed as described in Experimental section. Results are expressed as mean values of at least two independent assays (CV
% < 5.0%).
3. Discussion
We report here the rst and extensive comparative study
focussing on the activity and selectivity of three oxadiazolone
inhibitors, MmPPOX, MpPPOX and MPOX, towards each of the
digestive enzymes involved in gastrointestinal lipolysis. The results
obtained allowed to establish that these compounds are potent
inhibitors of lipases belonging to the acid lipase (DGL) and
pancreatic lipase (PPL, HPLRP2, GPLRP2) families, as well as human
carboxyl ester hydrolase (hCEH). These are new ndings, since
oxadiazolone compounds were previously assumed to exhibit some
selectivity only for the members of the HSL family [17] and
endocannabinoid-hydrolyzing enzymes [20,21]. The inhibition of
PPL conrms however the inhibitory effects of oxadiazolones
observed with crude PPE [19]; although PPL inhibition was only
supposed at that time and not demonstrated with the pure enzyme.
Like Orlistat, oxadiazolones can therefore inhibit enzymes from the
main lipase families.
The ability of each of the oxadiazolone compounds to inhibit the
catalytic activity of the aforementioned lipases was assessed in
Table 3
MALDI-TOF mass spectrometry analysis of inhibited and non-inhibited lipases by
MmPPOX (Mw, 284.3 Da).a
Lipolytic
enzymes
m/z Native
lipase ( )
m/z Inhibited
lipase ( )
Mass
modications (
HPLRP2
GPLRP2
DGL
hCEH
52,874
47,884
48,509
84,595
53,165
48,190
48,794
84,890
291
306
285
295
Da
Da
Da
Da
Da
Da
Da
Da
Da
Da
Da
Da
a
Molecular masses were measured to within a mean accuracy of 0.05% of the
total molecular mass of the sample.
457
Fig. 4. Peptide mass ngerprint spectra of the digested DGL, before (upper panels, A) and after (lower panels, B) a 30-min incubation period with MmPPOX at a molar ratio of
xI 40. Zoom on the region of interest. Left panels, region of the unmodied isotopic peptide L147-K168 (LHYVGHSQGTTIGFIAFSTNPK) containing the catalytic Ser153. Right panels,
region in which a new isotopic peptide was expected to occur, resulting from the covalent binding of compound MmPPOX to the catalytic serine. Mass shifts were calculated as the
difference between the experimental m/z of the modied peptide and the theoretical m/z of the unmodied peptide. The expected exact mass shift is 284.08 Da.
458
Fig. 5. Left panels: in silico molecular docking of Orlistat (yellow) and MmPPOX (green) into crystallographic structures of (A) HPL, (B) GPLRP2 and (C) DGL in a van der Waals
surface representation. Hydrophobic residues (alanine, leucine, isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline and methionine) are highlighted in white. Right
panels: superimposition of the top-scoring docking positions of: (D) MmPPOX (green and light blue) in HPL; (E) MpPPOX (blue and salmon) in GPLRP2; and (F) MPOX (pale green)
and MpPPOX (blue) in DGL active sites, respectively. Each inhibitor is represented in atom colour-code sticks (nitrogen, blue; oxygen, red); and the catalytic serine residues in each
protein are coloured in magenta. Structures were drawn with PyMOL Molecular Graphics System (Version 1.3, Schrdinger, LLC), using the following PDB les: 1LPB, 1GPL and 1K8Q,
for HPL, GPLRP2 and DGL, respectively. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
Fig. 6. Proposed structural basis for the selective inhibition of DGL by 3-phenyl substituted oxadiazolone derivatives.
459
460
(s), 130.2 (d), 129.6 (2 d), 123.2 (d), 119.0 (2 d), 110.8 (d), 107.7 (d),
103.5 (d), 52.8 (q).
5.1.3.2. Methyl
2-(4-phenoxyphenyl)hydrazinecarboxylate
(2b).
Prepared from 2a and methyl chloroformate applying a similar
method as described for the preparation of 1b. Light brown solid
(1.5 g, 78%). Analytical data for 2b: mp 102e104 C; IR (neat) n
3223 (NeH), 1722 (C]O), 1218 (CeO). Rf (AcOEt/hexane 3:7, v/v)
0.27; 1H NMR (200 MHz, CDCl3) d (ppm) 7.40e7.27 (m, 4H), 7.12e
7.03 (m, 1H), 7.03e6.94 (m, 4H), 6.53 (br s, 1H), 3.78 (s, 3H); 13C
NMR (50 MHz, CDCl3) d (ppm) 158.3 (s), 157.7 (s), 150.7 (s), 144.0
(s), 129.5 (2 d), 122.4 (d), 120.6 (2 d), 117.6 (2 d), 114.4 (br d),
52.8 (s).
5.1.3.3. Methyl 2-phenylhydrazinecarboxylate (3b). Prepared from
commercial phenylhydrazine 3a and methyl chloroformate
applying similar method described for the preparation of 1b. Light
yellow solid (3 g, 70%). Analytical data for 3b: mp 116e118 C; IR
(neat) n 3225 (NeH), 1721 (C]O), 1235 (CeO); Rf (AcOEt/hexane
2:8, v/v) 0.25; 1H NMR (200 MHz, CDCl3) d (ppm) 7.31e7.15 (m, 2H),
6.98e6.72 (m, 3H), 6.48 (br s, 1H), 5.73 (br s, 1H), 3.76 (s, 3H); 13C
NMR (50 MHz, CDCl3) d (ppm) 157.7 (s), 147.9 (s), 129.1 (2 d), 120.9
(d), 112.9 (d), 52.8 (s).
5.1.4. Synthesis of compounds 1c, 2c and 3c [16,21,36]
5.1.4.1. 5-Methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one
(1c MmPPOX). To a cold solution of 1b (1 g, 3.87 mmol, 1 equiv.)
in dry methylene chloride (3 mL) dry pyridine (0.63 mL, 7.74 mmol,
2 equiv.) was added dropwise, and the mixture was stirred 30 min
a 0 C. A solution of diphosgene in methylene chloride (0.51 mL,
4.25 mmol, 1.1 equiv.) was added dropwise over a period of 15 min
while cooling in an ice-salt bath under argon atmosphere. After the
addition was complete the reaction mixture was brought to room
temperature and stirred overnight. The reaction mixture was
washed with water, and the organic phase dried (Na2SO4) and
evaporated under reduced pressure. Crystallization in petroleum
ether gave the title compound as yellow crystals (824 mg, 75%).
Analytical data for 1c: mp 104e106 C; IR (neat) n 1797 (C]O), 1659
(C]N); Rf (AcOEt/hexane 1/9) 0.45; 1H NMR (200 MHz, CDCl3)
d (ppm) 7.59 (ddd, J 8.2, 2.0, 0.9 Hz, 1H), 7.50 (t, J 2.2 Hz, 1H),
7.42e7.29 (m, 3H), 7.18e7.08 (m, 1H), 7.08e6.99 (m, 2H), 6.83 (ddd,
J 8.2, 2.3, 0.9 Hz, 1H), 4.09 (s, 3H); 13C NMR (50 MHz, CDCl3)
d (ppm) 157.9 (s), 156.7 (s), 155.8 (s), 148.1 (s), 137.5 (s), 130.2 (d),
129.8 (d), 123.6 (d), 119.0 (d), 115.6 (d), 112.5 (d), 108.9 (d), 57.7 (q);
HPLC-MS: 285 (M 1).
5.1.4.2. 5-Methoxy-3-(4-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one
(2c MpPPOX). Prepared from 2b applying a similar method as
described for the preparation of 1c. Brown solid (490 mg, 70%).
Analytical data for 2c: mp 112e114 C; IR (neat) n 1785 (C]O), 1681
(C]N); Rf (AcOEt/hexane 1:9, v/v) 0.37; 1H NMR (200 MHz, CDCl3)
d (ppm) 7.80e7.69 (m, 2H), 7.41e7.29 (m, 2H), 7.19e6.96 (m, 3H),
4.10 (s, 3H); 13C NMR (50 MHz, CDCl3) d (ppm) 157.1 (s), 155.8 (s),
154.8 (s), 148.3 (s), 131.6 (s), 129.8 (d), 123.4 (d), 119.8 (d), 119.4 (d),
118.7 (d), 57.7 (q); HPLC-MS: 285 (M 1).
5.1.4.3. 5-Methoxy-3-phenyl-1,3,4-oxadiazol-2(3H)-one (3c MPOX).
Prepared from 3b applying a similar method as described for
the preparation of 1c. Yellow crystals (146 mg, 80%). Analytical
data for 3c: mp 97e99 C; IR (neat) n 1782 (C]O), 1662 (C]N);
Rf (AcOEt/hexane 1/9) 0.48; 1H NMR (200 MHz, CDCl3) d (ppm)
7.88e7.72 (m, 2H), 7.48e7.35 (m, 2H), 7.26e7.14 (m, 1H), 4.11 (s,
3H); 13C NMR (50 MHz, CDCl3) d (ppm) 155.7 (s), 148.2 (s), 136.1
(s), 129.0 (d), 125.4 (d), 117.8 (d), 57.6 (q); HPLC-MS: 193
(M 1).
461
(DMSO). The following lipases stock solutions were used: 3.2 mg/
mL PPE in 10 mM MES (pH 7.0), 150 mM NaCl; 0.2 mg/mL HPL in
10 mM MES (pH 7.0), 150 mM NaCl; 0.2 mg/mL PPL in 5 mM Tris (pH
8.0), 100 mM NaCl; 0.2 mg/mL HPLRP2 in 1 mM TriseHCl (pH 8.0),
150 mM NaCl, 5 mM CaCl2; 0.5 mg/mL GPLRP2 in 20 mM Tris (pH
8.0), 1 mM Benzamidine; 0.1 mg/mL DGL in 10 mM MES (pH 6.0),
150 mM NaCl; and 1 mg/mL hCEH in 10 mM MES (pH 6.5), 150 mM
NaCl. These enzyme solutions were used for enzyme-inhibitor preincubation for 30 min at 25 C at various inhibitor molar excess (xI).
Experiments with HPL or PPL were performed in the presence of
a ve-fold molar excess of colipase vs. pancreatic lipase. The presence of 4 mM NaTDC in the incubation medium was required for
the PPL, HPL, DGL and PPE inhibition assays [74]. Regarding the
inhibition by Orlistat, 2 mM NaTDC was used when HPLRP2 and
GPLRP2 were tested. The nal enzyme concentration in the incubation medium was constant and equal to: HPL or PPL y0.6 mM;
HPLRP2 y3.7 mM; GPLRP2 y10.0 mM; DGL y1.9 mM; hCEH
y14.1 mM; and PPE y23.0 mg/mL. During the inhibition experiments, samples of the incubation medium were collected and
injected into the pH-stat vessel in order to measure the residual
lipase activity at various times, and at various inhibitor molar
excess. The variation in the residual lipase activity was then used to
determine the molar excess of the inhibitor which reduced the
enzyme activity to 50% of its initial value (xI50) [38]. In addition, the
enzymes were pre-incubated at 25 C with each inhibitor at a xed
molar excess (xI 100 with HPL or PPL; xI 5 with HPLRP2; xI 2
with GPLRP2; xI 20 with DGL; and xI 4 with hCEH) for 1 h; and
the residual enzyme activity was measured at various times in
order to determine the half-inactivation time (t) corresponding to
50% residual enzyme activity [45]. The xI values used in these latter
experiments were chosen so that the average residual activities of
PPL, HPLRP2, GPLRP2, DGL and hCEH obtained after 30 min of
incubation were in the 15e20% range. In each case, control experiments were performed in the absence of inhibitor but with the
same volume of DMSO. It is worth noting that DMSO at a nal
volume concentration of less than 10% has no effect on the enzyme
activity.
5.2.5. Protein separation and digestion prior to MALDI-TOF mass
spectrometry analysis
Protein in-gel digestion was performed with sequencing grade
trypsin or chymotrypsin, in line with the manufacturers recommendations. Briey, bands were excised, placed in a clean Eppendorf tube and cut into small pieces with a sharp, clean scalpel blade.
All operations were performed at 37 C unless otherwise stated. Gel
pieces were destained by incubating them with 30% ethanol in
water and vortexed for 30 min. The supernatant was discarded and
the process repeated until complete discolouration had occurred.
The gel pieces were washed three times with 50% acetonitrile in
100 mM ammonium bicarbonate (pH 8.0) buffer for 10 min.
Disulde bridges were reduced with 10 mM dithiothreitol (DTT) in
100 mM ammonium bicarbonate (pH 8.0) buffer for 45 min at 56 C.
After removing the supernatant, gel pieces were treated with
55 mM iodoacetamide (IAA) in 100 mM ammonium bicarbonate
(pH 8.0) buffer for 30 min at room temperature in the dark.
Supernatants were discarded and gel pieces were washed twice
using 50% acetonitrile in 100 mM ammonium bicarbonate (pH 8.0)
buffer and dried in a SpeedVac. Dried samples were incubated with
10 mg/mL trypsin or chymotrypsin solution (20 mL) in 25 mM
ammonium bicarbonate (pH 8.0) buffer for 45 min in an ice bath.
Then, 25 mM ammonium bicarbonate (pH 8.0) buffer (50 mL) was
added and the sample was incubated overnight at 37 C for
digestion. Peptides were extracted several times with 0.1% TFA in
water and acetonitrile, pooled together and concentrated in
a SpeedVac for MALDI-TOF analysis.
462
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
and cinnarizine with the self emulsifying excipients Labrasol and Gelucire 44/
14, Pharm. Res. 26 (2009) 1901e1910.
C. Withers-Martinez, F. Carriere, R. Verger, D. Bourgeois, C. Cambillau,
A pancreatic lipase with a phospholipase A1 activity: crystal structure of
a chimeric pancreatic lipase-related protein 2 from guinea pig, Structure 4
(1996) 1363e1374.
A. Hjorth, F. Carrire, C. Cudrey, H. Wldike, E. Boel, D.M. Lawson, F. Ferrato,
C. Cambillau, G.G. Dodson, L. Thim, R. Verger, A structural domain (the lid)
found in pancreatic lipases is absent in the guinea pig (phospho)lipase,
Biochemistry 32 (1993) 4702e4707.
C. Eydoux, S. Spinelli, T.L. Davis, J.R. Walker, A. Seitova, S. Dhe-Paganon, A. De
Caro, C. Cambillau, F. Carriere, Structure of human pancreatic lipase-related
protein 2 with the lid in an open conformation, Biochemistry 47 (2008)
9553e9564.
J. De Caro, B. Sias, P. Grandval, F. Ferrato, H. Halimi, F. Carriere, A. De Caro,
Characterization of pancreatic lipase-related protein 2 isolated from human
pancreatic juice, Biochim. Biophys. Acta 1701 (2004) 89e99.
S. Amara, D. Lafont, B. Fiorentino, P. Boullanger, F. Carrire, A. De Caro,
Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic
enzymes using the pH-stat technique and a medium chain monogalactosyl
diglyceride as substrate, Biochim. Biophys. Acta 1791 (2009) 983e990.
B. Sias, F. Ferrato, P. Grandval, D. Lafont, P. Boullanger, A. De Caro, B. Leboeuf,
R. Verger, F. Carriere, Human pancreatic lipase-related protein 2 is a galactolipase, Biochemistry 43 (2004) 10138e10148.
D. Lombardo, O. Guy, C. Figarella, Purication and characterization of
a carboxyl ester hydrolase from human pancreatic juice, Biochim. Biophys.
Acta 527 (1978) 142e149.
O. Guy, C. Figarella, The proteins of human pancreatic external secretion,
Scand. J. Gastroenterol. Suppl. 67 (1981) 59e61.
J. Hyun, H. Kothari, E. Herm, J. Mortenson, C.R. Treadwell, G.V. Vahouny,
Purication and properties of pancreatic juice cholesterol esterase, J. Biol.
Chem. 244 (1969) 1937e1945.
D. Lombardo, J. Fauvel, O. Guy, Studies on the substrate specicity of a
carboxyl ester hydrolase from human pancreatic juice. I. Action on
carboxyl esters, glycerides and phospholipids, Biochim. Biophys. Acta 611
(1980) 136e146.
L.E. Kiss, H.S. Ferreira, A. Beliaev, L. Torrao, M.J. Bonifacio, D.A. Learmonth,
Design, synthesis, and structure-activity relationships of 1,3,4-oxadiazol2(3H)-ones as novel FAAH inhibitors, MedChemComm 2 (2011) 889e894.
S. Ransac, Y. Gargouri, F. Marguet, G. Buono, C. Beglinger, P. Hildebrand,
H. Lengsfeld, P. Hadvry, R. Verger, Covalent inactivation of lipases, Meth.
Enzymol. 286 (1997) 190e231.
S. Ransac, C. Rivire, C. Gancet, R. Verger, G.H. de Haas, Competitive inhibition
of lipolytic enzymes. I. A kinetic model applicable to water-insoluble
competitive inhibitors, Biochim. Biophys. Acta 1043 (1990) 57e66.
D.J.C. Pappin, P. Hojrup, A.J. Bleasby, Rapid identication of proteins by
peptide-mass ngerprinting, Curr. Biol. 3 (1993) 327e332.
O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
docking with a new scoring function, efcient optimization, and multithreading, J. Comput. Chem. 31 (2010) 455e461.
M.-P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, H. van Tilbeurgh, The 2.46 resolution structure of the pancreatic lipase-colipase
complex inhibited by a C11 alkyl phosphonate, Biochemistry 34 (1995)
2751e2762.
J. Hermoso, D. Pignol, B. Kerfelec, I. Crenon, C. Chapus, J.C. Fontecilla-Camps,
Lipase activation by nonionic detergents. The crystal structure of the porcine
lipase-colipase-tetraethylene glycol monooctyl ether complex, J. Biol. Chem.
271 (1996) 18007e18016.
S. Terzyan, C.S. Wang, D. Downs, B. Hunter, X.C. Zhang, Crystal structure of the
catalytic domain of human bile salt activated lipase, Protein Sci. 9 (2000)
1783e1790.
R. Verger, G.H. de Haas, Interfacial enzyme kinetics of lipolysis, Annu. Rev.
Biophys. Bioeng. 5 (1976) 77e117.
M.L.M. Mannesse, J.W.P. Boots, R. Dijkman, A.J. Slotboom, H.T.W.V. Vanderhijden,
M.R. Egmond, H.M. Verheij, G.H. Dehaas, Phosphonate analogues of triacylglycerols are potent inhibitors of lipase, Biochim. Biophys. Acta 1259 (1995)
56e64.
H.L. Brockman, Triglyceride lipase from porcine pancreas: EC 3.1.1.3 triacylglycerol acylhydrolase, Meth. Enzymol. 71 (1981) 619e627.
S. Fernandez, V. Jannin, J.-D. Rodier, N. Ritter, B. Mahler, F. Carriere,
Comparative study on digestive lipase activities on the self emulsifying
excipient labrasol(R), medium chain glycerides and PEG esters, Biochim.
Biophys. Acta 1771 (2007) 633e640.
J.-C. Bakala NGoma, S. Amara, K. Dridi, V. Jannin, F. Carrire, Understanding
the lipid-digestion processes in the GI tract before designing lipid-based drugdelivery systems, Ther. Deliv. 3 (2011) 105e124.
463