A reversible and selective inhibitor of monoacylglycerol lipase ameliorates multiple sclerosis

Article abstract of DOI:10.1002/anie.201407807

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacological tolerance. Hence, additional classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC₅₀ = 0.18 mm) which is active in vivo and ameliorates the clinical progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB₁-mediated side effects. These results support the interest in MAGL as a target for the treatment of MS.

Full text of DOI:10.1002/anie.201407807

Angewandte
Chemie
DOI: 10.1002/anie.201407807
Enzyme Inhibition
A Reversible and Selective Inhibitor of Monoacylglycerol Lipase
Ameliorates Multiple Sclerosis**
Gloria Hernꢀndez-Torres, Mariateresa Cipriano, Erika Hedꢁn, Emmelie Bjçrklund,
ꢂngeles Canales, Debora Zian, Ana Feliffl, Miriam Mecha, Carmen Guaza,
Christopher J. Fowler, Silvia Ortega-Gutiꢁrrez, and María L. López-Rodríguez*
Dedicated to the memory of Carlos F. Barbas III
Abstract: Monoacylglycerol lipase (MAGL) is the enzyme
responsible for the inactivation of the endocannabinoid 2-
arachidonoylglycerol (2-AG). MAGL inhibitors show analge-
sic and tissue-protecting effects in several disease models.
However, the few efficient and selective MAGL inhibitors
described to date block the enzyme irreversibly, and this can
lead to pharmacological tolerance. Hence, additional classes of
MAGL inhibitors are needed to validate this enzyme as
a therapeutic target. Here we report a potent, selective, and
reversible MAGL inhibitor (IC50 = 0.18 mm) which is active in
vivo and ameliorates the clinical progression of a multiple
sclerosis (MS) mouse model without inducing undesirable
CB1-mediated side effects. These results support the interest in
MAGL as a target for the treatment of MS.
such as pain, (neuro)inflammation-associated pathologies,
obesity, and cancer.[1] However, the use of direct-acting CB1
receptor agonists is associated with psychotropic effects, a fact
that has boosted the search for alternative strategies to
activate the ECS without impairing motor and cognitive
functions. To this end, one possibility is to increase the levels
of the endogenous cannabinoids (eCBs) anandamide (AEA)
and 2-arachidonoylglycerol (2-AG) by pharmacological
inhibition of the enzymes involved in their degradation.[2,3]
AEA is hydrolyzed by the well-characterized fatty acid amide
hydrolase (FAAH)[4,5] and its selective inhibition enhances
AEA levels and induces analgesia and anxiolytic effects
without concomitant psychoactivity.[6–8] However, the analge-
sic effects of an irreversible inhibitor of FAAH have not been
replicated in a phase II clinical trial.[9] Considering that 2-AG
is the major brain eCB, and acts as a full agonist for CB1 and
CB2 receptors, it has become a major focus of attention.[10]
The main enzyme involved in its inactivation is monoacyl-
glycerol lipase (MAGL), which accounts for 85% of the 2-AG
degradation in mouse brain.[11] It has been suggested that
MAGL inhibitors could be useful compounds for the treat-
ment of pain,[12,13] neuropsychiatric disorders,[14] cancer,[15] and
neurodegenerative diseases,[16–18] among others.[19] However,
potent and selective inhibitors of this enzyme are currently
scarce, a fact that has precluded its validation as a therapeutic
target.
T
he activation of the cannabinoid receptors CB1 and CB2
regulates many physiological processes. Therefore, modula-
tion of the endogenous cannabinoid system (ECS) holds great
therapeutic potential for the treatment of many disorders
[*] Dr. G. Hernꢀndez-Torres, Dr. ꢁ. Canales, M. Sc. D. Zian,
Prof. Dr. S. Ortega-Gutiꢂrrez, Prof. Dr. M. L. Lꢃpez-Rodrꢄguez
Department of Organic Chemistry I
Universidad Complutense de Madrid, 28040 Madrid (Spain)
E-mail: mluzlr@ucm.es
Homepage: http://www.ucm.es/info/quimicamedica
Although some MAGL inhibitors have been recently
disclosed[10,20–23] (Figure 1), they act in an irreversible manner.
This permanent inactivation of MAGL can lead to functional
M. Sc. A. Feliffl, Dr. M. Mecha, Dr. C. Guaza
Cajal Institute, CSIC
Dr. Arce 37, 28002 Madrid (Spain)
Dr. M. Cipriano, E. Hedꢂn, E. Bjçrklund, Prof. Dr. C. J. Fowler
Department of Pharmacology and Clinical Neuroscience
Umeå University, SE-901 87 Umeå (Sweden)
[**] This work was supported by grants from the Spanish Ministerio de
Economꢄa y Competitividad (MINECO, SAF2010-22198, SAF2013-
48271, and SAF2010-17501), Comunidad de Madrid (S2010/BMD-
2353 and S2010/BMD-2308), the Spanish Multiple Sclerosis Net-
work (RD201/0032/0008), the Swedish Research Council (grant no.
12158, medicine), and the Research Funds of the Medical Faculty,
Umea University. G.H.-T. and A.C. are Juan de la Cierva and Ramon
y Cajal Scholars, respectively, funded by MINECO and the European
Social Fund. M.C. is a recipient of a Laboratories For Chemical
Biology Umeå postdoctoral fellowship. D.Z. is a predoctoral fellow
funded by MINECO (FPU program). We also thank Linda Gabri-
elsson and Mona Svensson for their expertise in conducting the
COX and mouse brain MAGL/FAAH experiments.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201407807.
Figure 1. Representative irreversible MAGL inhibitors.
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antagonism of the ECS, pharmacological tolerance, and
receptor desensitization that eventually impairs the antinoci-
ceptive properties of the inhibitor.[24] Such problems with
tolerance are less likely to occur with reversible MAGL
inhibitors. To our knowledge, the only compounds described
as potent reversible MAGL inhibitors are the naturally
occurring terpenoids pristimerin and euphol.[25] The related
compounds a- and b-amyrin also inhibit MAGL,[26] but to our
knowledge the effects of these compounds upon the ECS in
vivo have not been investigated. Accordingly, there is an
Figure 3. Design of new MAGL inhibitors.
the arachidonic acid chain of 2-AG with 2-(4-benzylphenyl)-
acetate and 6-(biphenyl-4-yl)hexanoate subunits (compounds
1 and 2, respectively, Figure 3), whereas the glycerol moiety
could be mimicked with a methyloxirane group. These
modifications led to dual FAAH/MAGL inhibitors with IC50
values in the low micromolar range (Figure 3). In an attempt
to improve not only MAGL inhibition but also selectivity
against FAAH, we decided to explore whether the small
oxirane heterocycle of compounds 1 and 2 could be replaced
by different oxygenated cyclic subunits (C.S.), keeping as
optimized lipophilic moieties either the 4-benzylphenyl core
(series Ia) or the 1,1’-biphenyl scaffold (series Ib) (Figure 3).
Compounds 3–16 were prepared as described in the
Supporting Information (SI) and were evaluated for their
capacity to inhibit the 2-AG and the AEA hydrolytic
activities in brain homogenates. In the case of 2-AG, and as
previously described,[27] the most stable analogue, 2-oleoyl-
glycerol (2-OG), was used as a surrogate (see SI for
experimental details). First, we explored the influence of
Figure 2. A potent, selective, and reversible MAGL inhibitor would
increase the local 2-AG levels inducing therapeutic effects without CB1
side effects. AA=arachidonic acid.
important need for the develop-
Table 1: Influence of the cyclic subunit (C.S.) on the capacity of the compounds to inhibit 2-AG and AEA
ment of potent, selective, and rever-
sible MAGL inhibitors that can be
used to establish whether the bene-
ficial effects of MAGL inhibition
can be produced without concom-
itant undesirable side effects medi-
ated by central CB1 activation Cmpd.
(Figure 2). Towards this aim, we
hydrolysis.
Hydrolysis inhibition
Hydrolysis inhibition
IC50 [mm][a,b,c]
IC50 [mm][a,b,c]
C.S.
2-AG[d]
AEA
1.8
6.4
3.5
12
Cmpd.
10
C.S.
2-AG[d]
AEA
3.3
3
41
11
identified compounds 1 and 2 as
reversible dual MAGL/FAAH
2.5
5.8
4
48
11
inhibitors (Figure 3).[27] Using them
[93Æ2%]
[91Æ2%]
10
12
as a starting point, here we describe
5
20
12
[87Æ5%]
5.7
the structural exploration that led
7.1
to the identification of compound
21, which was characterized as
a potent, selective, and reversible
MAGL inhibitor. Moreover, deriv-
ative 21 is active in vivo in the
experimental autoimmune ence-
phalitis (EAE) mouse model of
multiple sclerosis (MS), in which it
clearly ameliorates the course of the
disease without inducing undesira-
ble CB1-agonist-like activity.
6
7
59
13
[68Æ2%]
[82Æ5%]
>100
6.8
>100
12
14
15
16
[25Æ5%]
[96Æ2%]
[27Æ2%]
[60Æ4%]
0.63
8
9
18
5.6
0.49
[97Æ1%]
9.3
[79Æ5%]
3.6
0.62
2.1
[60Æ3%]
[91Æ2%]
[a] The IC50 values were derived from the mean pI50 values. [b] When the data was better fitted to an
inhibition curve with a residual activity, the percentage of inhibitable component is given in the table as
[percentage of maximum inhibition Æstandard error of the mean (s.e.m.)]. [c] When compounds did not
produce ꢀ50% inhibition at the highest concentration tested (100 mm), the percentage of inhibition
seen at that concentration is indicated. [d] In these experiments, the most stable analogue 2-OG was
used as a surrogate of 2-AG (see SI for further details).
Our previous studies[27] indi-
cated that it was possible to replace
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the cyclic subunit (C.S.) in the capacity of the compounds to
inhibit the hydrolysis of both eCBs (Table 1).
The introduction of different oxygenated rings in the
previously optimized lipophilic moieties revealed that, in
general, compounds of series Ib (10–16) were more potent at
inhibiting 2-AG hydrolytic activity than their corresponding
analogues of series Ia (3–9). This effect is especially notable
in compounds 11 versus 4, and 13 versus 6 (Table 1).
However, all compounds of series Ib are still dual inhibitors,
as they display comparable IC50 values in both assays (see, for
example, derivatives 11–13) or even better inhibition of AEA
than of 2-AG hydrolysis (as is the case for derivatives 14–16).
Hence, we focused our efforts on series Ib by broadening
the exploration of the polar C.S. to increase selectivity.
Considering that FAAH has a relatively narrow binding
pocket compared to that of MAGL,[28] we envisioned that
compounds with bulkier C.S. groups could fit into the MAGL
but not into the FAAH active site. This idea led us to explore
benzofuran and benzodioxole scaffolds in compounds 18–22
(Table 2). Also, the requirement of the oxygen was assessed
by the small cyclopropane derivative 17 which shows around
a 10-fold decrease in its ability to inhibit 2-AG hydrolysis
when compared to its corresponding parent compound 2.
In general, all these new derivatives, with the exception of
22, were able to inhibit 2-AG hydrolysis (Table 2). Further-
more, compound 21 exhibited the best profile, with a sub-
micromolar IC50 value for MAGL inhibition and more than
50-fold selectivity versus FAAH [IC50 (MAGL) = 0.24 mm
versus IC50 (FAAH) = 18 mm; see Figure 4A for representa-
tive plots]. Finally, and in an attempt to obtain further insights
on the structural requirements needed for potent and
selective MAGL inhibition, we varied the distance between
the biphenyl group and the heterocycle (analogues 23 and 24),
the ester was replaced by an amide (25), and the polarity and
bulkiness of the lipophilic moiety was modified (compounds
26 and 27) (Table 3). Together, these results suggest that the
heterocyclic moiety of the inhibitor can modulate the
selectivity between MAGL and FAAH. Then, we performed
docking calculations to propose a likely binding mode for 21
!
~
Figure 4. A) Inhibition of the hydrolysis of AEA ( ) and 2-AG ( ) by
compound 21. The figure shows combined data for different independ-
ent dose–response experiments, shown are means (n=4–10). The
s.e.m. values are all enclosed within the symbols. B) The data show no
preincubation (white bars) and 60 min preincubation (gray bars)
expressed as means and s.e.m. (n=4). C) Bars show the hydrolysis for
preincubation followed by dilution (means and s.e.m., n=3), and
indicates the inhibition to be reversible. D) Hydrolysis kinetics
(meanÆs.e.m., n=3), indicative of noncompetitive inhibition with
a Ki value of 0.40 mm.
Table 3: Capacity of analogues of compound 21 to inhibit 2-AG and AEA
hydrolysis.
Table 2: Influence of the cyclic subunit (C.S.) on the capacity of the
compounds of series Ib to inhibit 2-AG and AEA hydrolysis.
Hydrolysis inhibition
IC50 [mm][a,b,c]
Cmpd.
R
n
X
2-AG[d]
AEA
Hydrolysis inhibition
IC50 [mm][a,b,c]
21
5
O
0.24
18
Cmpd.
17
C.S.
2-AG[d]
AEA
[94Æ1%]
10
13
11
27
23
24
3
1
O
O
17
[58Æ8%]
24
18
4.6
9.2
[70Æ6%]
[61Æ3%]
1.7
2.8
19
20
21
22
25
26
5
5
NH
O
15
80
15
[70Æ3%]
11
[60Æ4%]
[52Æ2%]
8.7
[63Æ9%]
43
0.24
18
[94Æ1%]
>100
[1Æ9%]
27
51
4.5
1.5
[88Æ4%]
[a–d] See footnotes for Table 1.
[a–d] See footnotes for Table 1.
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and to rationalize why other derivatives, such as 22 and 27 as
representative examples, were largely inactive at this enzyme.
These models (see SI for details) indicate that inhibitor 21 can
establish favorable polar and hydrophobic interactions with
MAGL whereas neither compound 22 nor 27 are completely
accommodated in the enzyme (Figure S1). In addition, 22 and
27 nicely fit in the FAAH enzyme cavity while derivative 21
shows a different binding mode with less polar interactions
and a different orientation of the benzodioxole ring, making
the overall interaction less favorable (Figure S2). These
models would be in agreement with the observed better
inhibition of FAAH of 22 and 27 compared to 21.
Figure 5. Competitive activity-based protein profiling (ABPP) for com-
pound 21. Proteomes from COS-7 cells transiently transfected with
human cDNAs for A) MAGL, B) ABHD12 (top), and ABHD6 (bottom)
were incubated with 21 (10 mm) for 10 min at RT and then with
fluorophosphonate-rhodamine (75 nm, 2.5 min). Then, proteins were
separated by SDS-PAGE, and analyzed by in-gel fluorescence scanning
to reveal enzyme inhibition. Fluorescent gel is shown in grayscale.
Note that MAGL migrates as several bands as reported previously.[30]
Further exploration of the cyclic subunit, different ester
replacements, and modifications on the biphenyl ring, has
been included, for the sake of clarity, in Tables S1 and S2 in
the Supporting Information. As none of the modifications
performed allowed us to improve the profile of derivative 21,
we assessed whether this inhibitor would fulfill the desired
features in terms of inhibition mechanism, kinetics, and
selectivity. First, we confirmed that 21 inhibited MAGL in
a reversible manner, as shown by preincubation and dilution
experiments. As expected for a noncovalent inhibitor, the
enzyme activity is not affected by preincubation with the
compound (Figure 4B). In addition, data from dilution
experiments (Figure 4C) are consistent with a reversible
character of the inhibitor, since the enzyme activity is fully
recovered after dilution of an initially inhibitory concentra-
tion of the compound. Kinetic studies indicated that 21 acts as
a noncompetitive inhibitor (Figure 4D) with a Ki value of
0.4 mm. In agreement with this result, NMR experiments
showed that the human recombinant MAGL (hrMAGL) was
not able to hydrolyze compound 21 whereas it did hydrolyze,
in the same time interval, competitive inhibitors such as
epoxide 2 (Figure S3). In order to estimate the half-life of
enzyme inhibition we carried out time course NMR studies.
After 13 h the enzyme activity is completely restored as the
inhibitor has been hydrolyzed by the enzyme (Figure S4).
Then, we assessed the selectivity of 21 against the CB1 and
CB2 receptors as well as other enzymes involved in the
degradation of 2-AG, mainly ABHD6 and ABHD12.[11] We
carried out competitive activity-based protein profiling
(ABPP) experiments in COS-7 cells transiently transfected
with the human serine hydrolases MAGL, ABHD6, and
ABHD12 in the presence of the serine hydrolase directed
probe fluorophosphonate-rhodamine[29] (FP-Rh, see SI for
details). These experiments confirmed MAGL inhibition
after treatment with compound 21 (Figure 5A). Under the
same conditions, quantification of the fluorescence intensity
did not show significant differences between ABHD12 and
ABHD6 enzymes (Figure 5B), a result that highlights the
selectivity of 21 over the rest of enzymes responsible for the
degradation of 2-AG. In addition, MAGL inhibitor 21 does
not bind CB1 or CB2 receptors (Ki > 10 mm). Furthermore, we
studied the selectivity of 21 in a broad panel that includes
a variety of receptors and enzymes. Compound 21 did not
inhibit significantly any of the analyzed targets (Table S3 and
Figure S5). Taken together, all these data show that com-
pound 21 fulfills the sought requirements of potency,
reversibility, and selectivity and, hence, it is a potentially
useful tool to validate MAGL as a useful target for drug
development. However, for that to be the case, the compound
needs to show in vivo activity.
MS is a chronic, inflammatory autoimmune disease
characterized by nerve demyelination. Disease onset usually
occurs in young adults and it affects up to 2.5 million people
worldwide.[31] Although considerable progress has been made,
there is no drug that prevents the progression of the disease in
patients with progressive forms of MS, and no means to repair
injured axons or protect neurons from further damage. Thus,
there is an important unmet need for new therapeutic
strategies.[32,33]
It has been proposed that the ECS can improve symptoms
commonly associated with progressive MS,[34] and eCBs have
been suggested to be neuroprotective in this context.[35] These
observations prompted us to examine the capacity of 21 to
ameliorate the progression of MS using the EAE mouse
model. Before in vivo administration, we determined the
inhibition of mouse MAGL and FAAH, and some pharma-
cokinetic parameters. Compound 21 inhibited mouse brain
MAGL and FAAH with IC50 values of 0.18 and 59 mm,
respectively (Figure S6). The compound bound to serum
albumin with a Kd value of 60 mm and after 120 min of
incubation in mouse serum, 42% of the compound still could
be detected. In addition, the pharmacokinetic profile showed
that the compound could be identified in mouse plasma after
4 h. Taken together, these values are sufficient to allow the in
vivo use of the compound.
EAE was induced as detailed in the Supporting Informa-
tion. Treatment started at day 6 post-immunization (p.i.) and
consisted of daily injections of compound 21 (5 mgkgÀ1,
intraperitoneal, i.p.) or vehicle for the following 21 days. All
mice were examined daily for clinical signs of EAE and were
killed at day 27 p.i. Administration of compound 21 clearly
ameliorated the progression of the disease, as assessed by the
significantly lower clinical score in the MS model (Fig-
ure 6A). This improvement correlated with an increase of the
2-AG levels in the spinal cord of treated animals (Figure 6B)
and with evident changes at the histological level, as
compound 21 significantly decreased leukocyte infiltration
and microglial response (Figure 6C,D), prevented axonal
damage, and partially restored myelin morphology in EAE
mice (Figures S7 and S8).
In order to rule out that the anti-inflammatory activity
observed could be due to direct inhibition of the prostaglan-
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ECS, including FAAH, ABDH6, and ABHD12 enzymes,
the CB1 and CB2 cannabinoid receptors, and also against
a broad panel of receptors and enzymes. The compound is
active in vivo, as it enhances the 2-AG levels in spinal cord,
and clearly ameliorates the progression of the disease in
a mouse model of MS by improving clinical symptoms and
decreasing tissue damage in the spinal cords of diseased
mice. Importantly, this therapeutic effect is not accompa-
nied by catalepsy or other motor impairments that have
been observed after the administration of potent and
irrreversible MAGL inhibitors previously described. These
results support the interest in MAGL as a target for the
treatment of MS and the potential clinical application for
MAGL inhibitors in the treatment of this disease.
Received: July 13, 2014
Published online: October 8, 2014
Keywords: endocannabinoids ·
.
endogenous cannabinoid system · enzyme inhibitors ·
monoacylglycerol lipase · multiple sclerosis
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~
shown as means ÆSD for the following groups: control animals (
,
&
^
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