Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis

Article abstract of DOI:10.1002/anie.201603746

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole–piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Full text of DOI:10.1002/anie.201603746

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Communications
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International Edition: DOI: 10.1002/anie.201603746
German Edition: DOI: 10.1002/ange.201603746
Medicinal Compounds
Hot Paper
Second-Generation Non-Covalent NAAA Inhibitors are Protective in
a Model of Multiple Sclerosis
Marco Migliore+, Silvia Pontis+, Angel Luis Fuentes de Arriba, Natalia Realini, Esther Torrente,
Andrea Armirotti, Elisa Romeo, Simona Di Martino, Debora Russo, Daniela Pizzirani,
Maria Summa, Massimiliano Lanfranco, Giuliana Ottonello, Perrine Busquet, Kwang-
Mook Jung, Miguel Garcia-Guzman, Roger Heim, Rita Scarpelli,* and Daniele Piomelli*
Abstract: Palmitoylethanolamide (PEA) and oleoylethanola-
mide (OEA) are endogenous lipid mediators that suppress
inflammation. Their actions are terminated by the intracellular
cysteine amidase, N-acylethanolamine acid amidase (NAAA).
Even though NAAA may offer a new target for anti-
inflammatory therapy, the lipid-like structures and reactive
Figure 1. NAAA hydrolyzes saturated and monounsaturated fatty acid
ethanolamides (for example, PEA (1)) into fatty acid (for example,
palmitic acid (2)) and ethanolamine.
warheads of current NAAA inhibitors limit the use of these
agents as oral drugs. A series of novel benzothiazole–
piperazine derivatives that inhibit NAAA in a potent and
selective manner by a non-covalent mechanism are described.
A prototype member of this class (8) displays high oral
bioavailability, access to the central nervous system (CNS), and
strong activity in a mouse model of multiple sclerosis (MS).
This compound exemplifies a second generation of non-
covalent NAAA inhibitors that may be useful in the treatment
of MS and other chronic CNS disorders.
Accordingly, small-molecule NAAA inhibitors restore
normal PEA and OEA levels in inflamed tissues and exert
marked anti-inflammatory effects in animal models, pointing
to NAAA as a potential target for therapy.[4]
Figure 2 illustrates the three most potent classes of
NAAA inhibitors described to date. Each class is defined by
the presence of a chemical warhead (b-lactone, b-lactam, or
isothiocyanate) that can react covalently with the catalytic
cysteine of NAAA to form a thioester bond.[5] While potent
and, in some cases, systemically active, these molecules share
two features that limit their use as oral drugs: 1) the presence
of a reactive warhead lowers their metabolic stability (for
example, b-lactone 3)[6] or increases risk of allergic reactions
(for example, b-lactam 4 and isothiocyanate 6);[7] and 2) the
hydrophobic fragment that ensures target recognition by
these agents negatively impacts drug-likeness.
NAAA is a cysteine hydrolase that catalyzes the bio-
degradation of PEA and OEA (Figure 1),[1] two lipids that
suppress inflammation by activating the ligand-operated
transcription factor, peroxisome proliferator-activated
receptor-a (PPAR-a).[2] Macrophages and other host defense
cells constitutively generate PEA and OEA in amounts that
are sufficient to fully engage PPAR-a.[3] This process is halted
during inflammation, leading to a decrease in PPAR-a
signaling and acceleration of the inflammatory response.[3]
Herein, we describe a series of benzothiazole–piperazine
derivatives that non-covalently inhibit NAAA (Figure 2).
Our experiments indicate that compound 8, a representative
[*] Dr. M. Migliore,[+] Dr. S. Pontis,[+] Dr. A. L. Fuentes de Arriba,
Dr. N. Realini, Dr. E. Torrente, Dr. A. Armirotti, Dr. E. Romeo,
Dr. S. Di Martino, Dr. D. Russo, Dr. D. Pizzirani, Dr. M. Summa,
Dr. M. Lanfranco, Dr. G. Ottonello, Dr. P. Busquet, Dr. R. Scarpelli,
Prof. D. Piomelli
Department of Drug Discovery and Development, Fondazione
Istituto Italiano di Tecnologia
via Morego 30, 16163 Genoa (Italy)
E-mail: rita.scarpelli@iit.it
piomelli@uci.edu
Dr. M. Garcia-Guzman, Dr. R. Heim
Anteana Therapeutics
11189 Sorrento Valley Road, Suite 104, San Diego, CA 92121 (USA)
Dr. K.-M. Jung, Prof. D. Piomelli
Departments of Anatomy and Neurobiology, Pharmacology and
Biological Chemistry, University of California
Irvine, CA 92697-4625 (USA)
[+] These authors contributed equally to this work.
Figure 2. Structures of published NAAA inhibitors (3,[6] 4,[5b] 6[8]),
activity-based NAAA probe (5)[9] (top), and compounds reported
herein (bottom).
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201603746.
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Table 3: Potencies of compounds 24–32 on hNAAA.
member of this class, is potent, selective for NAAA, and
orally available. Moreover, 8 crosses the blood–brain barrier,
elevates PEA and OEA levels in the CNS, and is strongly
active in a mouse model of MS.
A screening campaign to discover new scaffolds for
NAAA inhibition yielded the low-potency hit 7 (Table 1).
Compound
Y
hNAAA IC50 [mm][a]
24
25
26
27
28
29
30
31
32
H
0.43Æ0.03
1.55Æ0.29
7.2
6.87
5.73
0.55Æ0.20
2.95Æ0.84
0.30Æ0.02
0.71Æ0.21
6-Cl
6-CF3
6-CH3
6-OCH3
4-F
5-F
7-F
7-Cl
Table 1: Inhibitory potencies (IC50 in mm) of compounds 7 and 9–16 on
the activity of hNAAA expressed in HEK-293 cells.
Compound
X
hNAAA IC50 [mm][a]
[a] See Footnote for Table 1.
7
9
o-CH3
H
88.9
NA[b]
10
11
12
13
14
15
16
o-Cl
o-F
NA
NA
20.5
5.69Æ2.54
0.45Æ0.11
NA
substituent did not affect potency (24), whereas insertion of
electron-withdrawing (25, 26) or -donating (27, 28) groups was
detrimental. Moving a halogen to various positions of the ring
caused either minor effects or a decrease in potency (29–32).
The role of the piperazine was probed by replacing it with
alternatively oriented piperidines (Table 4). The compounds
obtained were either weakly (33) or moderately (34) active,
with 33 being approximately 60 times less potent than 14. Loss
of potency was also observed with 35, in which a methylene
bridge replaces the benzamide carbonyl of 14. To examine the
effect of conformational changes in the piperazine ring, we
introduced one or two methyl groups at various positions of
this moiety (36–39). These attempts did not lead to improve-
ments in potency, but confirmed the role of a mono-
substituted piperazine in this region, as shown by the drop
in activity caused by 2,2-dimethyl substitution (38). Support-
ing this, we noted that potency was highly sensitive to the
configuration of the methyl group, with 40 (the (R)-enantio-
mer of 36) being 15 times less potent than the (S)-enantiomer
8. Alkyl chain modifications on the sulfonyl substituent of 8
yielded no further improvement (41 and 42).
o-OCH3
o-SO2CH3
o-SO2CH2CH3
m-SO2CH2CH3
p-SO2CH2CH3
NA
[a] Values are the mean Æ SEM of three or more determinations, or the
mean of triplicate determinations in a single experiment. [b] Not Active,
<30% inhibition at 100 mm.
To improve potency, we launched
a structure–activity
relationship (SAR) study that started with the benzamide
portion of 7. Removal of the o-methyl group (9), or replace-
ment of such a group with a halogen (10, 11), caused
a complete loss of activity. By contrast, substitutions with
a methoxy (12), methylsulfonyl (13), or ethylsulfonyl (14)
group yielded compounds of progressively greater potency.
Since moving the ethylsulfonyl substituent to the meta- or
para-position of the phenyl ring strongly reduced activity (15,
16), we focused our exploration on o-sulfonyl derivatives
containing linear, branched, or cyclic alkyl groups (Table 2).
We found that potency was highly sensitive to length and size
of the alkyl fragment, with bulkier substituents producing
weaker inhibition (for example, 18 and 23).
We focused subsequent mechanistic work on 8 because of
its greater water solubility, compared to other similarly potent
compounds (Table 5; Supporting Information, Table S1). To
support this work, an easily scalable synthesis was developed
(Scheme 1).
Subsequently, we turned to the benzothiazole system,
using 14 as an entry point (Table 3). Removal of the 6-F
Current NAAA inhibitors react covalently with the
catalytic cysteine of NAAA.[5] We used four approaches to
probe the interaction of 8 with NAAA. First, we incubated
purified human (h) NAAA (2 mm) with 8 (50 mm), digested it
with trypsin, and searched for covalent adducts using liquid
chromatography mass spectrometry (LC-MS). As a positive
control, we included the b-lactam 4 (20 mm), for which
Table 2: Potencies of compounds 17–23 on hNAAA.
hNAAA IC50 [mm][a]
a
covalent interaction with NAAA is documented.[5b]
Compound
R
Incubating hNAAA with 4 yielded the expected acylated
peptide, whereas no such adduct was found when hNAAA
was exposed to either 8 or vehicle (Figure 3A). Similarly, no
covalent adducts were retrieved by a search through the
entire peptide map of hNAAA (Supporting Information,
Figure S1). Second, we incubated hNAAA (4.0 mm) with 4 or
17
18
19
20
21
22
23
(CH2)2CH3
(CH2)3CH3
i-propyl
c-propyl
c-butyl
0.39Æ0.06
8.80Æ2.40
0.50Æ0.09
0.19Æ0.03
0.32Æ0.00
1.10Æ0.09
38.11Æ5.49
c-pentyl
c-hexyl
8
(1.0 mm), precipitated the protein, and measured
[a] See Footnote for Table 1.
compounds in the supernatant by LC-MS. Whereas 4 was
2
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Table 4: Potencies of compounds 8 and 33–42 on hNAAA.
Compound
Structure
hNAAA
IC50 [mm][a]
33
26.17Æ7.15
34
35
36
37
38
1.77Æ0.18
1.82Æ0.32
0.50Æ0.075
0.72Æ0.12
NA[b]
Scheme 1. a) (2S)-2-methylpiperazine, NaHCO3, EtOH/H2O, reflux,
15 h, quantitative; b) EtI, NaOH (2m), EtOH, RT, 15 h, quantitative;
c) oxone, H2O, 408C, 15 h, 96%; d) HATU, Et3N, CH3CN, RT, 15 h,
43%.
precipitated with NAAA, as expected from its covalent
binding to the enzyme, 8 was entirely recovered in the
supernatant (Figure 3B). Third, we investigated whether 8
prevents the binding of the covalent activity-based probe 5 to
NAAA.[9] As previously shown,[9] 5 labeled hNAAA in cell
extracts, and this effect was blocked by incubation with 4
(Figure 3C). In contrast, incubation with 8 only partially
antagonized hNAAA labeling by 5 and at short incubation
times (Figure 3C). Lastly, kinetic analyses revealed that 8
39
1.44Æ0.14
40
8
3.43Æ0.50
0.23Æ0.04
0.20Æ0.05
0.18Æ0.04
41
42
[a,b] See Footnote for Table 1.
Table 5: In vitro metabolism and pharmacokinetic properties of 8 in
mice.[a]
Solubility in PBS [mm]
139Æ1
>120[b]
>60[c]
>60[d]
Plasma t1/2 [min]
MLM (NADPH) t1/2 [min]
MLM (UDPG) t1/2 [min]
3 mgkgÀ1 (iv)
3 mgkgÀ1 (oral)
Cmax [ngmLÀ1
Tmax [min]
]
1660Æ166
(5.0)
613Æ68
30
Cl [mLminÀ1 kgÀ1
]
33.2Æ1.6
73.9Æ3.7
3.5Æ0.2
1366.8Æ68.3
404.3Æ109.1
–
49Æ8
Figure 3. Compound 8 inhibits NAAA by a non-covalent mechanism.
A) LC-MS tracings showing (top) that the covalent inhibitor 4 forms
an adduct with a peptide containing the catalytic C126
t1/2 [min]
104Æ16
7.4Æ1.1
988Æ157
181Æ28
72Æ11
Vd [LkgÀ1
]
AUCplasma [hꢀngmLÀ1
]
(C126TSIVAQDSR) of NAAA (see inset), whereas (bottom) 8 or its
vehicle (DMSO) have no such effect. B) Covalent inhibitor 4 (top) or 8
(bottom) was incubated with NAAA (filled bars) or buffer alone (open
bars) and quantified in supernatant after protein precipitation. Bars:
mean Æ SEM, n=3. C) Lysosomal extracts of hNAAA-overexpressing
HEK293 cells were incubated with vehicle (2% DMSO), 4 or 8, for 2 h
before addition of probe 5. A rhodamine fluorophore was inserted by
click chemistry. The arrowhead indicates NAAA. Fluorescence (top),
Coomassie Blue staining (loading control; bottom).
AUCbrain [hꢀngmLÀ1
]
F [%]
[a] NADPH, reduced nicotinamide adenine dinucleotide phosphate;
PBS, phosphate-buffered saline; t1/2, terminal half-life; UDPG, uridine
diphosphate glucose; AUC, area under the curve; Cmax, maximal plasma
concentration; F%, oral bioavailability; Tmax, time at which Cmax is
reached. [b] 83Æ4% remaining at 120 min; [c] 70Æ7% remaining at
60 min; [d] 96% remaining at 60 min.
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Figure 5. PK and pharmacodynamic profiles of 8 in mice. A) Levels of
~
*
8 in the plasma (c c) or brain (c c) after oral admin-
istration (3 mgkgÀ1). B) Time course of the effects of 8 (30 mgkgÀ1
)
on PEA, OEA, and anandamide (AEA) levels in the brain. Results are
expressed as mean Æ SEM, n=3. * P<0.05; **, P<0.01; ***,
P<0.001, one-way ANOVA.
Figure 4. A) Michaelis–Menten analysis of the reaction of hNAAA in
~
*
the presence of vehicle (DMSO, c c) or 8 (50 nm, c c;
&
200 nm, c c). B) Lineweaver–Burk plot indicates uncompetitive
inhibition (inset: magnification of the plot close to its origin).
OEA.[12] PEA administration attenuates spasticity in the
experimental allergic encephalomyelitis (EAE) model of
MS.[13] Consequently, we tested whether accrual of PEA/OEA
by treatment with 8 might be beneficial in this model. EAE
mice and sham-immunized controls were treated with 8
(30 mgkgÀ1, twice daily) or vehicle for 28 days, while record-
ing clinical scores and body weight gain. Clinical scores were
generated using a symptom scale that ranged from 0 (no
clinical signs) to 5 (moribund).[14]
Figure 6 shows that treatment with 8 did not affect sham-
immunized mice, whereas it delayed disease onset, attenuated
symptom intensity, and normalized body weight in EAE
animals. Moreover, 8 reduced mononuclear cell infiltration
and microglia activation—two anatomical correlates of
disease—in the spinal cord of EAE mice (Supporting
Information, Figure S2). Further underscoring the superiority
of 8 relative to known NAAA inhibitors, the highly potent
covalent b-lactam derivative 4 had no effect in the EAE
model (Supporting Information, Figure S3).
In summary, the present study describes a novel class of
benzothiazole–piperazine derivatives that inhibit NAAA by
a non-covalent, uncompetitive mechanism. A representative
member of this class, 8, shows excellent oral PK properties
and good brain penetration. The strong protective activity of 8
in a mouse model of MS is similar to those of other agents
used to target this disease.[15] Compound 8 exemplifies a new
generation of non-covalent NAAA inhibitors that may find
therapeutic applications in the treatment of neuro-
inflammation.
inhibits NAAA by an uncompetitive mechanism
(Figure 4A,B). The results identify 8 as the first non-covalent
NAAA inhibitor disclosed to date.
The unprecedented mechanistic profile of 8 prompted us
to test the compoundꢀs usefulness as an oral agent. In vitro
studies demonstrated that 8 is soluble in aqueous buffer
(pH 7.4) and stable in mouse plasma (Table 5). Similarly, 8 is
stable in mouse liver microsomes (MLM).
Experiments in mice showed that 8 is extensively
absorbed after oral administration (Figure 5A). The pharma-
cokinetic (PK) parameters listed in Table 5 indicate that 8 had
excellent oral bioavailability. Importantly, 8 crossed the
blood–brain barrier, reaching a brain-to-plasma ratio of 0.2
and causing a substantial accumulation of PEA and OEA in
brain tissue (Figure 5A,B). No changes were seen in the
levels of the anandamide, an endocannabinoid lipid amide
that is degraded by fatty acid amide hydrolase (FAAH) rather
than by NAAA.[3]
A selectivity screen showed that 8 (10 mm) had little or no
effect on a panel of more than 50 receptors, ion channels, and
transporters (Supporting Information, Table S2). Moreover,
as expected from our target engagement studies (Figure 5B),
8 had only a weak inhibitory effect on FAAH (IC50
ꢀ 10 mm),[10] and no effect on either acid ceramidase (a
cysteine amidase that has 33–34% identity with NAAA) [1] or
monoacylglycerol lipase (a serine hydrolase that degrades the
endocannabinoid 2-arachidonoyl-sn-glycerol).[11]
MS is a neuroinflammatory disorder accompanied by
alterations in cerebrospinal and plasma levels of PEA and
4
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*
*
MOG(À)+8 (c c); Naive (c c).
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This work was partially supported by a grant from the
National Institute on Drug Abuse (to D.P.). The authors
thank Dr. Angelo Reggiani for discussion; Drs. Annalisa
Fiasella, Andrea Nuzzi, and Stefano Ponzano for the synthesis
of 4 and 5; Natasha Margaroli and Ilaria Penna for assay
support, and Silvia Venzano for compound handling. The
authors declare a competing financial interest: Daniele
Piomelli, Rita Scarpelli, Marco Migliore, Miguel Garcia-
Guzman, and Roger Heim are inventors on the patent
application that protects novel compounds disclosed in this
paper.
Keywords: cysteine hydrolase · fatty acylethanolamides ·
multiple sclerosis · N-acylethanolamine acid amidase ·
neuroinflammation
Received: April 18, 2016
Revised: May 11, 2016
Published online: && &&, &&&&
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Communications
Medicinal Compounds
M. Migliore, S. Pontis,
A. L. Fuentes de Arriba, N. Realini,
E. Torrente, A. Armirotti, E. Romeo,
S. Di Martino, D. Russo, D. Pizzirani,
M. Summa, M. Lanfranco, G. Ottonello,
P. Busquet, K.-M. Jung,
M. Garcia-Guzman, R. Heim,
R. Scarpelli,*
D. Piomelli*
Treating neuroinflammation: The cysteine
amidase N-acyl ethanolamine acid ami-
dase (NAAA) promotes inflammation by
degrading endogenous anti-inflammatory
lipid amides such as palmitoylethanola-
mide (PEA). A new class of non-covalent,
orally available NAAA inhibitors (see
example) interrupt NAAA-mediated lipid
amide degradation in the brain, and are
strongly protective in a mouse model of
neuroinflammation.
&&&& — &&&&
Second-Generation Non-Covalent NAAA
Inhibitors are Protective in a Model of
Multiple Sclerosis
6
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