β-Lactones inhibit N -acylethanolamine acid amidase by S-acylation of the catalytic N-terminal cysteine

Article abstract of DOI:10.1021/ml300056y

The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted β-lactones.

Full text of DOI:10.1021/ml300056y

Letter
pubs.acs.org/acsmedchemlett
β-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation
of the Catalytic N-Terminal Cysteine
Andrea Armirotti,† Elisa Romeo,† Stefano Ponzano,† Luisa Mengatto,† Mauro Dionisi,†
Claudia Karacsonyi,† Fabio Bertozzi,† Gianpiero Garau,† Glauco Tarozzo,† Angelo Reggiani,†
Tiziano Bandiera,† Giorgio Tarzia,∥ Marco Mor,§ and Daniele Piomelli*,†,‡
†Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy
‡Departments of Pharmacology, University of California, Irvine, 360 MSRII, California 92697-4625, United States
§Pharmaceutical Department, University of Parma, 43124 Parma, Italy
∥Dipartimento di Scienze Biomolecolari, Universita
̀
degli Studi di Urbino “Carlo Bo”, Piazza del Rinascimento 6, I-61029 Urbino, Italy
S
* Supporting Information
ABSTRACT: The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class
of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA
by substituted β-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed,
using high-resolution liquid chromatography−tandem mass spectrometry, that this compound forms a thioester bond with the
N-terminal catalytic cysteine in human NAAA.
KEYWORDS: NAAA, cysteine amidase, covalent inhibitors, high resolution mass spectrometry, proteomics
he fatty acid ethanolamides (FAEs) are a family of
bioactive lipid molecules that have attracted considerable
agonists of nuclear peroxisome proliferator-activated receptor-α
(PPAR-α), which is responsible for most of their analgesic and
anti-inflammatory properties.14−16 OEA and PEA are produced
in many mammalian tissues, including neurons17 and innate
immune cells,18 where a selective phospholipase D releases
them by cleaving the membrane precursor, N-acyl-phosphati-
dylethanolamine.19 They are deactivated either by FAAH,
which prefers anandamide and OEA over PEA, or by NAAA,
which conversely prefers PEA and OEA over anandamide.20
Despite its functional similarity with FAAH, NAAA shares no
homology with this or other enzymes of the same “amidase
signature” family. Rather, NAAA is an N-terminal nucleophile
(Ntn) hydrolase and belongs to the choloylglycine hydrolase
family of hydrolases, which are characterized by the ability to
cleave nonpeptide amide bonds. Like other Ntn enzymes, NAAA
is converted by proteolysis into a shorter active form upon
incubation at acidic pH.20 Considering its predominant
localization in lysosomes,21 it is conceivable that NAAA finds
within these organelles the acidic pH (≈5.0) necessary for
autocatalytic proteolysis and activation. Processing of NAAA
renders a cysteine residue (Cys131 in mice and rats and
Cys126 in humans) the N-terminal amino acid and catalytic
nucleophile.18,21,22 Pharmacological blockade of intracellular
NAAA activity results in a normalization of OEA and PEA
T
interest due to their potential role in the control of pain,
inflammation, and energy metabolism. Two structurally and
functionally distinct classes of FAEs have been described. Poly-
unsaturated FAEs, such as arachidonoylethanolamide (ananda-
mide) and eicosapentaenoylethanolamide, are endogenous
agonists of G protein-coupled cannabinoid receptors.1,2 These
compounds are thought to act as retrograde messengers at
synapses of the central nervous system and as local mediators in
peripheral tissues. In the brain, anandamide-mediated signaling
at CB1 type cannabinoid receptors has been implicated in the
regulation of anxiety,3 depression,4,5 stress-induced analgesia,6
and nausea.7 Outside the brain, anandamide released at sites of
tissue injury is thought to control the initiation of emerging
pain signals,8,9 presumably by reducing the local release of
proalgesic and proinflammatory factors from nociceptive nerve
terminals.10 The biological actions of anandamide are inter-
rupted by a two-step deactivation process consisting of carrier-
mediated transport into cells11,12 followed by intracellular
hydrolysis, which is catalyzed by the membrane-bound serine
hydrolase, fatty acid amide hydrolase (FAAH). Inhibitors of this
enzyme protect anandamide from deactivation and selectively
magnify its intrinsic actions, producing an array of pharmaco-
logical effects that include reduced anxiety, depression, and
pain.13 Saturated and monounsaturated FAEs, which include
oleoylethanolamide (OEA) and palmitoylethanolamide (PEA),
do not productively interact with cannabinoid receptors.
These compounds are, however, potent or moderately potent
Received: February 29, 2012
Accepted: April 6, 2012
Published: April 6, 2012
© 2012 American Chemical Society
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Letter
Figure 1. Chemical structures of β-lactone NAAA inhibitors, (S)-N-(2-
oxo-3-oxetanyl)-3-phenylpropanamide [(S)-OOPP], N-[(2S,3R)-2-
methyl-4-oxo-oxetan-3-yl] carbamate (ARN077), and N-[(2S,3R)-2-
methyl-4-oxo-oxetan-3-yl]-7-phenyl-heptanamide (ARN768).
Figure 3. (A) Extracted ion chromatograms of the native (540.26
m/z) and acylated (685.43 m/z) peptide (for clarity purposes, peaks
have not been normalized). (B and C) MS/MS spectra of m/z 540.26
and m/z 685.43. In both spectra, the y fragment ions series perfectly
match a CTSIVAQDSR sequence (bearing a side chain adduct on the
cysteine in the second case).
of substituted β-lactones, which shows promising potency and
target selectivity.18,26 The prototype of this class, (S)-N-(2-oxo-
3-oxetanyl)-3-phenylpropanamide [(S)-OOPP, Figure 1], is a
potent noncompetitive NAAA inhibitor that is highly effective
at preventing PEA and OEA hydrolysis in activated inflam-
matory cells and at reducing tissue reactions to various pro-
inflammatory stimuli.18
We have recently used the structure of (S)-OOPP as a
starting point to discover β-lactone-based NAAA inhibitors
with improved potency. This investigation lead us to identify
several new compounds that selectively inhibit NAAA at
low nanomolar concentrations, including 5-phenylpentyl
N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl] carbamate (ARN077,
Figure 1). The structure−activity relationship properties of
ARN077 will be described elsewhere (Ponzano, S.; et al.
Submitted for publication). In the present study, we used this
molecule as a probe to characterize the mechanism through
which substituted β-lactones inhibit NAAA. Our results indicate
that the N-terminal cysteine of human recombinant NAAA
(h-NAAA) is acylated by ARN077 and that the new bond
formed is a thioester between the cysteine thiol and the carbonyl
group of the β-lactone.
Figure 2. Inhibition of human NAAA by ARN077. (A) Concen-
tration−response curve for inhibition of recombinant hNAAA by
ARN077 after 30 min of incubation. (B) Michaelis−Menten analysis
of ARN077 (vehicle, ; 10 nM ARN077, ; and 30 nM ARN077, ).
(C) Preincbation time course of ARN077 (100 nM). (D) Reversibility
study of ARN077 (vehicle, open bars; ARN077, black bars). *p < 0.05
ARN077 postdialysis vs vehicle postdialysis; **p < 0.01 ARN077
postdialysis vs ARN077 predialysis; and ***p < 0.001 ARN077
predialysis vs vehicle predialysis.
■
●
▲
levels, which are markedly reduced in inflammatory
states,18,23,24 and exerts profound anti-inflammatory effects in
animal models.18 These findings suggest that NAAA inhibi-
tion might represent a novel mechanistic approach to control
inflammation. A few groups of NAAA inhibitors have been
developed to test this hypothesis.18,25,26 Among them is a class
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ACS Medicinal Chemistry Letters
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ARN077 (Figure 1) inhibited the activity of h-NAAA with a
median effective concentration (IC50) of 7.3 1.6 nM (n = 3)
(Figure 2A) and a Hill coefficient of −0.8. By contrast, at the
concentrations tested (up to 30 μM), the compound had no
significant effect on the activity of FAAH or acid ceramidase, a
cysteine amidase that exhibits 33−35% amino acid identity with
NAAA27 (Supporting Information, Table 1). Kinetic and time−
course experiments showed that the inhibitory effect of
ARN077 was noncompetitive (Figure 2B and Supporting
Information, Table 1) and reached half-maximal inhibition in
2.4 min and maximum inhibition within 10 min of incubation
with the enzyme (Figure 2C).
Scheme 1. Possible Nucleophylic Attacks of Cysteine on
ARN077
a
Following centrifugation through a dialyzing membrane, the
inhibition of h-NAAA activity produced by ARN077 (100 nM)
was markedly reduced but not completely reversed (Figure 2D).
The results indicate that ARN077 is a potent h-NAAA inhibitor
that acts through a noncompetitive and partially reversible
mechanism. Compounds that contain a β-lactone ring are
known to interact covalently with biological nucleophiles, and
some have been shown to inhibit Ntn enzymes such as
NAAA.26 This led us to hypothesize that ARN077 might
interact with h-NAAA through a covalent binding to the
enzyme's catalytic cysteine. h-NAAA is translated as a 331
amino acid protein, which undergoes autoproteolytic cleavage
at acidic pH 4.5 forming a new N-terminus, Cys126, which
serves as the catalytic nucleophile.28 We examined, therefore,
whether ARN077 might form a covalent adduct with the
N-terminal cysteine of the active form of h-NAAA. If present, this
modification should be carried by the corresponding N terminus
peptide produced by trypsin digestion (C126TSIVAQDSR;
MW 1078.51). The position of this peptide within the
primary sequence of the expressed h-NAAA is underlined
below, along with the position of the trypsin cleavage sites
(in bold):
a
Path a, thioether adduct; path b, thioester adduct; and path c,
hydrolysis of the carbamate, with two possible adducts (c and c′).
R = (CH2)5-Ph.
The adducts a and b are isomers of formula C58H95N15O21S
and are expected each to generate a doubly charged ion of m/z
685.43. A peptide with this m/z value was eluted approximately
20 min after the native, unmodified peptide (Figure 3A).
MS/MS analysis of this component (Figure 3C) produced a y
fragment ion series that matched that of the native peptide
(Figure 3B), clearly indicating that ARN077 covalently links to
the N-terminal cysteine of NAAA. The presence in the MS/MS
spectrum of an ion of m/z 292.16, together with the cor-
responding loss of water (m/z 274.14), indicate a fragmenta-
tion of the acylated cysteine side chain (C16H22NO4). This is
confirmed by the presence of the singly charged peptide ion
at m/z 1079.52. However, this fragmentation did not allow
us to conclusively determine whether the broken bond was a
thioester or a thioether. Diagnostic ions that would permit
to discriminate between S-alkylation and S-acylation are
expected to be formed upon C−C cleavage of the thioether
or α-cleavage of the thioester (Supporting Information, Figure 1).
The former, which should produce an ion of m/z 264.12
(C14H18NO4), was not detected in our analyses, while the latter,
which should produce an ion of m/z 264.16 (C16H24NO2), is
rather weak and was covered by the second isotopic peak
(m/z 264.16) of the very intense y2 ion of the backbone frag-
mentation at m/z 262.15. These results tentatively suggest that
ARN077 might form a thioester bond with the N-terminal
cysteine of h-NAAA, but additional evidence is needed to draw
such conclusion. To fill this gap, we tested the ability of the
compound ARN768 (Figure 1), an amide analogue of ARN077
MRTADREARPGLPSLLLLLLAGAGLSAASP-
PAAPRFNVSLDSVPELRWLPVLRHYDLDLVRAA-
MAQVIGDRVPKWVHVLIGKVVLELERFLPQPFT-
GEIRGMCDFMNLSLADCLLVNLAYESSVFC126TSIVA-
QDSRGHIYHGRNLDYPFGNVLRKLTVDVQFLKNG-
Q I A F T GT T F I GY V G L W TG Q S P HK FTVSG-
DERDKGWWWENAIAALFRRHIPVSWLIRATLSESEN-
FEAAVGKLAKTPLIADVYYIVGGTSPR EGV-
VITRNRDGPADIWPLDPLNGAWFRVETNYDHWK-
PAPKEDDRRTSAIKALNATGQANLSLEALF-
Q I L S V V P V Y N N L T I Y T T V M S A G S P D K Y M -
TRIRNPSRK
A control incubation of h-NAAA at pH 4.5 for 90 min yielded a
peptide of mass-to-charge ratio (m/z) 540.26 (Figure 3A),
which was unambiguously identified as the product of auto-
proteolytic cleavage of NAAA (Figure 3B), because the cor-
responding MS/MS spectrum shows a complete match of the
y fragment ions series.29 We next searched, in incubations
of intact h-NAAA with ARN077 (both at 5 μM), for adducts
that might be generated by the nucleophilic attack of the thiol
group of Cys126 on ARN077. As illustrated in Scheme 1, three
such adducts are expected to be produced: the thioether a,
formed through S-alkylation of the carbon in the 2-position of
the oxooxetan ring of ARN077; the thioester b, formed through
S-acylation of the oxooxetan carbonyl carbon of ARN077; and
the thioesters c and c′, formed by the attack of Cys 126 on the
carbamate group of ARN077.
that is a relatively potent h-NAAA inhibitor (IC50 314
52
nM ; n = 3), to interact covalently with h-NAAA. We found that
ARN0768 reacts with h-NAAA forming, as does ARN077, a
peptide adduct whose MS/MS spectrum shows a side chain
fragment ion at m/z 290.18 (C17H24NO3), the corresponding
loss of water at m/z 272.17, and a clearly detectable ion at m/z
262.18 (Figure 4).
The latter corresponds to the cleavage of a thioester
(C15H22NO3) rather than a thioether (C15H20NO3), whose
corresponding ion at m/z 262.14 was not detected. Notably,
attack on the carbamate group (mechanism c in Scheme 1) is
expected to produce the modified peptides c (C47H81N15O21S)
and c′ (C54H88N14O19S), with the alkyl-phenyl chain or the
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ACS Medicinal Chemistry Letters
Letter
essential tool for the direct validation of reaction mechanisms that
are important in drug discovery. In the present case, the high-
resolution tandem mass capabilities of the hybrid quadrupole-TOF
instrument allowed us to unequivocally assign the structure of side
chain fragment ions, which resulted in an unambiguous assignment
of the chemical structure of the modified peptide. Furthermore,
our results provide a solid mechanistic basis for the identification
of a second generation of β-lactone-based NAAA inhibitors with
improved potency, selectivity, and metabolic stability.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed description of the experimental procedures, MS/MS
fragmentation analysis, and pharmacological data of ARN077.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Prof. Daniele Piomelli Department of Drug Discovery and
Development, Istituto Italiano di Tecnologia, via Morego, 30,
16163 Genova. Email: Daniele.piomelli@iit.it.
Author Contributions
AA, GG, FB designed the experiments and analyzed the data.
SP synthesized β-lactones. GT, MD and LM performed
pharmacological profiling of ARN077. ER and CK produced
the recombinant h-NAAA. DP oversaw the project with
assistance from TB, AR, GT, MM. AA and DP wrote the
manuscript.
Funding
Funding for this research was provided by the Istituto Italiano
di Tecnologia.
Notes
The authors declare the following competing financial
interest(s): A patent protecting ARN077 and related com-
pounds was filed by the following authors: Piomelli, D.;
Bandiera, T.; Mor, M.; Tarzia, G.; Bertozzi, F.; Ponzano, S.
Figure 4. Zoomed MS/MS spectra of peptide CTSIVAQDSR acylated
by ARN077 and ARN768. Cysteine side chain fragment ions are
indicated along with the y2 fragment (262.15 m/z). S-Acylation
diagnostic fragment ion at 262.18 m/z is reported in the inset.
ACKNOWLEDGMENTS
■
We thank Sine Mandrup Bertozzi for reverse phase and chiral
HPLC purifications, Luca Goldoni for NMR technical support,
and Silvia Venzano for compounds handling.
threonine residue as respective leaving groups. These peptides
were not detected in our experiments, and the corresponding
mechanism was therefore tentatively excluded. Furthermore, no
evidence of adduct formation was found when the h-NAAA
holoenzyme was incubated with ARN077, indicating that only
the active form of the enzyme is subjected to S-acylation by this
compound. Together, these findings suggest that substituted
β-lactones, such as ARN077 and ARN768, react with Cys126
in h-NAAA to form a thioester bond. Moreover, we found no
evidence of other peptide adducts with either inhibitor, suggesting
that β-lactones selectively interact with the N-terminus cysteine.
The high-resolution mass spectrometry studies reported here
indicate that the substituted β-lactones, ARN077 and ARN768,
inhibit h-NAAA activity through a mechanism that requires
S-acylation of the catalytically active N-terminal cysteine residue
of this enzyme. These findings are consistent with the pharma-
cological properties of ARN077, which blocks h-NAAA activity in
a noncompetitive and partially reversible manner (as expected
from the covalent yet hydrolyzable nature of a thioester bond).
The results underscore the value of bottom-up proteomics as an
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