Design, Synthesis, and Evaluation of Acrylamide Derivatives as Direct NLRP3 Inflammasome Inhibitors

Article abstract of DOI:10.1002/cmdc.201600055

NLRP3 inflammasome plays a key role in the intracellular activation of caspase-1, processing of pro-inflammatory interleukin-1β (IL-1β), and pyroptotic cell death cascade. The overactivation of NLRP3 is implicated in the pathogenesis of autoinflammatory d

Full text of DOI:10.1002/cmdc.201600055

DOI: 10.1002/cmdc.201600055
Full Papers
Design, Synthesis, and Evaluation of Acrylamide
Derivatives as Direct NLRP3 Inflammasome Inhibitors
Mattia Cocco,[a] Gianluca Miglio,[a] Marta Giorgis,[a] Davide Garella,[a] Elisabetta Marini,[a]
[a]
[b]
[b]
[b]
¨
Annalisa Costale, Luca Regazzoni, Giulio Vistoli, Marica Orioli, Raıhane Massulaha-
Ahmed,[c] Isabelle Dꢀtraz-Durieux,[c] Marine Groslambert,[c] Bꢀnꢀdicte F. Py,[c] and
Massimo Bertinaria*[a]
NLRP3 inflammasome plays a key role in the intracellular acti-
vation of caspase-1, processing of pro-inflammatory interleu-
kin-1b (IL-1b), and pyroptotic cell death cascade. The overacti-
vation of NLRP3 is implicated in the pathogenesis of autoin-
flammatory diseases, known as cryopyrin-associated periodic
syndromes (CAPS), and in the progression of several diseases,
such as atherosclerosis, type-2 diabetes, gout, and Alzheimer’s
disease. In this study, the synthesis of acrylamide derivatives
and their pharmaco-toxicological evaluation as potential inhibi-
tors of NLRP3-dependent events was undertaken. Five hits
were identified and evaluated for their efficiency in inhibiting
IL-1b release from different macrophage subtypes, including
CAPS mutant macrophages. The most attractive hits were
tested for their ability to inhibit NLRP3 ATPase activity on
human recombinant NLRP3. This screening allowed the iden-
tification of 14, 2-(2-chlorobenzyl)-N-(4-sulfamoylphenethyl)a-
crylamide, which was able to concentration-dependently inhib-
it NLRP3 ATPase with an IC50 value of 74 mm. The putative
binding pose of 14 in the ATPase domain of NLRP3 was also
proposed.
Introduction
Inflammation is a key physiological response to harmful stimu-
li, including exogenous pathogens and endogenous danger
signals. Cells of the innate immune system recognize patho-
gen-associated molecular patterns (PAMPs) and danger-associ-
ated molecular patterns (DAMPs) through germline-encoded
pattern recognition receptors, such as Toll-like receptors (TLRs)
and nucleotide oligomerization domain (NOD)-like receptors
(NLRs).[1,2] A vast array of PAMPs and DAMPs has been recog-
nized so far, including microbial products (e.g., lipopolysac-
charide; LPS), molecules released after cell lysis (e.g., adenosine
triphosphate; ATP),[3,4] hypotonic stress,[5,6] and particles pro-
duced as a consequence of an altered metabolism, such as
cholesterol crystals,[7] sodium monourate crystals,[8] and b-amy-
loid aggregates.[9] Despite the well-established protective role,
uncontrolled and/or protracted inflammation is thought to
exert detrimental effects, by both exacerbating underlying
pathological processes and promoting the onset of new disor-
ders.
In the last decades, several studies have highlighted the piv-
otal role of inflammasomes, which are large intracellular pro-
tein complexes, in the molecular control of inflammatory pro-
cesses.[10–12] In particular, the NLR family pyrin-domain-contain-
ing 3 (NLRP3) inflammasome is the best characterized and the
most widely implicated pattern recognition receptor in cas-
pase-1-dependent pro-inflammatory events, including both in-
terleukin (IL)-1b maturation and cell death by pyroptosis. The
pathological role of NLRP3 inflammasome activation has been
better established in a subset of genetic disorders known as
cryopyrin-associated periodic syndromes (CAPS), also known as
cryopyrinopathies. CAPS are characterized by recurrent epi-
sodes of severe systemic inflammation and are related to
the presence of gain-of-function mutations in the NLRP3
gene.[13–15] NLRP3 and oligomeric ASC particles have also been
detected in the serum of patients with active CAPS, in which
they mediate the amplification of the inflammatory re-
sponse.[16] Moreover, compelling data have implicated inflam-
masome activation in the progression of several noncommuni-
cable diseases, such as atherosclerosis,[7] type-2 diabetes melli-
tus,[17] gout,[8] and Alzheimer’s disease.[18]
[a] M. Cocco, Dr. G. Miglio, Dr. M. Giorgis, Dr. D. Garella, Dr. E. Marini,
A. Costale, Prof. M. Bertinaria
Dipartimento di Scienza e Tecnologia del Farmaco, Universitꢀ degli Studi di
Torino, Via P. Giuria 9, 10125 Torino (Italy)
E-mail: massimo.bertinaria@unito.it
[b] Dr. L. Regazzoni, Prof. G. Vistoli, Prof. M. Orioli
Dipartimento di Scienze Farmaceutiche, Universitꢀ degli Studi di Milano,
Via Mangiagalli 25, 20133 Milano (Italy)
This evidence has increased the interest in the discovery of
agents able to prevent inflammasome activation, which is re-
garded as a promising therapeutic strategy to decrease chronic
inflammation and associated damage in different pathological
settings. To date, different approaches have been pursued,[19]
among which reversible or irreversible modification of reactive
cysteine (Cys) residues of relevant proteins seems to be the
[c] R. Massulaha-Ahmed, I. Dꢁtraz-Durieux, M. Groslambert, Dr. B. F. Py
CIRI, International Center for Infectiology Research, Inserm U1111, CNRS
UMR 5308, Ecole Normale Supꢁrieure de Lyon, Universitꢁ Lyon 1, 21 avenue
Tony Garnier, 69007 Lyon (France)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600055.
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prevalent one. Structure-based drug design would help in the
development of safer covalent drugs targeting suitably posi-
tioned Cys residues. However, structural knowledge of NLRP3
still needs to be fully understood: apart from the disulfide
bridge between Cys8 and Cys108 in the NLRP3 pyrin domain,
little is known of the other 43 Cys residues of NLRP3 (UniProt
ID: Q96P20-1).
Figure 2. Structures of approved acrylamide-based kinase inhibitors.
The crystal structure of the NLRP3 ATPase active site is not
yet available; nonetheless, this pocket in the NACHT domain
could be an interesting target to develop NLRP3 inhibitors.[20]
In fact, ATP hydrolysis is required to have active NLRP3 in the
cytosol.[21]
tives. The use of the acrylamide functionality should enable
the development of safer compounds by tuning down the re-
activity of the electrophilic warhead. The acrylamide function-
ality has already been exploited in the generation of approved
covalent drugs such as EGFR and BTK inhibitors, among others
(Figure 2).[23]
In a previous study, our group developed a series of electro-
philic warheads preventing the NLRP3-dependent and ATP-
triggered cell death of differentiated and primed THP-1 cells,
which is a cellular model of macrophage pyroptosis.[22] This
proof of concept study demonstrated that molecules endowed
with the ability to behave as Michael acceptors could efficient-
ly prevent pyroptotic cell death by inhibiting NLRP3 signaling.
A complex mechanism involving multitarget action owing to
the reactivity of the electrophile could explain this effect.
The most promising warheads identified (Figure 1, com-
pounds 1–3) also proved able to directly inhibit the NLRP3
The molecules depicted in Figure 3 were designed by modu-
lation of general structure I. In this series of compounds, we
maintained the o-chloro-substituted benzene ring, as it was
previously identified as the optimal aromatic portion in acry-
late-based inhibitors[22] in the western part of the molecules. To
investigate the role of the hydroxy group in the reactivity and/
or the cytotoxicity of this class of electrophilic compounds,
a small set of N-substituted compounds bearing a hydroxy
group in the benzylic position (i.e., compounds 4–7) was syn-
thesized and compared with a series of close analogues de-
prived of the hydroxy group (i.e., compounds 8, 9, 11). Remov-
al of the OH group was then coupled to different N-substitu-
tion patterns (i.e., compounds 10, 12–16) to explore the possi-
bility of increasing the activity through direct NLRP3 inhibition.
In particular, compounds 14 (INF58) and 15 were designed
by using a ligand-merging strategy. Both 14 and 15 share the
Michael-acceptor moiety present in compound 1 (INF4E) and
a sulfonamide or a sulfonylurea portion typical of compound
16673-34-0[24] and glyburide,[25] two known NLRP3-network in-
hibitors (Figure 4). Finally, the molecular pharmacophore dime-
rization strategy was considered in the design of compound
16 (Figure 3).
Figure 1. Electrophilic warheads that prevent NLRP3-dependent pyroptosis
and that inhibit NLRP3 ATPase activity.
ATPase activity of isolated enzyme. Unfortunately, compounds
1 and 3 exerted a certain degree of cytotoxicity,[22] which could
be related to their high reactivity. With the aim of limiting this
issue, in this work we developed a series of acrylamide deriva-
Figure 3. General structure I and structures of designed compounds 4–16.
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Figure 4. Ligand merging design of compounds 14 (INF58) and 15.
Results and Discussion
CF3CO2H to remove both the TBDMS and tert-butoxycarbonyl
(Boc) protecting groups to afford desired acrylamide 7.
Chemistry
Acrylamides 8–14, lacking the hydroxy group at the benzylic
position, were synthesized by using the route depicted in
Scheme 2. Commercially available 2-chlorobenzyl bromide was
treated with ethyl (diethoxyphosphoryl)acetate to afford phos-
phonate 20, which underwent Horner–Wadsworth–Emmons re-
action with paraformaldehyde to afford compound 21. Acrylic
ester 21 was hydrolyzed with NaOH to obtain acid 22, which
was then converted into activated ester by 23 using DCC and
NHS. N-Hydroxysuccinimidyl ester 23 was purified by flash
chromatography and was then treated with selected amines to
obtain derivatives 8–10, 12–14 in overall yields of 20–32%.
To obtain final compound 11, a deprotection step with the
use of 10% CF3CO2H in CH2Cl2 was required. Sulfonylurea de-
rivative 15 was synthesized by treating sulfonamide 14 with
cyclohexyl isocyanate in basic medium. Finally, derivative 16
bearing two electrophilic moieties was obtained by direct cou-
pling of 11 with a stoichiometric amount of 23 and an excess
amount of diisopropylethylamine (DIPEA) (Scheme 2).
Synthesis
To synthesize acrylamides 4–7 bearing a hydroxy group in the
benzylic position, we first followed the route previously de-
scribed for the synthesis of compound 4 by employing the 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU)/hydroxybenzotriazole (HOBt)-mediated cou-
pling of 2-[(2-chlorophenyl)(hydroxy)methyl]acrylic acid (2,
Figure 1) with n-propylamine.[22] However, the use of this route
with other primary amines gave rise to a complex mixture of
products with no possibility to obtain the desired compounds
with purities >95% by chromatography. This may have been
due to the presence of the highly reactive OH group in com-
pound 2. To overcome this inconvenience, we followed a differ-
ent route (Scheme 1). The hydroxy group in compound 1 was
protected by using tert-butyldimethylsilyl chloride (TBDMSCl),
and obtained intermediate 17 was subsequently hydrolyzed
with LiOH in a CH3CN/H2O mixture to afford 18 in 70% yield
from 1. Obtained acid 18 was coupled with the appropriate
amine by using dicyclohexylcarbodiimide (DCC) and N-
hydroxysuccinimide (NHS) to afford derivatives 19a–d. Deriva-
tives 19a–c were deprotected by using tetrabutylammonium
fluoride (TBAF) in THF to give desired compounds 4–6 in good
overall yields (41–49%). Compound 19d was treated with
Reactivity as Michael acceptor
The acrylamide functionality has been used in the develop-
ment of covalent kinase inhibitors.[26] Covalent inhibitors can
possess advantages over their reversible counterparts, such as
increased biochemical efficiency, longer duration of action, the
Scheme 1. Reagents and conditions: a) TBDMSCl (1.5 equiv), imidazole (2.5 equiv), DMF, RT, 16 h; b) LiOH (10 equiv), CH3CN/H2O 1:1, 608C, 16 h; c) 1. DCC
(1 equiv), NHS (1 equiv), DIPEA (1.5 equiv), THF, 08C, 15 min, RT, 2 h, 2. amine (2 equiv), RT, 16 h; d) TBAF (1.1 equiv), THF, RT, 1 h; e) CF3CO2H, CH2Cl2, RT, 2 h.
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Scheme 2. Reagents and conditions: a) ethyl (diethoxyphosphoryl)acetate (1.2 equiv), NaH (1.4 equiv), DMF, 08C, 1 h, RT, 18 h; b) paraformaldehyde (6.5 equiv),
K2CO3 (3 equiv), H2O, 908C, 16 h; c) NaOH, EtOH, RT, 16 h; d) DCC (1 equiv), NHS (1 equiv), THF, 08C, 16 h; e) for compounds 8–10, 12–14: H2NR (1.5 equiv),
CH2Cl2/DMF 2:1, Et3N (2 equiv), RT, 2–16 h; for compound 11: 1. H2NR (1.5 equiv), CH2Cl2/DMF 2:1, Et3N (2 equiv), RT, 3 h, 2. CF3CO2H (10%), CH2Cl2, RT, 1 h;
f) cyclohexyl isocyanate (1.6 equiv), K2CO3 (3 equiv), dry acetone, reflux, 16 h; g) DIPEA (1.2 equiv), DMF, RT, 1 h.
potential to avoid drug-resistance mechanisms, and the poten-
tial for improved efficacy, which could be reflected in lower
therapeutic doses.[23]
in the concentration of compound 14 was monitored by ultra-
high-performance liquid chromatography (UHPLC) for 7 h. Con-
sumption of the electrophile was detected and plotted as the
However, covalent protein modification has also been impli-
cated in immunotoxicity and idiosyncratic reactions. This kind
of toxicity is particularly evident if the covalent inhibitor is
highly reactive and/or lacks specificity.[27,28] To rationally design
electrophiles for covalent inhibition it is useful to tune both
their intrinsic reactivity and their noncovalent protein–inhibitor
interactions to optimize selectivity against the desired tar-
get(s).
natural log to check for linearity and pseudo-first-order reac-
[29]
tion kinetics, kpseudo1st
.
Under these conditions, compound 14
was indeed able to react slowly with CAM, with a kpseudo1st
value of 0.627Æ0.013 minÀ1 ꢂ10À3 (Figure S1, Supporting Infor-
mation).
Finally, to evaluate the potential of this class of compounds
to trigger idiosyncratic hypersensitivity reactions, all the com-
pounds were checked for their ability to bind human serum al-
bumin. Compounds were added into fresh human serum at
a 1 mm concentration (CH3CN <10% v/v) and were incubated
for 3 h at 378C. Serum aliquots were diluted 200-fold into H2O/
CH3CN/HCO2H (70/30/0.1 v/v/v), centrifuged, and analyzed by
ESI-MS as previously reported.[30] None of the acrylamide deriv-
atives was able to covalently react with albumin. On the con-
trary, compound 1 produced a modification of 65Æ5% of al-
bumin by generation of three covalent adducts. Collectively,
these data showed that the reactivity of this class of com-
pounds was decreased to a level that should not promote ad-
verse idiosyncratic reactions mediated by unspecific binding to
human serum albumin.
Accordingly, the electrophilic reactivities of synthesized com-
pounds 4–16 and reference compound 1 were checked by
using the kinetic cysteamine chemoassay previously de-
scribed.[22] Compounds were mixed with an equimolar amount
of cysteamine (CAM) in pH 7.4 phosphate-buffered solution at
378C by using CH3CN (12.5%) as the co-solvent. The progress
of the reaction was monitored by adding 5,5’-dithio-bis(2-nitro-
benzoic acid) (DTNB) at different time points over a period of
90 min. None of the tested acrylamides proved reactive under
the conditions used. Reference compound 1 showed a second-
order rate constant (k2) value of 0.824Æ0.017mÀ1 sÀ1 with ace-
tonitrile as the co-solvent (k2 =0.866Æ0.006mÀ1 sÀ1 with 2.5%
DMSO as the co-solvent),[22] whereas compound 4, which was
previously found to react slowly with CAM in DMSO (k2 =
0.126Æ0.005mÀ1
s
À1),[22] was unreactive with less-polar acetoni-
Pharmacology
trile. These data indicated that the reactivity of this series of
electrophilic compounds was efficiently tuned down by em-
ploying the acrylamide functionality.
Antipyroptotic activity and cytotoxicity
The synthesized compounds were initially evaluated for their
ability to prevent the NLRP3-dependent pyroptosis of phorbol
myristate acetate (PMA)-differentiated THP-1 cells. In this
model, the NLRP3 inflammasome-dependent events follow
a two-stage process. In the first stage (i.e., priming), NLRP3 ex-
To demonstrate the ability of these acrylamides to behave as
Michael acceptors, model compound 14 was treated with
10 molar equivalents of CAM in pH 7.4 phosphate-buffered so-
lution at 378C by using CH3CN as the co-solvent. The decrease
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pression is induced through NF-kB-mediated signaling. In the
second stage (i.e., activation), NLRP3 inflammasome is assem-
bled and activated following cell exposure to different stimuli.
In our experiments, THP-1 cells were primed with LPS and acti-
vated with ATP, as previously described.[22] Cell death was
quantified by measuring lactate dehydrogenase (LDH) activity
in the cell supernatants. First, to perform a preliminary compa-
rative evaluation, cells were exposed (1 h before the ATP pulse)
to the synthesized compounds (i.e., 4–16; all at 10 mm). The
antipyroptotic effects were determined and expressed as a de-
crease in pyroptosis relative to vehicle-alone-treated cells
(Table 1). Moreover, as structurally related compounds were
demonstrated to exert significant cytotoxicity,[22] the effects of
increasing concentrations of the newly synthesized derivatives
on the viability of THP-1 cells were evaluated (Table 1).
group at the a-carbon atom.[29] Notably, a marked decrease in
cell viability was measured for THP-1 cells that were cultured
in the presence of a-hydroxyalkyl-substituted acrylamides 4–6
the concentration required to decrease cell viability by 50%
(TC50) was in the range from 43.4Æ5.2 to 83.5Æ2.2 mm],
whereas higher TC50 values (>100 mm) were generally deter-
mined for compounds lacking the OH group in the X position
(Table 1). These results indicated that removing the OH group
in the a position decreased the cytotoxicity of these acrylam-
ide derivatives. Consequently, further development of OH-sub-
stituted compounds was discontinued, and chemical modula-
tion of the N substituent was performed by using compound 9
(antipyroptotic effect=28.0Æ6.6%; TC50 >100 mm) as the pre-
ferred scaffold. In addition, compound 8, lacking significant an-
tipyroptotic activity, was also considered as a model to gain
more complete insight into the activity of this class of com-
pounds.
The ATP-triggered cell death of THP-1 cells (48.0Æ6.8%) py-
roptosis vs. untreated cells was prevented by both compound
1 (positive control; 80.9Æ5.2%) and the new acrylamide deriv-
atives; their effects ranged from 7.5Æ5.6% to 69.8Æ2.6%.
Compounds 4, 5, and 7, bearing a hydroxy group in the X po-
sition (structure I), exerted higher antipyroptotic effects than
corresponding analogues 8, 9, and 11 lacking the hydroxy
group (see 4 vs. 8, 5 vs. 9, and 7 vs. 11 in Table 1). Higher in-
trinsic reactivity, although not measureable with the employed
assay, might be responsible for the enhanced antipyroptotic
effect of the oxygenated series of compounds. In agreement
with this hypothesis, the reactivity of a-methyl-N-arylacryla-
mides was recently shown to increase by introducing a hydroxy
To evaluate whether the higher activity of 9 relative to that
of 8 could be attributed to increased lipophilicity (clogP=3.78
and 3.06, respectively), N-phenylethyl derivative 10 (clogP=
4.10) was synthesized. Relative to that shown by compound 9,
this derivative exerted larger antipyroptotic effects (49.6Æ
5.7%), which thus confirmed the hypothesis that the relative
antipyroptotic activity of these related compounds could
depend on their lipophilicity. In addition, upon substituting the
N-propyl chain in 8 with an amino group at the terminal posi-
tion, less lipophilic, still poorly active, and more cytotoxic com-
pound 11 was obtained (clogP=1.94; anti-pyroptotic effect=
15.7Æ0.7%; TC50 =46.5Æ5.5 mm). The activity was slightly in-
creased upon introducing a methoxycarbonyl (see compound
12) or a carboxy group (see compound 13) in the same posi-
tion (Table 1). Of note, relative to compound 8, derivatives 11–
13 are more hydrophilic (clogP=1.94–2.77), which thus indi-
cates that factors other than lipophilicity alone are likely re-
sponsible for the pharmaco-toxicological activity of these de-
rivatives. Consistently, compound 16, formally obtained by di-
merization of 8, exerted only modest antipyroptotic effects
(25.2Æ8.1%), in spite of a high degree of lipophilicity (clogP=
4.75). Finally, to obtain more insight into the structure–activity
relationship of the acrylamide moiety, the effects exerted by
compounds 14 and 15 were studied. Interestingly, 16673-34-0-
derived compound 14 exerted effects (45.8Æ2.9% pyroptosis
inhibition) similar to those of 10 despite the decreased lipophi-
licity (clogP=2.27), whereas highly lipophilic glyburide-derived
compound 15 (clogP=4.40) exerted only modest antipyrop-
totic effects in our model (17.3Æ2.8%) pyroptosis inhibition.
To better characterize the antipyroptotic activity of these
acrylamides, the concentration–response curve of representa-
tive derivatives 8–10, 12, and 14 was studied (Figure 5). The
ATP-triggered pyroptosis of THP-1 cells was prevented by
these compounds in a concentration-dependent manner: the
IC50 values ranged from 12.7 to 53.1 mm. Collectively, these
data demonstrate that weak electrophiles, obtained by chemi-
cal modulation of structure I (X=H), can be efficiently used to
design and develop nontoxic NLRP3 inhibitors acting as anti-
pyroptotic agents.
Table 1. Inhibitory effect of INF4E (1) and compounds 4–16 on pyroptot-
ic death of THP-1 cells and cytotoxicity (TC50) in THP-1 cells.
Compd
Pyroptosis
decrease [%][a]
TC50 [mm][b]
clogP[c]
1 (INF4E)
4
5
6
7
8
9
10
80.9Æ5.2
31.2Æ6.8
69.8Æ2.6
45.9Æ5.2
35.8Æ5.3
7.5Æ5.6
67.0Æ3.4
83.5Æ2.2
44.5Æ1.2
43.4Æ5.2
>100
>100
>100
>100
46.5Æ5.5
>100
2.59
2.09
2.75
3.07
0.97[d]
3.06
3.78
4.10
1.94[d]
2.77
2.31[d]
2.27
4.40
4.75
28.0Æ6.6
49.6Æ5.7
15.7Æ0.7
34.7Æ3.5
38.5Æ10.8
45.8Æ2.9
17.3Æ2.8
25.2Æ8.1
11
12
13
>100
>100
>100
>100
14 (INF58)
15
16
[a] Determined by measuring LDH release in PMA-differentiated and LPS-
primed (5 mgmLÀ1; 4 h) THP-1 cells. Compounds were administered at
10 mm. After 1 h, pyroptosis was triggered with ATP (5 mm). LDH activity
was measured 1 h after ATP challenge. Data are the percentage of pyrop-
tosis decrease versus vehicle alone and are the meanÆSEM of three inde-
pendent experiments. [b] THP-1 cells were exposed to increasing concen-
trations (0.1–100 mm) of each compound, and cell viability was measured
at 72 h by MTT assay; TC50 is the molar concentration of compound re-
quired to decrease cell viability by 50%; data are the meanÆSEM of
three independent experiments. [c] clogP was calculated by using Chem-
BioDraw Ultra 12.0 (CambridgeSoft). [d] clogP was calculated for the neu-
tral form.
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All tested compounds significantly inhibited IL-1b secretion if
added simultaneously with LPS regardless of the type of mac-
rophage (Figure 6). Most compounds were also effective if
added 15 min only before ATP treatment, which suggests that
these compounds block NLRP3 activation in the ATP-driven
second step and not LPS-dependent priming. Consistently,
none of the compounds affected TLR4-dependent NLRP3-inde-
pendent tumor necrosis factor (TNF)-a release, which demon-
strates that they do not target activation of inflammatory
genes transcription triggered by LPS.
Inhibition of IL-1b release from CAPS mutant macrophages
Gain-of-function mutations in the NLRP3 gene cause hereditary
autoinflammation events referred to as CAPS that correspond
to a disease spectrum of three clinically defined disorders: fam-
ilial cold autoinflammatory syndrome (FCAS), Muckle–Wells
syndrome (MWS), and neonatal-onset multisystem inflammato-
ry disease (NOMID). To evaluate the potential of these com-
pounds in CAPS treatment, we tested their ability to dampen
the activity of CAPS-associated NLRP3 mutants. We reconstitut-
ed mouse immortalized NLRP3 KO BMDMs with murine NLRP3
R258W and A350V (corresponding to the human R260W and
A352V mutations, respectively, both associated with MWS) and
NLRP3 L351P (corresponding to the human L353P mutation as-
sociated with FCAS).[31–33] As previously described,[34,35] LPS pri-
ming is sufficient to trigger IL-1b secretion from macrophages
expressing NLRP3 R258W, A350V, and L351P.
Figure 5. Concentration–response curves of the antipyroptotic effects of
compounds 8, 9, 10, 12, and 14; IC50 values (meanÆSEM of three independ-
ent experiments) are reported in brackets; n.a.: not applicable.
Inhibition of IL-1b release from macrophages
We next verified the ability of the most interesting compounds
to inhibit NLRP3-dependent IL-1b secretion in macrophages.
Murine-immortalized
bone-marrow-derived
macrophages
(BMDMs), primary BMDMs, and primary inflammatory perito-
neal macrophages were primed with LPS and then treated
with extracellular ATP to activate NLRP3 (Figure 6). Compounds
5, 10, 12, 14, and 16 were added to the macrophages either si-
multaneously to LPS treatment or 15 min prior to ATP pulse.
Figure 6. Effects of compounds 5, 10, 12, 14, and 16 on IL-1b and TNF-a release in A) murine immortalized macrophages, B) bone-marrow-derived macro-
phages, and C) primary peritoneal macrophages. Macrophages were treated with LPS (50 ngmLÀ1, 8 h) and ATP (2 mm, 30 min). Compounds (20 mm) were
added simultaneously with LPS (8 h) or 15 min before ATP treatment (45 min total). Secretion of IL-1b (top row) and TNF-a (bottom row) in culture superna-
tants were measured by ELISA. *p<0.05, **p<0.01 versus LPS- and ATP-treated cells; t-test. Values are the meanÆSD; results are representative of two inde-
pendent experiments, each performed in duplicate.
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Figure 8. A) Inhibition of NLRP3 ATPase activity of selected compounds 5,
10, 12, and 14; values are the meanÆSEM of three independent experi-
ments. *p<0.05, **p<0.01 versus vehicle-treated enzyme; t-test. B) Concen-
tration–response curve for derivative 14 (INF58).
Figure 7. Effects of compounds 10, 12, and 14 on A) IL-1b and B) TNF-a re-
leased by BMDMs expressing NLRP3 WT, R258W, A350V, or L351P mutants.
Immortalized murine NLRP3 KO BMDMs reconstituted with wild-type or
mutant NLRP3 were treated with doxycycline (0.1 mgmLÀ1, 24 h), LPS
(50 ngmLÀ1, 8 h), and ATP (2 mm, 30 min). Compounds (20 mm) were added
simultaneously with LPS (8 h). Secretion of IL-1b and TNF-a in culture super-
natants were measured by ELISA. *p<0.05, **p<0.01 versus LPS-treated
cells in mutant BMDMs; *p<0.05, **p<0.01 versus LPS and ATP in wild-type
BMDMs; t-test. Values are the meanÆSD; results are representative of two
independent experiments, each performed in duplicate.
reported in Figure 8A. All the compounds inhibited NLRP3
ATPase activity upon testing at 100 mm, whereas only deriva-
tives 5 and 14 inhibited the enzyme at 50 mm. We next verified
the ability of compound 14, able to prevent pyroptotic cell
death and to inhibit IL-1b release from different macrophage
lines and devoid of significant toxicity, to concentration-de-
pendently inhibit ATPase activity of NLRP3 protein (Figure 8B).
The enzymatic activity was indeed decreased in a concentra-
tion-dependent manner with a calculated IC50 of 74 mm (95%
confidence interval: 63–86 mm), which demonstrated that
NLRP3 was a direct target of compound 14 (INF58).
Compounds 10, 12, and 14 selected for this kind of experi-
ment inhibited ATP-dependent IL-1b secretion in LPS-primed
macrophages expressing wild-type (WT) NLRP3 (Figure 7).
Compound 10 inhibited LPS-induced IL-1b secretion by macro-
phages expressing NLRP3 R258W, A350V, and L351P. Com-
pound 14 inhibited LPS-induced IL-1b secretion by macro-
phages expressing NLRP3 R258W and L351P, whereas the
effect of compound 12 was restricted to macrophages express-
ing R258W.
Computational studies
As discussed above, compound 14 and other analogues from
this class of acrylamide derivatives proved able to inhibit
NLRP3 ATPase activity. We then applied a computational ap-
proach to predict the putative binding mode of 14 and other
synthesized compounds in the ATP binding pocket in the
NACHT domain of NLRP3. As explained in the Computational
methods subsection of the Experimental Section below and as
depicted in Figure 9 (in orange), preliminary docking analysis
involved the natural ligand ATP with the view to assess the re-
liability of the modeled binding pocket further. Satisfactorily,
the computed complex is in line with the reported data,[36] as
the phosphate groups are seen to stabilize ion pairs with
Lys232 and Arg237 plus H-bonds with Thr233 and His522. The
Arg237 residue also contacts the ATP adenine base, the 6-
amino group of which elicits a reinforced H-bond with Lys238.
Inhibition of NLRP3 ATPase activity
The ability of compounds 5, 10, 12, and 14 to inhibit the
NLRP3 ATPase activity was tested on purified human recombi-
nant enzyme. Human recombinant NLRP3 was incubated at
378C in the presence of different concentrations (50 and
100 mm) of tested compounds for 15 min. ATP was then added,
and the mixture was incubated at 378C for another 40 min.
The amount of ATP converted into adenosine diphosphate
(ADP) was determined by luminescence by using the ADP-Glo
assay. The obtained results, expressed as percentage of residu-
al enzyme activity with respect to vehicle-treated enzyme, are
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ditional explanations for the measured ATPase activity. Indeed,
compounds 5, 10, and 12 reveal putative complexes that are
very similar to that observed for 14, in which the reinforced H-
bonds with Lys232 and Arg237 are replaced by extended
charge-transfer interactions with the distal phenyl ring in 5
and 10 and by H-bonds with the ester group in compound 12.
Conclusions
With the aim to obtain covalent NLRP3 inhibitors, we designed
and synthesized a series of acrylamide derivatives endowed
with low intrinsic electrophilicity that was reflected in avoided
unspecific idiosyncratic and cytotoxic effects. Most of the syn-
thesized compounds prevented ATP-triggered, NLRP3-depen-
dent pyroptotic cell death of PMA-differentiated THP-1 cells,
moreover, they showed no significant cytotoxicity. The ob-
tained results allowed the selection of 2-(2-chlorobenzyl)-N-
phenethylacrylamide (10), methyl 4-[2-(2-chlorobenzyl)acryla-
mide]butanoate (12), and 2-(2-chlorobenzyl)-N-(4-sulfamoyl-
phenethyl)acrylamide (14) able to prevent pyroptosis in a con-
centration-dependent manner and endowed with a promising
pharmaco-toxicological profile. Derivatives 10, 12, and 14 were
able to inhibit IL-1b release from different macrophage lines,
with no effect on TLR-4 dependent TNF-a production. Com-
pounds 10, 12, and 14 were also effective in inhibiting IL-1b
release from macrophages bearing CAPS-associated NLRP3
mutants. Compound 14 (INF58) inhibited NLRP3 ATPase activi-
ty with an IC50 value of 74 mm, which resulted in a good hit
compound to design new and improved direct NLRP3 inhibi-
tors. In silico prediction of the binding mode of 14 in the
ATPase catalytic pocket indicated that a putative interaction
with the Cys419 residue might account for this activity. Binding
studies to identify the binding site(s) of 14 (INF58) to human
recombinant NLRP3 are in progress, and the results will be re-
ported in due course.
Figure 9. Comparison of the putative poses as computed for ATP (orange)
and 14 (INF58, light blue). The dashed black line defines the path between
the supposedly reactive Cys419 residue and the b-carbon atom of the acryl-
amide group.
Lastly, the ATP sugar moiety is engaged in H-bonds with
Tyr381 and to a minor extent with Trp416.
As shown in Figure 9 (in light blue), compound 14 is accom-
modated in the regions of the binding site that harbor the
ribose ring and the phosphate groups of ATP, where it can
elicit a set of key interactions that can be schematized as fol-
lows.
The 2-chlorophenyl moiety is engaged in extended p–p
stacking interactions with surrounding aromatic residues such
as Tyr381, Tyr385, and Trp416. This last residue also contacts
the reactive vinyl group of the acrylamide moiety and might
have a key role in approaching the reactive ligand moiety to
Cys419 (see below). The amide oxygen atom is involved in
a clear H-bond with Tyr381, whereas the phenyl sulfonamide
moiety mimics the ATP phosphate groups by stabilizing a rich
set of charge-transfer interactions and reinforced H-bonds in-
volving Lys232, Thr233, Arg237, and His522. Such an extended
network of contacts should maintain the inhibitor in a pose
stably conducive to the formation of a covalent adduct with
the protein. In detail, the reactive acrylamide is surrounded by
three rather close cysteine residues (i.e., Cys409, Cys415, and
Cys419). Among them, Cys419 seems to be the most reactive
one for two main reasons. First, it is the closest residue, as its
distance to the acrylamide is 8.3 ꢃ (the corresponding distan-
ces for Cys409 and Cys415 are 14.9 and 11.1 ꢃ, respectively)
and between Cys419 and the acrylamide there are no residues
that can obstruct the approaching, whereas the hypothetic
path for Cys409 and Cys415 is hindered by other residues as
exemplified by Pro412. Second, Cys419 is surrounded by resi-
dues, such as Tyr385, which should enhance its reactivity by
stabilizing its thiolate form with a mechanism already observed
for albumin and glutathione transferase enzymes.[37]
Experimental Section
General procedures
All reactions were monitored by TLC on Merck 60 F254 (0.25 mm)
plates, which were visualized by UV inspection and/or by spraying
with KMnO4 (0.5 g in 100 mL 0.1n NaOH). Flash chromatography
(FC) purifications were performed by using silica gel Fluka with
60 mesh particles. 1H NMR and 13C NMR spectra were registered
with a Bruker Avance 300 spectrometer at 300 and 75 MHz, respec-
tively. Coupling constants (J) are given in Hertz (Hz) and chemical
shifts (d) are given in ppm calibrated to tetramethylsilane as an in-
ternal standard. The following abbreviations are used to describe
multiplicities: s=singlet, d=doublet, t=triplet, q=quadruplet,
m=multiplet, and br=broad signal. Low-resolution mass spectra
were recorded with a Finnigan-MAT TSQ700 in chemical ionization
(CI) mode by using isobutane. Melting points were measured with
a capillary apparatus (Bꢄchi 540). The purities of the compounds
were checked by UHPLC (PerkinElmer) Flexar 15, equipped with
a UV/Vis diode array detector by using an Acquity UHPLC CSH
Phenyl-Hexyl 1.7 mm 2.1ꢂ50 mm column (Waters) and H2O/CH3CN
and H2O/CH3OH solvent systems. Detection was performed at l=
The other simulated derivatives show similar poses and all
are characterized by the capacity to insert the 2-chlorophenyl
ring in the above-described subpocket lined by several aromat-
ic residues that have the dual role of stabilizing the complex
and of constraining the acrylamide moiety in a pose conducive
to Michael addition. Notably, the obtained docking results and
in particular the observed interactions stabilized by the varying
moieties linked to the acrylamide nitrogen atom can offer ad-
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200, 215, and 254 nm. The analytical data confirmed that the puri-
ties of the products were ꢀ95%.
mine (0.190 mL, 2.78 mmol) to obtain 19b as a pale-yellow oil
(0.420 g, 74%): Rf =0.72 (petroleum ether/EtOAc 9:1); 1H NMR
(300 MHz, CDCI3): d=7.67 (d, J=7.6 Hz, 1H), 7.35–7.23 (m, 3H),
6.26 (br, 1H), 5.85 (s, 1H), 5.76 (s, 1H), 5.33 (s, 1H), 1.93–1.83 (m,
2H), 1.75–1.58 (m, 3H), 1.41–1.32 (m, 2H), 1.22–1.13 (m, 4H), 0.96
(s, 9H), 0.24 (s, 3H), À0.21 ppm (s, 3H); 13C NMR (75 MHz, CDCl3):
d=168.6, 144.8, 141.9, 131.2, 129.4, 128.5, 128.0, 127.1, 122.6, 74.1,
51.6, 43.3, 31.0, 25.9, 25.7, 24.0, À0.5, À4.0 ppm; MS (CI, isobutane):
m/z (%): 410 (32), 408 (100) [M+H]+.
Synthesis
Ethyl
2-{[(tert-butyldimethylsilyl)oxy](2-chlorophenyl)methyl}-
acrylate (17): Imidazole (1.70 g, 25 mmol) was added to a stirred
solution of 1[22] (2.41 g, 10 mmol) in DMF (3 mL). After complete
dissolution, tert-butyldimethylsilyl chloride (2.26 g, 15 mmol) was
added portionwise, and the solution was stirred overnight at RT.
Water (20 mL) was added, and the mixture was extracted with
EtOAc (3ꢂ30 mL), washed with brine (30 mL), dried (Na2SO4), fil-
tered, and concentrated under reduced pressure. Purification by
silica gel chromatography (petroleum ether/EtOAc 9:1) gave 17 as
a colorless oil (2.91 g, 82%): Rf =0.87 (petroleum ether/EtOAc 9:1);
1H NMR (300 MHz, CDCI3): d=7.44–7.11 (m, 4H), 6.28 (s, 1H), 6.05
(s, 1H), 5.82 (s, 1H), 4.09 (q, J=7.1 Hz, 2H), 1.17 (t, J=7.1 Hz, 3H),
0.85 (s, 9H), 0.09 (s, 3H) À0.12 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=168.5, 146.2, 142.9, 135.6, 132.2, 132.1, 131.5, 129.6,
127.9, 71.9, 63.5, 28.6, 21.0, 17.0, À2.0, À2.1 ppm; MS (CI, isobu-
tane): m/z (%): 357 (32), 355 (100) [M+H]+.
N-Benzyl-2-{[(tert-butyldimethylsilyl)oxy](2-chlorophenyl)meth-
yl}acrylamide (19c): The reaction was run with benzylamine
(0.304 mL, 2.78 mmol) to obtain 19c as a pale-yellow oil (0.468 g,
81%): Rf =0.70 (petroleum ether/EtOAc 9:1); 1H NMR (300 MHz,
CDCI3): d=7.39–7.15 (m, 9H), 6.23 (br, 1H), 5.79 (s,1H), 5.31 (s, 1H),
5.18 (s, 1H), 3.87 (s, 2H), 0.94 (s, 9H), 0.22 (s, 3H), À0.33 ppm (s,
3H); 13C NMR (75 MHz, CDCl3): d=168.2, 143.1, 138.9, 136.3, 134.6,
131.5, 130.0, 129.1, 128.4, 128.0, 127.8, 127.3, 121.1, 73.8, 44.1, 32.0,
25.7, À1.5, À3.9 ppm; MS (CI, isobutane): m/z (%): 418 (32), 416
(100) [M+H]+.
tert-Butyl [3-(2-{[(tert-butyldimethylsilyl)oxy](2-chlorophenyl)me-
thyl}acrylamido)propyl]carbamate (19d): The reaction was run
with N-Boc-1,3-propanediamine (0.484 g, 2.78 mmol) to obtain 19d
as a off-white oil (0.403 g, 60%): Rf =0.61 (petroleum ether/EtOAc
2-{[(tert-Butyldimethylsilyl)oxy](2-chlorophenyl)methyl}acrylic
acid (18): LiOH (1.96 g, 82.0 mmol) was added to a solution of
compound 17 (2.91 g, 8.20 mmol) in CH3CN/H2O (1:1, 20 mL). The
mixture was stirred at 608C overnight. The solvent was evaporated
under reduced pressure. The residue was diluted with 1n HCl
(10 mL) and extracted with EtOAc (4ꢂ25 mL). The combined or-
ganic phase was washed with brine (30 mL), dried (Na2SO4), fil-
tered, and concentrated under reduced pressure. Purification by
silica gel chromatography (CH2Cl2/MeOH 97:3 to 9:1) gave 18 as
a colorless oil (2.28 g, 85%): Rf =0.68 (CH2Cl2/MeOH 9:1); 1H NMR
(300 MHz, CDCI3): d=10.04 (br, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.32–
7.16 (m, 3H), 6.42 (s, 1H), 6.03 (s, 1H), 5.85 (s, 1H), 0.87 (s, 9H),
0.10 (s, 3H), À0.09 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=
171.2, 142.9, 140.2, 133.3, 130.0, 129.7, 129.4, 128.6, 127.4, 69.5,
26.4, 28.8, À4.2, À4.3 ppm; MS (CI, isobutane): m/z (%): 329 (37),
327 (100) [M+H]+.
1
9:1); H NMR (300 MHz, CDCI3): d=7.35–7.13 (m, 4H), 7.03 (br, 1H),
5.79 (s,1H), 5.63 (s, 1H), 5.25 (br, 1H), 5.10 (s, 1H), 3.33 (t, J=
7.1 Hz, 2H), 3.08 (t, J=8.1 Hz, 2H), 1.61–1.57 (m, 2H), 1.42 (s, 9H),
0.96 (s, 9H), 0.18 (s, 3H), À0.32 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=168.7, 156.6, 142.4, 136.3, 134.0, 131.1, 129.5, 128.0,
126.7, 119.7, 79.2, 74.2, 37.0, 36.3, 31.1, 30.0, 28.5, 25.7, À1.5,
À3.8 ppm; MS (CI, isobutane): m/z (%): 485 (32), 483 (100)
[M+H]+.
General procedure for synthesis of compounds 4–7: Tetrabutyl-
ammonium fluoride (1.0m in THF, 0.77 mL, 0.77 mmol) was added
to a solution of compound 19a–d (0.70 mmol) in dry THF (5 mL) at
08C. The mixture was stirred at RT for 1 h and then diluted with
water (15 mL) and extracted with CH2Cl2 (3ꢂ20 mL). The organic
phase was washed with brine (20 mL) and water (20 mL), dried
(Na2SO4), filtered, and concentrated. The products were purified by
silica gel chromatography (petroleum ether/EtOAc 95:5).
General procedure for the synthesis of compounds 19a–d: Car-
boxylic acid 18 (0.456 mg, 1.39 mmol) was dissolved in THF
(10 mL); then, DIPEA (0.364 mL, 2.10 mmol) and DCC (0.288 g,
1.39 mmol) were added. The mixture was stirred for 15 min at 08C
and then at RT for another 2 h. The amine (2 equiv) was then
added, and the mixture was stirred at RT overnight. The mixture
was filtered, and the liquid phase was extracted with EtOAc (4ꢂ
30 mL). The organic phase was washed with saturated NH4Cl solu-
tion (20 mL), dried (Na2SO4), filtered, and concentrated. The prod-
uct was purified by silica gel chromatography (petroleum ether/
EtOAc 9:1).
2-[(2-Chlorophenyl)(hydroxy)methyl]-N-propylacrylamide (4): Ob-
tained as a white solid (0.169 g, 95%) starting from 19a: character-
ization data were in agreement with the previously reported
data.[20]
N-Benzyl-2-[(2-chlorophenyl)(hydroxy)methyl]acrylamide (5): Ob-
tained as a white solid (0.169 g, 80%) starting from 19b: Rf =0.52
(petroleum ether/EtOAc 85:15); mp: 94.3–95.68C; 1H NMR
(300 MHz, CDCI3): d=7.63 (d, J=7.6 Hz, 1H), 7.33–7.14 (m, 8H),
6.78 (br, 1H), 5.85 (d, J=4.9 Hz, 1H), 5.78 (s, 1H), 5.30 (s, 1H), 3.57
(d„ J=5.1 Hz,1H), 3.16 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=
168.4, 143.2, 138.5, 136.6, 134.8, 131.7, 129.9, 129.1, 128.6, 128.1,
127.7, 127.4, 120.1, 66.7, 44.0 ppm; MS (CI, isobutane): m/z (%): 304
(32), 302 (100) [M+H]+.
2-{[(tert-Butyldimethylsilyl)oxy](2-chlorophenyl)methyl}-N-
propylacrylamide (19a): The reaction was run with propylamine
(0.228 mL, 2.78 mmol) to obtain 19a as a colorless oil (0.317 g,
62%): Rf =0.74 (petroleum ether/EtOAc 8:2); 1H NMR (300 MHz,
CDCI3): d=7.55 (d, J=7.8 Hz,1H),7.29–7.19 (m, 3H), 6.68 (br, 1H),
5.95 (s, 1H), 5.92 (s, 1H), 5.53 (s, 1H), 3.24–3.21 (m, 2H), 1.55–1.48
(m, 2H), 0.90 (s, 9H), 0.87 (t, J=7.1 Hz, 3H), 0.12 (s, 3H),
À0.02 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=166.6, 144.4,
138.9, 132.2, 129.6, 128.8, 128.3, 126.7, 121.6, 72.1, 41.1, 25.7, 22.8,
18.1, 11.5, 0.0, À5.0 ppm; MS (CI, isobutane): m/z (%): 370 (32), 368
(100) [M+H]+.
2-[(2-Chlorophenyl)(hydroxy)methyl]-N-cyclohexylacrylamide (6):
Obtained as a white solid (0.179 g, 87%) starting from 19c: Rf =
0.41 (petroleum ether/EtOAc 9:1); mp: 119.4–120.88C; 1H NMR
(300 MHz, CDCI3): d=7.69 (d, J=7.6 Hz, 1H), 7.34-.7.23 (m, 3H),
6.27 (br, 1H), 5.84 (s, 1H), 5.76 (s, 1H), 5.30 (s, 1H), 4.28 (br, 1H),
1.94–1.87(m, 2H), 1.75–1.60 (m, 3H), 1.41–1.32 (m, 2H), 1.22–
1.13 ppm (m, 4H); 13C NMR (75 MHz, CDCl3): d=168.6, 144.6, 142.1,
2-{[(tert-Butyldimethylsilyl)oxy](2-chlorophenyl)methyl}-N-cyclo-
hexylacrylamide (19b): The reaction was run with cyclohexyla-
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131.2, 129.3, 128.3, 127.7, 127.0, 122.3, 66.7, 51.5, 43.3, 25.7,
NH4Cl solution (15 mL), dried (Na2SO4), filtered, and concentrated.
Purification by silica gel chromatography (CH2Cl2/EtOAc 99:1) gave
23 as a white solid (3.00 g, 84%): Rf =0.37 (CH2Cl2/EtOAc 99:1);
mp: 131.7–133.28C; 1H NMR (300 MHz, CDCl3): d=7.38–7.18 (m,
4H), 6.55 (s, 1H), 5.62 (s, 1H), 3.80 (s, 2H), 2.82 ppm (s, 4H);
13C NMR (75 MHz, CDCl3): d=169.7, 135.3, 134.9, 134.2, 131.6,
130.3, 130.1, 129.4, 128.5, 127.3, 35.7, 26.0 ppm; MS (CI, isobutane):
m/z (%): 296 (32), 294 (100) [M+H]+.
24.2 ppm; MS (CI, isobutane): m/z (%): 296 (32), 294 (100) [M+H]+.
3-{2-[(2-Chlorophenyl)(hydroxy)methyl]acrylamido}propan-1-
aminium 2,2,2-trifluoroacetate (7): Trifluoroacetic acid (0.500 mL,
6.49 mmol) was added dropwise to a solution of 19d in CH2Cl2
(5 mL). The mixture was stirred at RT for 2 h and then concentrated
to dryness. The white solid was washed several times with CH2Cl2
(2ꢂ20 mL) and diethyl ether (3ꢂ20 mL), and 7 was obtained as
1
a white amorphous solid (140.6 mg, 99%); H NMR (300 MHz, D2O):
General procedure for the synthesis of compounds 8–14: The
amine (2.00 mmol) was added to a stirred solution of 23 (0.29 g,
1.00 mmol) in CH2Cl2/DMF (2:1, 6 mL) at RT; this was followed by
the addition of triethylamine (3.00 mmol). The mixture was stirred
at RT for 2 h-24 h. The mixture was diluted with water (30 mL),
acidified with 1n HCl (20 mL), and then extracted with EtOAc (4ꢂ
30 mL). The organic phase was washed with brine (35 mL), dried
(Na2SO4), filtered, and concentrated to obtain the crude product.
d=7.47–7.29 (m, 4H), 5.83 (s,1H), 5.76 (s, 1H), 5.26 (s, 1H), 3.17–
3.11 (m, 2H), 2.71 (t, J=7.1 Hz, 2H), 1.69–1.55 ppm (m, 2H);
13C NMR (75 MHz, D2O): d=172.5, 142.2, 136.1, 134.1, 131.3, 129.9,
128.7, 127.5, 121.2, 118.5, 114.8, 68.9, 40.5, 34.4, 26.9 ppm.
Ethyl 3-(2-chlorophenyl)-2-(diethoxyphosphoryl)propanoate (20):
60% NaH in mineral oil (1.64 g, 41.0 mmol) was added to a stirred
solution of ethyl (diethoxyphosphoryl)acetate (7.86 g, 35.1 mmol)
in DMF (30 mL) at 08C, and the mixture was stirred at 08C for 1 h.
Then, 2-chlorophenylmethyl bromide (6.00 g, 29.3 mmol) was
added at 08C, and the mixture was stirred at RT for 18 h. The reac-
tion was quenched with water, and the mixture was extracted with
EtOAc (50 mLꢂ3). The combined organic layer was washed with
water (50 mL) and brine (50 mL), dried (Na2SO4), and concentrated.
Purification by silica gel chromatography (petroleum ether/EtOAc
from 7:3 to 1:1) gave 20 as a colorless oil (7.25 g, 71%): Rf =0.29
(petroleum ether/EtOAc 7:3); 1H NMR (300 MHz, CDCI3): d=7.36–
7.09 (m, 4H), 4.26–4.06 (m, 6H), 3.52–3.27 (m, 3H), 1.39–1.33 (m,
6H), 1.15 ppm (t, J=7.0 Hz, 3H).[38]
2-(2-Chlorobenzyl)-N-propylacrylamide
(8):
Propylamine
(118.2 mg, 2.00 mmol) was used as the amine, and the mixture was
stirred for 6 h. Purification by silica gel chromatography (CH2Cl2 to
CH2Cl2/EtOAc 99:1) gave 8 as a pale-yellow oil (183.5 mg, 77%):
Rf =0.63 (CH2Cl2/EtOAc 9:1); 1H NMR (300 MHz, CDCl3): d=7.37–
7.17 (m, 4H) 5.92 (br, 1H), 5.71 (s,1H), 5.11 (s, 1H), 3.78 (s, 2H), 3.24
(t, J=7.1 Hz, 2H), 1.51 (m, 2H), 0.87 ppm (t, J=7.2 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=166.9, 140.4, 137.3, 134.3, 128.6,
128.5, 127.2, 126.9, 124.3, 40.8, 37.1, 23.2, 11.2 ppm; MS (CI, isobu-
tane): m/z (%): 240 (32), 238 (100) [M+H]+.
N-Benzyl-2-(2-chlorobenzyl)acrylamide
(9):
Benzylamine
Ethyl 2-(2-chlorobenzyl)acrylate (21): A solution of potassium car-
bonate (8.62 g, 62.4 mmol) in water (60 mL) was added to a stirred
mixture of 20 (7.25 g, 20.8 mmol) and paraformaldehyde (4.13 g,
137 mmol) in water (60 mL) at RT. The mixture was stirred at 908C
overnight. After cooling to RT, the mixture was extracted with
EtOAc (3ꢂ50 mL), and the organic layer was washed with brine
(50 mL), dried (Na2SO4), filtered, and concentrated. Purification by
silica gel chromatography (petroleum ether/EtOAc 7:3) gave 21 as
a colorless oil (3.06 g, 66%): Rf =0.45 (petroleum ether/EtOAc
(214.3 mg, 2.00 mmol) was used as the amine, and the mixture
was stirred for 8 h. Purification by silica gel chromatography
(CH2Cl2 to CH2Cl2/EtOAc 98:2) gave 9 as a white solid (157.2 mg,
56%): Rf =0.81 (CH2Cl2/EtOAc 9:1); mp: 87.1–88.08C; 1H NMR
(300 MHz, CDCl3): d=7.38–7.21 (m, 9H), 6.23 (br, 1H), 5.76 (s,1H),
5.18 (s, 1H), 4.50 (s, 2H), 3.84 (s, 2H); 13C NMR (75 MHz, CDCl3): d=
168.4, 143.2, 138.5, 136.4, 134.8, 131.7, 130.0, 129.1, 128.5, 128.1,
127.9, 127.4, 120.1, 44.1, 36.3 ppm; MS (CI, isobutane): m/z (%): 288
(32), 286 (100) [M+H]+.
1
95:5); H NMR (300 MHz, CDCI3): d=7.50–6.99 (m, 4H), 6.27 (d, J=
2-(2-Chlorobenzyl)-N-phenethylacrylamide (10): Phenethylamine
(242.4 mg, 2.00 mmol) was used as the amine, and the mixture
was stirred for 12 h. Purification by silica gel chromatography
(CH2Cl2/EtOAc 9:1) gave 10 as a white solid (158.9 mg, 53%): Rf =
0.71 (CH2Cl2/EtOAc 9:1); mp: 66.5–68.08C; 1H NMR (300 MHz,
CDCl3): d=7.34–7.09 (m, 9H), 5.90 (br, 1H), 5.61 (s, 1H), 5.07 (s,1H),
3.73 (s, 2H), 3.52 ppm (t, J=7.1 Hz, 2H), 2.78 ppm (t, J=7.1 Hz,
2H); 13C NMR (75 MHz, CDCl3): d=168.8, 140.2, 139.3, 137.4, 134.2,
130.6, 129.3, 128.7, 128.5, 127.7, 127.2, 125.9, 124.2, 40.8, 37.1,
35.2 ppm; MS (CI, isobutane): m/z (%): 302 (32), 300 (100) [M+H]+.
0.9 Hz,1H), 5.33 (d, J=1.2 Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 3.76 (s,
2H), 1.29 ppm (t, J=7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
166.8, 138.4, 136.4, 134.5, 131.2, 129.6, 127.9, 126.8, 126.3, 60.4,
35.4, 14.2 ppm; MS (CI, isobutane): m/z (%): 227 (32), 225 (100)
[M+H]+.
2-(2-Chlorobenzyl)acrylic acid (22): 2.5m NaOH (1.3 mL) was
added to a stirred solution of 21 (3.06 g, 19.6 mmol) in EtOH
(5 mL), and the mixture was stirred at RT overnight. The mixture
was diluted with 10% NaHCO3 (15 mL) and extracted with EtOAc
(15 mL). The aqueous phase was acidified with 1m HCl (to pH<1)
and then extracted with EtOAc (3ꢂ20 mL). The organic phase was
dried (Na2SO4), filtered, and concentrated. Compound 22 was ob-
tained as a white solid (2.54 g, 95%): mp: 90.5–91.58C; 1H NMR
(300 MHz, CDCI3): d=11.84 (br, 1H),7.37–7.14 (m, 4H), 6.41 (s,1H),
5.44 (s, 1H), 3.74 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=172.4,
137.9, 136.5, 134.9, 131.6, 130.1, 129.4, 128.5, 127.3, 35.4 ppm; MS
(CI, isobutane): m/z (%): 199 (32), 197 (100) [M+H]+.
N-(3-Aminopropyl)-2-(2-chlorobenzyl)acrylamide trifluoroacetate
(11): tert-Butyl (3-aminopropyl)carbamate (348.5 mg, 2.00 mmol)
was used as the amine, and the mixture was stirred for 3 h. Purifi-
cation by silica gel chromatography (petroleum ether/EtOAc 1:1)
gave Boc-protected 11 as a white solid (264.7 mg, 75%). This inter-
mediate (148.7 mg, 0.422 mmol) was dissolved in CH2Cl2 (5 mL)
and trifluoroacetic acid (0.500 mL, 6.49 mmol) was added. The mix-
ture was stirred at RT for 1 h and then concentrated to dryness.
The white solid was washed several times with CH2Cl2 (2ꢂ20 mL)
and diethyl ether (3ꢂ20 mL), and 11 was obtained as a white crys-
talline solid (134.5 mg, 87%): Rf =0.23 (CH2Cl2/MeOH 1:1); mp:
2,5-Dioxopyrrolidin-1-yl-2-(2-chlorobenzyl)acrylate (23): N,N’-Di-
cyclohexylcarbodiimide (2.50 g, 12.1 mmol) was added to a stirred
solution of 22 (2.39 g, 12.1 mmol) in dry THF (25 mL) at 08C. After
10 min, N-hydroxysuccinimide (1.39 g, 12.1 mmol) was added at
the same temperature, and the mixture was stirred at RT overnight.
The mixture was filtered, and the liquid phase was extracted with
EtOAc (3ꢂ30 mL). The organic layer was washed with saturated
1
118.1–119.08C; H NMR (300 MHz, D2O): d=7.30 (d, J=7.5 Hz, 1H),
7.13–7.10 (m, 3H), 5.54 (s,1H), 5.24 (s, 1H), 3.61 (s, 2H), 3.11 (t, J=
6.5 Hz, 2H), 2.54 (t, J=6.6 Hz, 2H), 1.67–1.55 (m, 2H), 1.66–1.50 (m,
4H), 1.23–0.87 ppm (m, 6H); 13C NMR (75 MHz, D2O): d=172.5,
&
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142.2, 136.1, 134.1, 131.3, 129.9, 128.7, 127.5, 121.2, 118.5, 114.8,
40.5, 36.6, 34.4, 26.9 ppm.
sure. Purification by silica gel chromatography (CH2Cl2/MeOH 98:2)
gave 16 as a white solid (75.0 mg, 84%): Rf =0.30 (CH2Cl2/MeOH
1
98:2); mp: 138.8–140.38C; H NMR (300 MHz, CDCl3): d=7.61–6.94
Methyl 4-[2-(2-chlorobenzyl)acrylamido]butanoate (12): Methyl
4-aminobutanoate hydrochloride (307.2 mg, 2.00 mmol) was used
as the amine, and the mixture was stirred for 16 h. Purification by
silica gel chromatography (petroleum ether/EtOAc 7:3 to 1:1) gave
12 as a white solid (162.7 mg, 55%): Rf =0.64 (petroleum ether/
EtOAc 1:1); mp: 69.5–71.08C; 1H NMR (300 MHz, CDCl3): d=7.38–
7.18 (m, 4H), 6.16 (br, 1H), 5.72 (s,1H), 5.12 (s, 1H), 3.77 (s, 2H),
3.67 (s, 3H), 3.34 (t, J=7.1 Hz, 2H), 2.32 (t, J=7.1 Hz, 2H), 1.88–
1.79 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=174.4, 168.6, 143.2,
136.5, 134.7, 131.6, 130.0, 128.5, 127.4, 119.9, 52.2, 39.6, 36.2, 31.9,
24.8 ppm; MS (CI, isobutane): m/z (%): 298 (32), 296 (100) [M+H]+.
(m, 8H), 6.61 (br, 2H), 5.79 (s, 2H), 5.17 (s, 2H), 3.69 (s, 4H), 3.19 (t,
J=6.2 Hz, 4H), 1.67–1.46 ppm (m, 2H); 13C NMR (75 MHz, CDCl3):
d=168.7, 142.7, 136.1, 134.4, 131.2, 129.6, 128.1, 126.9, 119.7, 35.9,
35.5, 29.5 ppm; MS (CI, isobutane): m/z (%): 432 (64), 430 (100)
[M+H]+.
Kinetic cysteamine chemoassay: The thiol assay was performed in
96-well plates by using 100 mm phosphate buffer (pH 7.4) with
500 mm ethylenediaminetetraacetic acid (EDTA) as the solvent
system. The DTNB reagent was prepared by dissolving DTNB
(0.014 mmol, Sigma–Aldrich, St. Louis, MO, USA) and sodium hy-
drogen carbonate (0.5 mmol) in 100 mm phosphate buffer (pH 7.2,
25 mL). All measurements were done with a Multilabel Plate
Reader (Victor X4, PerkinElmer, Waltham, MA, USA) at 378C. To per-
form the assay, CH3CN solutions of the compounds (10 mm) and
a water solution of cysteamine (CAM) (Sigma–Aldrich) (10 mm)
were diluted in the phosphate buffer to give a concentration of
0.5 mm. Equal amounts of both solutions were combined and
mixed, and the kinetic measurements were started immediately. At
various time points (over a time of 90 min), the DTNB reagent
(150 mL) was added, and after 1 min the absorption at l=405 nm
was measured. The concentration of the remaining reduced CAM
was determined by a CAM calibration curve of thiol content versus
absorbance (CAM concentration ranging from 0.03 to 0.35 mm).
Rate constants of the reaction between CAM and the electrophilic
compounds were determined as reported.[22]
4-[2-(2-Chlorobenzyl)acrylamido]butanoic acid (13): 4-Aminobu-
tanoic acid (206.2 mg, 2.00 mmol) was used as the amine, and the
mixture was stirred in CH2Cl2/DMF (1:1, 10 mL) for 24 h. Purification
by silica gel chromatography (CH2Cl2/MeOH 99:1) gave 13 as
a white solid (128.9 mg, 66%): Rf =0.80 (CH2Cl2/MeOH 1:1); mp:
68.5–70.38C; 1H NMR (300 MHz, CDCl3): d=10.60 (br, 1H), 7.33–
7.17 (m, 4H), 6.56 (br, 1H), 5.72 (s, 1H), 5.09 (s, 1H), 3.73 (s, 2H),
3.32 (t, J=7.1 Hz, 2H), 2.31 (t, J=7.1 Hz, 2H), 1.85–1.76 ppm (m,
2H); 13C NMR (75 MHz, CDCl3): d=178.2, 168.3, 142.7, 136.4, 134.7,
131.6, 130.0, 128.6, 127.4, 120.5, 39.6, 36.1, 31.9, 24.7 ppm.
2-(2-Chlorobenzyl)-N-(4-sulfamoylphenethyl)acrylamide (14): 4-
(2-Aminoethyl)-benzenesulfonamide (400.5 mg, 2.00 mmol) was
used as the amine, and the mixture was stirred in DMF (10 mL) for
12 h. Purification by silica gel chromatography (CH2Cl2 to CH2Cl2/
EtOAc 7:3) gave 14 as a white solid (223.5 mg, 59%): Rf =0.43
Reaction of compound 14 with cysteamine: The electrophilic re-
activity of compound 14 was quantified in terms of the pseudo-
first-order reaction rate constant, kpseudo1st, by employing cystea-
mine (CAM) as a nucleophile. The reaction vessel contained
500 mm electrophile and 5 mm CAM in 100 mm potassium phos-
phate buffer (pH 7.4) with 25% acetonitrile as co-solvent. The
stirred mixture was maintained at (37Æ0.5)8C for 7 h. At different
time intervals, an aliquot (500 mL) of this solution was analyzed by
reversed-phase UHPLC by using a Flexar UHPLC (PerkinElmer)
equipped with a Flexar Solvent Manager 3-CH-Degasser, a Flexar-
FX UHPLC autosampler, a Flexar-FX PDA UHPLC detector, a Flexar-
LC column oven, and a Flexar-FX-15 UHPLC pump. The analytical
column was an Acquity CSH (2.1ꢂ100 mm, 1.7 mm particle size)
(Waters) column. The samples were analyzed by using an isocratic
method by employing a mobile phase consisting of methanol/
buffer (70:30, flow rate 0.2 mLminÀ1). The column effluent was
monitored at l=204 nm referenced against l=360 nm. Quantifi-
cation was done by using calibration curves of compound 14 chro-
matographed under the same conditions. The linearity of the cali-
bration curves was determined in the concentration range of 100
to 1000 mm (r2 >0.98). Data analysis was performed by using Chro-
mera Manager (PerkinElmer). All experiments were run in triplicate.
The pseudo-first-order rate constant was determined by plotting
the natural log of the concentration of 14 as a function of time.
The negative slope of the straight line is the pseudo-first-order
rate constant. The value of kpseudo1st was then calculated according
to the reported method.[29]
1
(CH2Cl2/EtOAc 1:1); mp: 150.3–151.18C; H NMR (300 MHz, CD3OD):
d=7.79 (d, J=8.0 Hz, 2H), 7.44–7.28 (m, 4H), 7.22 (d, J=9.1 Hz,
2H), 5.66 (s,1H), 5.11 (s, 1H), 3.72 (s, 2H), 3.47 (t, J=7.1 Hz, 2H),
2.88 ppm (t, J=7.1 Hz, 2H); 13C NMR (75 MHz, CD3OD): d=168.5,
142.9, 141.6, 140.4, 135.0, 132.9, 129.7, 128.0, 127.9, 126.8, 125.6,
124.7, 117.9, 39.2, 34.2, 33.5 ppm; MS (CI, isobutane): m/z (%): 381
(32), 379 (100) [M+H]+.
2-(2-Chlorobenzyl)-N-{4-[N-(cyclohexylcarbamoyl)sulfamoyl]phe-
nethyl}acrylamide (15): Compound 14 (98.7 mg, 0.261 mmol) was
dissolved in dry acetone (15 mL) under a N2 atmosphere and K2CO3
(108.0 mg, 0.732 mmol) was added portionwise. After stirring at
reflux for 1.5 h, cyclohexyl isocyanate (52.0 mg, 0.417 mmol) dis-
solved in dry acetone (20 mL) was added dropwise to the mixture.
The mixture was heated at reflux overnight. After cooling, water
(15 mL) was added, and the mixture was acidified to pH 1 with 1n
HCl. The obtained white precipitate was collected and recrystal-
lized from methanol to afford 15 as a white solid (51.0 mg, 39%):
1
Rf =0.43 (CH2Cl2/EtOAc 1:1); mp: 166.7–168.58C; H NMR (300 MHz,
[D6]DMSO): d=10.34 (br, 1H), 8.25 (s, 1H), 7.79 (d, J=8.0 Hz, 2H)
7.44–7.28 (m, 6H), 6.39 (d, J=7.7 Hz, 1H), 5.69 (s,1H), 5.03 (s, 1H),
3.65 (s, 2H), 3.38 (t, J=6.9 Hz, 2H), 3.33–3.30 (m, 1H), 2.84 (t, J=
6.8 Hz, 2H), 1.66–1.50 (m, 4H), 1.23–0.87 ppm (m, 6H); 13C NMR
(75 MHz, [D6]DMSO): d=168.1, 150.5, 146.1, 139.0, 137.4, 134.2,
131.9, 130.1, 129.9, 129.1, 128.1, 128.0, 127.7, 120.0, 48.9, 40.7, 36.1,
35.5, 33.2, 22.8, 25.1 ppm; MS (CI, isobutane): m/z (%): 505 (32),
503 (100) [M+H]+.
N,N’-(Propane-1,3-diyl)bis[2-(2-chlorobenzyl)acrylamide]
(16):
Albumin modification test: The analytic platform was composed
of a Surveyor LC system, which was connected to a TSQ Quantum
Ultra mass spectrometer through a Finnigan IonMax electrospray
ionization (ESI) source assembled with a low flow stainless steel
emitter (Thermo Fisher Scientific, Rodano, MI, Italy). Each com-
pound was dissolved in acetonitrile and tested separately. Com-
pounds were spiked into fresh human serum down to a concentra-
DIPEA (51 mL, 0.299 mmol) was added dropwise to a solution of 11
(91.0 mg, 0.249 mmol) in DMF (5 mL). The mixture was stirred at RT
for 10 min and then 23 (61.0 mg, 0.208 mmol) was added. The mix-
ture was stirred at RT for 1 h. The mixture was diluted with 1n HCl
(10 mL) and extracted with CH2Cl2 (3ꢂ25 mL). The organic phase
was dried (Na2SO4), filtered, and concentrated under reduced pres-
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tion of 1 mm. The spiked volume was less than 10% of serum
volume to avoid protein precipitation. The temperature was kept
at 378C throughout the incubation time (3 h). Before the analysis,
serum aliquots were diluted 200-fold into H2O/CH3CN/HCO2H
(70:30:0.1 v/v/v). Samples were then centrifuged for 10 min at
18000 g, and the supernatant was placed in clear glass vials and
kept at 48C in the autosampler compartment. The analyses were
performed by an automated loop injection method, and sampling
was programmed as a 50 mL-partial-loop injection performed by
the HPLC system. Once loaded into the sample loop, samples were
pushed at a flow rate of 25 mLminÀ1 through a peek tube directly
connected to the ESI source. The mobile phase isocratic flow was
delivered by the pump at the final composition H2O/CH3CN/HCO2H
(70:30:0.1 v/v/v). The analyzer was operating under conditions sim-
ilar to those reported.[27] Briefly, MS spectra were acquired for
5 min by a TSQ Quantum Ultra mass spectrometer in positive-ion
mode by using the following settings: ESI voltage, 3.5 KV; capillary
temperature, 3008C; sheath gas, 35%; Q3 scan range, m/z=1410–
1500; Q3 power, 0.4 amu; scan time, 1 s; Q2 gas pressure, 200 Pa;
skimmer offset, 10 V; microscan set to 3. Full instrument control
and extraction of albumin ESI mass spectra were provided by Xcali-
bur software (version 2.0.7, Thermo Fisher Scientific, Rodano, MI,
Italy). Mass spectra deconvolution was provided by MagTran soft-
ware (version 1.02).[39] Covalent adducts were detected by the ex-
pected molecular weights [i.e., Mr (adduct)=Mr (albumin)+
Mr (compound)]. The amount of modified albumin was then calcu-
lated from the relative abundance of unmodified protein and ad-
ducts.
mNLRP3_R258W_F:
mNLRP3_R258W_R:
mNLRP3_A350V_F:
mNLRP3_A350V_R:
mNLRP3_L351P_F:
mNLRP3_L351P_R:
ctatttgttctttatccactgctgggaggtgagcctcag-
gac,
gtcctgaggctcacctcccagcagtggataaagaa-
caaatag,
cataacgacgaggccggtagtcttggagaaactgcag-
catc,
gatgctgcagtttctccaagactaccggcctcgtcgt-
tatg,
cgacgaggccggtagccccggagaaactgcag-
catctc,
gagatgctgcagtttctccggggctaccggcctcgtcg.
WT, R258W, A350V, and L351P NLRP3 cDNAs were cloned in GFP
encoding pInducer21 under a doxycycline-dependent promotor for
lentiviral vector production.
Cell culture: Immortalized wild-type and NLRP3 KO bone-marrow-
derived macrophages (BMDMs) were a kind gift from Dr. E. Alnemri
(Thomas Jefferson University). Immortalized NLRP3 KO BMDMs
were reconstituted with WT, R258W, A350V, or L351P NLRP3 by len-
tiviral transduction followed by flow cytometry sorting of green
fluorescent protein (GFP)-positive cells. Inflammatory peritoneal
macrophages and immortalized BMDMs were cultured at 0.5–1ꢂ
106 cellsmLÀ1 in Dulbecco’s modified Eagle’s Medium (DMEM) sup-
plemented with 1ꢂ penicillin/streptomycin (PS) and 10% fetal
bovine serum (FBS) (Gibco). Primary BMDMs were cultured at 0.5–
1ꢂ106 cellsmLÀ1 in DMEM supplemented with 10% FBS, 10% mac-
rophage colony-stimulating factor (M-CSF)-conditioned media, 1%
HEPES, 1% sodium pyruvate, 1% glutamine. Cells were treated
with doxycycline (Sigma), LPS from Escherichia coli 0111:B4 (Sigma)
and ATP (Sigma).
ELISA: mIL-1b and mTNF-a assays were performed by using the
DuoSet ELISA kits (R&D Systems).
Biological studies
In vitro models of pyroptosis: Pyroptosis was studied as previously
described.[22] The day before each experiment, cells were plated in
48-well culture plates (75ꢂ103 cellswellÀ1) and were differentiated
into monocyte-macrophages-like cells by treatment with PMA
(50 nm; 24 h; Sigma–Aldrich). PMA-differentiated THP-1 cells were
washed with phosphate-buffered saline (PBS, 2ꢂ) and primed with
LPS (5 mgmLÀ1; 4 h; Sigma–Aldrich) in serum-free medium. Cell
death was triggered with ATP (5 mm; 1 h; Sigma–Aldrich). Cell
death was quantified by using the CytoTox 96 Non-Radioactive Cy-
totoxicity Assay (Promega Corporation, Madison, MI, USA), based
on a colorimetric measurement of LDH activity in the collected su-
pernatants. Cell death was expressed according to the manufactur-
er’s instruction.
Measurement of NLRP3-ATPase activity: Human recombinant NLRP3
(0.105 mg; BPS Bioscience, San Diego, CA, USA) was incubated with
the assessed compounds in the reaction buffer (20 mm Tris-HCl,
pH 7.8, 133 mm NaCl, 20 mm MgCl2, 3 mm KCl, 0.56 mm EDTA,
0.5% DMSO) for 15 min at 378C. ATP (250 mm, Ultra Pure ATP) was
added, and the mixtures were further incubated for 40 min at
378C. The hydrolysis of ATP by NLRP3 was determined by a lumi-
nescent ADP detection performed with ADP-Glo Kinase Assay
(Promega, Madison, MI, USA) according to the manufacturer’s pro-
tocol.
Computational methods
Cytotoxicity assay: THP-1 cells were plated in 96-well culture plates
(5ꢂ103 cellswellÀ1) and exposed to increasing concentrations (0.1–
100 mm) of each compound. The cultures were maintained at 378C,
95% air/5% CO2 in a fully humidified incubator. Cell viability was
measured at 72 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay.
Docking simulations involved the NACHT domain of the human
NLPR3 protein (residues 220–536, Entry Id: Q96P20, Entry Name:
NLRP3_HUMAN), the homology model of which was generated by
using the resolved structure of NLRC4 (PDB ID: 4KXF). Briefly, ho-
mology modeling was performed by Modeller 9.10 by using the
default parameters;[36] among the 20 generated models, the best
structure was selected according to the computed scores (i.e.,
DOPE and GA341) as well as to the percentage of residues falling
in the allowed regions of the Ramachandran (91.2%) and chi plots
(95.8%). The selected model was carefully checked to avoid un-
physical occurrences such as cis peptide bonds, wrong configura-
tions, improper bond lengths, nonplanar aromatic rings, or collid-
ing side chains. To remain compatible with physiologic pH, Asp,
Glu, Lys, and Arg residues were considered in their ionized forms,
whereas His and Cys residues were maintained neutral by default.
The so-completed model underwent a minimization procedure by
Mice: C57Bl/6J mice were housed at the PBES Facility (ENS Lyon).
Experiments were performed in accordance with European and in-
stitutional guidelines. Inflammatory peritoneal macrophages were
elicited by the intraperitoneal injection of 4% thioglycolate broth
for 4 days.
Constructs: Mouse NLRP3 cDNA was amplified by PCR from cDNAs
of C57Bl/6J mouse macrophages. cDNAs coding for R258W, A350V,
and L351P NLRP3 mutants were obtained from mouse NLRP3
cDNA by PCR (QuickChange II site-directed mutagenesis kit, Agilent
Technologies) by using the following oligonucleotides:
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Full Papers
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keeping the backbone atoms fixed to preserve the predicted fold-
ing, and the so-obtained final structure was used in the following
docking simulations that involved ATP, taken as a reference ligand,
plus the herein-reported inhibitors. The ATP structure was retrieved
from the resolved structure (PDB ID: 4AFF), which showed the best
resolution among those co-crystallized with ATP, and its conforma-
tion was optimized by the PM7 semiempirical method as imple-
mented in MOPAC2012. In contrast, the conformational profile of
the here-reported inhibitors was explored by Monte Carlo simula-
tions, which generated 1000 minimized geometries by randomly
rotating the rotatable bonds. The so-obtained lowest energy struc-
ture underwent the following docking simulations that were per-
formed by using PLANTS and arranged in two steps.[40] The first
step involved the docking of ATP; its search was focused on a 12 ꢃ
radius sphere around the highly conserved Lys232 residue, the key
role of which was confirmed by previous studies. The so-computed
complex was then minimized by keeping all atoms outside a 12 ꢃ
radius sphere fixed around the bound ATP, and the so-optimized
NLPR3 structure was used in the docking analyses of the reported
inhibitors by focusing the search on a 12 ꢃ radius sphere around
the bound ATP. In all docking simulations, 20 poses were generat-
ed and scored by using the ChemPLP score function with speed
equal to 1. The computed best complexes for the proposed inhibi-
tors were optimized with the same protocol already described for
the ATP complex.
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Keywords: acrylamide derivatives
inflammasomes · inflammation · pyroptosis
·
drug design
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Published online on && &&, 0000
&
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ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
CAPS block is on! Cryopyrin-associated
periodic syndromes (CAPS) are rare ge-
netic diseases caused by gain-of-func-
tion mutations in NLRP3. Overactivation
of the NLRP3 inflammasome is also in-
volved in other metabolic diseases.
Herein, a series of acrylamides that in-
hibit NLRP3-dependent pyroptosis and
IL-1b release from CAPS-mutant macro-
phages are reported. Direct inhibition of
NLRP3 ATPase in human isolated NLRP3
has emerged as a potential target for
this compound class.
M. Cocco, G. Miglio, M. Giorgis,
D. Garella, E. Marini, A. Costale,
L. Regazzoni, G. Vistoli, M. Orioli,
R. Massulaha-Ahmed, I. Dꢁtraz-Durieux,
M. Groslambert, B. F. Py, M. Bertinaria*
&& – &&
Design, Synthesis, and Evaluation of
Acrylamide Derivatives as Direct
NLRP3 Inflammasome Inhibitors
ChemMedChem 2016, 11, 1 – 15
www.chemmedchem.org
15
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ