Development of an Acrylate Derivative Targeting the NLRP3 Inflammasome for the Treatment of Inflammatory Bowel Disease

Article abstract of DOI:10.1021/acs.jmedchem.6b01624

Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1β release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.

Full text of DOI:10.1021/acs.jmedchem.6b01624

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Article
Development of an Acrylate Derivative Targeting the NLRP3
Inflammasome for the Treatment of Inflammatory Bowel Disease
Mattia Cocco, Carolina Pellegrini, Helios Martínez Banaclocha, Marta Giorgis, Elisabetta Marini, Annalisa
Costale, Gianluca Miglio, Matteo Fornai, Luca Antonioli, Gloria Lopez-Castejon, Ana Tapia-Abellan, Diego
Angosto, Iva Hafner-Bratkovi#, Luca Regazzoni, Corrado Blandizzi, Pablo Pelegrin, and Massimo Bertinaria
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01624 • Publication Date (Web): 14 Apr 2017
Downloaded from http://pubs.acs.org on April 15, 2017
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Development of an Acrylate Derivative Targeting the NLRP3 Inꢀ
flammasome for the Treatment of Inflammatory Bowel Disease.
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Mattia Cocco,†ǁ Carolina Pellegrini,# §,ǁ Helios MartínezꢀBanaclocha,‡,ǁ Marta Giorgis,† Elisabetta
Marini,† Annalisa Costale,† Gianluca Miglio,† Matteo Fornai,# Luca Antonioli,# Gloria LópezꢀCastejón,§
Ana TapiaꢀAbellán,‡ Diego Angosto,‡ Iva HafnerꢀBratkovič,¶ Luca Regazzoni,Ψ Corrado Blandizzi,#
Pablo Pelegrín,‡,* Massimo Bertinaria†,*
†Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P. Giuria 9 – 10125 Torino, Italy.
#Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 55 – 56126 Pisa, Italy.
§Faculty of Biology, Medicine and Health, The University of Manchester, R4.004 AV Hill Building, Oxford Rd, Manchesꢀ
ter, UK.
‡Molecular Inflammation Group, Biomedical Research Institute of Murcia (IMIBꢀArrixaca), University Clinical Hospital
“Virgen de la Arrixaca”, University of Murcia, Carretera Buenavista s/n, 30120, El Palmar, Murcia, Spain.
¶Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, SIꢀ1000 Ljubljana, Sloꢀ
venia
ΨDipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy.
KEYWORDS: NLRP3, Bioluminescence Resonance Energy Transfer assay, Pyroptosis, Inflammatory Bowel Disease, Acryꢀ
lates.
ABSTRACT: Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of Inflamꢀ
matory Bowel Disease (IBD). In this work, we report the design, the synthesis, and the biological screening of a series of acrylate
derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotꢀ
ic properties allowed the selection of 11 (INF39), a nonꢀtoxic, irreversible NLRP3 inhibitor able to decrease interleukinꢀ1β release
from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interꢀ
fere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of DNBSꢀ
induced colitis in rats after oral administration.
INTRODUCTION
Human inflammatory bowel diseases (IBD), the most important entities being ulcerative colitis and Crohn’s disease, are chronic
relapsingꢀremitting inflammatory conditions that result from a chronic dysregulation of the mucosal immune system in the gastroinꢀ
testinal tract.1 Ulcerative colitis is a recognized risk factor for colitisꢀassociated colon cancer.2 In vivo models of colitis showed that
a key role in the onset and maintenance of chronic inflammation is played by a multiprotein complex known as the nucleotideꢀ
binding oligomerization domain, leucineꢀrich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome.3 NLRP3 inꢀ
flammasome is a cytosolic complex involved in the production of proꢀinflammatory cytokines such as interleukin (IL)ꢀ1β and ILꢀ
18. NLRP3 inflammasome is formed by oligomers of NLRP3 that recruit via pyrinꢀpyrin homotypic interactions the adaptor protein
apoptosisꢀassociated speckꢀlike protein containing a caspase recruitment domain (ASC). Then, ASC oligomerizes in filamentꢀlike
structures that recruit and activate the proꢀinflammatory caspaseꢀ1.4ꢀ6 The NLRP3 inflammasome activation is tightly regulated and
requires two different signals to exert its biological response: the first signal (priming) is provided by the activation of the Tollꢀlike
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receptorꢀnuclear factorꢀκB (TLRꢀNFꢀκB) pathway which upregulates the expression of NLRP3 and the inactive form of ILꢀ1β (proꢀ
ILꢀ1β); the second signal (activation and assembly) is triggered by different pathogenꢀassociated molecular patterns (PAMPs) or
damageꢀassociated molecular patterns (DAMPs).7, 8 After NLRP3 inflammasome is assembled and oligomerizes ASC, procaspaseꢀ1
is recruited and autocleaves itself releasing active caspaseꢀ1, which is the final effector in interleukin maturation and pyroptotic cell
death.8 Therefore, NLRP3 is emerging as a promising target to develop novel and specific antiꢀinflammatory compounds.9, 10 Inꢀ
deed, although NLRP3 is considered a pivotal player in regulating the integrity of intestinal homeostasis, it also shapes innate imꢀ
mune responses against commensal bacteria, and its overactivation during intestinal inflammation is associated with a breakdown
of the intestinal immune balance, with consequent detrimental effects for the host.11
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Of note, current clinical evidence has documented an increased ILꢀ1β secretion from colonic tissues and macrophages of IBD paꢀ
tients, these patterns being correlated with the severity of the disease, thus suggesting an involvement of inflammasome pathways
in the pathogenesis of IBDs.12ꢀ14 Moreover, single nucleotide polymorphism in the regulatory region of NLRP3 gene has been assoꢀ
ciated with the susceptibility to Crohn’s disease.15
In this setting, given the involvement of inflammasome pathways in the pathophysiology of intestinal inflammation, current reꢀ
search efforts are focusing on the potential therapeutic benefits, in terms of antiꢀinflammatory activity, resulting from the pharmaꢀ
cological blockade of NLRP3 inflammasome. The most explored strategies in the discovery of new experimental drugs targeting
NLRP3 pathways in IBD are: (i) inhibition of activation of the transcription factor NFꢀκB; (ii) protection from mitochondrial damꢀ
age; (iii) activation of Keapꢀ1/ Nrf2 antioxidant pathway; (iv) inhibition of proꢀcaspaseꢀ1 cleavage through undefined interactions
with NLRP3 inflammasome (Figure 1).16 Currently there are no specific NLRP3 inhibitors used in IBD therapy; to date few moleꢀ
cules acting at the aforementioned pathways have been studied (structures are summarized in Figure S1). None of the studied moleꢀ
cules showed direct inhibition of NLRP3 ATPase activity.16ꢀ23
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Figure 1. Experimental NLRP3 inflammasome pathway inhibitors studied for the treatment of IBD and their putative mechanism
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of action (see supporting info for references).
According to these observations, in the present study we explored the use of direct NLRP3 inflammasome inhibitors as potential
new tools for the treatment of bowel inflammation.
To design NLRP3 inhibitors suitable for further development we relied on our previous experience with electrophilic warheads.
In a proof of concept work we demonstrated that α,βꢀunsaturated carbonyl derivatives, behaving as Michael acceptors, can signifiꢀ
cantly prevent NLRP3ꢀdependent pyroptosis of THPꢀ1 cells. These effects were associated with the inhibition of the NLRP3
ATPase activity. Among the studied compounds those based on the acrylate scaffold showed the most favorable pharmacoꢀ
toxicological profile, with ethyl 2ꢀ((2ꢀchlorophenyl)hydroxyl)methyl)acrylate (INF4E, 1)24 emerging as a potential lead amenable
of further optimization (Figure 2).25 Compound 1 still suffered from some drawbacks: its reactivity being not perfectly tuned, leadꢀ
ing to a certain degree of cytotoxicity in THPꢀ1 cells. As a matter of fact, 1 was later shown to bind human serum albumin in fresh
human serum by 65 ± 5% through the formation of three covalent adducts.26 The structure of 1 needed further chemical optimizaꢀ
tion. In order to modulate the acrylate scaffold toward less toxic compounds, in this work, we followed a simple but straightforward
strategy as depicted in Figure 2; in an early phase of the project the chemical modulation of the 2,4ꢀdichlorophenylꢀsubstituted
acrylate 2, a previously identified close analogue of 1 showing good antipyroptotic properties,25 was also considered.
Compounds 3 and 4 (Figure 2) were designed to verify whether the presence of a free carboxylic group could lead to active or inꢀ
active derivatives. The functionalization (derivatives 5–8, 10) and the removal (derivatives 11–14) of the benzylic hydroxyl group
was performed to investigate the role played by this moiety in the reactivity and the cytotoxicity of this class of compounds. Finalꢀ
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ly, compounds 13 and 14, deprived of the terminal double bond, were synthesized to test their biological activity and allow for
mechanistic considerations.
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In this work, we describe the selection of 11 (INF39)27 a nonꢀcytotoxic, acrylateꢀbased NLRP3 inhibitor after assessment of the
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Michael acceptor reactivity, cytotoxicity, in vitro NLRP3 ATPase inhibition, and antipyroptotic properties of a small series of acryꢀ
late electrophiles. Furthermore, novel insights into the mechanism of NLRP3ꢀinflammasome inhibition by 11 are given thanks to its
ability to inhibit ILꢀ1β release from macrophages and to affect the activation kinetic profile of NLRP3 measured by bioluminesꢀ
cence resonance energy transfer (BRET). In vitro metabolic stability studies and ex vivo intestinal permeation experiments give a
preliminary insight into compound 11 pharmacokinetic profile. Finally, in vivo pharmacological studies of 11 in a rat model of coliꢀ
tis show a relevant reduction of gutꢀassociated inflammation.
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Figure 2. Design of acrylates as NLRP3 inhibitors.
RESULTS AND DISCUSSION
Chemistry. Derivatives 1 and its dichloro analogue 2, obtained by MoritaꢀBaylisꢀHillman (MBH) reaction, 25 were chosen as the
starting materials for the synthesis of the designed compounds (Schemes 1–3). The corresponding acids 3 and 4 were obtained by
NaOHꢀmediated hydrolysis of the ester group in quantitative yields at room temperature (rt) (Scheme 1). Oꢀfunctionalyzed derivaꢀ
tives 5 and 6, which were previously obtained in trace amounts as byꢀproducts of the MBH reaction between the corresponding
chlorobenzaldehydes and ethylacrylate,25 were synthesized from 1 using an oxaꢀMichael reaction. This reaction was carried out in
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CH3CN employing ethylacrylate as the electrophile, 1,5ꢀdiazabiciclo[5.4.0]undecꢀ5ꢀene (DBU), and a stoichiometric amount of 4ꢀ
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(dimethylamino)pyridine (DMAP) as the bases affording desired derivatives 5 and 6 in about 30% unoptimized yields (Scheme 1).
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Scheme 1. Synthesis of compounds 3–6
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aReagents and conditions: (a) NaOH 2.5 M, 1,4ꢀdioxane/H2O 1/1, rt, 16 h; (b) ethyl acrylate, DMAP, DBU, CH3CN, rt, 48 h.
To obtain the methoxy derivative 7, the oxygen atom of 1 was alkylated using an excess of methyl iodide and Ag2O (Scheme 2).
After 4 days at rt derivative 7 was obtained in 40% yield after chromatography. The acetoxymethyl acrylate 8 was efficiently obꢀ
tained by dropwise addition of acetic anhydride to a mixture of 1 containing 0.2 molar equivalents of DMAP. Compound 8 was
then converted into the Oꢀallyl derivative 9 through displacement of the highly reactive acetoxy group with 2ꢀpropenol in dry THF
using two equivalents of DABCO as the base. These intermediates were then submitted to a ringꢀclosing metathesis (RCM) reaction
using the 2nd generation HoveydaꢀGrubbs catalyst 1,3ꢀbisꢀ(2,4,6ꢀtrimethylphenyl)ꢀ2ꢀimidazolidinylidene)dichloro(oꢀ
isopropoxyphenylmethylene)ruthenium to yield 10 in fair yields. Unfortunately, after isolation, 10 decomposed either in solution or
on overnight standing giving rise to a complex mixture. From this mixture only about 34% of 10 could be recovered by silicagel
column chromatography after 20 h of storage at ꢀ18 °C. Consequently, we were able to chemically characterize 10 by NMR and
MS, however, due to its chemical instability, in the continuation of this work it was possible to use derivative 10 only for the deꢀ
termination of its reactivity toward thiol groups. NaBH4ꢀmediated reduction of 8, conducted in a mixture of THF/H2O (1/1) in the
presence of DABCO, afforded 11 in 84% yield. This compound was then hydrolyzed using the above reported method to furnish 12
(Scheme 2). Finally, Pdꢀcatalyzed hydrogenation of 1 and subsequent hydrolysis allowed us to obtain probe derivatives 13 and 14
in high yields (Scheme 3).
Scheme 2. Synthesis of compounds 7–12
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aReagents and conditions: (a) Ag2O, CH3I, CH2Cl2, rt,4 d; (b) Ac2O, DMAP, CH2Cl2, rt, 40 min; (c) 2ꢀpropenol, DABCO, dry THF, rt,
10 min then 60 °C, 48 h; (d) 2nd generation HoveydaꢀGrubbs catalyst, dry CH2Cl2, reflux 3 h, rt, 16 h; (e) NaBH4, DABCO, THF/H2O 1/1,
rt, 30 min; (f) NaOH 2.5 M, 1,4ꢀdioxane/H2O 1/1, rt, 16 h.
Scheme 3. Synthesis of compounds 13–14
aReagents and conditions: (a) H2, Pd/C (10 %), abs EtOH, rt, 30 min; (b) NaOH 2.5 M, 1,4ꢀdioxane/H2O 1/1, rt, 16 h.
Reactivity as Michael acceptor. The ability of the reference compound 1 and of the synthesized compounds 2−8, 10−14 to react
with thiol groups was determined using the kinetic cysteamine chemoassay previously described.25, 26 Compounds were mixed with
an equimolar amount of cysteamine (CAM) in pH 7.4 phosphateꢀbuffered solution at 37 °C using CH3CN as the cosolvent. The
progress of the reaction was monitored adding 5,5′ꢀdithioꢀbis(2ꢀnitrobenzoic acid) (DTNB reagent) at different time points over a
period of 90 min, obtained results are collected in Table 1. The reference compound 1 showed a k2 value of 0.824 ± 0.017 Mꢀ1 sꢀ1,
surprisingly its dichloroꢀanalogue 2 proved more reactive with a k2 value of 1.340 ± 0.220 Mꢀ1 sꢀ1. As expected, the corresponding
acrylic acids 3 and 4 did not show any reactivity in the test conditions. The series of compounds bearing a substituted oxygen atom
in benzylic position maintained the ability to react with thiols. Derivatives 5 and 6 showed k2 values (0.734 ± 0.047 Mꢀ1 sꢀ1; 0.292 ±
0.014 Mꢀ1 sꢀ1, respectively) lower than that shown by parent 1. The methoxyꢀsubstituted compounds 7 showed an increased electroꢀ
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philic ability (k2 = 1.60 ± 0.056 Mꢀ1 sꢀ1) which was maximized when the acetoxy group was introduced in benzylic position as in
compound 8 (k2 = 56.2 ± 5.4 Mꢀ1 sꢀ1). When the oxygen atom was enclosed in a dihydrofuran ring (10) a compound with about tenꢀ
fold the reactivity of parent 1 was obtained. Finally, removal of the OH group afforded a compound (11) endowed with no apparent
Michael reactivity in our assay conditions. Moreover, when the assay was repeated using glutathione as a model of biological releꢀ
vant thiol, no reactivity of 11 was detected both at pH 7.4 and pH 9.0 (data not shown). To ascertain the electrophilicity of the
acrylic ester 11, its ability to react with thiols under different test conditions was then checked. Compound 11 was reacted with an
excess of CAM in pH 7.4 phosphateꢀbuffered solution at 37 °C using CH3CN as the cosolvent. The decrease in 11 concentration
was monitored by UHPLC for 4 h. In these conditions, the concentration of 11 decreased to 89.4 ± 1.5%, 83.3 ± 2.1%, and 77.5 ±
3.2% after 30 min, 60 min, and 4 h, respectively, thereby indicating a slow in vitro reactivity with thiol groups (Figure S2).
While 11 proved to be a weak electrophile (a necessary requirement for the development of safe covalent drugs), other known
NLRP3 inflammasome inhibitors such as BAY 11ꢀ7082 (BAY),28 3,4ꢀmethylenedioxyꢀβꢀnitrostyrene (MNS),29 and dimeꢀ
thylfumarate (DiMF) showed a markedly higher reactivity as nucleophiles than 11. In particular MNS showed a k2 = 2.50 ± 0.12 Mꢀ
1 sꢀ1 while DiMF a k2 = 39.01 ± 0.57 Mꢀ1 sꢀ1. Noteworthy, the reactivity of BAY with CAM was too fast to determine a reliable k2
value.
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To further characterize the reactivity of 11 toward biological nucleophiles in quasiꢀphysiological conditions we tested its reactiviꢀ
ty toward human serum albumin (HSA) in fresh human serum. This method was recently reported as a valid test for monitoring the
reactivity of Michael acceptors with unspecific endogenous targets, relying on HSA as a multifunctional probe that contains a set of
selfꢀactivated nucleophiles with diverse chemistries and accessibilities.30 Unlike compound 1, compound 11 (which proved stable in
human serum; Figure S3) showed no reactivity toward HSA. In fact, a 3ꢀhour incubation of compound 1 into human serum resulted
in a modification of the mass spectrum of HSA due to covalent binding, while the corresponding spectra from serum incubated with
compound 11 and from a control sample were superimposable. In detail, Figure 3 reports the mass spectra of HSA obtained from
serum (Figure 3A), serum treated with compound 11 (Figure 3B) and serum treated with compound 1 (Figure 3C). As reported in
the spectra in Figure 3 AꢀB, in the 1400ꢀ1500 m/z region there are 3 main signals corresponding to HSA multiply charged ions
[HSA+zH+]z+ with charge (z) values between 45+ and 47+, accompanied by the corresponding ions of cysteinylated HSA (HSAꢀSꢀ
Cys) and glycated HSA (HSAꢀNꢀGlc). The additional signals in figure 3C have m/z values corresponding to +240 Da increments of
the molecular weight of HSA, which is the expected value for compound 1 covalent adducts. Therefore, compound 11 can be reꢀ
garded as a weak “safe” electrophile suggesting the possible absence of unspecific reactivity toward biological nucleophiles and a
reduced possibility to trigger idiosyncratic hypersensitivity reactions.
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Figure 3. ESIꢀmass spectra (1400ꢀ1500 m/z region) of human serum albumin (HAS) after 3 h incubation of (a) human serum; (b) human
serum treated with compound 11 (1 mM); (c) human serum treated with compound 1 (1 mM).
Assessment of cytotoxicity in THPꢀ1 cells. To select candidates for further in vitro and in vivo studies, cytotoxic activity was
evaluated. Human THPꢀ1 cells were exposed to increasing concentrations of the synthesized compounds (0.1–100 µM, 72 h), then
cell viability was evaluated by MTT assay.26 As shown in Table 1, a TC50 (the concentration able to decrease cell viability by 50%)
value ≤100 µM was determined for the parent compounds 1, 2 and the highly reactive compound 8. A TC50 value >100 µM was
instead observed for the other compounds, thus indicating that a decreased cytotoxicity was achieved by introducing focused strucꢀ
tural modulations. These data demonstrate that the αꢀhydroxy group likely plays a dual role in αꢀbenzyl acrylates electrophiles reguꢀ
lating both reactivity, due to its effect on the carboxylate moiety,31 and toxicity. Owing to the differences in the TC50 values among
1 vs compounds 5 and 7 (less and more reactive than 1, respectively), it could be suggested that the cytotoxicity of this class of
compounds does not entirely depend on their reactivity toward thiols.
The cytotoxicity of other electrophilic NLRP3 pathway inhibitors, namely MNS, BAY, and DiMF was determined in THPꢀ1
cells using the same assay. As shown in Table 1, compared to 11 (TC50 > 100 µM) both MNS and BAY resulted markedly more
cytotoxic (TC50 3.2 ± 0.8, and 1.5 ± 0.3 µM, respectively) while DiMF showed a slightly higher toxicity (TC50 95.5 ± 1.2 µM). Reꢀ
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sults obtained with reference NLRP3 pathway inhibitors showed that, also in this case, cytotoxicity is related not only to the elecꢀ
trophilic reactivity but also to other structural parameters as evidenced by comparing reactivity and cytotoxicity for MNS vs DiMF
(Table 1).
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Inhibition of NLRP3 ATPase activity. The ability of nonꢀtoxic compounds to inhibit the NLRP3 ATPase activity was tested on
purified human recombinant protein. Human recombinant NLRP3 was incubated at 37 °C in the presence of two different concenꢀ
trations (100 µM, 50 µM) of tested compounds for 15 min. ATP (250 µM) was then added and the mixture incubated at 37 °C for
further 40 min. The amount of ATP converted to ADP was determined by luminescence using the ADPꢀGlo assay.26 The obtained
results, expressed as percentage of enzyme inhibition with respect to vehicle treated enzyme, are reported in Table 1. All the tested
acrylates inhibited NLRP3 ATPase activity at 100 µM; as expected, propanoic acid derivatives 13 and 14, showed a negligible acꢀ
tivity suggesting that the presence of electrophilic acrylate moiety is essential for NLRP3 ATPase inhibition for this series of comꢀ
pounds. The inhibition of NLRP3 ATPase appeared to be concentrationꢀdependent. Derivatives 1–3, 5, 7, 11, and 12 showed the
most interesting activities at 50 µM, with an inhibition spanning the 19–32 % range.
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Collectively these data indicate that the acrylate derivatives with reduced reactivity inhibit the NLRP3 ATPase activity which is
necessary for the NLRP3 inflammasome assembly and activation,32 consequently they can be effectively used as NLRP3 blockers.
Antipyroptotic activity. To assess the ability of the synthesized compounds to affect a NLRP3ꢀdependent cellular response,
their effect on the ATPꢀtriggered pyroptosis of THPꢀ1 cells was evaluated.25 Pyroptosis was significantly decreased by compounds
1ꢀ7, 11, and 12 (all at 10 µM; Table 1). No effect was exerted by derivatives 13 and 14, lacking the acrylate substructure. Comꢀ
pounds were classified into two subclasses: highly active (70–80% pyroptosis inhibition), such as αꢀhydroxymethyl acrylates 1 and
2; and moderately active (30–60% inhibition), including derivatives 3–7, 11, and 12. Of note, an antipyroptotic activity was deterꢀ
mined for the carboxylic acids 3, 4, and 12, suggesting that these compounds, being the putative cellular metabolites of parent ester
derivatives 1, 2, and 11 respectively, can maintain the in cell activity; this is not surprising taking into account their ability to act as
direct NLRP3 ATPase inhibitors. Compared with the monochloro derivatives 1, 3, and 5, the more lipophilic dichloroꢀsubstituted
derivatives 2, 4, and 6 showed similar or lower NLRP3 ATPase inhibition and antipyroptotic properties, therefore, the development
of dichloroꢀsubstituted compounds was discontinued.
Analysis of the overall data, together with synthetic considerations, prompted us to choose 11 for further studies. 11 is a nonꢀ
toxic ethyl acrylate derivative showing an acceptable degree of reactivity toward thiol groups. Compound 11 inhibits NLRP3
ATPase in a concentrationꢀdependent manner and prevents pyroptosis of THPꢀ1 cells; moreover, it can be easily synthesized in
high yield (74%) from 1 allowing the possibility to produce multigram amounts of compound for in vivo studies.
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1
2
3
Table 1. Reactivity with cysteamine, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic activity of synthesized compounds
1–8, 10–14, and reference inhibitors MNS, BAY, DiMF.
4
5
6
Compd
Reactivity
k2 (Mꢀ1 sꢀ1)a
Cytotoxicity TC50 (ꢁM)b
[% viability]
NLRP3 ATPase inhibition
(%)c
Pyroptosis decrease (%)d cLogPe
7
8
9
100 µM
72 ± 14
35 ± 8
50 µM
32 ± 11
19 ± 5
0.824 ± 0.017
1.340 ± 0.220
Not Reactive
Not Reactive
0.734 ± 0.047
0.292 ± 0.014
1.596 ± 0.056
56.2 ± 5.4
65.0 ± 5.0
68.3 ± 5.1
80.9 ± 5.7f
74.4 ± 2.1
45.0 ± 6.9g
40.2 ± 6.6
58.3 ± 7.6g
43.9 ± 7.8g
37.2 ± 6.7
Not Tested
Not Tested
33.6 ± 7.1
35.8 ± 2.0
3.8 ± 2.1
2.59
3.30
1.53h
2.24h
4.06
4.77
3.44
3.42
3.40
3.92
2.94h
3.83
2.92h
2.18
0.94
0.78
1
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
> 100 [80.7 ± 3.2]
> 100 [88.1 ± 4.2]
> 100 [95.7 ± 6.2]
> 100 [87.7 ± 7.2]
> 100 [78.1 ± 3.7]
33.3 ± 7.7
69 ± 12
43 ± 1
21 ± 7
3
11 ± 6
4
56 ± 2
27 ± 1
5
Not soluble
60 ± 11
Not Tested
Not Tested
52 ± 10
44 ± 9
10 ± 3
6
24 ± 6
7
Not Tested
Not Tested
29 ± 8
8
8.182 ± 0.043
Not Reactive.
Not Reactive
Not Reactive
Not Reactive
2.50 ± 0.12
Not Tested
10
11
12
13
14
MNS
BAY
DiMF
> 100 [96.6 ± 2.4]
> 100 [102.1 ± 4.5]
> 100 [86.1 ± 4.2]
> 100 [98.7 ± 3.2]
3.2 ± 0.8
22 ± 4
12 ± 2
inactive
inactive
Not Tested
Not Tested
inactive
15 ± 6
5.2 ± 3.3
Not Tested
Not Tested
inactive
Not Tested
Not Tested
65.2 ± 3.1
Not Applicaplei
1.5 ± 0.3
39.01 ± 0.57
95.5 ± 1.2
aCompounds (250 µM) were reacted with equimolar amount of CAM in pH 7.4 PBS containing 12.5% CH3CN at 37 °C. The decrease
in free CAM was monitored using DTNB reagent at different time points during 90 min. Secondꢀorder rate constants, calculated according
b
ͤ
to the equation 1/͗
= 1/͗
+ kt, were determined from 3ꢀ10 data points per rate plot. THPꢀ1 cells were exposed to increasing conꢀ
ꢀꢁꢂ
ꢀꢁꢂ
centrations (0.1–100 µM) of each compounds and cell viability was measured at 72 h by MTT assay. TC50 represents the concentration of
compound which decreases cell viability by 50%. The percentage of viable cells at 72 h exposed to compound (100 µM) is in brackets.
DMSO used as the cosolvent had no effect. cCompounds were incubated with isolated NLRP3 for 20 min, then ATP 250 µM was added
d
and its conversion to ADP was measured by the ADPꢀGlo assay
.
Data are expressed as percentage of inhibition vs vehicle alone. PMAꢀ
differentiated and LPSꢀprimed THPꢀ1 cells were preꢀtreated with either vehicle alone or each compound (10 µM; 1 h). Pyroptosis was
triggered with ATP (5 mM) and LDH activity was measured in the collected supernatant 1 h after ATP exposure by a colorimetric assay.
Data are expressed as percent of pyroptosis decrease vs vehicle alone. k2 values and biological data are expressed as mean ± SEM of at
least three separate experiments. DMSO used as the cosolvent had no effect. eCalculated with ChemBioDraw Ultra 12.0, CambridgeSoft.
fRef. 26. gRef 25.; hcLogPN. iToo high to be measured.
Compound 11 inhibits ILꢀ1β release. We next compared the ability of 11 with other inhibitors of NLRP3 inflammasome sigꢀ
naling pathway to block ILꢀ1β release from macrophages after NLRP3 activation. LPSꢀprimed mouse bone marrowꢀderived macroꢀ
phages (BMDM) were exposed for 1 h to different concentrations of 11 and thereafter NLRP3 was activated with the classical actiꢀ
vating stimuli extracellular ATP or nigericin. 11 was able to significantly inhibit ATPꢀ and nigericinꢀinduced ILꢀ1β release at 10
ꢁM (Figure 4A,B). The inhibition of ILꢀ1β release by 11 was similar or even greater than that observed by DiMF but less than that
of BAY (Figure 4A,B). Furthermore, 11 reduced caspaseꢀ1 activation and pyroptosis (measured as extracellular LDH) in these
macrophages (Figure 4C–F). Compared to DiMF and BAY, compound 11 was more effective in blocking pyroptosis (Figure
4C,D).
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We next assessed the ability of 11 to block LPS induced proꢀinflammatory gene expression. When the macrophages were exꢀ
posed to 11 1 h before LPS priming, the expression of Tnfa, Il6 and Il1b genes was significantly decreased (Figure 4G), suggesting
that 11 could not only block NLRP3 activation, but also the NFꢀκB pathway. Next, as compound 11 could potentially react with
CysꢀSH residues in the active site of cysteine protease caspaseꢀ1, we next assessed the ability of 11 to inhibit caspaseꢀ1 activity.
Recombinant human caspaseꢀ1 activity was unaffected by different concentrations of 11 (Figure 4H), suggesting that 11 did not diꢀ
rectly target caspaseꢀ1 activity.
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This result can be considered a further improvement in terms of selectivity of 11 compared to 1, previously found to irreversibly
inhibit caspaseꢀ1.25 This behavior confirms that highly reactive acrylates (i.e. 1) can interact unspecifically with biological nucleoꢀ
philes (such as HSA or caspaseꢀ1), while better selectivity is achieved by reducing the electrophilicity as with compound 11, thus
compensating the loss of potency that is usually obtained with weak electrophiles.
Dexamethasone (DEX), a standard steroidal drug used in the treatment of IBD, was also studied. Interestingly, DEX exerted sigꢀ
nificant antiꢀpyroptotic effects at 100 and 250 nM in mouse BMDM without any effects on ILꢀ1β release.
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Figure 4. Effect of 11 on ILꢀ1β release and caspaseꢀ1 activation in macrophages. (A, B) ELISA for ILꢀ1β release from LPSꢀprimed
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BMDM (1 ꢀg/mL, 3 h), then incubated with 11, DEX, BAY, DiMF or vehicle (1 h) and stimulated with ATP (5 mM, 30 min, panel A) or
nigericin (10 ꢀM, 30 min, panel B). (C, D) Percentage of extracellular LDH vs total intracellular LDH from macrophages treated as in A or
B. (E, F) Western blot of mature caspaseꢀ1 (p10 fragment) and mature ILꢀ1β (p17 fragment) in macrophage supernatants treated as in A or
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B. (G) Relative gene expression (2ꢀꢁCt) for Tnfa, Il6 and Il1b determined by quantitative RTꢀPCR from BMDMs incubated with 11 at indiꢀ
cated concentrations (30 min) and then treated or not with LPS (1 ng/mL, 4 h). (H) Recombinant human caspaseꢀ1 (10 U) activity incubatꢀ
ed with AcꢀYVADꢀCMK (100 ꢀM) or the indicated concentrations of 11. Western blots are a composite from separated lanes from the
same blot/image exposed the same time (as denoted by a dotted line). Each column in A–D, G, H represents the mean ± SEM. (n = 3 indeꢀ
pendent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (P > 0.05); Oneꢀway ANOVA with Bonferroni’s postꢀtest.
Mechanism of NLRP3 inhibition by 11. To further investigate the mechanism of action of 11, we used HEK293 cells expressꢀ
ing NLRP3 fused to Luciferase and the yellow fluorescence protein (YFP) (Figure S4A) to record BRET signal that allowed us to
follow NLRP3 conformational changes during its activation.33, 34 The recombinant receptor NLRP3 was functional as it was able to
recover nigericinꢀinduced ILꢀ1β release when expressed in NLRP3ꢀdeficient macrophages (Figure S4B). After 24 h incubation, 11
was able to reduce the steady state (or basal) BRET signal of NLRP3 (Figure 5A) without affecting viability of cells (Figure S4C),
meaning that 11 could interfere with the basal NLRP3 conformation. After nigericin treatment, BRET signal for NLRP3 decreases
and then recovers with time (Figure 5B).34 These changes result from the conformational changes of NLRP3 protein during activaꢀ
tion as a consequence of the intracellular K+ decrease induced by nigericin, since they could be avoided by applying nigericin in a
buffer with 140 mM of K+ (Figure 5B). 11 did not affect the initial drop in BRET signal, but was able to impair the recovery of
BRET (Figure 5B). As a control, high K+ buffer or 11ꢀcontaining vehicle buffer did not change NLRP3 BRET signal over time
(Figure S4D). When measuring the slope of the three phases for the BRET variation after nigericin treatment (Figure S4E), we
found that 11 was able to significantly affect the second phase of conformational changes upon stimulation (Figure 5C). Taken toꢀ
gether, 11 did not block the initial conformational changes suffered by NLRP3 upon sensing the decrease of intracellular K+, howꢀ
ever it affected a second step of NLRP3 conformational change that could be related with the ATPase activity of the receptor and
being independent on the decrease of intracellular K+. To confirm the independency of NLRP3 ATPase activity from K+ the previꢀ
ously described NLRP3 ATPase assay was performed using high K+ concentration (140 mM) in the test buffer. In these conditions
the ATPase activity of NLRP3 protein was unaffected (data not shown).
Compounds 11 and its close nonꢀelectrophilic analogue 13 (used as a probe derivative) were further tested to verify the irreversiꢀ
ble inhibition of the NLRP3 ATPase activity by 11. The HisꢀFLAGꢀtags NLRP3 protein was immobilized on a 96ꢀwell plate using
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Journal of Medicinal Chemistry
Flag Ab. NLRP3 ATPase activity was assayed introducing a washing step after preincubation with tested compounds, before ATP
addition. In these conditions 11 behaved as a covalent inhibitor (45 ± 1% inhibition without washing; 40 ± 4% with washing), while
its close analogue 13 showed a fully restored ATPase activity after the washing step (10 ± 1% inhibition without washing; no deꢀ
tectable inhibition with washing). According to the obtained results, despite the limited potency on isolated enzyme of compound
11, its irreversible NLRP3 inhibition can offer pharmacological advantages such as a prolonged time of action typical of targeted
covalent inhibitors.35
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Figure 5. Compound 11 affects K+ꢀindependent conformational changes of NLRP3 during activation. (A) Basal BRET signal from
HEK293 cells expressing recombinant YFPꢀNLRP3ꢀLuc receptor and incubated for 24 h with vehicle or the indicated concentrations of 11
(n = 10 independent experiments). (B) Kinetics of the NLRP3 BRET signal in response to 10 ꢁM nigericin in vehicle buffer (black circles),
high K+ buffer (white circles), or with 11 (100 ꢁM). The arrow indicates when nigericin was added (n = 5–7 independent experiments). (C)
Slope of the BRET signal in the three phases denoted in B (n = 5–12 independent experiments). Each column in A, C or circle in B repreꢀ
sent the mean ± SEM. of the indicated number of experiments. **P < 0.01; ***P < 0.001; ns: not significant (P > 0.05); Oneꢀway ANOVA
with Bonferroni’s postꢀtest.
Characterization of in vitro preliminary ADME profile of compound 11
Compound 11, able to efficiently inhibit NLRP3ꢀdependent ILꢀ1β release, was selected for in vivo studies in a model of colitis.
However, the measured solubility of 11 in PBS (pH 7.4) was very low (0.0060 ± 0.0008 mg/mL), therefore we considered to
adopt oral administration using olive oil as the preferred vehicle. Accordingly, we checked the stability of 11 suspended in olive oil
in simulated gastric fluid and simulated intestinal fluid to verify potential early metabolic instability of 11.36 Compound 11 (1.56
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mg in 100 µL olive oil) was incubated at 37 °C in simulated gastric fluid (SGF) for 2 h, subsequently in simulated intestinal fluid
(SIF) for further 2 h. Samples of the fluids were withdrawn at fixed time (0, 30, 60, and 120 min) and analyzed by UHPLCꢀMS
compared to a control sample.37 Compound 11 proved stable in both conditions tested with >99.5 % of 11 being detected after 2 h
of incubation in both fluids.
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The permeability of 11 through rat small intestine was then evaluated ex vivo with the nonꢀeverted intestinal sac method. Despite
some shortfalls (e.g. interruption of normal blood flow, lack of a nervous system) this method is widely used to study passive abꢀ
sorption of molecules.38 Furthermore, the presence of a mucus layer, the expression of transport proteins and drug metabolism alꢀ
low this model to provide additional useful data.39
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A suspension of 11 (12.5 mg in 200 µL olive oil) was syringed into intestinal sacs obtained from different segments of rat intesꢀ
tine (duodenum, jejunum, ileum). The filled tissues were incubated at 37 °C for 2 h. Sample solutions were withdrawn from the seꢀ
rosal side and the amount of analytes was determined by UHPLCꢀMS. Analysis of the solution withdrawn from the serosal side
during the incubation of intestinal sacs filled with 11 revealed only the presence of the acid derivative 12, while no detectable
amount of 11 was found. As reported in Figure 6, the concentration of the acid derivative 12 increased with time in all the intestinal
segments, with differences in time onset: ileum ˃ duodenum > jejunum. Cumulative amount of 12 from sacs filled with 11 resulted
significantly higher in ileum tract than in duodenum and jejunum (Figure 6). Obtained data indicates that 11 is absorbed and activeꢀ
ly metabolized to the corresponding acid 12 in intestinal cells; the absorption and/or metabolism of 11 resulted faster and higher in
the ileum tract with respect to the other intestinal segments studied.
Figure 6. Timeꢀcourse measurements of permeation of compound 11 in rat small intestine. Nonꢀeverted intestinal sacs were
filled with a suspension of 11 (12.5 mg / 0.2 mL olive oil) and incubated in oxygenated KrebsꢀRinger buffer at 37 °C with smooth
shaking. Sample solution (0.5 mL) was withdrawn from the serosal side at fixed time intervals and replaced with fresh buffer.
Compound 11 was not detected at the serosal side by UHPLCꢀMS analysis, while the concentration of its metabolite (12) was quanꢀ
tified. Tests were performed in triplicate on intestinal segments from three different rats. Cumulative amount of 12 at 120 min: * P
< 0.05 ileum vs duodenum, P < 0.05 ileum vs jejunum, tꢀtest.
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Journal of Medicinal Chemistry
Next, we assessed the in vitro stability in rat microsomal fractions40 of the metabolite 12, the sole species identified to pass
through gut epithelium in intact intestine. Remarkably, compound 12 showed a similar in vitro activity and cytotoxicity profile with
respect to 11 (Table 1). Since in IBD patients the intestinal barrier is usually damaged and endowed with a higher degree of permeꢀ
ability41 we cannot exclude that, in IBD setting, compound 11 can be absorbed through the loosen cell junctions without undergoing
epithelial cleavage. Therefore, the metabolism of 11 in rat microsomal fractions was also studied. Compounds 11 and 12 in DMSO
solution (final conc. 100 µM with 2% DMSO) were incubated at 37 °C for 2 h in 100 mM PBS (pH 7.4) with rat liver microsomes
(0.5 mg proteins/mL) in the presence of a NADPHꢀgenerating system. The reaction was stopped at different times (0, 15, 30, 60,
and 120 min) and analyzed by UHPLCꢀMS. In these conditions compound 12 proved stable (>99% of compound detected) giving
rise to no detectable metabolites after 2 h of incubation, while compound 11 was quantitatively converted to 12 within a very short
time (<1 min), giving no possibility to measure rate of microsomal clearance of 11. Of note, when the experiment was repeated
with 11 in the absence of NADPHꢀgenerating system the same results were obtained, thus indicating that cytochromeꢀmediated meꢀ
tabolism is not responsible for the generation of 12.
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Collectively, the obtained results suggest that compound 11 reaches the intestinal epithelium without undergoing chemical modiꢀ
fications. After absorption into epithelial cells it is likely to act locally at the mucosal epithelial level where it is also converted to
the nonꢀtoxic metabolite 12. This latter compound can diffuse into the blood flow and act systemically without undergoing further
hepatic metabolism (Figure 7).
Figure 7. Metabolic fate of compound 11 and its putative mechanism of action. Following oral administration, 11 reaches the inꢀ
testinal epithelium without undergoing metabolic cleavage. After absorption into intestinal epithelial cells, 11 inhibits NLRP3
pathways thanks to both direct NLRP3 inhibition and interference with NFꢀκB ꢀmediated gene expression. Epithelial intestinal cells
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metabolize compound 11 to its active acid metabolite 12. This metabolite crosses the basal membrane and diffuses into the blood
flow. Compound 12 is stable to liver metabolism (2 h). In the presence of a damaged intestinal barrier (as in IBD), compound 11
could potentially diffuse into the blood flow. Once delivered to the liver it is rapidly metabolized to generate the acid 12.
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Oral administration of 11 reduces systemic and colonic inflammation in DNBSꢀtreated rats. To verify the therapeutic poꢀ
tential of using an NLRP3 inhibitor in colonic inflammation, 11 was tested in a rat model of colitis induced by 2,4ꢀ
dinitrobenzenesulfonic acid (DNBS)42 in comparison with standard drug dexamethasone (DEX). Briefly, colitis was induced in rats
via intrarectal administration of DNBS (15 mg in 0.25 mL of 50% ethanol). 11 (12.5, 25.0, 50.0 mg/kg/day in 0.2 mL olive oil) or
DEX (1 mg/kg day in 0.2 mL 1% methylcellulose43) were administered intragastrically for 6 days, starting the same day of colitis
induction. Rats treated with different vehicles did not display differences as compared to control group (DNBSꢀuntreated animals)
and colitis group (DNBSꢀtreated rats), therefore only one control group and one colitis group were included. At the end of treatꢀ
ments, colonic tissues were excised, and scored for macroscopic damage, in accordance with the criteria previously reported.42
Macroscopic score was evaluated on the whole colon. The macroscopic damage was scored for each rat on a 0ꢀ to 6ꢀpoint scale,
based on the system displayed in Table S1, by two observers blinded to the treatment. At the time of experiment, the body weight,
the weight of spleen and colonic length were also measured.
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Effect of oral administration of 11 on body weight and spleen weight. Six days after DNBS administration, rats displayed a deꢀ
crease of 63 ± 4.1 g in their body weight, whereas control rats showed a weight gain (+20 ± 1.7 g) (Figure 8A). Significant increꢀ
ments of body weight were observed in inflamed rats under treatment with 11 (12.5, 25 and 50 mg/kg), while DEX (1 mg/kg) was
without effects (Figure 8A). Measurement of spleen weight was assumed as an index of systemic inflammation.44 Treatment with
DNBS resulted in a significant increment of spleen weight (+39.3%) (Figure 8B). Such an increase was significantly reduced by
administration of 11 (+2.2, +4.3 and +4.8% at 12.5, 25, 50 mg/kg, respectively), or DEX (+1.6%) (Figure 8B).
Effect of oral administration of 11 on colon length and macroscopic damage score. Six days after DNBS administration, the
inflamed rats were characterized by a shortened colonic length (ꢀ34.4%). The inhibition of NLRP3 inflammasome complex with 11
doseꢀdependently attenuated the decrease in colonic length (ꢀ19, ꢀ13 and ꢀ8% at 12.5, 25, 50 mg/kg, respectively) (Figure 8C).
Six days after DNBS administration, the distal colon appeared thickened and ulcerated with evident areas of transmural inflamꢀ
mation (Figure 9). Adhesions were often present, and the bowel was occasionally dilated, with a macroscopic damage accounting
for 8.6 ± 0.6. Rats treated with 11 displayed a significant reduction of macroscopic damage score (4.7 ± 0.9 at 12.5 mg/kg, 3.1 ± 0.7
at 25 mg/kg, and 2.8 ± 0.4 at 50 mg/kg, Figure S5), DEXꢀtreated animals also showed a decrease in macroscopic damage score (2.0
± 0.1) (Figures 9, S5).
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Figure 8. Effects of 11 (12.5, 25, and 50 mg/kg) or dexamethasone (DEX, 1 mg/kg) on body weight (A), spleen weight (B), colonic length
(C), tissue myeloperoxidase (MPO) (D), ILꢀ1β (E), and TNF levels (F) at day 6 after induction of colitis with DNBS. Each column repreꢀ
sents the mean ± SEM. (n = 10). *P < 0.05 vs control group; aP < 0.05, vs DNBS group
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Oral administration of 11 reduces colonic myeloperoxidase, ILꢀ1β and TNF levels in DNBSꢀtreated rats. Biochemical assays
were performed on specimens taken from a region of the inflamed colon immediately adjacent and distal to the gross necrotic damꢀ
age. In particular, since several inflammatory mediators, including ILꢀ1β and TNF cytokines, contribute to the pathogenesis of
bowel inflammation,14, 45 we assessed the effects of 11 on inflammatory infiltrate as well as ILꢀ1β and TNF levels in colonic tissues
from DNBSꢀtreated rats.
The levels of tissue myeloperoxidase (MPO), regarded as a quantitative index to estimate the degree of bowel wall infiltration by
inflammatory polymorphonuclear cells, were assessed as previously described.46 Specimens of colonic tissues (30 mg) were hoꢀ
mogenized and MPO concentration was determined by means of an enzymeꢀlinked immunosorbent (ELISA) assay. The results
were expressed as ng of MPO per mg of colonic tissue (Figure 8D). Rats with DNBSꢀinduced colitis showed a marked increase in
colonic MPO levels (48.1 ± 4.7 ng mgꢀ1 tissue) as compared with control animals (3.9 ± 0.2 ng mgꢀ1 tissue). Treatments with 12.5,
25 and 50 mg/kg of 11, or 1 mg/kg of DEX significantly prevented the increments of colonic MPO levels associated with DNBS
administration (Figure 8D). ILꢀ1β and TNF levels in colonic tissue homogenates were measured by ELISA as previously deꢀ
scribed47 and expressed as ng and pg per mg of tissue, respectively (Figure 8E,F). The induction of colitis was associated with a
significant increment of colonic ILꢀ1β levels (22.3 ± 2.9 ng/mg tissue) confirming previous work where increase of ILꢀ1β was
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found in a mouse model of postinfectious irritable bowel syndrome.48 Treatment with 11 resulted in a doseꢀdependent decrease in
tissueꢀassociated ILꢀ1β levels (16.4 ± 0.5, 12.8 ± 2.5, and 6.6 ± 0.2 ng/mg tissue at 12.5, 25, 50 mg/kg, respectively) (Figure 8E).
Colonic inflammation induced by DNBS was associated also with a significant increase in tissue TNF levels (5.9 ± 0.6 pg/mg tisꢀ
sue). Treatments with 11 significantly decreased the concentration of this cytokine in colonic tissues (3.5 ± 0.2, 3.3 ± 0.2, and 2.7 ±
0.1 pg/mg tissue at 12.5, 25, 50 mg/kg, respectively, Figure 8F). DEXꢀtreated animals showed a significant decrease in ILꢀ1β and
TNF colonic tissue levels (6.7 ± 0.3 ng/mg tissue and 2.2 ± 0.2 pg/mg tissue, respectively, Figure 8E,F). This effect confirms our in
vitro data where 11 is able to block expression of both cytokines, as well as inhibiting the NLRP3 inflammasome.
Taken together, our findings show that 11 can exert beneficial effects on colitis, by reducing MPO, ILꢀ1β and TNF proꢀ
inflammatory cytokine levels in colonic tissues from inflamed rats, thus suggesting that the blockade of NLPR3 activation could
represent a suitable pharmacological target for the management of intestinal inflammation.
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Of note, 11, at the dose of 50 mg/kg was able to elicit similar beneficial effects as dexamethasone, although at a higher dose.
However, 11 appears to be more advantageous than dexamethasone, since it showed a good general tolerability up to the dose of 50
mg/kg, and, most importantly, it increased significantly the body weight in inflamed rats, while dexamethasone treatment was assoꢀ
ciated with body weight loss both in control and in DNBSꢀtreated rats. In addition, it is widely recognized that steroids are associatꢀ
ed with several adverse effects, which limit their use as longꢀterm therapy. Indeed, steroidꢀfree remission presently represents a
primary endpoint in the treatment of IBD. According to the obtained results, 11, in particular at the dose of 25 and 50 mg/kg, was
able to attenuate colonic inflammation. In addition, 11 at 50 mg/kg exerted beneficial effects on colonic inflammation comparable
with DEX, without deleterious effects on the body weight.
Figure 9. Macroscopic appearance of colonic tissues from control rats, DNBSꢀtreated rats, and rats treated with DNBS and either 11 (25,
and 50 mg/kg), or dexamethasone (DEX, 1 mg/kg).
CONCLUSIONS
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Journal of Medicinal Chemistry
Through the chemical modulation of a previously identified acrylate derivative we were able to obtain 11, a nonꢀcytotoxic moleꢀ
cule able to target the activation of the NLRP3 inflammasome. Compound 11 was able to counteract NLRP3 activation through diꢀ
rect irreversible interaction with NLRP3 and partial inhibition of LPSꢀdriven proꢀinflammatory gene expression. Preliminary in
vitro ADME studies showed that 11, following oral administration, is stable and is absorbed into the intestinal epithelium where it
can act locally and generate the nonꢀtoxic metabolite 12. The in vivo ability of 11 to alleviate the outcomes of DNBSꢀinduced coliꢀ
tis after oral administration was established. Compound 11, counteracting intestinal inflammation, may represent a lead compound
for the development of novel NLRP3 inflammasome inhibitors, characterized by good degree of potency and reduced toxicity, suitꢀ
able for treatment of inflammatory intestinal disorders. Further medicinal chemistry and pharmacological studies aimed at increasꢀ
ing NLRP3 inhibition and characterizing the in vivo pharmacokinetics and mechanism of action of the identified lead are in proꢀ
gress.
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EXPERIMENTAL SECTION
General methods. All the reactions were monitored by TLC on Merck 60 F254 (0.25 mm) plates, which were visualized by UV
inspection and/or by spraying KMnO4 (0.5g in 100 ml 0.1 N NaOH). Na2SO4 was used as drying agent for the organic phases. Flash
chromatography (FC) purifications were performed using silica gel Fluka with 60 mesh particles. 1H and 13C NMR spectra were
registered on Bruker Avance 300 spectrometer, at 300 and 75 MHz respectively. Chemical shifts (δ) are given in ppm, calibrated to
tetramethylsilane (TMS) as internal standard. Lowꢀresolution mass spectra were recorded on a FinniganꢀMAT TSQ700 in chemical
ionization mode (CI) using isobutane. ESIꢀmass spectra were recorded on a Waters Micromass Quattro Micro equipped with an ESI
source. Melting points were measured with a capillary apparatus (Büchi 540). Purity of compounds was checked by UHPLC (Perꢀ
kinꢀElmer) Flexar 15, equipped with UVꢀVis diode array detector using an Acquity UPLC CSH PhenylꢀHexyl 1.7 µm 2.1×50 mm
column (Waters) and H2O/CH3CN and H2O/CH3OH solvent systems. Detection was performed at λ = 200, 215 and 254 nm. The
analytical data confirmed that the purity of the products was ≥95%. Solubility and stability of compounds in biological fluids were
determined using an Acquity UPLCꢀMS instrument, equipped with binary solvent manager, sample manager, column manager,
PDA detector (Waters) e Micromass Quattro Micro (API). The analytical conditions are reported in the following sections. Derivaꢀ
tives 1–3,25 1226 were obtained according to the reported methods.
2ꢀ((2,4ꢀDichlorophenyl)(hydroxy)methyl)acrylic acid (4). To a stirred solution of 2 (229.2 mg, 0.839 mmol) in 1,4ꢀdioxane/water
1/1 (2 mL), 2.5 M NaOH was added and the reaction was stirred at rt for 18 h. The mixture was diluted with 10% NaHCO3 (15 mL)
and extracted with EtOAc (15 mL). The organic layer was removed and the aqueous phase was acidified with 1M HCl and extractꢀ
ed with EtOAc (3 x 20 mL). The organic phases were dried and evaporated. The product was purified by FC eluting with
CH2Cl2/MeOH 9.5/0.5. Compound 4 was obtained as a white solid (203.1 mg, 0.822 mmol, 98% yield). Mp: 113.1ꢀ114.4 °C;
MS/ESI: [MꢀH]ꢀ 245; 1H NMR (CDCl3): δ, 7.45ꢀ7.22 (m, 3H, ArH), 7.14 (br, 1H, COOH), 6.44 (s, 1H, C=CH), 5.87 (s, 1H, CH),
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5.64 (s, 1H, C=CH), 3.54 (br, 1H, OH); 13C NMR (CDCl3): δ, 171.1, 139.7, 136.6, 134.3, 133.4, 129.7, 129.3, 129.0, 127.4, 68.5.
Anal. (C10H8Cl2O3) C, H
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2ꢀ((2ꢀChlorophenyl)(3ꢀethoxyꢀ3ꢀoxopropoxy)methyl)acrylate (5). To a stirred solution of 1 (240.7 mg, 1.00 mmol) in CH3CN (2
mL) DMAP (122.2 mg, 1.00 mmol) and DBU (0.448 mL, 3.00 mmol) were added at rt. After 10 min ethyl acrylate (1.10 mL, 10.0
mmol) was added dropwise and the mixture was stirred at rt for 48 h. The reaction was diluted with 2N HCl (15 mL) and then exꢀ
tracted with CH2Cl2 (3 x 15 mL). The combined organic phases were washed with brine (20 mL), dried, and evaporated. The crude
mixture was purified by FC eluting with PE/EtOAc 95/5 to afford compound 5 as a colorless oil (115.2 mg, 0.338 mmol, 34%
yield). Characterization in accordance with literature data.25
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2ꢀ((2,4ꢀDichlorophenyl)(3ꢀethoxyꢀ3ꢀoxopropoxy)methyl)acrylate (6). Compound 6 was synthesized as described above starting
from 2 (298.2 mg, 1.08 mmol). Colorless oil (118.3 mg, 0.315 mmol, 29% yield). Characterization in accordance with literature
data.25
Ethyl 2ꢀ((2ꢀchlorophenyl)(methoxy)methyl)acrylate (7). To a stirred solution of 1 (392.6 mg, 1.63 mmol) in CH2Cl2 (10 mL)
Ag2O (755.5 mg, 6.52 mmol) was added. CH3I (0.732 mL, 11.8 mmol) was added portionwise and the reaction was stirred at rt for
96 h. The mixture was filtered through Celite® and the solvent removed under reduced pressure. The mixture was purified by FC
eluting with PE/EtOAc 98/2 to give compound 7 (168.0 mg, 0.660 mmol, 40% yield) as a colorless oil. MS/ESI [M+H]+ 255; 1H
NMR (CDCl3): δ, 7.51ꢀ7.11 (m, 4H, ArH), 6.39 (s, 1H, C=CH), 5.69 (s, 1H, C=CH), 5.61 (s, 1H, CH), 4.18 (q, 2H, J= 7.1 Hz,
OCH2), 3.38 ( s, 3H, OCH3), 1.24 (t, 3H, J= 7.1 Hz, CH3); 13C NMR (CDCl3): δ, 166.2, 140.5, 137.3, 134.6, 129.9, 129.5, 128.8,
127.4, 126.7, 77.6, 61.2, 58.1, 14.5. Anal. (C13H15ClO3) C, H
Ethyl 3ꢀ(acetoxy(2ꢀchlorophenyl)methyl)ꢀ2ꢀoxobutꢀ3ꢀenoate (8). To a stirred solution of 1 (2.07 g, 8.60 mmol) and DMAP (210.2
mg, 1.72 mmol) in CH2Cl2 (40 mL) acetic anhydride (0.974 mL, 10.3 mmol) was added portionwise in 1 h. The reaction was stirred
at rt for 1 h. The mixture was washed with water (3 x 30 mL), 10% NaHCO3 (10 mL), and brine (15 mL). The organic phase was
dried, and the solvent removed under reduced pressure. The crude product was purified by FC eluting with PE/EtOAc 9/1 to obtain
compound 8 (2.14 g, 7.57 mmol, 88% yield) as a colorless oil. MS/CI (isobutane): [M+H]+ 283; 1H NMR (CDCl3): δ, 7.47ꢀ7.26 (m,
4H, ArH), 7.06 (s, 1H, CH), 6.47 (s, 1H, C=CH), 5.63 (s, 1H, C=CH), 4.19 (q, 2H, J= 7.1 Hz, OCH2), 2.12 ( s, 3H, COCH3), 1.23
(t, 3H, J= 7.1 Hz, CH3); 13C NMR (CDCl3): δ, 169.6, 165.3, 138.9, 135.8, 134.1, 130.2, 130.0, 128.9, 127.8, 127.3, 70.5, 61.5, 21.1,
14.4. Anal. (C14H15ClO4) C, H.
Ethyl 2ꢀ((allyloxy)(2ꢀchlorophenyl)methyl)acrylate (9). To a stirred solution of 8 (299.7 mg, 1.06 mmol) in dry THF (5 mL)
DABCO (237.8 mg, 2.12 mmol) was added and the solution stirred at rt for 10 min. 2ꢀPropenol (0.198 mL, 3.18 mmol) was then
added and the reaction was stirred at 60 °C for 48 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20
mL), washed with brine (15 mL), dried, and the solvent removed under reduced pressure. The crude product was purified by FC
eluting with PE/EtOAc 95/5 to obtain compound 9 (193.4 mg, 0.689 mmol, 65% yield) as a colorless oil. MS/ESI: [M+H]+ 281; 1H
NMR (CDCl3): δ, 7.45ꢀ7.14 (m, 4H, ArH), 6.39 (s, 1H, C=CH), 5.99ꢀ5.87 (m, 1H, HC=CH2), 5.76 (s, 1H, CH), 5.72 (s, 1H,
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Journal of Medicinal Chemistry
C=CH), 5.30ꢀ5.14 (m, 2H, HC=CH2), 4.18 (q, 2H, J= 7.1 Hz, OCH2CH3), 4.06ꢀ4.00 (m, 2H, OCH2), 1.23 (t, 3H, J= 7.1 Hz, CH3);
13C NMR (CDCl3): δ, 165.7, 140.4, 137.2, 134.4, 133.9, 129.5, 129.0, 128.7, 126.9, 126.4, 117.1, 75.0, 70.7, 60.8, 14.1.
Ethyl 2ꢀ(2ꢀchlorophenyl)ꢀ2,5ꢀdihydrofuranꢀ3ꢀcarboxylate (10). To a stirred solution of 9 (80.7 mg, 0.287 mmol) in dry CH2Cl2
(12 mL) under N2, 1,3ꢀbisꢀ(2,4,6ꢀtrimethylphenyl)ꢀ2ꢀimidazolidinylidene)dichloro(oꢀisopropoxyphenylmethylene) ruthenium (4.0
mg) was added. The reaction was heated at reflux for 3 h and then at rt for 16 h. The solvent was removed under reduced pressure
and the crude product was purified by FC eluting with PE, then PE/EtOAc 98/2. Compound 10 (30.9 mg, 0.123 mmol, 43% yield)
was obtained as a yellow solid. MS/ESI [M+H]+ 253; 1H NMR (CDCl3): δ, 7.48ꢀ7.15 (m, 4H, ArH), 7.14ꢀ7.00 (m, 1H, C=CH),
6.45ꢀ6.37 (m, 1H, CH), 5.05ꢀ4.66 (m, 2H, OCH2), 4.06 (q, 2H, J= 7.3 Hz, OCH2CH3), 1.13 (t, 3H, J= 7.1 Hz, CH3); 13C NMR
(CDCl3): δ, 162.5, 140.0, 138.1, 135.4, 134.3, 130.1, 129.8, 129.2, 127.3, 83.7, 75.6, 61.1, 14.4.
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Ethyl 2ꢀ(2ꢀchlorobenzyl)acrylate (11). To a stirred solution of 8 (968 mg, 3.43 mmol) and DABCO (384 mg, 3.43 mmol) in
THF/H2O 1/1 (40 mL), NaBH4 (129 mg, 3.43 mmol) was added portionwise and the reaction was stirred at rt for 1 h. The mixture
was diluted with water (30 mL), and extracted with EtOAc (3 x 50 mL). The organic phase was washed with brine (50 mL), dried,
and evaporated under reduced pressure. After purification by FC eluting with PE/EtOAc 95/5 compound 11 (649 mg, 2.88 mmol,
84% yield) was obtained as a colorless oil. MS/CI (isobutane): [M+H]+ 225; 1H NMR (300 MHz, CDCI3) δ= 7.50ꢀ6.99 (m, 4H),
6.27 (d, J= 1.1 Hz,1 H), 5.33 (d, J= 1.1 Hz, 1H), 4.22 (q, J= 7.1 Hz, 2H), 3.76 (s, 2H), 1.29 (t, J= 7.1 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ= 166.8, 138.4, 136.4, 134.5, 131.2, 129.6, 127.9, 126.8, 126.3, 60.4, 35.4, 14.2. Characterization was in accordance with
literature data.26
Ethyl 3ꢀ(2ꢀchlorophenyl)ꢀ2ꢀmethylpropanoate (13). To a stirred solution of 1 (240.7 mg, 1.00 mmol) in abs EtOH (5 mL), 10%
Pd/C (24.1 mg) was added. The atmosphere was then changed to H2 and the mixture was stirred at rt for 30 min. The reaction was
filtered through Celite® and the filtrate evaporated to obtain 13 as a pale yellow oil (219.2 mg, 0.967 mmol, 97% yield); MS/CI
(isobutane): [M+H]+ 227; 1H NMR (CDCl3): δ, 7.37−7.11 (m, 4H, ArH), 4.07 (q, 2H, J= 7.0 Hz, OCH2), 3.01 (dd, 1H, J= 11.9, 5.5
Hz, CH), 2.78ꢀ2.56 (m, 2H, CH2), 1.29ꢀ1.08 (m, 6H, 2CH3); 13C NMR (CDCl3): δ, 178.2, 138.3, 135.1, 131.1, 130.6, 128.8, 127.3,
61.8, 41.7, 36.4, 16.7, 14.7. Anal. (C12H15ClO2) C, H
3ꢀ(2ꢀChlorophenyl)ꢀ2ꢀmethylpropanoic acid (14). Compound 14 was obtained starting from 13 (119.5 mg, 0.527 mmol) using
the same procedure described for 4. Pale yellow solid (100.5 mg, 0.506 mmol, 96% yield); Mp: 36.7 – 37.4 °C. MS/ESI: [MꢀH]ꢀ
197; 1H NMR (CDCl3): δ, 11.63 (bs, 1H, COOH), 7.38−7.24 (m, 4H, ArH), 3.07 (dd, 1H, J= 13.0, 6.6 Hz, CH), 2.85ꢀ2.56 (m, 2H,
CH2), 1.16 (d, 3H, J= 6.6 Hz CH3); 13C NMR (CDCl3): δ, 182.8, 139.4, 138.5, 135.2, 129.4, 128.8, 126.8, 41.6, 39.7, 16.7. Anal.
(C10H11ClO2) C, H.
Biological screening
Reactivity as Michael acceptor, modification of human serum albumin, cytotoxicity in THPꢀ1 cells, and Antipyroptotic acꢀ
tivity were performed according to the previously reported methods.25, 26
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Measurement of NLRP3ꢀATPase Activity: Human recombinant NLRP3 (0.105 ꢀg; BPS Bioscience, San Diego, USA) was inꢀ
cubated with the assessed compounds in the reaction buffer (20 mM TrisꢀHCl, pH 7.8, 133 mM NaCl, 20 mM MgCl2, 3 mM KCl,
0.56 mM EDTA, DMSO 0.5 %) for 15 min at 37 °C. ATP (250 ꢀM, Ultra Pure ATP) was added and the reaction mixtures were
further incubated for 40 min at 37 °C. The hydrolysis of ATP by NLRP3 was determined by a luminescent ADP detection perꢀ
formed with ADPꢀGlo Kinase Assay (Promega, Madison, USA) according to the manufacturer’s protocol.
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Irreversible inhibition of NLRP3 ATPase activity. NALP3 (CIAS1), HisꢀFLAGꢀtags protein was immobilized on a well plate
using Flag Ab. Flag Ab (250 ng) diluted in PBS (pH 7.4), was added in each well of a Maxisorpꢀ96ꢀwell plateꢀwhite and incubated
for 3 h at 4 °C. After washing the remaining protein binding sites of the plate were saturated with blocking solution containing boꢀ
vine serum albumin (BSA) 2% overnight at 4 °C. The next day NALP3 protein was bound (105 ng per well) incubating the protein
diluted in the ATPase assay buffer for 3 hours at 4 °C. After washing the activity of NALP3ꢀimmobilized protein and the inhibition
of compounds 11 and 13 were measured. Compounds 11 and 13 (100 µM final concentration, 2% DMSO) were added to wells conꢀ
taining immobilized NALP3 protein and preꢀincubated for 55 min at 37 °C to mimic normal experiment time (15 min preincubation
+ 40 min incubation with ATP); in the control wells a mixture of buffer and DMSO was added. After preꢀincubation time the wells
were washed three times with reaction buffer and ATP (250 µM) was added for 40 min at 37 °C. ADP formation was measured
with ADPꢀGloꢀAssay using the reported method.26 The activity level of the enzyme was comparable to that obtained with nonꢀ
immobilized NALP3 protein. An experiment of ATPase activity without washing step was also repeated incubating 11 and 13 on
NLRP3 immobilized protein to confirm percentage of inhibition of compounds 11 and 13 as previously measured. The total amount
of immobilized NALP3 was also checked by ELISA using an antiꢀHis Tag antibody and equal amount of enzyme were detected in
all labeled wells (99.6 ± 0.7 mean of % vs control ± S.D.).
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Solubility of 11 in pH 7.4 PBS. Compound 11 (1 mg) was added to 1 mL of phosphate buffer solution (10mM with KCl 10 mM,
pH 7.4). The samples were shaked with an orbital shaker at 25 °C for 24 h. These suspensions were filtered through a PES 0.2 ꢀm
filter (Agilent) and the solutions were chromatographically analyzed.
The concentration of compounds in the PBS was quantified by UHPLCꢀMS using an Acquity Waters C18 BEH (1.7µm, 2.1 x 50
mm) column. The samples were analyzed using an isocratic method employing a mobile phase consisting of acetonitrile/water +
0.1% formic acid (60/40). The flow rate is 0.5 mL/min. The injection volume is 5 µL. The column effluent was monitored at λ =
235 nm referenced against 360 nm. The linearity of the calibration curves was determined in a concentration range of 10–500 µM
(r2 > 0.99). Data analysis was performed by using Mass Linx software (Waters).
Stability of 11 in simulated gastric fluid and simulated intestinal fluid. In vitro simulated digestion was performed according to
the reported method with some modifications.36 A simulated gastric fluid (SGF, pH 2) containing pepsin (3.2 mg/mL) and a simuꢀ
lated intestinal fluid (SIF, pH 7) containing pancreatin (8xUSP; 2 mg/mL) were prepared as described previously.36 Compound 11
(1,56 mg) was suspended into 100 µL of olive oil (dose and vehicle used for oral rat administration) and mixed with 100 µL of
SGF. The mixture was incubated at 37 °C in a shaker at 350 rpm for 2 h. For intestinal phase 200 µL of SIF were added at the samꢀ
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Journal of Medicinal Chemistry
ples derived from gastric phase and they were incubated at 37 °C in a shaker at 350 rpm for further 2 h. For both gastric and intestiꢀ
nal phases four different times were analyzed (0, 30, 60, and 120 min). A dedicated sample was prepared for each incubation time
considered. At each timeꢀpoint 200 µL of acetonitrile were added and mixed for 30 min. The samples were then centrifuged (3200
rpm, 10 min), and the water/acetonitrile phase was diluted with acetonitrile to a theoretic 0.1 mM concentration and immediately
analyzed by UHPLCꢀMS.37 Compounds concentration in the gastro intestinal fluid was quantified by UHPLCꢀMS using a Zorbax
Eclipse C18 (4.6 x 150 mm) column. The samples were analyzed using an isocratic method employing a mobile phase consisting of
acetonitrile/water + 0.1% formic acid (80/20). The flow rate is 0.5 mL/min. The injection volume is 5 µL. The column effluent was
monitored at λ = 235 nm referenced against 360 nm. Data analysis was performed by using Mass Linx software (Waters). To check
for chemical stability, a suspension of compound 11 in olive oil was also incubated with water alone, acidic solution (pH 2) without
pepsin, and intestinal solution (pH 7) without pancreatin. Each experiment was independently repeated at least three times.
Permeation measurements across excised rat small intestine. Ex vivo absorption evaluation was carried out by permeation
measurements in excised rat small intestine. Male Sprague Dawley rats (200–250 g) were anaesthetized with isoflurane, decapitated
and exsanguinated. Freshly excised rat duodenum, jejunum and ileum tissues were washed with KrebsꢀRinger buffer (KRB) and cut
into pieces of 4–5 cm. A suspension (0.2 mL) containing compound 11 (12.5 mg / 0.2 mL olive oil) was syringed into intestinal
sacs; the filled tissues were incubated in oxygenated KRB (10 mL) at 37 °C with smooth shaking. Sample solution (0.5 mL) was
withdrawn from the serosal side at fixed time intervals up to 120 minutes and replaced with fresh buffer. Tests were performed in
triplicate for each compound on the three different intestinal segments from three different rats. Compound concentration in the inꢀ
cubation buffer was quantified by UHPLCꢀMS using a Zorbax Eclipse C18 (4.6 x 150 mm) column. The samples were analyzed
using an isocratic method employing a mobile phase consisting of acetonitrile + 0.1% formic acid/water + 0.1% formic acid (80/20)
(flow rate 0.5 mL/min). The column effluent was monitored at λ = 235 nm. Quantitation was done using calibration curves of comꢀ
pound 12 chromatographed under the same conditions. The linearity of the calibration curves was determined in a concentration
range of 2.5–500 µM (r2 > 0.99). Data analysis was performed by using Mass Linx software (Waters).
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Microsomal stability assay. Compounds 11 and 12 in DMSO solution (final concentration 100 µM) were incubated in a shaker
at 350 rpm at 37 °C for 120 min in 100 mM phosphate buffer (pH 7.4), with rat liver microsomes (0.5 mg proteins / mL), in the
presence of a NADPHꢀgenerating system at a final volume of 1 mL (DMSO did not exceed 2%). The reaction was stopped at difꢀ
ferent times (0, 15, 30, 60, and 120 min) by adding to 100 µL of incubation mixture, 100 µL of acetonitrile and cooling in ice. The
samples were then centrifuged (3200 rpm, 10 min) and analyzed by UHPLCꢀMS. Compounds concentration in the incubation miꢀ
crosomes was quantified by UHPLCꢀMS using a Zorbax Eclipse C18 (4.6 x 150 mm) column. The samples were analyzed using an
isocratic method employing a mobile phase consisting of acetonitrile/water + 0.1% formic acid (80/20) for compound 11 and of acꢀ
etonitrile/water + 0.1% formic acid (70/30) for compound 12. The flow rate is 0.5 mL/min. The injection volume is 5 µL. The colꢀ
umn effluent was monitored at λ = 235 nm referenced against 360 nm. The linearity of the calibration curves was determined in a
concentration range of 2.5–500 µM (r2 > 0.99). Data analysis was performed by using Mass Linx software (Waters). To check for
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microsomal or NADPHꢀgenerating system interferences in analytical analysis, samples containing microsomes without compounds
and boiled microsomes were also incubated and analyzed. Finally, microsomes without NADPH generating system were incubated
to analyze the presence of nonꢀcytochromeꢀdependent reactions. Each experiment was independently repeated at least three times.
Cells and treatments. Primary BMDMs were in vitro differentiated from C57BL/6 mice with 20% of Lꢀcell media as reported
elsewhere,49 primed with LPS (1 ꢁg/mL, 3 h, Sigma) and subsequent treated with the indicated concentrations of dexamethasone
(Sigma), Bay 11ꢀ7082 (Sigma), dimethylfumarate (Sigma) or compound 11 or vehicle (1 h), and finally stimulated with either ATP
(5 mM, 30 min, Sigma) or nigericin (10 ꢁM, 30 min, Sigma). Immortalized BMDMs from NLRP3ꢀdeficient mice were a kind gift
of K. A. Fitzgerald.50 Retroviral TetꢀON 3G inducible system (Clontech) was used to introduce YFPꢀNLRP3ꢀLuc gene. First, cells
were transduced with tetracycline transactivatorꢀencoding retrovirus. Selected clone was further transduced by retrovirus encoding
the YFP(Nt)ꢀNLRP3ꢀLuc(Ct) fusion reporter or empty vector. After selection, these macrophages were treated with doxycycline
(0.5 ꢁg/ml, Sigma) to induce the expression of YFPꢀNLRP3ꢀLuc and LPS (100 ng/mL) for 24 h and subsequently activated with
nigericin (10 ꢁM, 1 h) to assess functionality of the NLRP3 BRET sensor. 293 cells stably expressing the BRET sensor YFPꢀ
NLRP3ꢀLuc receptor were maintained in DMEM supplemented with 10% FCS, 2 mM Glutamax, and 1% penicillin–streptomycin.
Bioluminescence resonance energy transfer measurements. 293 cells expressing the NLRP3 BRET sensor were plated on a
polyꢀLꢀlysine–coated white opaque 96ꢀwell plate; after adhesion, cells were incubated for 15 h with the indicated doses of comꢀ
pound 11 or vehicle, washed with PBS with calcium and magnesium, and BRET readings were performed immediately after the
addition of 5 ꢁM coelenterazineꢀH substrate (Invitrogen). Signals were detected with two filter settings (Renillaꢀluciferase (Luc)
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filter [485 ± 20 nm] and YFP filter [530 ± 25 nm]) at 37 ̊
C using the Synergy Mx plate reader (BioTek) as described before.33, 34
Lactate dehydrogenase–release measurements. The presence of lactate dehydrogenase (LDH) in the medium was measured
using the Cytotoxicity Detection kit (Roche), following the manufacturer’s instructions. It was expressed as the percentage of the
total amount of LDH in the cells.
Caspaseꢀ1 activity assays. Caspaseꢀ1 activity was measured by incubating human recombinant caspaseꢀ1 (10 U, Merckꢀ
Millipore) with different concentrations of 11 or the caspaseꢀ1 inhibitor AcꢀYVADꢀCMK (100 ꢀM, MerckꢀMillipore) and monitorꢀ
ing the cleavage of the fluorescent substrate zꢀYVADꢀAFC (Promokine) at 400 nm and 505 nm with a Synergy Mx plate reader
(BioTek) for 3 h at 10 min intervals. Results are presented as the relative fluorescence units (RFU) per min of reaction.
Quantitative reverse transcriptaseꢀPCR analysis. The detailed methods used for quantitative reverse transcriptaseꢀPCR have
been described previously.51 Specific primers were purchased from Qiagen (QuantiTech Primer Assays). For each primer set, the
efficiency was >95%, and a single product was obtained on melt curve analysis. The presented relative gene expression levels were
calculated using the 2ꢀꢁCt method normalizing to Hrpt expression levels for each treatment.
ELISA and Western blot to detect ILꢀ1β release and caspaseꢀ1 activation from macrophages. ILꢀ1β and caspaseꢀ1 were deꢀ
tected from clarified macrophage supernatants using standard procedure for Western blot.49 ELISA kits for mouse ILꢀ1β were from
R&D Systems and were used according to the manufacturer’s instructions.
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Animals. Male SpragueꢀDawley rats, 200–250 g body weight, were used throughout the study. The animals were fed standard
laboratory chow and tap water ad libitum and were not employed for at least one week after their delivery to the laboratory. They
were housed, three in a cage, in temperatureꢀcontrolled rooms on a 12ꢀh light cycle at 22–24 °C and 50–60% humidity. Their care
and handling were in accordance with the provisions of the European Community Council Directive 86ꢀ609, recognized and adoptꢀ
ed by the Italian Government. All efforts were made to minimize animals' suffering and to reduce the number of animals used.
Induction of Colitis and Drug Treatments. Colitis was induced in accordance with the method previously described.42 During a
short anesthesia with isoflurane (Abbott, Rome, Italy), 15 mg of 2,4ꢀdinitrobenzenesulfonic acid (DNBS) in 0.25 mL of 50% ethaꢀ
nol was administered intrarectally via a polyethylene PEꢀ60 catheter inserted 8 cm proximal to the anus. Control rats received 0.25
mL of 50% ethanol. Animals underwent subsequent experimental procedures 6 days after DNBS administration to allow a full deꢀ
velopment of histologically evident colonic inflammation. Test drugs were administered by intragastric route for 6 days, starting the
same day of colitis induction. DNBSꢀuntreated and DNBSꢀtreated animals were assigned to the following treatment groups: 11
(12.5, 25, 50 mg/kg/day) or dexamethasone (DEX, 1 mg/kg/day). 11 and dexamethasone were suspended in olive oil and 1%
methylcellulose, respectively, and administered in a volume of 0.2 mL/rat. DNBSꢀuntreated animals (control group) and DNBSꢀ
treated rats (colitis group) received drug vehicle to serve as controls. Body weight was monitored daily starting from the onset of
drug treatments.
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Effective doses of 11 were selected by preliminary experiments designed to assay increasing doses of this compound (6, 12.5, 25,
50 and 100 mg/kg) on food intake, body weight, spleen weight, and macroscopic score in the model of DNBSꢀinduced colitis. The
dose of dexamethasone was selected on the basis of a previous study performed on a rat model of colitis.43 Macroscopic score was
evaluated on the whole colon, while biochemical assays were performed on specimens taken from a region of the inflamed colon
immediately adjacent and distal to the gross necrotic damage.
Assessment of Colitis. At the end of treatments, colonic tissues were excised, rinsed with saline, and scored for macroscopic
damage. The macroscopic damage was scored on a 0ꢀ to 6ꢀpoint scale, based on the system illustrated in Table S1. The presence of
adhesions between colonic tissue and other organs (0, none; 1, minor; 2, major adhesions) and the consistency of colonic fecal maꢀ
terial (0, formed; 1, loose; 2, liquid stools) were also scored.
Evaluation of tissue myeloperoxidase levels (MPO). MPO, regarded as a quantitative index to estimate the degree of bowel
wall infiltration by inflammatory polymorphonuclear cells, was assessed as described.46 Specimens of colonic tissues (30 mg) were
homogenized on ice with a polytron homogenizer (QIAGEN, Milan, Italy) in 0.6 mL of iceꢀcold lysis buffer containing 200 mM
NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerine, 1 mM phenylmethylsulfonyl fluoride, 1 g/mL leupeptin and 28 g/mL aprotinin
(pH 7.4). The homogenate was centrifuged 2 times at 4 °C for 15 min at 1,500 g. The supernatant was diluted 1:5 and used for the
determination of MPO concentration by means of an enzymeꢀlinked immunosorbent assay (ELISA) (Hycult Biotech, Uden, Netherꢀ
lands). The results were expressed as nanograms of MPO per milligram of colonic tissue.
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Evaluation of tissue TNF and ILꢀ1β levels. TNF and ILꢀ1β levels in colonic tissues were measured by an ELISA kit (Abcam),
as previously described.47 For this purpose, colonic tissue samples, stored previously at ꢀ80 °C, were weighed, thawed, and homogꢀ
enized in 0.4 mL of pH 7.2 PBS/20 mg of tissue at 4 °C, and centrifuged at 10,000 g for 5 min. Aliquots (100 ꢁL) of the supernaꢀ
tants were then used for assay. Tissue TNF and ILꢀ1β levels were expressed as pg and ng per mg of tissue, respectively.
Statistical analysis. The results are presented as mean ± SEM. unless otherwise stated. The significance of differences was evalꢀ
uated by Student tꢀtest for unpaired data or oneꢀway analysis of variance (ANOVA) followed by postꢀhoc analysis with Studentꢀ
NewmanꢀKeuls or Bonferroni tests. P values < 0.05 were considered significantly different. All statistical procedures were perꢀ
formed by commercial software (GraphPad Prism, version 3.0 from GraphPad Software Inc., San Diego, CA, USA).
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ANCILLARY INFORMATION
Supporting information: Molecular formula strings (.CSV file); structures of NLRP3 pathway inhibitors studied in IBD models and releꢀ
vant references, reactivity of 11 with cysteamine, stability of 11 in human serum; BRET assay approach; table of criteria used for scoring
of colonic macroscopic damage, and damageꢀscore determination in 11ꢀtreated animals. The Supporting Information is available free of
charge on the ACS Publications website at DOI:……..
Corresponding Author information
*M.B. eꢀmail, massimo.bertinaria@unito.it; phone, +39 011 6707667; *P. P. pablo.pelegrin@ffis.es; phone, +34 868885038
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ǁM.
C., ǁC. P. and ǁH. M.ꢀB. contributed equally to this work.
Notes
Authors declare no competing financial interest.
Acknowledgment
We thank M. C. Baños and A. I. Sánchez (IMIBꢀArrixaca, Murcia, Spain) for molecular and cellular technical assistance, Prof. L. Lazzaraꢀ
to for assistance in UHPLCꢀMS experiments, and Dr. Elisa Lupino for assistance in NLRP3 ATPase experiments. This work was supportꢀ
ed by Università degli Studi di Torino (Ric. Loc. 2015, and Ric. Loc. 2015 QUOTA B), by grants from Instituto Salud Carlos IIIꢀFEDER
(PS13/00174 to PP) and European Research Council (ERCꢀ2013ꢀCoG 614578 to PP), Juan de la Cierva postdoctoral fellowship from Minꢀ
isterio de Economía y Competitividad (FJCIꢀ2014ꢀ22041 to DA), Rio Hortega fellowship from Instituto Salud Carlos III (CM14/00008 to
HMꢀB), PP and IHꢀB would like to acknowledge networking support by the COST Action BMꢀ1406.
Abbreviations Used
ASC, apoptosisꢀassociated speckꢀlike protein containing a CARD; BAY, BAY 11ꢀ7082; BRET, Bioluminescence Resonance Energy
Transfer; BSA, bovine serum albumin; CAM, cysteamine; DEX, dexamethasone; DiMF, dimethyl fumarate; HSA, human serum albuꢀ
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min; Keap1, Kelchꢀlike ECHꢀassociated protein 1; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; LRR, leucineꢀrich repeat doꢀ
main; MNS,4ꢀmethylenedioxyꢀβꢀnitrostyrene; Nrf2, NFꢀE2ꢀrelated factor 2; TLR, Tollꢀlike receptor.
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