Development and Characterization of a Hydroxyl-Sulfonamide Analogue, 5-Chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a Novel NLRP3 Inflammasome Inhibitor for Potential Treatment of Multiple Sclerosis

Article abstract of DOI:10.1021/acschemneuro.7b00124

In our efforts to develop novel small-molecule inhibitors for the NOD-like receptor family pyrin-domain-containing 3 (NLRP3) inflammasome as potential disease-modifying agents to treat neurological disorders including multiple sclerosis (MS), a hydroxyl sulfonamide analogue JC-171 has been rationally designed and biologically characterized both in vitro and in vivo. Our studies established that JC-171 dose dependently inhibited LPS/ATP-induced interleukin-1β (IL-1β) release from J774A.1 macrophages with an IC₅₀ of 8.45 ± 1.56 μM. Selective inhibition of the NLRP3 inflammasome induced IL-1β release by this compound was also confirmed using mouse bone-marrow-derived macrophages and LPS-challenged mice in vivo. Furthermore, immunoprecipitation study revealed that JC-171 interfered with NLRP3/ASC interaction induced by LPS/ATP stimulation. More importantly, JC-171 treatment delayed the progression and reduced the severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, in both prophylactic and therapeutic settings. This coincided with blocking of IL-1β production and a pathogenic Th17 response. Collectively, these results suggest that JC-171 is a selective NLRP3 inflammasome inhibitor with biological activity in vivo, thus strongly encouraging further development of this lead compound as a potential therapeutic agent for human MS.

Full text of DOI:10.1021/acschemneuro.7b00124

Article
Development and characterization of a hydroxyl-sulfonamide
analog, 5-chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-
methoxy-benzamide, as a novel NLRP3 inflammasome
inhibitor for potential treatment of multiple sclerosis
Chunqing Guo, Jacob W Fulp, Yuqi Jiang, Xia Li, Jeremy Edward
Chojnacki, Jingde Wu, Xiang-Yang Wang, and Shijun Zhang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00124 • Publication Date (Web): 27 Jun 2017
Downloaded from http://pubs.acs.org on June 29, 2017
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Development and characterization of a hydroxylꢀsulfonamide analog, 5ꢀchloroꢀNꢀ[2ꢀ(4ꢀ
hydroxysulfamoylꢀphenyl)ꢀethyl]ꢀ2ꢀmethoxyꢀbenzamide, as a novel NLRP3 inflammasome
inhibitor for potential treatment of multiple sclerosis
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Chunqing Guo aǂ, Jacob W. Fulpbǂ, Yuqi Jianga, Xia Lib, Jeremy E. Chojnackib, Jingde Wuc,
XiangꢀYang Wanga,*, Shijun Zhangb,*
aDepartment of Human & Molecular Genetics, bDepartment of Medicinal Chemistry, Virginia
Commonwealth University, Richmond, Virginia, USA, cDepartment of Medicinal Chemistry,
School of Pharmaceutical Sciences, Shandong University, Jinan, China.
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ABSTRACT: In our efforts to develop novel small molecule inhibitors for the NODꢀlike
receptor family pyrin domain containing 3 (NLRP3) inflammasome as potential diseaseꢀ
modifying agents to treat neurological disorders including multiple sclerosis (MS), a hydroxyl
sulfonamide analog JCꢀ171 has been rationally designed and biologically characterized both in
vitro and in vivo. Our studies established that JCꢀ171 dose dependently inhibited LPS/ATP
induced interleukinꢀ1β (ILꢀ1β) release from J774A.1 macrophages with an IC50 of 8.45 ± 1.56
ꢁM. Selective inhibition of the NLRP3 inflammasome induced ILꢀ1β release by this compound
was also confirmed using mouse bone marrow derivedꢀmacrophages and LPSꢀchallenged mice
in vivo. Furthermore, immunoprecipitation study revealed that JCꢀ171 interfered with
NLRP3/ASC interaction induced by LPS/ATP stimulation. More importantly, JCꢀ171 treatment
delayed the progression and reduced the severity of experimental autoimmune encephalomyelitis
(EAE), a mouse model of MS, in both prophylactic and therapeutic settings. This coincided with
blocking of ILꢀ1β production and a pathogenic Th17 response. Collectively, these results suggest
that JCꢀ171 is a selective NLRP3 inflammasome inhibitor with biological activity in vivo, thus
strongly encouraging further development of this lead compound as a potential therapeutic agent
for human MS.
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KEYWORDS: NLRP3 inflammasome, rational design, small molecule inhibitor, experimental
autoimmune encephalomyelitis, multiple sclerosis.
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INTRODUCTION
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Multiple sclerosis (MS) is considered an immuneꢀmediated neurodegenerative disease of
central nervous system (CNS) and is the leading cause of chronic neurological disability among
young adults.1 Epidemiological studies estimated 400,000 cases of MS in the United States with
200 new cases being added every week. Three types of MS exist: Relapsingꢀremitting MS,
primary progressive MS, and secondary progressive MS. Although several classes of drugs have
been successfully developed to ameliorate symptoms, speed recovery and slow the progression
of the disease, unfortunately, there is currently no cure for MS patients. Furthermore, not all MS
patients are responsive to the available treatment agents. Therefore, there is still urgent need to
develop new agents with novel mechanism of action for this CNS disease.
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Neuroinflammation persists as one of the pathologic hallmarks of MS throughout the
course of MS and is closely associated with axonal injury observed in MS. The
immunopathology of MS is characterized by the infiltration of myelinꢀreactive T cells into the
CNS and induction of demyelination which disrupts the communication of the nervous system.2
Although the exact etiology and pathogenesis of MS remain elusive, emerging evidence supports
a critical role for caspaseꢀ1, ILꢀ1β and ILꢀ18 in the pathogenesis of MS, as demonstrated by
studies from both MS patients and experimental autoimmune encephalomyelitis (EAE) mouse
model, an animal model of MS.3ꢀ6 Notably, activation of caspaseꢀ1 and maturation of cytokine
interleukin (IL)ꢀ1β with biological activity are closely associated with a multimeric protein
platform, termed inflammasome, in response to damage associated molecular pattern molecules
or pathogenꢀassociated molecular patterns.7, 8 Among the known inflammasomes, the NODꢀlike
receptor family pyrin domain containing 3 (NLRP3) inflammasome, composed of NLRP3, ASC
(apoptosisꢀassociated speckꢀlike protein containing a CARD), caspaseꢀ1, is the most extensively
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studied and widely implicated regulator of caspaseꢀ1 activation, the maturation and production of
ILꢀ1β and ILꢀ18.8ꢀ11 There are a number of reports that strongly suggest the involvement of the
NLRP3 inflammasome in the development of MS. Intriguingly, MSꢀlike lesions were observed
in a MuckleꢀWells syndrome (MWS) patient who had a diseaseꢀsusceptible mutation in the
Nlrp3 gene.12 Expression of caspaseꢀ1 is elevated in MS plaques and peripheral blood
mononuclear cells of MS patients.3, 4 A correlation between severity of MS and ILꢀ1β has been
well documented.4, 5 Additionally, increased NLRP3 expression has been observed in the spinal
cord of EAE mice, and NLRP3 deficiency substantially delayed onset and reduced severity of
EAE symptoms, decreased neuroinflammation, demyelination and oligodendrocyte loss
progression,13ꢀ15 thus strongly suggesting the general involvement of NLRP3 inflammasome in
the pathogenesis of MS. Importantly, it has been demonstrated that the effectiveness of IFNꢀβ, a
drug that has been used for more than 15 years as a firstꢀline treatment for MS, is NLRP3
inflammasome dependent.16 The presence of an NLRP3 inflammasomeꢀindependent subset in
EAE,16 indicates a multifactorial and heterogeneous nature of this disease. However, given the
fact that many MS patients fail to respond to currently available MS treatments, including IFNꢀβ
and the demonstrated critical role of NLRP3 inflammasome in MS, development of novel small
molecule inhibitors that selectively target NLRP3 inflammasome may provide new opportunities
to MS intervention for therapeutic benefits in the clinic.
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Recently, we started development of small molecule compounds based on the structure of
glyburide, a FDA approved antiꢀdiabetic agent and has been shown to inhibit NLRP3
inflammasome,17 as NLRP3 inflammasome inhibitors. Structural modification of glyburide led to
a sulfonamide analog JCꢀ21 (Figure 1A, referred as 16673ꢀ34ꢀ0 in previous reports).18, 19
Biological characterization demonstrated that JCꢀ21 selectively targets NLRP3 inflammasome,
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suppresses the production of ILꢀ1β, and inhibits the activation of caspaseꢀ1 in vitro. Furthermore,
JCꢀ21 was shown to mitigate the pathology and protect animals from injuries potentially
involving NLRP3 inflammasome in acute myocardial infarction (AMI) mouse models.19, 20
Notably, our results suggested that JCꢀ21 specifically blocks the assembly of the NLRP3
inflammasome protein complex,19 thus representing a novel mechanism of action for this
chemotype as NLRP3 inflammasome inhibitors. Herein, we report the design and biological
characterization of a completely new analog, JCꢀ171, for the first time as a potential agent for
treatment of MS.
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RESULTS AND DISCUSSION
Design of JCꢀ171. Although JCꢀ21 has shown promising activities both in vitro and in vivo as a
NLRP3 inflammasome inhibitor,18ꢀ20 solubility was recognized an issue for further development.
Therefore, we designed a new analog JCꢀ171 with a hydroxamic acid on the sulfonamide moiety
with an intention to increase the polarity of this compound (Figure 1A). The LogP value for JCꢀ
21 and JCꢀ171 is 0.19 and 0.81, respectively, thus reflecting the significant difference in polarity.
Furthermore, Nꢀhydroxylation has been indicated as active metabolite of sulfonamide analogs
and Nꢀhydroxy sulfonamide analogs have been shown to exhibit antiꢀinflammatory effects.21, 22
In addition, another goal of designing this analog was that the results will provide insight into
whether structural modifications are tolerated at this specific position, thus facilitating further
structureꢀactivity relationship studies to develop more potent analogs. The chemical synthesis of
JCꢀ171 is relatively straightforward as outlined in Scheme 1.
JCꢀ171 is an active NLRP3 inflammasome inhibitor. Our previous studies have established
that mouse J774A.1 macrophages upon stimulation with lipopolysaccharide (LPS) and adenosine
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triphosphate (ATP) represent a reliable model for testing NLRP3 inflammasome activation and
ILꢀ1β release.18, 19 After chemical synthesis, we first examined the cytotoxic effects of JCꢀ171
on J774A.1 cells. As shown in Figure 1B, JCꢀ171 did not show any significant cytotoxicity on
J774A.1 cells themselves at up to 30 µM concentration. Upon challenge with LPS/ATP,
significant cell death was observed for J774A.1 cells and JCꢀ171 dose dependently protected
J774A.1 cells from LPS/ATP induced cell death (Figure 1C). We next tested whether JCꢀ171
inhibits the release of ILꢀ1β by J774A.1 cells upon stimulation with LPS/ATP. As shown in
Figure 1D, JCꢀ171 reduced the release of ILꢀ1β due to NLRP3 inflammasome activation after
LPS and ATP challenge. The inhibition potency of JCꢀ171 (IC50: 8.45 ± 1.56 ꢁM) is about two
and half fold less than that of JCꢀ21 (IC50: 3.25 ± 1.35 ꢁM). Analysis of the effects of JCꢀ171 on
caspaseꢀ1 activation by Western blotting also confirmed that treatment of J774A.1 cells with JCꢀ
171 resulted in the inhibition of caspaseꢀ1 activation as evidenced by the decreased level of
cleaved caspaseꢀ1 fragments (p20 and p10) (Figure 1E). On the other hand, no significant change
was observed for the level of NLRP3, proꢀILꢀ1β, or proꢀcaspaseꢀ1 upon treatment of J774A.1
cells with JCꢀ171 under the experimental conditions (Figure 1E). Thus the results rule out the
possibility that the observed suppression of ILꢀ1β or caspaseꢀ1 cleavage by JCꢀ171 is due to the
interference of expression of NLRP3, proꢀILꢀ1β, or proꢀcaspaseꢀ1.
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We next employed primary mouse bone marrowꢀderived macrophages (BMDMs) to
confirm the inhibitory effects of JCꢀ171 on the production of ILꢀ1β and to also test the effect of
JCꢀ171 on the production of other cytokines. As shown in Figure 2A, upon treatment of
BMDMs with JCꢀ171 following the same experimental condition as used in J774A.1 cells, the
release of ILꢀ1β upon stimulation with LPS/ATP was significantly blocked in a dose dependent
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manner. Notably, the production of TNFꢀα and ILꢀ6 was not affected by JCꢀ171 even at high
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concentration (Figure 2B), indicating its selective effect on the NLRP3 inflammasome.
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Our previous studies employing various cellular models including BMDMs carrying
constitutively active NLRP3 suggested that this chemical scaffold might function as inhibitor by
specifically blocking the assembly of the NLRP3 inflammasome protein complex.19 To test this,
coꢀimmunoprecipitation (IP) study was performed using primary BMDMs after stimulation with
LPS and ATP. As shown in Figure 2C, treatment of BMDMs with JCꢀ171 blocked the
recruitment of ASC during NLRP3 activation, thus providing further evidence to the mechanistic
hypothesis of this novel chemical scaffold as NLRP3 inflammasome inhibitor.
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Given the fact that JCꢀ171 retained an inhibitory effect on the NLRP3 inflammasome
activation, although slightly less potent than JCꢀ21 in vitro, and the fact that it is a potential
metabolite of JCꢀ21, we decided to evaluate the biological effect of JCꢀ171 in vivo. We
pretreated the mice with JCꢀ171 before intraperitoneal injection of LPS, which has been shown
to trigger ILꢀ1β production in a NLRP3ꢀdependent manner.23 Serum levels of ILꢀ1β was sharply
reduced by JCꢀ171 treatment, whereas the amount of TNFꢀα was not significantly altered,
further indicating the in vivo selective activity of JCꢀ171 on the NLRP3 inflammasome (Figure
2D).
JCꢀ171 treatment suppresses disease progression of EAE. Since CNS inflammation
represents one of the pathologic hallmarks of MS, and NLRP3 inflammasome and ILꢀ1 signaling
13, 24, 25
have been suggested to play a critical role in MS,
we decided to test JCꢀ171 in mouse
EAE model as a proofꢀofꢀconcept for such inhibitors as potential agents for treatment of human
MS. As shown in Figure 3A, prophylactic treatment of EAE mice with JCꢀ171 (treatment started
on the day of immunization) substantially delayed the disease onset and reduced the severity of
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EAE. We next examined the effect of JCꢀ171 treatment on a pathogenic Tꢀhelper 17 (Th17)
response that is known to be involved in the pathogenesis of EAE, 26, 27 and plays a causative role
in autoimmune damage to the CNS in MS.28, 29 Intracellular cytokine staining and flow
cytometry analysis of lymph node cells showed significantly reduced frequencies of ILꢀ
17A+CD4+ Th17 cells in JCꢀ171ꢀtreated mice as compared to the vehicleꢀtreated mice (Figure
3B). Consistent with the attenuated Th17 response, as shown in Figure 3C, the transcription of
rora and rorc, two transcription factors that are critical for Th17 cell differentiation, was also
substantially decreased upon treatment with JCꢀ171. These data also further support a critical
role of NLRP3 and ILꢀ1β in the induction of ILꢀ17ꢀproducing T cells that mediate EAE.13, 24
Notably, treatment with JCꢀ171 also reduced serum levels of ILꢀ1β in EAE mice, but not the
levels of TGFꢀβ and ILꢀ6, indicating the in vivo selectivity of JCꢀ171 on the NLRP3
inflammasome (Figure 3D). Given a wellꢀdocumented role of ILꢀ1β signaling in promoting the
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differentiation of Th17 cells,30 we demonstrate that inhibition of ILꢀ1β production via blocking
activation of the NLRP3 inflammasome by JCꢀ171, can significantly reduce a pathogenic Th17
response and alleviate the disease severity. While these data have clearly established the activity
of JCꢀ171 in suppressing the activation of NLRP3 inflammasome as well as its downstream
effects (e.g., Th17 response) in the context of EAE, future studies involving genetic
manipulation of NLRP3 inflammasome are necessary for precisely delineating its contribution.
We next evaluated the therapeutic potency of JCꢀ171 by administration of the compound
starting when the clinical scores of individual mice have reached 1 (flaccid tail). Despite the use
of a low dose of JCꢀ171 (10 mg/kg), treatment of mice with JCꢀ171 efficiently suppressed EAE
progression compared with vehicle treatment (Figure 4A). Notably, the therapeutic effect of JCꢀ
171 treatment was quite durable and lasted throughout the chronic phase of EAE. JCꢀ171
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exhibited an in vivo therapeutic activity comparable with MCC950, a small molecular inhibitor
for NLRP3 inflammasome recently reported to block EAE development.31 Histologic analysis
showed that, consistent with the reduced clinical score, myelin injury was reduced in the white
matter of spinal cords from JCꢀ171ꢀtreated EAE mice, indicated by Luxol fast blue staining
(Figure 4B). Treatment with JCꢀ171 also resulted in a substantial decrease in the frequency of
MOG35–55ꢀspecific Th17 cells in the spleens and spinal cords of EAE mice (Figure 4C).
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In summary, we rationally designed a hydroxylꢀsulfonamide analog, JCꢀ171, from parent
compound JCꢀ21. In vitro biological characterization using mouse J774A.1 macrophage and
primary BMDMs demonstrated that JCꢀ171 is a selective inhibitor of the NLRP3 inflammasome,
which is further supported by in vivo data from LPSꢀchallenged mice. The inhibition potency of
JCꢀ171 on ILꢀ1β release by LPS/ATP stimulation in J774A.1 cells is comparable to the parent
compound JCꢀ21, thus indicating that structural modifications at the sulfonamide moiety can be
tolerated. Furthermore, inhibition of the NLRP3 inflammasome by JCꢀ171 administration
effectively blocks disease progression and ameliorates the clinical symptoms in EAE mice,
which is associated with significantly reduced levels of ILꢀ1β. Collectively, the results of JCꢀ
171 together with that of JCꢀ21 suggest that this chemical scaffold represents a promising
template to develop novel small molecule inhibitors for the NLRP3 inflammasome. These data
also wanrrant further development of JCꢀ171 and analogs as potential agents for treatment of
human MS as well as other inflammatory diseases involving NLRP3 inflamamsome.
MATERIALS AND METHODS
Chemical synthesis
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Reagents and solvents were obtained from commercial suppliers and used as received unless
otherwise indicated. Reactions were monitored by thinꢀlayer chromatography (TLC) (precoated
silica gel 60F254 plates, EMD Chemicals) and visualized with UV light or by treatment with
phosphomolybdic acid (PMA). Flash chromatography was performed on silica gel (200ꢀ300
mesh, Fisher Scientific) using solvents as indicated. 1HNMR and 13CNMR spectra were routinely
recorded on a Bruker ARX 400 spectrometer. The NMR solvent used was CDCl3 or DMSOꢀd6
as indicated. Tetramethylsilane (TMS) was used as the internal standard.
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀphenethylbenzamide (3). 5ꢀChloroꢀ2ꢀmethoxybenzoic acid 1 (1 mmol)
and trimethylamine (Et3N, 2 mmol) were dissolved in dimethyl formaldehyde (DMF, 25 mL)
and cooled to 0 ˚C. Nꢀ(3ꢀdimethylaminopropyl)ꢀN’ꢀethylcarbodiimide hydrochloride (EDC, 1.5
mmol) was added and after 30 min, hydroxybenzotriazole (HOBt, 1.5 mmol) was added. After 1
h, 2ꢀphenylethanamine 2 (1 mmol) was added to the mixture and the reaction was allowed to
warm to room temperature for overnight. The solution was concentrated under reduced pressure
and H2O was added. The solution was extracted with ethyl acetate (EtOAc) and combined
organic phase was concentrated and purified by column chromatography (EtOAc/Hexane 20/80)
1
to give compound 3 as a viscous oil (yield: 82%). H NMR (400 MHz, DMSOꢀd6) δ 8.22 (br. s.,
1H), 7.66 (br. s., 1H), 7.50 (dd, J = 3.00, 8.80 Hz, 1H), 7.32 (d, J = 7.22 Hz, 2H), 7.28 (d, J =
8.03 Hz, 2H), 7.20 ꢀ 7.24 (m, 1H), 7.16 (d, J = 9.46 Hz, 1H), 3.81 (s, 3H), 3.54 (q, J = 7.06 Hz,
13
2H), 2.85 (t, J = 6.20 Hz, 2H); C NMR (100 MHz, DMSOꢀd6) δ 163.4, 155.7, 139.4, 131.5,
129.6, 128.7, 128.3, 126.1, 124.7, 124.3, 114.1, 56.2, 40.7, 34.9.
4ꢀ(2ꢀ[(5ꢀchloroꢀ2ꢀmethoxyphenyl)formamido]ethyl)benzeneꢀ1ꢀsulfonyl chloride (4). Compound
3 (1.73 mmol) was dissolved in methylene chloride (2 mL). To this, excess chlorosulfonic acid
(1 mL) was added, and the solution stirred at 70 °C for 2 h. The reaction was cooled to room
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temperature, and then poured over crushed ice. The product was extracted into methylene
chloride, and then concentrated under reduced pressure. The product was purified by column
chromatography (EtOAc/Hexanes: 20/80 to 50/50) yielding compound 4 as a white solid (yield:
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65%). H NMR (400 MHz, DMSOꢀd6) δ 8.20 (br. s., 1H), 7.66 (s, 1H), 7.56 (d, J = 8.28 Hz,
2H), 7.50 (dd, J = 2.76, 8.78 Hz, 1H), 7.22 (d, J = 8.53 Hz, 2H), 7.16 (d, J = 9.04 Hz, 1H), 3.80
(s, 3H), 3.51 (q, J = 7.30 Hz, 2H), 2.84 (t, J = 6.80 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ
163.5, 155.7, 146.1, 139.8, 131.5, 129.5, 128.0, 125.6, 124.7, 124.3, 114.2, 56.2, 40.6, 34.6.
5ꢀchloroꢀNꢀ(2ꢀ[4ꢀ(hydroxysulfamoyl)phenyl]ethyl)ꢀ2ꢀmethoxybenzamide
(JCꢀ171).
Hydroxylamine (6.44 mmol) and Nꢀmethylmorpholine (0.2 mL) were dissolved in MeOH (2
mL). To this, compound 4 (1.29 mmol) was added, and the solution was stirred for 4 h. H2O was
added, and the product was extracted into methylene chloride and concentrated. The product
was purified by column chromatography (dichloromethane/methanol: 94/6) to give compound
1
JCꢀ171 as a white solid (yield: 56%). H NMR (400 MHz, DMSOꢀd6) δ 9.56 (d, J = 3.26 Hz,
1H), 9.52 (d, J = 3.26 Hz, 1H), 8.27 (t, J = 5.40 Hz, 1H), 7.78 (d, J = 8.28 Hz, 2H), 7.64 (d, J =
2.76 Hz, 1H), 7.48 ꢀ 7.53 (m, 3H), 7.15 (d, J = 8.78 Hz, 1H), 3.81 (s, 3H), 3.55 (q, J = 6.70 Hz,
13
2H), 2.94 (t, J = 6.90 Hz, 2H); C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 145.2, 135.3,
131.5, 129.5, 129.2, 128.2, 124.8, 124.3, 114.2, 56.2, 40.2, 34.7.
Cell culture
J774A.1 murine macrophage cells were purchased from Americal Type Cell Culture (ATCC),
Manassas, VA and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10%
fetal bovine serum (FBS), 1% penicillin/streptomycin. Bone marrow cells from C57BL/6 mice
was differentiated for
8
days in DMEM supplemented with 10% FBS, 1%
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penicillin/streptomycin and 30% L929 mouse fibroblast conditioned media that contains
macrophage colonyꢀstimulating factor.
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Mice
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C57BL/6 mice were purchased from the National Cancer Institute (Bethesda, MD). All
experiments and procedures involving mice were approved by the Institutional Animal Care and
Use Committee of Virginia Commonwealth University.
Antibodies and reagents
Fluorochromeꢀconjugated mouse monoclonal antibodies (mAbs) for flow cytometry analysis of
immune cells, including FITC–CD4 (GK1.5), Percp/Cy5.5–ILꢀ17A (TC11ꢀ18H10.1),
CD16/CD32 (2.4G2), isotype control rat IgG2b (RTK4530) and IgG1 (RTK2071) were
purchased from BioLegend (San Diego, CA). Mouse TNFꢀα, ILꢀ6 and ILꢀ1β enzymeꢀlinked
immunosorbent assay (ELISA) kits were purchased from Biolegend. Mouse ILꢀ1β ELISA kits
was from eBioscience (San Diego, CA). Monoclonal antibodies for ASC (clone 2EIꢀ7, 04ꢀ147,
EMD Millipore, Billerica, MA), and NLRP3 (clone D4D8T, Cell Signaling Biotechnology) were
purchased from commercial resources as indicated. AntiꢀNLRP3 (abcam, USA), antiꢀILꢀ1β
(EMD Millipore, CA, USA), and antiꢀcaspaseꢀ1 (p20 and p10) (Santa Cruz Biotechnology, Santa
Cruz, USA) for Western blotting analysis were purchased from commercial resources as
indicated and used per manufacturer’s instruction.
Cell viability assays
J774A.1 cells were plated into a 96ꢀwell plate (1 × 105 cells/well) for 24 h in growth medium.
Cells were then treated with different concentrations of JCꢀ171 for 0.5 h. After treatment, 3ꢀ(4.5ꢀ
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dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT, 0.5% solution) was added. After
incubation for 4 h, DMSO (100 µL) was added for 5 min. Absorbance at 570 nm was then
immediately recorded using a FlexStation 3 plate reader.
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Cell death rescue assays
J774A.1 cells were plated into a 96ꢀwell plate (1 × 105 cells/well) for 24 h in growth medium.
Cells were primed with Escherichia coli 0111:B4 LPS (SigmaꢀAldrich) (final concentration: 1
µg/mL) for 4.5 h. Next, JCꢀ171 was added for 30 min. ATP (5 mM) was added at the same time
when JCꢀ171 was added to induce NLRP3 inflammasome formation, and cells were incubated
another 30 min. Cell viability was measured by MTT assay as described above.
In vitro NLRP3 activation assays
J774A.1 cells were primed and activated with LPS/ATP as described above. JCꢀ171 was added
at the time when ATP was added for 30 min. The supernatants were collected and levels of ILꢀ
1β were measured with a mouse ILꢀ1β Enzymeꢀlinked Immunosorbent Assay (ELISA) kit
following the manufacturer’s instructions. BMDMs were similarly treated as described with
J774.A1 cell line followed by ELISA analysis of ILꢀβ production. To assess effect of JCꢀ171 on
induction of cytokine TNFꢀα or ILꢀ6, BMDMs were pretreated with JCꢀ171 (400 ꢀM) for 2 h
and then stimulated LPS (100 ng/mL) for 5.5 h, followed by ATP (5 mM) treatment for 30 min.
Western blotting assays
J774A.1 cells were seeded on 6ꢀwell plates (2 × 106 cells/well) in RPMI 1640 medium for 24 h.
Cells were then primed and activated with LPS/ATP and treated with JCꢀ171 as described above.
After treatment, cells were collected and wash with iceꢀcold PBS twice. Cells pellets were lysed
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by sonication in a radioimmunoprecipitation (RIPA) buffer solution. Protein samples were
quantified by the Bradford method. Equal amounts of protein (20.0 ꢁg) were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSꢀPAGE) and transferred onto a
polyvinylidene fluoride (PVDF) membrane (BioꢀRad). Proteins were probed with primary
antibodies overnight at 4 °C: antiꢀNLRP3 (1:800, abcam, USA), antiꢀIL1β (1:500, EMD
Millipore, CA, USA), antiꢀcaspaseꢀ1 (P20) (1:1000, Cell Signaling Technology, MA, USA),
antiꢀcaspaseꢀ1 (P10) (1:200, Santa Cruz Biotechnology, Santa Cruz, USA) and incubated with a
1:1000 dilution of horseradish peroxidaseꢀconjugated rabbit or mouse secondary antibodies (Cell
Signaling Technology, USA). After washing three times in TBSꢀTween 20 for 10 min, the
proteins were visualized by employing chemiluminescent reagent (Thermo Fischer Scientific,
Waltham, MA). The blots were also probed with antibodies against αꢀtubulin to ensure equal
loading of proteins.
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Immunoprecipitation and immunoblotting
BMDM cells were washed with iceꢀcold phosphateꢀbuffered saline (PBS) and lysed in modified
lysis buffer (50 mM TrisꢀCl (pH 7.4), 1% Nonidet Pꢀ40, 150 mM NaCl, 1 mM
ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml
each of aprotinin and leupeptin, and 1 mM Na3VO4). 1 mg protein of cell extracts was incubated
with 2 µg of antiꢀNLRP3 antibodies for 2 h at 4 °C, followed by incubation with 40 µl of Protein
A/G PlusꢀSepharose beads (Santa Cruz Biotechnology, Inc.) overnight at 4 °C. The beads were
washed with lysis buffer, and immune complexes were eluted by boiling in 2 × sodium dodecyl
sulfate (SDS) Laemmli loading buffer for 5 min prior to immunoblotting analyses with indicated
antibodies. For immunoblotting, the immune complexes were resolved by SDSꢀPAGE and
transferred to nitrocellulose membranes. Membranes were immunoblotted with antibodies for
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NLRP3 or ASC, followed by HRPꢀconjugated secondary antibodies. Specific bands were
visualized using Pierce™ ECL Western Blotting Substrate from ThermoFisher Scientific
(Waltham, MA). Band intensities of immunoprecipitated ASC or NLRP3 panel were quantified
using Image Studio LITE (LIꢀCOR biotechnology) software. The ratios of ASC/NLRP3
association were calculated with PBS control group set as 1.
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LPS challenge in vivo and JCꢀ171 treatment
C57BL/6 mice were injected intraperitoneally (i.p.) with 20 mg/kg LPS (SigmaꢀAldrich) or PBS
one hour after JCꢀ171 (100 mg/kg) or vehicle treatment. 2.5 h after LPS injection, serum levels
of ILꢀ1β and TNFꢀα were measured by ELISA.
EAE induction and JCꢀ171 treatment
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EAE was induced as we previously described. Briefly, mice were immunized subcutaneously
with 200 ꢁg Myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (ProSpec, East Brunswick,
NJ) emulsified in Complete Freund's Adjuvant (CFA) on day 0 followed by injection of 200 ng
pertussis toxin (SigmaꢀAldrich, St. Louis, MO) i.p. on days 0 and 2. After the first immunization,
the severity of EAE was monitored and graded daily in a blinded fashion on a scale of 0–5 as we
previously described. For prophylactic treatment, JCꢀ171 was administered to mice (100 mg/kg,
i.p.) on days 0, 1 and 2; and every other days thereafter. Therapeutic treatment with JCꢀ171 (10
mg/kg, i.p.) or MCC950 (SigmaꢀAldrich, 10 mg/kg, i.p.) was initiated when the clinical scores of
individual mice have reached 1 (flaccid tail), and given every other days. Upon termination of
the experiment, mice were transcardially perfused with iceꢀcold PBS followed by 4%
paraformaldehyde. Spinal cords with vertebrae were removed. Paraffinꢀembedded sagittal
sections of cervicothoracic spinal cord were stained with Luxol fast blue/periodic acid–Schiff to
determine demyelination.
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Analyses of immune response in EAE mice
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Mononuclear cells (MNCs) were isolated from spinal cords of EAE mice using percoll gradient
centrifugation after perfusion with PBS as we previously described. 32 MNCs were stimulated for
5 h in the presence of phorbol 12ꢀmyristate 13ꢀacetate (10 nM; SigmaꢀAldrich), ionomycin (1
ꢁM; EMD Biosciences) with the addition of brefeldin A (5 ꢁg/ml, Biolegend) during the last 3
hours of culture. After surface staining with antiꢀCD4 mAbs for 30 min at 4°C, cells were fixed,
permeabilized, and stained with anti–ILꢀ17A mAbs for 30 min at 4°C. For analysis of Th17 cells
in lymphoid organs, cells were stimulated with MOG35–55 (1 ꢁg/ml) overnight at 37°C, 5% CO2
first and stained with the mAbs as decribed above. Data were acquired using BD FACSCalibur
and analyzed using FlowJo software (Tree Star, Ashland, OR). Cytokine levels in the serum
were assayed using the ELISA kits according to the manufacturer’s instructions.
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AUTHOR INFORMATION
#Equal Contributions: C.G. and J.F.
*Corresponding author: Shijun Zhang, Eꢀmail: szhang2@vcu.edu. Telephone: 804ꢀ628ꢀ8266.
Fax: 804ꢀ828ꢀ7625 and XiangꢀYang Wang, Email: xywang@vcu.edu. Telephone: 804ꢀ628ꢀ2679
Author contributions: Chemical synthesis was completed by J.F. and J.C.; Cellular assays with
J774A.1 cells were completed by J.F.; Western blotting studies with J774A.1 cells were
completed by Y.J.; Studies employing mouse BMDMs and immunoprecipitation study were
completed by X.L.; Animal studies were completed by C.G.; Experiment design, data analysis,
writing, and editing were completed by C.G.; J.F.; J.W.; X.Y.W.; and S.Z.
ACKNOWLEDGEMENTS
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This work was supported in part by the National Institutes of Health Grants R01AG041161
(S.Z.), CA175033 (X.Y. W.), CA154708 (X.Y.W.) and Alzheimer’s Drug Discovery Foundation
(S.Z.), and the VCU Presidential Research Quest Fund (S.Z., X.Y.W.)
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ABBREVIATIONS
Acute myocardial infarction (AMI), apoptosisꢀassociated speckꢀlike protein containing a CARD
(ASC), adenosine triphosphate (ATP), central nervous system (CNS), Dulbecco’s Modified
Eagle Medium (DMEM), dimethyl formaldehyde (DMF), experimental autoimmune
encephalomyelitis (EAE), Nꢀ(3ꢀdimethylaminopropyl)ꢀN’ꢀethylcarbodiimide hydrochloride
(EDC), ethylenediaminetetraacetic acid (EDTA), Enzymeꢀlinked Immunosorbent Assay (ELISA),
ethyl acetate (EtOAc), fetal bovine serum (FBS), hydroxybenzotriazole (HOBt),
immunoprecipitation (IP), interleukinꢀ1β (ILꢀ1β), intraperitoneally (i.p.), lipopolysaccharide
(LPS), monoclonal antibodies (mAbs), mononuclear cells (MNCs), multiple sclerosis (MS),
myelin oligodendrocyte glycoprotein (MOG), NODꢀlike receptor family pyrin domain
containing 3 (NLRP3), phosphateꢀbuffered saline (PBS), phosphomolybdic acid (PMA),
phenylmethylsulfonyl fluoride (PMSF), polyvinylidene fluoride (PVDF),
radioimmunoprecipitation (RIPA), sodium dodecyl sulfate (SDS), sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDSꢀPAGE), Tꢀhelper 17 (Th17), trimethylamine (Et3N).
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Scheme 1a
SO2NHOH
SO2Cl
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8
COOH
NH2
a
c
b
H3CO
+
Cl
9
O
NH
O
NH
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O
NH
H3CO
2
1
H3CO
H3CO
JC-171
4
Cl
3
Cl
Cl
aReagents and conditions: a) EDC, HOBt, Et3N, methylene chloride; b) Chlorosulonic acid,
methylene chloride; c) NH2OH, N-methylmorpholine, MeOH.
Figure 1. JCꢀ171 inhibits the release of ILꢀ1β in J774A.1 cells upon stimulation with
LPS/ATP. Rational design of JCꢀ171 (A). J774A.1 cells were treated with JCꢀ171 at indicated
concentrations in the absence (B) or presence (C) of LPS priming (1 µg/mL, 4.5 h) and ATP (5
mM, 30 min). JCꢀ171 was added at the time of ATP addition. Cell viability was then measured
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by MTT assay. J774A.1 cells were activated with LPS/ATP and treated with JCꢀ171 as described
above. Supernatant were collected and level of ILꢀ1β was assayed with mouse ELISA kit per
manufacture’s instruction (D). J774A.1 cells were primed with LPS/ATP and treated with JCꢀ
171 as described above. Cells were collected and lysed and the lysates were analyzed by Western
blotting with corresponding antibodies (The image represents the results from one of three
independent experiments). Error bar represents SEM. **, p < 0.01
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Figure 2. JCꢀ171 blocks NLRP3 inflammasome activation and ILꢀ1β production in primary
macrophages and LPSꢀchallenged mice. BMDMs were primed with LPS (1 ꢀg/mL) for 4.5 h
and then treated with indicated doses of JCꢀ171 when adding ATP (5 mM) stimulation for 30
min. ILꢀβ in the culture media was assayed by ELISA (A). JCꢀ171 treatment has no effect on the
production of TNFꢀα and ILꢀ6. BMDMs were preꢀtreated with JCꢀ171 (400 µM) for 2 h
followed by stimulation with LPS and ATP. Cytokines in the culture media was assayed by
ELISA (B). JCꢀ171 interferes with association of NLRP3 and ASC during activation of NLRP3
inflammasome. Cells were also collected after LPS/ATP stimulation for IP assay using antiꢀ
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NLRP3 antibodies (C, left). Pullꢀdowned immunocomplexes were subjected to immunoblotting
with antiꢀASC antibodies to detect association of ASC with NLRP3. Whole cell lysates were also
used as input controls. The ratios of ASC and NLRP3 band intensities in the complex were
quantified and calculated, with PBS control group set as 1 (C, right). Serum levels of ILꢀ1β and
TNFꢀα from C57BL/6 mice pretreated with JCꢀ171 (100 mg/kg) or vehicle control were
measured by ELISA 2.5 h after i.p. injection of LPS (20 mg/kg) (D). *, p < 0.05; **, p < 0.01;
NS, not significant.
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Figure 3. Administration of JCꢀ171 delays disease progression in EAE mice by blocking ILꢀ
1β production. C57BL/6 mice (n = 5) were immunized with MOG peptide (200 ꢀg) emulsified
in CFA on day 0. JCꢀ171 treatment (100 mg/kg) was started on day 0 for three consecutive doses
followed by treatment on every other day. EAE development in mice was followed, and clinical
scores were recorded (A). Lymph nodes were collected after the 10th treatment, the frequency of
ILꢀ17A+CD4+ Th17 cells was assayed by flow cytometry after stimulation with MOG peptide for
3 days (B). Transcription of rorc and rora genes in the spleen was determined by realꢀtime PCR
(C). ILꢀ1β, TGFꢀβ and ILꢀ6 levels in the serum were assayed by ELISA on day 17 after
immunization (D). NS, not significant
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Figure 4. Therapeutic JCꢀ171 treatment reduces EAE severity and a Th17 response in EAE
mice. C57BL/6 mice (n = 5) were immunized with MOG peptide (200 ꢀg) emulsified in CFA on
day 0. JCꢀ171 or MCC950 treatment (10 mg/kg) was started when the clinical scores of
individual mice have reached 1 (flaccid tail) and continued every other day. EAE development in
mice was followed, and clinical scores were recorded (A). Twentyꢀeight days after immunization,
spinal cords were dissected from mice and subjected to Luxol fast blue/periodic acid–Schiff
staining for analysis of demyelination (B). The frequency of ILꢀ17A+CD4+ Th17 cells in the
spleen and spinal cord was determined by flow cytometry (C). Bar=50 µm
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