1521-0103/353/3/539–550$25.00 http://dx.doi.org/10.1124/jpet.115.222570THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:539–550, June 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Curcumin Analog L48H37 Prevents Lipopolysaccharide-InducedTLR4 Signaling Pathway Activation and Sepsis via Targeting MD2

Yi Wang, Xiaoou Shan, Yuanrong Dai, Lili Jiang, Gaozhi Chen, Yali Zhang, Zhe Wang,Lili Dong, Jianzhang Wu, Guilong Guo, and Guang LiangChemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang,China (Y.W., G.C., Y.Z., Z.W., J.W., G.L.); Department of Paediatrics (X.S., L.J., L.D.) and Department of Respiratory Medicine(Y.D.), the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China; and Department ofOncological Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China (G.G.)

Received January 5, 2015; accepted April 9, 2015

ABSTRACTEndotoxin-induced acute inflammatory diseases such as sepsis,mediated by excessive production of various proinflammatorycytokines, remain the leading cause of mortality in critically illpatients. Lipopolysaccharide (LPS), the characteristic endotoxinfound in the outer membrane of Gram-negative bacteria, caninduce the innate immunity system and through the myeloiddifferentiation protein 2 (MD2) and Toll-like receptor 4 (TLR4)complex, increase the production of inflammatory mediators. Ourprevious studies have found that a curcumin analog, L48H37[1-ethyl-3,5-bis(3,4,5-trimethoxybenzylidene)piperidin-4-one], wasable to inhibit LPS-induced inflammation, particularly tumornecrosis factora and interleukin 6 production and gene expressionin mouse macrophages. In this study, a series of biochemical

experiments demonstrate L48H37 specifically targets MD2 andinhibits the interaction and signaling transduction of LPS-TLR4/MD2. L48H37 binds to the hydrophobic region of MD2 pocket andforms hydrogen bond interactions with Arg90 and Tyr102. Sub-sequently, L48H37was shown to suppress LPS-inducedmitogen-activated protein kinase phosphorylation and nuclear factor kBactivation in macrophages; it also dose dependently inhibits thecytokine expression in macrophages and human peripheral bloodmononuclear cells stimulated by LPS. In LPS-induced septic mice,both pretreatment and treatment with L48H37 significantlyimproved survival and protected lung injury. Taken together, thiswork identified a newMD2 specific inhibitor, L48H37, as a potentialcandidate in the treatment of sepsis.

IntroductionSepsis, or systemic inflammatory response syndrome, is

a severe condition marked by an overwhelming immuneresponse to a serious infection and results in the excessiveproduction of various proinflammatory cytokines and cellularinjury. In critically ill patients, sepsis is the leading cause ofmortality, with hospital mortality rates between 15–30%(Gaieski and Goyal, 2013), and is responsible globally formillions of deaths each year (Balk, 2014). Bacteria are the

most common culprits in infections that develop into sepsis,particularly Gram-negative bacteria, owing to lipopolysaccha-ride (LPS), the major glycolipid found in their outer membrane.Following infection with Gram-negative bacteria, the host isexposed to microbial LPS through ancillary proteins, such asLPS-binding protein and CD14, which transport the microbialLPS to bind a surface receptor complex composed of Toll-likereceptor 4 (TLR4) and myeloid differentiation protein 2 (MD2)in specific target cells. Subsequent TLR4 activation leads to therecruitment of myeloid differentiation primary-response gene88 (MyD88), activating downstream nuclear factor (NF)-kB andmitogen-activated protein kinase (MAPK) pathways. It is wellknown that LPS from Gram-negative bacteria is a potentstimulant of the immune response (Rossol et al., 2011), and theactivation of the NF-kB and MAPK pathways induces theupregulation and increased expression of proinflammatorygenes, contributing to multiple organ dysfunction and systemic

This study was supported by the National Natural Science Funding of China[Grants 81472307 and 21272179], High-Level Innovative Talent Funding ofZhejiang Department of Health (to G.L.), Zhejiang Natural Science Funding[Grant LQ14H310003], and Zhejiang College Students’ Science and Technol-ogy Innovation Activities Program [Grant 2014R413071].

Y.W. and X.S. contributed equally to this work.dx.doi.org/10.1124/jpet.115.222570.

ABBREVIATIONS: bis-ANS, 1,19-bis(anilino)-4,49-bis(naphthalene)-8,89-disulfonate; CRX-526, (2S)-2-[[(3R)-3-hexanoyloxytetradecanoyl]amino]-3-[(2R,3R,4R,5S,6R)-3-[[(3R)-3-hexanoyloxytetradecanoyl]amino]-4-[(3R)-3-hexanoyloxytetradecanoyl]oxy-6-(hydroxymethyl)-5-phosphonooxyoxan-2-yl]oxypropanoic acid; DMSO, dimethyl sulfoxide; E5531, (6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-enoyloxydecyl]-2-[3-oxo-tetrade-canoylamido]-b-O-phosphono-a-D-glucopyranose tetrasodium salt; ELISA, enzyme-linked immunosorbent assay; ERK, extracellularsignal-related kinase; FITC, fluorescein isothiocyanate; IL, interleukin; JNK, c-Jun N-terminal kinase; JSH, 29,4-dihydroxy-69-isopentyloxychalcone;JTT-705, S-[2-({[1-(2-ethylbutyl)cyclohexyl]carbonyl}amino)phenyl] 2-methylpropanethioate; L48H37, 1-ethyl-3,5-bis(3,4,5-trimethoxybenzylidene)piperidin-4-one; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MPM, mouse primary peritoneal macrophage; NF, nuclearfactor; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; qPCR, quantitative PCR;rhMD2, recombinant human MD2; RT-qPCR, reverse transcription qPCR; SPR, surface plasmon resonance; TAK-242, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate; TNF, tumor necrosis factor.

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inflammatory response syndrome (Park et al., 2012; Cighettiet al., 2014).Interestingly, rather than TLR4, it is MD2 that recognizes

the lipid A moiety of LPS, and MD2 is absolutely required totrigger LPS-induced TLR4 activity and primarily responsiblefor determining the specificity of different LPS chemotypes(Park et al., 2012). Since MD2 plays a critical role in LPSrecognition, increasing studies reveal that MD2 can be thepotential therapeutic target of acute inflammatory disorders,including sepsis. So far, several MD2 inhibitors have beenreported (Fig. 1A). For example, Peluso et al. (2010) showedthat a chalcone derivative, xanthohumol, competitively dis-placed LPS from MD2, which inhibited LPS-induced TLR4activity. Two natural compounds, caffeic acid phenethyl esterand JSH [29,4-dihydroxy-69-isopentyloxychalcone], containingthe same moiety 3-(4-hydroxyphenyl) acrylaldehyde in thestructures, were also found to inhibit LPS-induced TLR4activation partly by interfering with LPS binding to MD2(Roh et al., 2011; Kim et al., 2013). Curcumin, isolated fromthe natural spice turmeric, is a pleiotropic molecule thatexhibits many pharmacological effects, including anti-inflammatory, antitumor, antioxidative, and cardiovascularprotective effects (Prasad et al., 2014). It has been shown thatcurcumin is able to regulate a variety of molecular targets incells (Hasima and Aggarwal, 2012). With the moiety 3-(4-hydroxyphenyl) acrylaldehyde in its structure, curcumin hasbeen shown to bind at submicromolar affinity to MD2,competing with LPS for the same binding site and resultingin the pharmacological outcomes in suppression of theinflammation caused by LPS (Gradisar et al., 2007).

In the past decade, our laboratory has been engaged inmedicinal chemistry, turning to natural products, such ascurcumin, in efforts to discover new anti-inflammatory drugs.Our laboratory and others observed that owing to curcumin’spoor stability under physiologic conditions, its clinical applica-tion was extremely limited (Joe et al., 2004). Therefore, inefforts to increase the stability of curcumin, we designed andsynthesized in our previous studies a series of mono-carbonylcurcumin analogs without the b-diketone moiety of curcumin,which showed enhanced chemical stability (Fig. 1B) (Wu et al.,2013). Among these curcumin analogs, 1-ethyl-3,5-bis(3,4,5-trimethoxybenzylidene)piperidin-4-one (L48H37; Fig. 1B),exhibited high chemical stability and strong anti-inflammatoryability. The aim of the present study was to find the moleculartarget of L48H37 as well as the underlying mechanism of itsanti-inflammatory actions. On the basis of the structuralsimilarity, we hypothesized that L48H37 exerted its anti-inflammatory actions by directly targeting MD2. This studyidentified L48H37 as a novel and specific MD2 inhibitor that candirectly bind to MD2, block the LPS-TLR4/MD2 signalingactivation, and suppress the expression of inflammatorycytokine in vitro and endotoxin-induced septic shock in vivo.

Materials and MethodsCells, Materials, and Reagents. Mouse primary peritoneal

macrophages (MPMs) were prepared and cultured from C57BL/6mice using the method described in our previous paper (Pan et al.,2013). Human peripheral blood mononuclear cells (PBMCs) werepurified as described by Goodall et al. (2014). Briefly, the whole blood

Fig. 1. Design, synthesis, and stability assay of curcumin analog L48H37. (A) Structures of current MD2 inhibitors. (B) Design and synthesis ofcurcumin analog L48H37. (C) UV-visible absorption spectra of curcumin and L48H37. Curcumin or L48H37 were dissolved in phosphate buffer (pH 7.4)to a final concentration of 20 mM. Absorbance readings were taken from 250 to 600 nm using a SpectraMaxM5. The UV absorption spectra were collectedfor over 25 minutes at 5-minute intervals at 25°C.

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was overlayered on Ficoll Hypaque (GE Healthcare, Buckinghamshire,UK) at a 2:1 ratio of blood to Ficoll before separation via centrifugation at1600 rpm without braking for 30 minutes at room temperature. Afterthe layers were separated, the PBMC layer was directly removed fromabove the Ficoll layer and washed three times with phosphate-bufferedsaline (PBS). Collected PBMCs were resuspended in RPMI for furtheranalysis. Curcumin, LPS (from Salmonella typhosa), TLR2 agonistPam3CK, and fluorescein isothiocyanate (FITC)–labeled LPS (LPS-FITC; fromEscherichia coli 055:B5) were purchased fromSigma-Aldrich(St. Louis, MO). Antibodies against p38, p-p38, c-Jun N-terminal kinase(JNK), p-JNK, extracellular signal-related kinase (ERK), p-ERK, IkBa,and b-actin were purchased from Santa Cruz Biotechnology (Dallas,TX). Recombinant human TLR4 protein was purchased from SinoBiologic Inc. (Beijing, China). Anti-human MD2 antibody and mousetumor necrosis factor (TNF)-a and interleukin (IL)-6 enzyme-linkedimmunosorbent assay (ELISA) kits were obtained from eBioscience(eBioscience, San Diego, CA).

Synthesis of L48H37. The procedure for synthesis of compoundL48H37 is briefly described: The compounds 1-ethylpiperidin-4-one(2 mmol) and 3,4,5-trimethoxybenzaldehyde (4 mmol) were dissolvedin ethanol and catalyzed by NaOH at 5–8°C. Silica gel CXC(Sinopharm Chemical Reagent Co, LTD, Shanghai, China) was usedto monitor the reactions, and at the end of the reaction distilled waterwas added to the reaction mixture to precipitate the product. Furtherpurification was accomplished through column chromatography withpetroleum ether/ethyl acetate. The chemical structure of syntheticL48H37was well characterized by proton nuclearmagnetic resonance(1H NMR) and electrospray ionization mass spectroscopy. Before usein biologic experiments, high-performance liquid chromatographywas employed to determine the purity (99.12%). In in vitro experi-ments, L48H37 was dissolved in dimethyl sulfoxide (DMSO) solutionwith DMSO as a vehicle control. In in vivo studies, L48H37 was firstdissolved in water with macrogol 15 hydroxystearate (a nonionicsolubilizer for injection from BASF, Florham Park, NJ). Theconcentration of L48H37 in the water solution was 2 mg/ml, whereasthe concentration of solubilizer ranged 7.5% in final solution. A 7.5%solubilizer/water solution was used as the vehicle control.

Protein Expression and Purification of MD2 Proteins.Recombinant human MD2 (rhMD2; residue 17–160, PDB ID 2E59)cDNA was synthesized from Invitrogen (Shanghai, China). Thesynthesized fragment was then ligated into pET28a vector (Invitro-gen, Carlsbad, CA). The rhMD2 mutations R90A and Y102A wereintroduced into the pET28a vector by polymerase chain reaction(PCR)–based mutagenesis (primers for mutations were listed inSupplemental Material). The three expression vectors were clonedintoE. coliBL21(DE3). Onemillimolar isopropyl-b-D-(–)thiogalactosidewas added to induce protein expression, and the culture was allowedto incubate at 28°C for 8 hours. After extraction from E. coli cellsusing a combination of lysozyme and sonication, the inclusion bodieswere harvested and dissolved with 50 mM Tris-HCl, 0.6 M NaCl,and 8 M urea (pH 8.0), and kept at room temperature for 12 hours.Centrifugation at 12,000 rpm for 30 minutes removed any residualinsoluble matter, and the supernatant was filtered through a 0.22-mmfilter (EMD Millipore, Billerica, MA). The diluted supernatant wasapplied to Ni-IDA Sepharose 6 Fast Flow (General Electric, Fairfield,CT) according to the manufacturer’s instructions. Refolding of rhMD2,rhMD2/R90A, and rhMD2/Y102A peptides were carried out by gradientdialysis against 6–0.5 M urea in 50 mM Tris-HCl and 0.6 mM NaCl(pH 8.0) at 4°C. Bradford method was used to assess the concentrationof purified protein.

Animals. Male C57BL/6 mice (6–8 weeks age) were obtained fromthe Animal Center of WenzhouMedical University (Wenzhou, China).Animal experiments were performed in accordance with the Guide forthe Care and Use of Laboratory Animals. All animal experimentalprocedures were approved by the Wenzhou Medical UniversityAnimal Policy and Welfare Committee.

UV-Visible Absorption Spectra of Curcumin and L48H37.Absorbance readings from 250 to 600 nm were taken using

a SpectraMax M5 (Molecular Devices, Sunnyvale, CA). A stocksolution of 1 mM curcumin or L48H37 was prepared and diluted byphosphate buffer (pH 7.4) to a final concentration of 20 mM. The UVabsorption spectra were collected over 25 minutes at 5-minuteintervals at 25°C. All spectral measurements were carried out ina 1-cm path-length quartz cuvette.

Coimmunoprecipitation Assay. Mouse peritoneal macrophageswere obtained as previous described (Pan et al., 2013). Beforetreatment, MPMs were cultured in 60-mm plates and incubatedovernight at 37°C. After overnight incubation, MPMs were pretreatedwith L48H37 (10 mM) or vehicle control (DMSO) for 30 minutes andthen incubated with LPS (1 mg/ml) for 5 minutes. Total cells were lysedin an extraction buffer (containing mammalian protein extractionreagent supplemented with protease and phosphatase inhibitor cock-tails) and centrifuged at 12,000 rpm for 10 minutes at 4°C. Anti-MD2antibody was then added to 400 mg of protein and gently shaken at 4°Covernight. The immunocomplex was collected with protein A1Gagarose, and the precipitates were washed five times with ice-coldPBS. Finally, proteins were released by boiling in sample buffer andanalyzed through Western blotting with anti-TLR4 antibody.

Flow Cytometric Analysis. Cellular binding of LPS-FITC (fromE. coli 055:B5; Sigma-Aldrich) was measured as described previously(Roh et al., 2011). Briefly, HUVEC304 cells (1 � 105) were incubatedwith LPS-FITC (50 mg/ml) for 30 minutes with or without thepresence of L48H37 (0.1, 1, or 10 mM). After washing, the cells withbound LPS-FITC were analyzed by flow cytometry.

LPS Binding Assay. Anti-humanMD2 antibody (eBioscience) wascoated to a 96-well plate overnight at 4°C in 10 mMTris-HCl buffer (pH7.5). The plate was washed with PBS–Tween-20 and blocked with3% bovine serum albumin for 1.5 hours at room temperature. rhMD2,rhMD2/R90A, or rhMD2/Y102A (4 mg/ml, respectively) in 10 mMTris–HCl buffer (pH 7.5) was added to a precoated plate and incubatedfor 1.5 hours at room temperature. After washing with PBS–Tween-20,biotin-labeled LPS (InvivoGen, SanDiego, CA) was incubated for 1 hourat room temperaturewith or without the presence of compound L48H37(1 mM). After further washing, streptavidin-conjugated horseradishperoxidase (Beyotime, Shanghai, China) was added for 1 hour at roomtemperature. The horseradish peroxidase activity was determinedusing 3,39,5,59-tetramethylbiphenyl-4,49-diamine substrate solution(eBioscience). The optical density of each well was measured at 450 nm.

Fluorescence Measurements of Competition Displacement.Fluorescence measurements were performed with a SpectraMax M5(Molecular Devices). Briefly, 1,19-bis(anilino)-4,49-bis(naphthalene)-8,89-disulfonate (bis-ANS; 5 mM) and rhMD2 protein (5 nM) weremixed in PBS (pH 7.4) and incubated until fluorescence valuesfollowing excitation at 385 nm stabilized. Nonfluorescent L48H37 (at2.5, 5, 10, 20, 30 mM) was then treated for 5 minutes, and the relativefluorescence units emitted at 430–590 nm were measured.

Docking of L48H37 to the MD2 Structural Model. Dockingsimulation of L48H37 with MD2 protein (PDB ID 2E56) was carriedout with the Tripos molecular modeling package Sybyl-2.0 (Tripos,St. Louis, MO). The ligand-receptor complex went through energyminimization using the Tripos force field and Gasteiger-Hückelelectrostatic charges, according to the protocol previously indicated.To allow for flexible docking and production of over 100 structures, theligand-binding groove on MD2 was kept fixed, whereas all torsiblebonds of L48H37 were kept free. Final docked conformations wereclustered within the tolerance of 1 Å root-mean-square deviation.

Surface Plasmon Resonance Analysis. The binding affinity ofL48H37 was determined using a ProteOn XPR36 Protein InteractionArray system (Bio-Rad Laboratories, Hercules, CA) with anHTE sensorchip (ProteOn). Briefly, rhMD2, rhTLR4, rhMD2/R90A, or rhMD2/Y102A (in acetate acid buffer pH 5.5) was loaded to the sensor, whichwas activated with 10mMNiSO4, and the L48H37 samples (at 100, 50,25, 12.5, and 6.25 mM) were prepared with running buffer (PBS, 0.1%SDS, 5% DMSO). Sensor and sample plates were placed on theinstrument. The L48H37 samples were then captured in the first flowcells, and the second flow cell was left as a blank. Five concentrations

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were simultaneously injected at a flow rate of 30 mM/min for 120seconds of association phase, followed with 120 seconds of dissociationphase at 25°C. The final graphs represent the difference between theduplex or quadruplex sensorgrams and the blank sensorgrams. Dataanalysis was done using the ProteOn Manager Software, and the KD

was calculated by aligning the kinetic data from various concentrationsof L48H37 to the 1:1 Langmuir binding model.

Western Blot. Collected cells were lysed, and 30mg of lysates wasseparated by 10% SDS-PAGE and electrotransferred onto a nitrocel-lulose membrane. Following preincubation for 1 hour at roomtemperature in Tris-buffered saline (pH 7.6) with 0.05% Tween-20and 5% nonfat milk, each membrane was then incubated with specificantibodies. Following incubation with a secondary antibody conjugatedwith horseradish peroxidase, the membrane was visualized usingenhanced chemiluminescence reagents (Bio-Rad), and the immunore-active bands were then detected. The protein levels were analyzed usingImageJ software version 1.38e (NIH, Bethesda, MD) and normalized totheir respective control.

Assay of Cellular NF-kB p-65 Translocation. Using a CellularNF-kB p-65 Translocation Kit (Beyotime Biotech, Nantong, China), thecells were immunofluorescence-labeled in accordance with the manufac-turer’s instructions. Briefly, culturedMPMswere pretreatedwith L48H37(10 mM) or the vehicle control (DMSO) for 2 hours, and then stimulatedwith LPS (0.5 mg/ml) for 1 hour. After 1 hour of treatment, the cells wereincubated with p65 antibody and Cy3 fluorescein-conjugated secondaryantibody, and nuclei were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. The images (200� magnification) were obtained by fluo-rescence microscope. The experiment was repeated independently threetimes with similar results, and the results were quantified.

ELISA. ELISA kits (eBioscience) were used to measure theprotein levels of TNF-a and IL-6 in the culture medium. The totalamount of cytokines in the cell medium was normalized to the totalamount of protein in the viable cell pellet. The experiments wereperformed in triplicate.

RNA Extraction and Real-Time Quantitative PolymeraseChain Reaction. Cells were homogenized in TRIzol (Invitrogen)according to the manufacturer’s protocol for extraction of RNA. A two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit(Invitrogen) was used for both quantitative (qPCR) and reversetranscription qPCR (RT-qPCR), and Eppendorf’s Mastercycler realplexdetection system (Eppendorf, Hamburg, Germany) was used for qPCRanalysis. The primers of genes used are shown as follows:

mouse TNF-a forward 59- TCTCATTCCTGCTTGTGGCAG-39and reverse 59- TCCACTTGGTGGTTTGCTACG-39;mouse IL-6 forward 59-CCAAGAGGTGAGTGCTTCCC-39and reverse 59- CTGTTGTTCAGACTCTCTCCCT-39;mouse IL-1b forward 59-ACTCCTTAGTCCTCGGCCA-39and reverse 59-CCATCAGAGGCAAGGAGGAA-39;mouse IL-10 forward 59-GGTTGCCAAGCCTTATCGGA-39and reverse 59-ACCTGCTCCACTGCCTTGCT-39;mouse COX-2 forward 59-TGGTGCCTGGTCTGATGATG-39and reverse 59-GTGGTAACCGCTCAGGTGTTG-39;mouse iNOS forward 59-CAGCTGGGCTGTACAAACCTT-39and reverse 59-CATTGGAAGTGAAGCGTTTCG-39;mouse b-actin forward 59-TGCACCACCAACTGCTTAG-39and reverse 59-GGATGCAGGGATGATGTTC-39;human TNF-a forward 59-CCCAGGGACCTCTCTCTAATC-39and reverse 59-ATGGGCTACAGGCTTGTCACT-39;human IL-6 forward 59-GCACTGGCAGAAAACAACCT-39and reverse 59-TCAAACTCCAAAAGACCAGTGA-39;human b-actin forward 59-CCTGGCACCCAGCACAAT-39and reverse 59-GCCGATCCACACGGAGTACT-39.

All primers were synthesized and purchased from Invitrogen(Shanghai, China). The gene expression levels were normalized tothe amount of b-actin.

Treatment of Mice with L48H37 in Endotoxic Mouse Model.Male C57BL/6 mice weighing 18–22 g were injected with 200 ml of

LPS (at 20 mg/kg i.v. and through tail vein) 15 minutes before (fortreatment) or after (for prevention) injection of L48H37 (at 10 mg/kgi.v. and through tail vein), respectively. LPS was used for intravenousinjection in a 0.9% saline. Body weight change and mortality wererecorded for 7 days. In another experiment, male C57BL/6 miceweighing 18–22 g were injected with 200 ml of LPS (at 20 mg/kgi.v. and through tail vein) 15 minutes after injection of L48H37 (at10 mg/kg i.v. and through tail vein). Two or eight hours after LPSinjection, mice were anesthetized with diethyl ether and sacrificed.Lung samples were harvested and fixed in 10% formalin for 24 hours.The formalin-fixed lung samples were then embedded in paraffin,sectioned, stained with H&E, and observed 200� under a lightmicroscope. In the above in vivo studies, mice in both the vehiclecontrol group and LPS alone group received 100 ml solution of 7.5%macrogol 15 hydroxystearate in water, and mice in vehicle controlgroup also received 200 ml saline.

Statistical Analysis. All values are represented asmeans6S.E.M.from three independent experiments. Data analysis, including statisticalanalysis with the student’s t test or one-way analysis of variance, wasdone using GraphPad Prism 5.0. A P value of ,0.05 was considered tobe statistically significant.

ResultsChemical Stability of L48H37 Was Improved In

Vitro. The chemical stability of L48H37 and curcumin wastested using an absorption spectrum assay. Figure 1C showsthat the UV-visible absorption spectrum of curcumin dis-played a significant peak with a maximum absorption of closeto 425 nm. However, the intensity of curcumin’s absorptionspectrum significantly decreased over time in the phosphatebuffer (pH 7.4). In contrast, L48H37 showed no degradationunder the same conditions (Fig. 1C), suggesting that a chemicalmodification to L48H37 significantly increased curcumin’sstability and attenuated in vitro degradation.L48H37 Blocks the Interaction between MD2 and

LPS. We first determined the effect of L48H37 on LPS-induced TLR4/MD2 complex conformation by immunoprecipi-tation assay. As shown in Fig. 2A, the complex of TLR4/MD2profoundly increased in LPS-stimulatedmacrophages, whereastreatment with L48H37 significantly inhibited LPS-inducedTLR4/MD2 complex. It is unclear whether L48H37 directlyaffects the interaction of LPS-MD2 or that of TLR4-MD2. SinceMD2 is mainly located in the cell membrane, where LPS caninteract with MD2, we further tested if L48H37 is able toreduce the LPS-MD2 binding in cell surface. MPMs, with orwithout L48H37, were incubated with FITC-marked LPS(FITC-LPS) and then subjected to flow cytometry analysis.Figure 2B shows that FITC-LPS binds to the cell surface witha median fluorescence intensity of 17.4, and treatment withL48H37 dose dependently reduced the interaction of FITC-LPSwith the receptor on the cell surface. To validate the effects ofL48H37 on the interaction between LPS and MD2, weestablished a biotin-streptavidin–based ELISA system at themolecular level. The results in Fig. 2C show that biotin-markedLPS was able to bind to recombinant human MD2 (rhMD2)protein in the plates, whereas coincubation with L48H37significantly blocked the interaction of biotin-marked LPSand rhMD2.L48H37 Directly Binds to MD2 Protein. The direct

interaction of L48H37 and rhMD2 protein was determinedusing fluorescence spectroscopy and surface plasmon reso-nance (SPR) assay. As shown in Fig. 2D, fluorescence values ofbis-ANS, a fluorescent probe used to map the hydrophobic

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Fig. 2. Antagonistic effect of L48H37 on LPS binding toMD2. (A) Coimmunoprecipitation ofMD2.MPMswere pretreated with L48H37 (10 mM) orDMSO for30 minutes and then incubated with LPS (1 mg/ml) for 5 minutes. Cells were lysed and the total protein was collected. Four hundred micrograms of the totalproteinwas incubatedwith beads and anti-MD2antibody overnight at 4°C. The immunoprecipitated proteins and precipitatedMD2proteins, were resolved bySDS-PAGE and detected using anti-TLR4 antibody. The column figures represent the mean optical density ratio of three independent experiments (**P ,0.01). (B) Flow cytometric analysis. HUVEC304 cells were incubatedwithmedia alone (Ctrl), L48H37 (10mM), LPS-FITC (50mg/ml), andLPS-FITC (50mg/ml)plus L48H37 (0.1, 1, and 10 mM), respectively. These cells were subjected to flow cytometry analysis, in which the values for the median fluorescence intensitywere also provided. Conc., concentration. (C) In vitro assays for LPS binding to MD2. rhMD2 antibody was coated to a 96-well plate at 4°C overnight. rhMD2(4mg/ml) in 10mMTris-HCl bufferwas added to the precoated plate for 1.5 hours at room temperature. After washingwith PBS–Tween-20, biotin-labeled LPSwas added to the plate with or without the presence of L48H37 (1.5 mM). LPS ability to bind to rhMD2 was determined using ELISA, represented byabsorbance values at 450 nm (A450). Data are mean values (6S.E.M.) of three separate experiments, each performed in duplicate. **P , 0.01 versus bufferalone–added group. (D) Fluorescence measurements. bis-ANS (5 mM) was preincubated with rhMD2 (5 nM) to reach stable fluorescence values underexcitation at 380 nM and to reach stable relative fluorescence units emitted at 430–590 nm under excitation at 385 nm. Nonfluorescent L48H37 (at 2.5, 5, 10,20, 30 mM) was then treated for 5 minutes, and the relative fluorescence units emitted at 430–590 nm were measured. (E) SPR analysis showed that L48H37could not directly bind to rhTLR4 protein. (F) The binding affinity of L48H37 with rhMD2 was determined using a SPR assay. Conc., concentration; IB,immunoblot; IP, immunoprecipitation; MFI, mean fluorescence intensity; RU, response unit.

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binding sites in proteins, were markedly enhanced uponbinding to cell-free rhMD2 protein, whereas incubation withL48H37 dose dependently decreased the fluorescence intensityof bis-ANS, suggesting that L48H37 competitively binds torhMD2. Next, the SPR experiments showed no interactionbetween L48H37 and recombinant human TLR4 proteins

(Fig. 2E), whereas Fig. 2F shows that L48H37 directly bindsrhMD2 protein in a dose-dependent manner and with a veryhigh affinity (KD 5 0.0000113 M). These data indicate thatL48H37 is a novel and MD2-specific inhibitor.L48H37 Acts on Arg90 and Tyr102 Residues in MD2

Protein Pocket. We further predict the underlying binding

Fig. 3. Antagonistic mechanism of L48H37 on LPS binding toMD2. (A)Molecular docking of L48H37 with rhMD2 (PDB ID 2E56) was analyzed with theSybyl-2.0 molecular modeling software from Tripos. hTLR4 and rhMD2 are shown in white and green, respectively; (B and C) Surface plasmonresonance analysis. rhMD2R90A or rhMD2Y102A was biotinylated with biotin, and L48H37 was diluted to 100, 50, 25, 12.5, or 6.25 mM. The bindingaffinity of L48H37 was determined using a fortéBio Octet Red (Menlo Park, CA) equipped with a super streptavidin sensor; (D and E) In vitro assay forLPS binding to MD2 variants. rhMD2 antibody was coated on a 96-well plate at 4°C overnight. rhMD2R90A or rhMD2Y102A (4 mg/ml) in 10 mM Tris-HClbuffer was added to the precoated plate for 1.5 hours at room temperature. After washing with PBS–Tween-20, biotin-labeled LPS was added to the platewith or without the L48H37 treatment (1.5 mM). LPS binding to rhMD2 was determined by ELISA and represented by absorbance values at 450 nm(A450). Data are mean values (6S.E.M.) of three separate experiments, each performed in duplicate. RU, response unit.

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mode of L48H37 in MD2 protein using a molecular simulationof L48H37-MD2 complex. As shown in Fig. 3A, L48H37 wasfitted into the hydrophobic pocket of MD2, interacting with theresidues, including Tyr102, Phe121, Leu61, Cys133, and Arg90, inthe most energetically favorable configuration (Fig. 3A). Thewhole molecule of L48H37 is buried inside the lipid-bindingpocket and overlaps to a large extent with the binding sites ofLPS, indicating the structural mechanism behind L48H37’sobserved competitive inhibition of LPS. The computer-assistedsimulation also shows that the two amino residues Arg90 andTyr102 are the most probable candidates to form hydrogenbonds with L48H37 (Fig. 3A). Thus, to confirm the importanceof Arg90 and Tyr102 in L48H37 binding to rhMD2, two newrhMD2 mutations, rhMD2R90A or rhMD2Y102A, were prepared,respectively. SPR assay indicated that L48H37 no longer bindsto these two mutations (Fig. 3, B and C), and the ELISAmethod also found that L48H37 could not inhibit the binding ofBiotin-LPS with either rhMD2R90A or rhMD2Y102A (Fig. 3, D

and E). These results present the possible binding sites ofL48H37 in the MD2 protein pocket, which we believe will behelpful in the design of new MD2 inhibitors.L48H37 Inhibited LPS-Induced MAPKs and NF-kB

Activation in Macrophages. We then determined theeffects of L48H37 on LPS-activated downstream signaling inthe TLR4/MD2 cascade, including the representative MAPKspathway and the transcriptional factor NF-kB. The MAPKfamily consists of ERK, p38, and JNK. Figure 4A shows thatall three pathways were activated by LPS stimulation inMPMs, whereas the LPS-induced phosphorylations of ERK,p38, and JNK were markedly decreased following pretreat-ment with L48H37 in a dose-dependent manner. After IkBdegradation, NF-kB p65 translocates from the cytoplasm tothe nucleus, binds to the target promoters, and inducestranscription. Using Western blotting, we first evaluated theeffect of L48H37 on IkB degradation in total cell proteinextracts. LPS exposure for 1 hour induced an 84% degradation

Fig. 4. L48H37 inhibited LPS-inducedMAPK phosphorylation and NF-kB activation. (A and B)MPMs were pretreated with the vehicle control (DMSO)or L48H37 (1, 2.5, 5, or 10 mM) for 2 hours followed by incubation with LPS (0.5 mg/ml) for 1 hour. The protein levels of p-ERK, ERK, p-p38, p38, p-JNK,JNK, I-kB were examined by Western blot. The column figures represent the mean optical density ratio of three independent experiments. *P , 0.05,**P , 0.01, versus the LPS-treated group. (C) Cultured MPMs were pretreated with L48H37 (10 mM) or vehicle control (DMSO) for 2 hours, and thenstimulated with LPS (0.5 mg/ml). After 1 hour of treatment, the cells were incubated with p65 antibody and Cy3 fluorescein-conjugated secondaryantibody (red), and the nuclei were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (blue). The images (200�magnification) were obtainedby fluorescence microscope and overlay. Similar results were obtained for three independent experiments. The column figure for the p65 translocationrepresents the mean optical density ratio in three independent experiments. *P, 0.05, **P, 0.01, versus LPS-treated group. GAPDH, glyceraldehyde3-phosphate dehydrogenase.

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of IkB, whereas pretreatment with L48H37 reversed LPS-induced IkB degradation in MPMs in a dose-dependentmanner (Fig. 4B). Consequently, as shown in Fig. 4C, LPSstimulation could increase NF-kB p65 nuclear translocation(red point within blue nucleus, as indicated by arrows),whereas in L48H37 pretreated cells, LPS-induced nuclearlevels of p65 were significantly decreased.L48H37 Strongly Inhibits LPS-Induced Inflamma-

tory Cytokine Expression in Macrophages. MPMs andhuman PBMCs were used to examine the anti-inflammatory

activity of L48H37. As shown in Fig. 5, A and B, the LPS-induced increases in TNF-a and IL-6 levels were dosedependently inhibited by L48H37 in MPMs. Here, a TLR2agonist Pam3CK was used as a comparison. Interestingly,although 0.1 mg/ml Pam3CK significantly induced TNF-aoverexpression in MPMs, L48H37 could not inhibit theinflammatory response induced by Pam3CK (Fig. 5A). SinceMD2 is not required in TLR2 signaling pathway activation,the fact that L48H37 failed to fight TLR2-related inflamma-tion validates the specificity of L48H37 as an MD2 inhibitor.

Fig. 5. L48H37 inhibited LPS-induced inflammatory cytokine expression in mouse macrophages and human PBMCs. (A and B) MPMs were pretreatedwith the vehicle control (DMSO) or L48H37 (1, 5, or 10 mM) for 2 hours followed by incubation with LPS (0.5 mg/ml) or Pam3CK (0.1 mg/ml) for 22 hours.The protein levels of TNF-a (A) and IL-6 (B) in the culture medium were measured by ELISA. The total amount of cytokines in the cell medium wasnormalized to the total amount of protein in the viable cell pellet. The results are expressed as a percentage of the LPS-alone group (solid dark bar). Eachbar represents mean6 S.E.M. of three to five independent experiments. (C) MPMs were pretreated with vehicle control (DMSO) or L48H37 (10 mM) for2 hours followed by incubation with LPS (0.5 mg/ml) for 6 hours. ThemRNA levels of inflammatory cytokines, including TNF-a, IL-6, IL-1b, IL-10, COX-2,and iNOS were quantified by RT-qPCR. The mRNA values were normalized to the internal control b-actin mRNA and are expressed as a percentage ofthe values for the LPS control. Each bar represents mean 6 S.E.M. of three to five independent experiments. (D) Human PBMCs were pretreated withthe vehicle control (DMSO) or L48H37 (1, 5, or 10 mM) for 2 hours followed by incubation with LPS (0.5 mg/ml) for 6 hours. The mRNA levels ofinflammatory cytokines, including TNF-a and IL-6 were quantified by RT-qPCR. The mRNA values were normalized to the internal control b-actinmRNA and are expressed as a ratio of the LPS-alone group (solid dark bar). Each bar represents mean 6 S.E.M. of three to five independentexperiments. *P , 0.05, **P , 0.01, versus LPS-treated group; ns, not significant.

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The anti-inflammatory activity of L48H37 was also observedat the mRNA level. MPMs treated with LPS (0.5 mg/ml) for6 hours were examined through qPCR for the expression ofproinflammatory genes in the presence or absence of L48H37.As shown in Fig. 5C, L48H37 at 10 mM potently inhibitedLPS-induced upregulation of TNF-a (54.7%, P , 0.01), IL-6(82.3%, P , 0.01), IL-1b (91.2%, P , 0.01), cycloxygenase-2(COX-2, 57.5%, P , 0.05), and iNOS (50.9%, P , 0.01)transcripts in MPMs. As expected, L48H37 upregulated theexpression of the anti-inflammatory cytokine IL-10. Further-more, similar results were observed in human PBMCs, andL48H37 also significantly and dose dependently suppressedLPS-increased TNF-a and IL-6 expression (Fig. 5D).L48H37 Effectively Protects Mice from LPS-Induced

Septic Shock and Lung Injury. Male C57BL/6 mice wereinjected with LPS (20 mg/kg i.v.) in the presence or absence ofL48H37 pretreatment (intravenous), and the survival rateswere monitored for 7 days. Figure 6A shows that animalstreated with LPS alone all died within 48 hours. In contrast,treatment with L48H37 at 10mg/kg either 15minutes prior toLPS injection (prevention group) or 15 minutes after LPSinjection (treatment group) significantly improved the sur-vival rates compared with those of the control group (P, 0.01

in both groups versus LPS group). Also, the weight lost inboth groups improved slowly 2–7 days after LPS injection(Fig. 6B).We also examined the beneficial effects of L48H37 on lung

injury in LPS-treated mice. Two or eight hours after adminis-tration with LPS (20 mg/kg i.v.), histopathological changesin the lungs of C57BL/6 mice were observed using H&E stain-ing. L48H37 pretreatment at 10 mg/kg significantly improvedpulmonary damage and amended the LPS-injured tissue struc-ture of pulmonary lobules (Fig. 6C). These data demonstratethe anti-inflammatory effects of L48H37 in septic mice.

DiscussionSepsis can be caused by trauma, infection, or burns and can

lead to septic shock and organ failure. More than 30pharmaceutical candidates for the treatment of sepsis havebeen developed or are currently in the developmental stage;however, most treatments brought to clinical use have failedbecause of the complicated nature of sepsis, which remainsthe most common cause of death in intensive care units (Kinget al., 2014). Xigris was used to treat sepsis as a recombinanthuman–activated protein C that attenuates the development

Fig. 6. IUPAC chemical name to list of abbreviations LPS (i.v.). Survival rates (A) and body weight (B) were recorded for 7 days after LPS injection at theinterval of 12 hours. **P , 0.01 versus LPS-treated group. (C) C57BL/6 mice (n = 10/group) were treated with 10 mg/kg L48H37 15 minutes beforeinjection of 20 mg/kg LPS (i.v.). Two or eight hours after LPS injection, five mice were anesthetized with diethyl ether and sacrificed. Lunghistopathological analysis was performed using H&E staining as described in Materials and Methods. The representative images are shown.

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of organ failure attributable to sepsis. However, no significantimprovement was observed in clinical uses, and in 2012 use ofXigris was suspended (Opal et al., 2014). Although statinshave some nonspecific anti-inflammatory effects, they arecurrently not being considered as therapeutic options forsepsis (Gazzerro et al., 2012; Ou et al., 2014). Therefore, thereis an urgent need to find novel and effective therapeuticapproaches for sepsis.One potential approach to treating and preventing septic

shock and its associated diseases is the intervention of theTLR/MD2-mediated inflammatory response (Savva andRoger, 2013). LPS is presented to TLR4/MD2 complex viathe LPS-binding protein and CD14 (Park and Lee, 2013). MD2recognizes the lipid A domain of LPS, leading to the formationof the TLR4/MD2/LPS complex and activation of the down-stream cellular response (Park et al., 2012; Oblak and Jerala,2015). Both TLR4 and MD2 are essential for the LPS-inducedinflammatory response and sepsis. Both TLR42/2 and MD22/2

mice fail to respond to LPS and survive endotoxic shock (Duanet al., 2014). Therefore, TLR4 and MD2 are proposed aspotential targets for therapy that neutralizes the toxic effects ofendotoxin.The growth in our understanding of the structure and function

of the TLR4/MD2 complex has provided a new direction in thedevelopment of new drug targets in the treatment and pre-vention of sepsis (Peri and Calabrese, 2014). Because of theseemingly higher importance of TLR4, researchers paid moreattention to TLR4 thanMD2 in the past decades (Wittebole et al.,2010; �Svajger et al., 2013). However, the clinical trials ofTAK-242 [(6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate], a TLR4-specific inhibitor, have failed to treatsevere sepsis and related respiratory disease in patients (Riceet al., 2010). In addition, blocking TLR can lead to severe sideeffects, “inappropriate” immune responses, such as allergic Th2responses, or immunologic tolerance (Ishii et al., 2006; Nakamotoand Kanai, 2014). On the other hand, a series of MD2 antagonistswith lipid A structure (mimics LPS) targeting the MD2 protein,such as fatty acid chain–containing E5531 [(6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-enoyloxydecyl]-2-[3-oxo-tetrade-canoylamido]-b-O-phosphono-a-D-glucopyranosetetrasodium salt] (Bryant et al., 2007), CRX-526 [(2S)-2-[[(3R)-3-hexanoyloxytetradecanoyl]amino]-3-[(2R,3R,4R,5S,6R)-3-[[(3R)-3-hexanoyloxytetradecanoyl]amino]-4-[(3R)-3-hexanoyloxytetradecanoyl]oxy-6-(hydroxymethyl)-5-phosphonooxyoxan-2-yl]oxypropanoicacid] (Lin et al., 2013), and eritoran (Rallabhandi et al., 2012),have been evaluated in clinical and preclinical studies.Unfortunately, the most studied one, eritoran, failed in phaseIII clinical trials in 2011 because there was no significantimprovement in eritoran-treated patients compared with theplacebo group (Barochia et al., 2011).Recently, some natural active compounds that do not contain

the structure of lipid A or fatty acids have been found to be ableto targetMD2 directly (Fig. 1A). These smallmolecules, such asxanthohumol (Peluso et al., 2010), caffeic acid phenethyl ester(Kim et al., 2013), JSH (Roh et al., 2011), and curcumin(Gradisar et al., 2007), bind directly to the MD2 pocket, andblock the TLR4/MD2’s recognition of LPS, resulting in theprevention of proinflammatory signaling and septic shock.Although their specificities for targeting other proteins remainto be defined, these natural compounds provide us theimportant structural information for the design and discoveryof new synthetic MD2 inhibitors. As shown in Fig. 1A, the

structures of the MD2 inhibitors share the same 3-(4-hydroxyphenyl) acrylaldehyde skeleton. Thus, it is hypothe-sized that our new synthetic compound, L48H37, which showsexcellent anti-inflammatory activity and contains the structureof 3-(4-hydroxyphenyl) acrylaldehyde, may target MD2 andserve as an antisepsis candidate.Hence, the interaction between L48H37 and MD2 was

investigated at both cell-free molecular and cellular levels.Fluorescence spectroscopy and SPR assay demonstrated thatL48H37 was able to dose dependently bind to rhMD2 protein(Fig. 2, D and E). The interaction of L48H37 with MD2remarkably affected the LPS binding to rhMD2 (Fig. 2C),suggesting that the binding site for L48H37 in MD2 pocketoverlaps that for LPS, which is also consistent with themolecular docking results (Fig. 3A). At the cellular level, flowcytometry (Fig. 2B) and immunoprecipitation (Fig. 2A)revealed the inhibitory effects of L48H37 on LPS-MD2interactions and MD2-TLR4 complex formation, respectively.Interestingly, our data also showed that L48H37 is a specificMD2 inhibitor, since L48H37 could not inhibit the Pam3CK-induced TLR2 activation, which shares the MAPKs/NF-kB–involved proinflammatory signaling pathway with TLR4(Fig. 5A) but is independent on MD2. There, this studydemonstrates that MD2 is a molecular target of L48H37 andthat L48H37 can downregulate TLR4 activation and in-flammatory gene expression, as well as attenuate LPS-induced sepsis, by interrupting the association of LPS withMD2 (Fig. 6).

Fig. 7. Proposed model of signaling pathway involved in L48H37prevented LPS-induced TLR4 signaling pathway activation and sepsis.

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Molecular modeling of the crystal structure of MD2provided further support for the binding of L48H37 to MD2.The X-ray diffraction–based structural information and exactbinding mechanism for nonlipid compounds binding to MD2protein are still unclear. The MD2 binding sites of nonlipidcompounds have been predicted by computer-assisted simu-lation, and the Cys133 in theMD2 binding pocket is considereda molecular target of several natural inhibitors. Small-molecule inhibitors with a,b-unsaturated ketones are capableof forming covalent bonds with Cys133 via a Michael-typereaction. JTT-705 [S-[2-({[1-(2-ethylbutyl)cyclohexyl]carbonyl}amino)phenyl] 2-methylpropanethioate] (Mancek-Keber et al.,2009) and caffeic acid phenethyl ester (Kim et al., 2013) havebeen predicted to covalently bind Cys133 residue and showedan irreversible inhibition against MD2. However, thea,b-unsaturated ketone–containing curcumin interacts withMD2 via a noncovalent mechanism, a finding supported bystudies showing that it can be removed from the complex boundto MD2 by chloroform extraction and that it can still inhibitLPS from binding to the mutant MD2 Cys133Phe in the samemanner as the wild-type (Gradisar et al., 2007). In addition,some residues, Lys122, Tyr102, Gly123, Ser120, Lys130, andPhe126, in theMD2 pocket were predicted to play a possible rolein the interaction between MD2 protein and natural small-molecule inhibitors such as JSH (Roh et al., 2011), taxanes(Resman et al., 2008), and xanthohumol (Peluso et al., 2010). Inthis study, we found the possible binding mechanism ofL48H37-MD2 using the molecular dockingmethod. The resultsindicated that the binding site for L48H37 in the MD2 pocketoverlapped that for LPS, rather than TLR4 (Fig. 3A), which isalso evidenced by the experimental data at the molecular andcellular levels. Using further molecular dynamics, we showedthat L48H37 may form hydrogen bonds with two key residues,Arg90 and Tyr102, which also play a role in the binding of LPS(Fig. 3A). To validate this prediction, we replaced the twoamino residues Arg90 and Tyr102 with Ala in rhMD2mutations.As expected, the SPR analysis andELISA showed that L48H37could not interact with the rhMD2 mutations, indicating thatArg90 and Tyr102 play a critical role in L48H37-MD2 inter-actions. Although the Tyr102 residue has been predicted to be ofimportance in isoxanthohumol-MD2 interactions, the authorsfailed to demonstrate the possible hydrogen bond formationwith Tyr102 (Peluso et al., 2010). In addition, this is the firsttime that Arg90 has been highlighted as an importantmolecular target for MD2 inhibitors. Thus, the results of thisstudy provide the important structural information andunderstanding of the amino residue sites that support theuse and further design of MD2 inhibitors as anti-inflammatoryagents.L48H37’s inhibition of MD2 resulted in a series of anti-

inflammatory activities in macrophages. MAPKs and NF-kBhave been demonstrated to be the main mediators in the LPS-TLR4/MD2 proinflammatory signaling pathway. L48H37prevented TLR4-mediated MAPKs and NF-kB activation in LPS-stimulated macrophage, as evidenced by a dose-dependentdecrease in the levels of ERK/p38/JNK phosphorylation, IkBdegradation, and p65 translocation (Fig. 4). Figure 5 furthershows the inhibitory effects of L48H37 on LPS-inducedinflammatory cytokine overexpression in both mouse MPMsand human PBMCs. In vivo, either pretreatment or post-treatment with L48H37 significantly increased survival in theLPS-induced septic mice (Fig. 6A). Lung histologic changes in

the LPS-injected mice were also suppressed by L48H37pretreatment (Fig. 6C). These results validated the potentialof the MD2-targeting L48H37 as a therapeutic agent in boththe prevention and treatment of acute inflammatory diseases.Collectively, our data reveal that MD2 is the anti-

inflammatory target of novel compound L48H37 and can leadto the blockage of LPS-TLR4/MD2 complex formation anddecrease of downstream signal activation and inflammatorymediator expression. A schematic for the protection of L48H37from LPS-induced sepsis is illustrated in Fig. 7. Arg90 andTyr102 in the MD2 protein play an important role in L48H37’sinteraction withMD2 via two hydrogen bonds. In vivo, L48H37improved survival and protected lungs against LPS-inducedinjury in septic mice. This study suggests that MD2 is animportant therapeutic target against inflammatory disordersand proves that a new MD2 inhibitor, L48H37, can bedeveloped as a potential agent in the treatment of sepsis.

Authorship Contributions

Participated in research design: Y. Wang, Liang, Shan, Guo.Conducted experiments: Y. Wang, Jiang, Chen, Zhang, Z. Wang.Contributed new reagents or analytic tools: Dong, Wu.Performed data analysis: Y. Wang, Dai, Liang.Wrote or contributed to the writing of the manuscript: Y. Wang,

Jiang, Liang.

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Address correspondence to: Dr. Guang Liang, Chemical Biology ResearchCenter, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou,Zhejiang, 325035, China. E-mail: wzmcliangguang@163.com; or Dr. Guilong Guo,Department of Oncological Surgery, the First Affiliated Hospital of WenzhouMedical University, Wenzhou, Zhejiang, China. E-mail: guoguilong@sina.com

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