Benzoxathiole Derivative Blocks Lipopolysaccharide-Induced Nuclear Factor- B Activation and Nuclear Factor- B-Regulated Gene Transcription through Inactivating Inhibitory B Kinase

Benzoxathiole derivatives have been used in the treatment of acne and have shown cytostatic, antipsoriatic, and antibacterial properties. However, little is known about the molecular basis for these pharmacological properties, although nuclear factor (NF)B activation is closely linked to inflammation and cell proliferation. Here, we demonstrate that the novel small-molecule benzoxathiole 6,6-dimethyl-2-(phenylimino)-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (BOT-64) inhibits NFB activation with an IC50 value of 1 M by blocking inhibitory B (I B) kinase (IKK ), and suppresses NFB-regulated expression of inflammatory genes in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. BOT-64 inhibits IKK -mediated I B phosphorylation in LPS-activated macrophages, resulting in sequential prevention of downstream events, including proteolytic degradation of I B , DNA binding ability, and transcriptional activity of NFB. BOT-64 inhibits LPS-inducible IKK activity in the cells and catalytic activity of highly purified IKK . Moreover, the effect of BOT-64 on cell-free IKK was abolished by substitution of Ser-177 and Ser-181 residues in the activation loop of IKK to glutamic acid residues, indicating a direct interaction site of benzoxathiole. BOT-64 attenuates NFB-regulated expression of inflammatory genes such as inducible nitric-oxide synthase, cyclooxygenase-2, tumor necrosis factor, interleukin (IL)-1 , and IL-6 in LPS-activated or expression vector IKK -transfected macrophages. Furthermore, BOT-64 dosedependently increases the survival rates of endotoxin LPSshocked mice. Nuclear factor (NF)B is a family of eukaryotic transcription factors that play an important role in the regulation of immune and inflammatory processes, cell proliferation and survival, and cellular stress responses (O’Sullivan et al., 2007; Olivier et al., 2006). Mammalian NFB consists of homoor heterodimers of the Rel protein family. In normal cells, NFB is sequestered in the cytoplasm as an inactive complex bound to inhibitory B (I B) proteins such as I B , I B , and I B (Baeuerle and Baltimore, 1988). In response to lipopolysaccharide (LPS), an endotoxin recognized by tolllike receptor-4 and its accessory protein MD-2 on immune cells (Nagai et al., 2002), cellular I B kinase (IKK) complex is activated and phosphorylates cytoplasmic I Bs (Zandi et al., 1998). In the case of I B , the most studied member of the class, Ser-32 and Ser-36 residues are phosphorylated by the IKK complex (DiDonato et al., 1996). Analogous serine residues have been identified in both I B and I B . This phosphorylation is essential for subsequent ubiquitination folThis work was financially supported by a grant (R05-2004-000-10241-0, E00207) from the Korea Research Foundation, and another grant (KRF-2005005-J15001) funded by the Korean Government (MOEHRD, Basic Research Promotion Fund). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.041251. ABBREVIATIONS: NF, nuclear factor; BOT-64, 6,6-dimethyl-2-(phenylimino)-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one; PTN, parthenolide; I B, inhibitory B; IKK, I B kinase; LPS, lipopolysaccharide; iNOS, inducible nitric-oxide synthase; COX-2, cyclooxygenase-2; TNF, tumor necrosis factor; IL, interleukin; ERK-1/2, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEAP, secretory alkaline phosphatase; NPT, neomycin phosphotransferase; PGE2, prostaglandin E2; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; WB, Western blot; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; RFU, relative fluorescence unit; Luc, luciferase; TNF, tumor necrosis factor; AT buffer, HEPES, Triton X-100, glycerol, EDTA, EGTA, NaF, Na4P2O7, dithiothreitol, Na3VO4, phenylmethylsulfonyl fluoride, leupeptin, and pepstatin; PS-1145, XXX; IRFI 042, mono[2-[2-(acetylthio)ethyl]-2,3-dihydro-4,6,7-trimethyl-5-benzofuranyl] ester; YS 51, 1-( -naphtylmethyl)-6,7-dihydroxy-1,2,3,4,-tetrahydroisoquinoline; BMS-345541, (4(2 -aminoethyl)amino-1,8-dimethylimididazo(1,2-a)quinoxaline)4,5-dihydro-1,8-dimethylimidazo(1,2-a)quinoxalin-4-one-2-carboxylic acid; PS-1145, N-(6-chloro-9H-carbolin-8-yl)-nicotinamide. 0026-895X/08/7304-1309–1318$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 4 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 41251/3320953 Mol Pharmacol 73:1309–1318, 2008 Printed in U.S.A. 1309 at A PE T Jornals on July 1, 2017 m oharm .aspeurnals.org D ow nladed from lowed by proteasome-mediated degradation of I Bs, after which NFB moves into the nucleus (Karin and Ben-Neriah, 2000). There NFB binds to the B sequences for transcriptional regulation of immune and inflammatory genes, including inducible nitric-oxide synthase (iNOS), cyclooxygenase (COX)-2, and cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1 , and IL-6 (Tian and Brasier, 2003). IKK complex-catalyzed I B phosphorylation is a prerequisite for the activation and nuclear translocation of NFB (Karin and Ben-Neriah, 2000). The IKK complex consists of catalytic subunits of IKK and IKK and a regulatory subunit of NEMO/IKK . Although inducible serine/threonine kinase activity of IKK complex is regulated by NEMO/IKK , it is known that phosphorylation of Ser-177 and Ser-181 residues in the activation loop of IKK is crucial (Delhase et al., 1999). Genetic study of IKK subunit knockout mice has provided evidence that almost all inflammatory stimuli, including LPS, require the IKK subunit for NFB activation (Li et al., 1999b). Dominant-negative IKK but not dominant-negative IKK blocks NFB-regulated gene transcription in inflammatory states (Aupperle et al., 1999). IKK does not seem to play a major role in the classic pathway of NFB activation but instead is important for developmental processes (Li et al., 1999a). Benzoxathiole derivatives have been used in the treatment of acne and are reported to have cytostatic, antipsoriatic, and antibacterial properties (Goeth and Wildfeuer, 1969; Lius and Sennerfeldt, 1979). However, the molecular basis of these pharmacological properties remains to be defined. We postulated that benzoxathiole derivatives could mediate some of their pharmacological effects by modulating NFB activation, which is closely linked to inflammation and cell proliferation. In this study, we demonstrate that a novel benzoxathiole derivative (BOT-64) of 6,6-dimethyl-2-(phenylimino)-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (Fig. 1A) is an efficient inhibitor of IKK in NFB activation, and this mechanism of action down-regulates NFB-regulated expression of inflammatory genes in macrophages and increases the survival rate of endotoxin LPS-shocked mice. Materials and Methods Reagents, Antibodies, and Plasmids. LPS (Escherichia coli 055:B5), parthenolide (PTN), and anti-FLAG M2 affinity gel freezersafe beads were purchased from Sigma-Aldrich (St. Louis, MO), and Lipofectamine was from Invitrogen (Carlsbad, CA). Antibodies against I B , IKK , iNOS, COX-2, extracellular signal-regulated kinase (ERK)-1/2, or c-Jun N-terminal kinase (JNK) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and those against p-I B , p-ERK-1/2, or p-JNK were from Cell Signaling Technology (Danvers, MA). pSV-galactosidase control vector was purchased from Promega (Madison, WI). A reporter plasmid of pNFB-secretory alkaline phosphatase (SEAP)-neomycin phosphotransferase (NPT) expression vectors encoding IKK , IKK , IKK (C/A), IKK (SS/EE), NFB p65 or p50, and luciferase (Luc) reporter plasmids of piNOS ( 1592/ 183)-Luc, pTNF( 1260/ 60)-Luc, pIL-1 ( 1856/ 1)-Luc, or pIL-6 ( 250/ 1)-Luc have been described previously (Hiscott et al., 1993; Lowenstein et al., 1993; Zhang et al., 1994; Yao et al., 1997; Moon et al., 2001; Kim et al., 2007). Novel Benzoxathiole BOT-64. 5,5-Dimethylcyclohexa-1,3-dione (1 mM) in 10 ml of methylene chloride was added drop-wise to a solution of iodosylbenzene (1 mM) in 15 ml of methylene chloride and stirred for 1 h at room temperature. After the methylene chloride was evaporated under vacuum, the reaction mixture was dissolved in 15 ml of benzene and then added with a solution of rhodium (II) acetate dimer (20 mg) and phenylisothiocyanate (1 mM) in 15 ml of benzene. The resulting mixture was refluxed for 5 h, concentrated under vacuum, and then subjected to column chromatography (hexane/ethyl acetate 8:1) to separate BOT-64 with 95% purity. BOT-64: yield, 35%; yellow solid; melting point, 91 to 93°C; IR (KBr) 2950, 2920, 2870, 1660, 1620 cm ; NMR (CDCl3): 1.19 (s, 6H), 2.42 (s, 2H), 2.67 (s, 2H), 7.04 (d, J 7.6 Hz, 2H), 7.17 (t, J 7.2 Hz, 1H), 7.38 (t, J 7.6 Hz, 2H). Cell Culture. RAW 264.7 and THP-1 cells are murine and human macrophages, respectively (American Type Culture Collection, Manassas, VA). They were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, benzylpenicillin potassium (143 U/ml), and streptomycin sulfate (100 g/ml) at 37°C in a 5% CO2 atmosphere. RAW 267.4 cells harboring pNFB-SEAPNPT reporter construct were cultured in the same media supplemented with Geneticin (500 g/ml). Cell Proliferation Assay. RAW 264.7 cells were seeded at 1 10 cells/well in 96-well culture plates and incubated overnight. After cells were treated with BOT-64 for various periods, they were exposed to 10 l of WST-1 reagent (Dojindo Laboratories, Kumamoto, Japan) for 3 h, and the absorbance values were measured at a wavelength of 450 nm. SEAP Assay. RAW 264.7 cells harboring pNFB-SEAP-NPT reporter construct or THP-1 cells transfected with pNFB-SEAP reporter construct were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 16 h. SEAP activity was measured as described previously (Moon et al., 2001). In brief, aliquots of the culture media were heated at 65°C for 5 min and reacted with 4-methylumbellifery phosphate (500 M) in the dark for 1 h. SEAP activity was measured as relative fluorescence units (RFUs) with emission at 449 nm and excitation at 360 nm. In another experiment, Lipofectamine was used to transiently transfect RAW 264.7 cells harboring the pNFB-SEAP-NPT construct with pSV-galactosidase control vector in combination with expression vectors encoding IKK , IKK , NFB p65, or p50. The transfected cells were treated with BOT-64 for 16 h and then subjected to the SEAP assay. Electrophoretic Mobility Shift Assay. RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) Fig. 1. Effect of BOT-64 on cell proliferation. A, chemical structure of BOT-64. B, RAW 264.7 cells were incubated with various concentrations (1–10 M) of BOT-64 for the indicated times. Proliferation of the cells was analyzed by WST-1 method. Values are represented as percentage of the control, media alone-treated group. Data are means S.E. from three separate experiments. 1310 Kim et al. at A PE T Jornals on July 1, 2017 m oharm .aspeurnals.org D ow nladed from for 1 h. The cells were disrupted in a lysis buffer (10 mM HEPES, pH 7.9, 2 mM MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF) and incubated on ice for 10 min. After centrifugation, cell pellets were resuspended in a lysis buffer (20 mM HEPES, pH 7.9, 50 mM MgCl2, 420 mM KCl, 20% glycerol, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF) and incubated on ice for 30 min. After centrifugation, supernatants were used as the sources of nuclear extracts. A double-stranded oligonucleotide (5 -AGTTGAGGGGACTTTCCCAGGC-3 , in which the NFB binding site is underlined) was P-end labeled using [ -P]ATP and polynucleotide kinase and then reacted with nuclear extracts in a binding buffer [10 mM Tris, pH 7.5, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 0.05 g/ l poly(dI-dC), and 4% glycerol] on ice for 30 min. The DNA-protein complex was resolved on native 6% acrylamide gel by electrophoresis, and radioactive bands from the dried gels were visualized by exposure to X-ray film. Supershift assays were performed by incubating the DNA-binding reactions with 4 g each of anti-NFB p65 antibody or anti-NFB p50 antibody for an additional 20 min on ice before electrophoresis. Western Blot Analysis. RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for various times. Cell extracts were prepared in an AT buffer (20 mM HEPES, pH 7.9, 1% Triton X-100, 20% glycerol, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na4P2O7, 1 mM DTT, 1 mM Na3VO4, 0.5 mM PMSF, 1 g/ml leupeptin, and 1 g/ml pepstatin). Equal amounts of protein were resolved on SDS-acrylamide gels by electrophoresis and transferred to PVDF membrane. Either 5% nonfat milk in phosphatebuffered saline with Tween 20 or 5% bovine serum albumin in Tris-buffered saline with Tween 20 was used as the blocking buffer. The blots were incubated at 4°C overnight with primary antisera. The antisera and dilutions are as follows: anti-I B (1:300), anti-pI B (1:1000), anti-IKK (1:200), anti-p-ERK-1/2 (1:1000), anti-pJNK (1:500), anti-iNOS (1:1500), and anti-COX-2 (1:200). The blots were then incubated at room temperature for 2 h with appropriate horseradish peroxidase-labeled secondary antisera. Immune complexes on the blots were visualized by exposure to X-ray film after reacting with an enhanced chemiluminescence reagent (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Enzyme Assay of IKK . The catalytic activity of IKK was measured as described previously (Kim et al., 2007). In brief, either highly purified IKK (Millipore Corporation, Billerica, MA) or immunoprecipitated enzyme was incubated with GST-I B (2 g) as the substrate and [ -P]ATP (5 Ci) in an assay buffer (20 mM HEPES, pH 7.7, 2 mM MgCl2, 50 M ATP, 10 mM -glycerophosphate, 10 mM NaF, 300 g/ml Na3VO4, 2 M PMSF, 10 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, and 1 mM DTT) at 30°C for 1 h. The reaction mixtures were resolved on SDS-10% acrylamide gel by electrophoresis, and radioactive bands from the dried gels were visualized by exposure to X-ray film. For preparation of IKK immunoprecipitates, RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 10 min. Cell extracts were incubated with anti-IKK antibody (2 g) and protein A beads (GE Healthcare). To obtain IKK immunoprecipitates, RAW 264.7 cells were transfected with FLAG-tagged expression vectors encoding IKK (C/A) or IKK (SS/EE). Cell extracts were incubated with anti-FLAG affinity gel freezer-safe beads on ice for 2 h. Beads were washed three times with the AT buffer, twice with 20 mM HEPES, pH 7.7, and then subjected to the kinase assay. Nitrite Quantification. RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 24 h. Amounts of nitrite were measured as described previously (Archer, 1993). In brief, aliquots (100 l) of the culture media were reacted with 1:1 mixture (100 l) of 1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine, and the absorbance values were measured at 540 nm. Enzyme-Linked Immunosorbent Assay. RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 24 h. Amounts of prostaglandin E2 (PGE2), TNF, IL-1 , or IL-6 in the culture media were measured using enzyme-linked immunosorbent assay (ELISA) kits (GE Healthcare). Reverse Transcription-Polymerase Chain Reaction. RAW 264.7 cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 4 to 6 h. Total RNA of the cells was subjected to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using an RNA PCR kit (Bioneer Co., Daejeon, Korea). In brief, total RNA was reverse-transcribed at 42°C and then subjected to 25 to 30 cycles of PCR consisting of 30-s denaturation at 94°C, 30-s annealing at 50 to 60°C, and 90-s extension at 72°C. The sequences of primers for RT-PCR and the sizes of PCR products are as follows: iNOS: sense, 5 -GTCAACTGCAAGAGAACGGAGAC-3 , and antisense, 5 -GAGCTCCTCCAGAGGGTAGGCTTG3 , 457 bp; COX-2: sense, 5 -ACTCACTCAGTTTGTTGAGTCATTC-3 , and antisense, 5 -TTTGATTAGTACTGTAGGGTTAATG-3 , 583 bp; TNF: sense, 5 -AGGTTCTGTCCCTTTCACTCACTGG-3 , and antisense, 5 -AGAGAACCTGGGAGTAGACAAGGT-3 , 487 bp; IL-1 : sense 5 -CCTGTCCTGTGTAATGAAAGACGGC-3 , and antisense, 5 -GTGCTGCCTAATGTCCCCTTGAATC-3 , 525 bp; IL-6: sense, 5 -ATGAAGTTCCTCTCTGCAAGAGACT-3 , and antisense, 5 -CCTTCTGTGACTCCAGCTTATCTGT-3 , 549 bp; and -actin: sense, 5 -CACCACACCTTCTACAATGAGCTGC-3 , and antisense, 5 -GCTCAGGAGGAGCAATGATCTTGAT-3 , 745 bp. RT-PCR products were resolved on agarose gels by electrophoresis and stained with ethidium bromide. Luciferase Assay. RAW 264.7 cells were transiently transfected with pSV-galactosidase control vector in combination with each Luc reporter construct of p-iNOS ( 1592/ 183)-Luc, p-TNF( 1260/ 60)-Luc, p-IL-1 ( 1856/ 1)-Luc, or pIL-6 ( 250/ 1)-Luc, using Lipofectamine. The transfected cells were treated with BOT-64 for 2 h and then stimulated with LPS (1 g/ml) for 16 h. Cell extracts were subjected to luciferase assay and -galactosidase assay using the appropriate kits (Promega). In another experiment, the cells were transiently transfected with pSV-galactosidase control vector and expression vector encoding IKK in combination with each of the Luc reporter constructs. The transfected cells were treated with BOT-64 for 16 h and subjected to the luciferase assay. Septic Shock in Mice. Male ICR mice (20 2 g body weight, 10 mice per group) were purchased from Samtako Animal Center (Kyeongki, Korea) and acclimated under semi-specific pathogen-free conditions with free access to standard diet and water ad libitum. The mice were administered BOT-64 (3–30 mg/kg, i.p.), and septic shock was induced 2 h after by injection of LPS (1.5 mg/mouse, i.p.). Survival rates of the mice were monitored over the next 48 h. Animal studies have been carried out in a veterinary facility of Chungbuk National University, Korea, in accordance with all institutional and federal ethical regulations for experimental animal care. Statistical Analysis. Data are expressed as means S.E., and were analyzed by analysis of variance followed by Dunnett’s test apart from survival rates in septic shock of mice, which were analyzed by the logrank test. Different values of P 0.01 were consid-