PCT WO 2007/114296 Al
Contents
1. cErp 2 3 4 2 WO 2007 114296 42 PCT JP2007 056962 2 Chem Pharm Bull 38 2792 2796 199 0 LpL E giimib o Mrp Scap FxR KEEL 16 7 MMp2. J Antibiotics 38 877 885 1985 WO 2007 114296 39 PCT JP2007 056962 B Gp dv EBM 1R 2R ode 2 ese 1 3 ILH 1 2 4
3. 11 WO 2007 114296 41 PCT JP2007 056962 0105 12 12 1
4. 8 WO 2007 114296 21 PCT JP2007 056962 0055 0066 KA RB ERE IN RR BR gt SH EIS Re GAL HOR OL AB me ofa RRA SAAR ARNO BUR HK RE SB A DNA DNA TLR4 DNA cDNA DNA RINNA mRNA Li Polymerase Chain Reaction PcR
5. 6 000 UT WO 2007 114296 51 PCT JP2007 056962 0113 50 Fog
6. TLR4 Ce CES TLR4 BRE
7. 4 WO 2007 114296 40 PCT JP2007 056962 0104 5 AN RF MIL C II gt 6
8. 45 LTR ead E 46
9. TLR4 WO 2007 114296 32 PCT JP2007 056962 oo oo 4 007 TLR4 TS OS MOTLR TLR TLR TNF e I 1 IL 0
10. 1g TLR4 01 100mgs TLR4 LH HSN
11. 5 WAX AN BR AA WERE XIX BR FE OS OA AOR AA SR TAR RAB OM BT IRR BH 2
12. TLR TLR4 TLR4 CES WO 2007 114296 16 PCT JP2007 056962 oo d 0 1 0052 0053 TLR4
13. LpS NF kB IRF 3 WO 2007 114296 12 PCT JP2007 056962 0041 T LR4 0 OL lt 1 00 0 1 1 0 0 5 2 TLR4
14. BE S1 Dsg
15. a Yyy HIY ZDRU SPP TY DLYA Fry ANR ee PAF iACE
16. t Bz1 CI Bzl 2 B Tos 4 2 3 6 DNP Bum Boc Trt Fm oc WO 2007 114296 19 PCT JP2007 056962 0058 0059 006 d HR Pd Pd
17. TLR4 TLR4 UER A Al fit HS VER BA CLTH ocew REL pH Ol RB
18. S PR Biaco re ORME K OAD OG MALDI MS ESI MS FAB MS ENIF Y CE EB BDA
19. ADP F 7 AT Proc Natl Acad Sci USA 69 21 1 0 1972 Ge ne 17 1 07 1982 Molec ula amp General Genetics 168 111 1979 Methods in Enzymology 194 182 187 1991 AT Proc Natl Acad Sci USA 75 1929 1978 WO 2007 114296 27 PCT JP2007 056962 0082 Bio Technology 6 47 55 1988
20. 1 OF JL 7 FH E WO 2007 114296 38 PCT JP2007 056962 D E MARAR OE vb HIV
21. TLR4 2 TLR4 664 7 06 747 831 1 lt 7 OF 747
22. OR R 2 Rg 7 R OK BEERS R R R R RAIL 1 2 WO 2007 114296 2 PCT JP2007 056962 0007 0008 0009 001 0 0011 0012 0013 3 OR R 4 1 lt 4
23. TLR4 TIR TLR4 K OSM CORSE OR N C GS Hi MBP Flag
24. 2 UC M 0C 1 4 1 2 2 4 1 2 1 4
25. WO 2007 114296 31 PCT JP2007 056962 003 Lom te VEE ARE TLR4 2 CH C lt BKM
26. 1 1 TLR N c
27. DNA TLR4 lt WO 2007 114296 23 PCT JP2007 056962 0072 0073 oo4 TLR4 2 TLR4 664 7 05 747 8 3 1 1 7 05 747 gt TLR4
28. 7 8 9 10
29. TLR4 DNA TLR4DNA lt o hol 9 N DNA TLR4 TLR4 2 TL R4 664 7 OF 747 8 3 1 1 7 5 747
30. Molecular Cloning 2 J Sambrook ct al Cold Spring Harbor Lab Press 1989 lt 19 lt 4 MM lt 19 2 OnM 5 0 lt 7 0C 6 0 lt 65C 1smM 65C
31. Flas AROS ERI Bl AE H s P TLR4 2 lt TLR4S 9 WO 2007 114296 10 PCT JP2007 056962 0037 OFF RAL BU AS ARAM REA A RR A B
32. TLR4 TLR4 N N Gin WO 2007 114296 15 PCT JP2007 056962 0047 0048 0049 ENRE TLR4 8 TLR 4 SOWA
33. MANR 25 WO 2007 114296 44 PCT JP2007 056962 C c K Ns ACE ACE
34. r amp j 2 9 1 SA J P ai ie 100 8915 4 3 03 3581 1101 Am 3448 PCT ISA 2 1 0 2 2005 4 PC JP2007 056962 c VERSTAK B et al Toll like receptor signalling and the clinical benefits that lie within Inflamm Res 2007 vol 56 pp 1 10 cr Aisa 4210 2 2005 4
35. Escherichia coli K12 DH1 Proc Natl Acad Sci USA 6 OF 16 01968 JM1 3 yA Nucleic Acids Resea rch 9 3 1981 JA221 Journal of Molecular Biology 12 0 517 197 8 HB101 ELF 4 1 459 1969 C6 00 Ge netics 39 44 01954 Bacillus subtilis MI1 14 24 255 1983 2 07 21 ju of Biochemist y 95 87 1984 Saccharomyces cerevisiae A H22 AH22R NA87 11A DKD 5D 2 0B 12 WO 2007 114296 26 PCT JP2007 056962 0081 Schizosaccha romyces pombe NCYC1913 NCYC2 Bo
36. vil MAEDA BE ii m Nl W Cm gt S Ww W N 2 W N N YE W 4 co gt N AS N
37. 5 x 0 MEM Science 122 5 01 1952 DMEM Virology 8 396 1959 RPMI 164 O tt STF The Journal of the Am erican Medical Association 199 519 1967 199 4AY Proceeding of the Society for the Biological Medicine 73 1 195 0 pH lt 6 3 UC 4 0C 15 0 TLR4 TLR4 TLR4
38. B vib Vi A x OEE Ci Ci PRM OM OER ov xvi xMi viii avy DIC 3 4 BAERE BRO BRL E BNF G 6 B RRARS BR SE BHAA
39. TLR4 Cik HAE 2 1 2 1 1 3 0 1 1 2 1 2 1 0 1 0 1 SM 0 1 2 1 2 1 0 1 3 0 1 OER 1 SM 2 WO 2007 114296 13 PCT JP2007 056962 00 gt oo 1 1 lt 1 OFF E 1 M 0 1
40. D C 5 42
41. 1 eR gt pNA 3 pNA TIR Ap 5 GCGCGGATCCATGGCATCATCGACCTCCCT 3 5 GCGCGCGGCCGCTCAAA GTAGATCAGATA 3 6 TLR2 TIR 5 GGATCCTATGATGCATTTGTTTCTTACAGT 3 7 5 AAGCGGCCGCCTAGGACTTTATCGCAGCTCTCAG 3 5 8 TLR4 TIR 5 GGATCCTATGATGCCTTTGTTATCTACTCA 3 5 AAGCGGCCGCTCAGATAGATGTTGCTTCCTGCCA 3 0 DpNA TAKA 1 Ex Taq Hos C2 95 C Cs 0 ss CT gt 1 3 0 BE C72 CTC 0 pNA TA Cloning Kit Invitrogen S phaglas BandP rep Kit Amersh am
42. TLR4 1 TLR4 D D lt 4 TLR4 TLR4 TLR4 WO 2007 114296 49 PCT JP2007 056962 0110
43. s 263 267 los Virology 52 456 1973 DRED Vata ey BR AEM NLD h
44. EGP HC Bl a E 34 35
45. TLR4 1 TLR4 2 TLR4 2 3 TLR4 2 4 TLR4 2 ARRE O TLR4
46. E WO 2007 114296 20 PCT JP2007 056962 0061 0062 0063 0064 COME NOT NE E C TLR4 TLR4 TLR4 STLR4
47. 36 TAK 147 WO 2007 114296 46 PCT JP2007 056962 0O EVR FY SOMBER ERS 37 L WY 38 ONE I ARR E 39 3 4 0 41 6 O Q
48. 12 2 o 13 MRAM 14 15 HMo CoA38
49. BE coli S3 0extract system Promega RTS 5 00 Rapid Tranlation Syst em Roche Rabbit Reticuloc lt Lysate System Promega pRorglo S Toy ogo o johnston F B ect al Nature 179 16 0 161 ios7 W Erickson A H et al Meth Enzymol 96 38 0 i996 E Pratt M et al 1984 Spirin A S et al Science 242 1162 1164 1988 OR gt VIp6 pRorEloS Wheat ge rm cell ttee protein synthesis core kit TOYOBO
50. 1 FCS DMEM A 6R C IN 2 4 1 1 1 B a e N 2 4 1 1 C 6 N 2 4 1 1 FSMD qd 6 4 1 1 0 3 0 100 3 00nmol 37 C 5 C O 2 1 00ng mL 3LPS 4 Dual Glo Luciferase Assay System Promega BE IS firefly luciferase FL NF kB ren illa luciferase RL FL cpS counts per second xL DCPS TH E LEIE FL RL ratio ER OK 3
51. 5 WO 2007 114296 29 PCT JP2007 056962 0086 0087 0088 00s9 oo d x 1 00 TLR4 BAUS DS
52. TLR4 TLR4 DNA RNA CES RNA TLR4 DNA Pratt JM et al Transcription and Tra WO 2007 114296 30 PCT JP2007 056962 oo 1 002 nlation 179 2 Hames B D amp Higgins S J eds IRL Press Oxford 1984
53. pNA ligation kit Ver 2 TAKARA hox 4T Amersham WO 2007 114296 54 PCT JP2007 056962 0117 0mL ampicillin LB 7 C 18 4 mL 13 0mL ampicilin Lp 3 o D 09 1 mol L isopropyl B D thiogalactopy rnoside i3 0 Le 4 0mL Lysis Bu r 10m mol L Trig pH7 3 15 Onmol L Nac l 05 triton 1 00 1 mmo lp L PMSF Lic 3 00u LOGlutathione Sepharo se 4B Amersham L 4 CTs 0 cGSr 01 Triton x 1 00 TBS Trig buffered saline 5 M Tris HCI pH7 5 15 mM NaCl TH cSr A C 0 1 Triton x 1 00 TBS 3 2 mercapt oethanol ME As odium dodecyl sulfate SpS
54. WO 2007 114296 50 PCT JP2007 056962 0111 0112 1 99 98 1 0 9 0 TLR4 TLR4 CES TLR4
55. 3 TLR 1 09 40242 2 01 1 0826 3 0984527 TLR4
56. TLR4 TLR4 TLR4 WO 2007 114296 37 PCT JP2007 056962 ol 018 OES ENM RRR A AR TLR4 ATO BE WO HMR MARR SEAGER MRR BH ALAR GBR RBM RRA JARE SMEAR Dine HDL
57. HERB BR BRE EE HIV DNA PRO KG ANA BEAR Ze
58. TLR4 TLR4 TLR4 CES 4 WO 2007 114296 17 PCT JP2007 056962 oo4 0 5 4 PAM 4 4 2 4 2 4
59. dhf r TLR 4 DNA 5 PhoA ompA x MEF a S UC2 x
60. WO 2007 114296 14 PCT JP2007 056962 0044 0045 0046 C EDC NN OH SH C C TLR4 TLR4 TLR4 TLR4
61. 1 2 2 0 TLR4 TLR4 TLR4 LA TLR4 TLR4 TLR4 OBR NO TN
62. H A 2 OR 747 3 HEk2o3 1 FcS pMEM ye gt x 1 0cells well 7 s c o o pTI MEM Invitrogen 5 0u L well FLAG TLR4 wr 0CA ca PH IKR SKR 05 ns wcll pLUS nvitrosen Lipo ctAMINE inVitrosen LA 37 C 5 c O 2 FCS DMEM 1 00L all WO 2007 114296 58 PCT JP2007 056962 oiio ol2 dl DNA pNiFty NF kB plasmiq 15 ng we Il phRL TK plasmiq 15 ns well MD 2 plasmid 1 0ns well CD14 plasm id 1 0ng well TLR4
63. TLR lt COOH COO_ CONH COOR R n lt 6 lt gs w lt gw lt lt TLR lt TLR CB lt TLR N
64. ccc Ga P Myers Miler CABIOS 4 11 17 1988 cec a LIGn ve rsi on 2 0 Pea rson S Proc Natl Acad Sci USA 85 2444 244 s i9ss ccG FA STA lt 2 7 0 s 0 9 0O 9s 9s 2 BABI CH
65. 2 7 06 747 2 1 4 1 1 TLR4 NF B lt IRF 3 1 TLR4 1 ChE NF B lt IRF 2 2 1 2 O 1 2 4 1 2 2 096
66. eH DONA rLR4 1CA WO 2007 114296 55 PCT JP2007 056962 5 TGATGCTTCTTGCTGGCGC CATAAAGTATGGTAGAG 3 11 5 CTCTACCATACTTTATGGC GCCAGCAAGAAGCATCA 3 Ma 5 12 2Ca 5 CCTCCATTTCAGCTCGCCCTTCACTACAGAGA 3 13 5 TCTCTGTAGTGAAGGGC GAGCTGAAATGGAGG 3 14 3 CA 5 ATCCAGAGCCGCTGGGCTATCTTTGAATATGA 3 15 5 TCATATTCAAAGATAGC CCAGCGGCTCTGGAT 3 16 4 CA 5 ACAGTGGGTACAGGAGC CAATTGGCAGGAAGC 3 17 5 GCTTCCTGCCAATTGGCTCCTGTACCCACTGT 3 18 PH 5 TACAGAGACTTTATTCACGGTGTGGCCATTGC 3 15 5 GCAATGGCCACACCGTGAATAAAGTCTCTGTA 2 2 0 lke 5 CAGTTCTGGTCTATAGGTTCTATTTTCACCTG 3 2 1 5 CAGGTGAAAATAGAACCTATAGACCAGAACTG 3 2 2KR 5 TGCTGGCTGCATAAGGTATGGTAGAGGTG 3 23 5 CACCTCTACCATACCTTATGCAGCCAGCA 3 24 3KR 5 GGAATGAGCTAGTAAGGAATTTAGAAGAAGG 3 25 s CCTTCTTCTAAATTCCTTACTAGCTCATTCC 3 ft A amp 26 4KR 5 CATGAAGGTTTCCATAGAAGCCGAAAGGTGATT_3
67. TLR4 2 TLR4 652 839 TLR4 NF IRF3 TLR4 TLR4 TLR4 1 TLR4 1 2 TLR4 664 7 05 747 83 1
68. 47 WO 2007 114296 48 PCT JP2007 056962 0109 48 49 rBpl 21 BI 51017 IID Sc 59735 FFpD r PAF Ly 20sss r JER TOF 1 Vc MTNF CD14 c Fab LpS Al JrE 007 E 3531 g 5564 S S 92 0 FR 1eess ONO 1714 ONO 5S H6 sivelestat GW 273629 rw 67657 c r 270773 NOx 1 00 GR 27073 NOx 1 00 5 0 gt HT3 1 G2
69. 1 s T WS WO 2007 114296 18 PCT JP2007 056962 0056 0057 PREE LLTI B ZIE Z Boc 4 C1 Z B 2
70. 2 TLR4 TLR4 OS 0 01 99 8 B 0 1 5 OB BM 0 5 2 0 01 99 8 lt 0 1 5 0B 0 5 2 0
71. TLR4 DNA DNA 6 0 7 0 s O 0 DNA NcB I BLAST N ational Center for Biotechnology Information Basic Local Alignment Searah Too 0 oN WO 2007 114296 22 PCT JP2007 056962 0057 008 00s9 oo d 001 3
72. 2 4 5 2 4 HONB N N HOBt TAT E FS C C C NN BO N C
73. LER OR 1 0 95 lt OR SA 0 001 95 TLR4 R e Twee n 8 0 EB HCO6 0 LIE Roe TLR4
74. ERA E T LR4 TLR WO 2007 114296 36 PCT JP2007 056962 1 AVI BRS ARR OY 2 PTCA DCA iy y
75. 7 6 747 WO 2007 114296 9 PCT JP2007 056962 065 oe lt 1 0 5 O 100 2 00 TLR4 TLR4 839
76. TLR4 1 0 01 1000mg kg lt 0 01 1 Oomg kg 0 1 100mg kg 0 1 5 0 mg kg 1 5 3 One keg 1 1 HS 1kg 0 001 00Qng lt 0 OL 00ng lt
77. Tol IL 1 receptor TIR TLR4 2 674 839 1 2 7 05 747 TLR4 TLR4 TL R4 lt 2
78. Burkholder Bostian K L WO 2007 114296 28 PCT JP2007 056962 0083 0684 00s 5 Proc Natl Acad S ci USA 77 45 0 198 0 0 5 9 SD Bitter G A Proc Natl Acad Sci USA 81 533 0 1984 REM RU pH 2 0C 85C 24 2 PH ZT DTH Grace s Insect Medium Grace T C C Nature 195 788 196 2 1 0 pH 6 2 6 4 27 C 3 lt
79. IFN w IFN RT CR RNA TLR TLR TLR4 UE TLR DNA BAIS BROS MORE CLG TORRES 0 TAY Z TLR TLR NF TLR
80. TLR4 TNF a IL 1 IL 6 IFN y IFN 8 IFN w IP 1 0 GA RG16 IRG 1 NF IRF3 AP 1 C EBP He SPR CES
81. TLR4 DNA RNA DNARNA DNA E D NA RNA DNA RNA TLR4 DNA DNA UT A R A A B FF
82. A 2 3 3 8 4 1 2 vp vi dD Kx
83. 2 7 02 8 09 9 0 95 98 MRM CS 2 Phe Trp Ty Ala Leu mle Val Gln Asn Lys Arg His Glu Asp Ser Thr Gly Ala Ser
84. Fmoc 2 4 4 4 BE Nb NER ELK CE
85. L NF lt IRF3 1 TLR4 1 ChE NF B lt IRF3 2 2 1 2 O 1 2 4 1 2 2 0 TLR4 5 2 7 OF 747
86. lt LpS NF IRF3 TLR4 TLR4 TLR4 COOH COO_ K CONH COOR R TLR4 Bo
87. Ke pH CHS Ms Miller ELF Journal of Expe riments in Molecular Ge netics 431 433 Cold Spring Harbor Laboratory New York 1972 lt 3 B 15 3 C 3 4 3 Om OCT Ho 4
88. 26 j oth Hie BE NM 27 28 NN Z NIF RJ FRU EER OO IEF C 29 RN
89. WO 2007 114296 52 PCT JP2007 056962 0114 TLR4 6 kg TLR4 1 0 01 1000mg kg lt 0 01 100mg kg lt 0 1 100mg kg 0 1 5 Ong kg 1 5 3 Ong kg 1 1 NS 6 kg
90. Fmoc CES co E Ce Dece N WN N N 3 HOBt HOOBt
91. AcAr Av imib o Enucimib o o AI Milano i gt A4 ABcA1 Lx s ER 17 HDL cErp LPL 18 MMp AcAr 2 19 ATP KA OR DOF VU VERE 2 0 R S 21 gt 22
92. LPS lt NF GGGRNNYYCC 3 IRF 3 GIV SSGIV NY 4 NF lt IRF GFp
93. LR4 COC MA 1 M Bodanszky MA Ondetti Peptide Synthesis In tor once Publishor New York 1966 2 Schroeder Luebke RThe Peptide Academic Press New York 1965 O 1973 1 IV 2 5 1977 O 14 TLR4
94. HOBL HOOBt N N N N N 2 0C 0C
95. L Ar 7 3 ae 4 n SS A Hon A n 1 4 CRENS SW BAH ii 65 i l a R GL Ra le SON Ar R OR R CHEN 6 WO 2007 114296 3 PCT JP2007 056962 0014 0015 0016 0017 0018 0019 or d R R R R R Ar E7
96. 2 0 TLR4 1 TLR4 WO 2007 114296 34 PCT JP2007 056962 ol oo ol 01 2 TLR4 2 TLR4 TLR4
97. 2 FcS JIDMEM 65 mL gi sh gt 1 FcS pMEM sn H 00nmol 3 7C s c O FB Co psS 5 mL 1 1mL qisnh lysis buffer 1 Ommo 1 L Trig pH 7 5 15 0mmol L Nac l 1 Nonidet P 40 0 3 3 cholamjdopropy dimethyl ammo njo 1 Propanesy fonate c HAPS 3 Ommol L NaF 1 mmol L Na VO Protease inhibitor cocktail Sigma Lysis bu r is 3 00pm 4 10min WO 2007 114296 57 PCT JP2007 056962 0118 t t l cell lysate total cell lysate FLAG anti FLAG M2 Sigma 3 Ou L 4 Ss 50 u LOprotein A Se phgpose beads 4 C lysis bu r 3 s gt ME
98. DNA DNA DNA DNA s ATG L 3 TA A TGA TAGQ DNA CES WO 2007 114296 24 PCT JP2007 056962 0075 0076 0077 0078 TLR4 DNA TLR4 DNA TLR4 DNA DNA DN
99. 4 6 4 7 1 2 TLR4 1 TLR4 NF lt IRF3 2 TLR4 1 NF lt IRF 3 8 TLR4 1
100. OERSVAR A A DESSY DE D D D D D_ Gi ERS VER oc 6 WO 2007 114296 47 PCT JP2007 056962 0108 ZIF Yd e iv K ERE K K K K MI B B B B B B B_ vi ERS YH i E 43 6 2 5 1 1 D 5 6 D 44
101. WO 2007 114296 59 PCT JP2007 056962 2 006 095936 KK 1 2 3 4 5 6 7 WO 2007 114296 60 PCT JP2007 056962 TLR4 1 2 652 839 1 1
102. A ACE Ca BRR Va YEW a AII 23 WO 2007 114296 43 PCT JP2007 056962 0106 K BEE K 24 co RRR A
103. ARIPO BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW X 3 XT AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE E FL FR GB GR HU IE IS IT LT LU LV MC MT NL PL PT Ra SE SI SK TR OAPI BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG 2 PC7 57 Abstract Disclosed is a method for screening of a prophylactic therapeutic agent for at least one disease selected from the group consisting of a heart disease an autormmune disease an inflammatory disease a cent nervous system disease an Infec tious disease sepsis severe sepsis and septic shock The method is characterized by selecting a substance capable of binding to an intercellular part of TLR4 and inhibiting the signaling from that molecule Also disclosed is a kit for the method which can detect a signal from TLR4 by utilizing the expression of a reporter gene as an indicator The kit comprises 1 a cell capable of expressing wild type TLR4 and 2
104. WO 2007 114296 7 PCT JP2007 056962 oo d oo 1 0032 7 TLR4 lt 1TTIR P TLR2 TIR TLR4 OTIR GST LEDE CBB A B 1 GST 2 GST TIR P 3 GS TLR2 ATIR 4 GS TLR4 ATIR 2 TLR4 TLR4 HH
105. Reverse Transcr ijptase PCR RT PCR TLR4 DNA cDNA ADNA RNA lt j nRNA ADNA cDNA lt p cR TLR4 DNA DNA 2 CH
106. RNA gt 000 333673 TLR4 OSD TLR4 OS WORMS Ik OSD G Ss Hi MBP
107. T7 SPO1 SPO2 penP PHO5 PGK G AP ADH RUA P1 0 A SV4 0 SV4 Obri WO 2007 114296 25 PCT JP2007 056962 009 oo d dhfr MTX Amp Neo G4 18 dh
108. s AATCACCTTTCGGCTTCTATGGAAACCTTCATG 3 F 28 5KR 5 CCATAAAAGCCGAAGGGTGATTGTTGTGG 3 y WO 2007 114296 56 PCT JP2007 056962 5 CCACAAcaa TCACC CTTCGGCTITTATGG 3 3 0 6KR 5 CATTGTCCTGCAGAGGGTGGAGAAGAcc 3 31 5 GGTCTTCTCCACCCTCTGCAGGACAAr Gg 3 32 TIKES 5 CAGAAcctGGAGA GGACCCTGCTCAGGC 3 33 5 GCCTGAGCAGGGTCCTCTCCACCTTCTG 3 34 8KR 5 GAGACGACTCAGA AgaaccccTGcTGGATG 3 35 5 CATCCAGCAGGGCTCTTCTGAGTCGTCTC 3 36 SKR 5 CCTGCTGGATGGTAGATCATGGAMAccAGA 3 37 5 TCTGGATTCCATGATCTACCATCCAGCAGG 3 38 Enao Free R Plasmid Maxi Kit Quiag en 5 00 00 g c OS o PTI MEM Invitrogen 2 1 OOF CSM 3 10 u g mL streptomyci n 1 00U mL penicilin pMEM Invitrosen So pTI MEM sm L qish FLAG ILR4 wr pLOS mvitrog en Lipo ctAMINE nyitrosen c OS 37 s c O
109. IFN IRF ECS IFN TLR NF IRF TLRy TLR Lp s Lp s TLR4 OFF WO 2007 114296 33 PCT JP2007 056962 ows 009
110. OA We TLR4 2 7 06 747 4 4 1 AD TLR4 1 TLR4 NF lt IRF3 8 WO 2007 114296 61 PCT JP2007 056962 2 TLR4 1 NF lt IRF 3
111. Pichia pastoris KM71 AcNpV EAB Spodoptera tugiperda cell St Trichoplusia ni MG1 T richoplusia ni High Five Mamestra brassicae Estigme na acrea BmNpV Bombyx mori N BmN Sf sfs ATCC CRLI71D sf21 Vaushn JL 5 In Vivo 13 213 217 0977 Nature 315 592 1985 COS 7 V ro CHO dhfr fd CHO dhfr L At 20 GH3 HEFL HEK293 He La
112. 0 1 00mg 1 1 4 TLR4 TLR4 WO 2007 114296 53 PCT JP2007 056962 ois 0116 1 3 1 0 1 1 1 TLR4 TLR4 TLR4 1 1 1 0 lt 1 1
113. SpS PAG E ample bu r i odc 1 0 SpS PAGE buff er is SpS polyvinylidene dinuoride PVDP FOS pvp F pvp FAR 3 w V nonfat milk Tris HCI buffer 5 0 mmo 1 L Tris 138 mmo 1 L NaC 1 2 7 mmol L KCl pHs 0 anti FLAG M2 10 g g mL 3 milk Tris HCI bu r gt gt HRP anti mouse IgG i EcL HRP E x Kodak Biom x MS amp AX 2 pyp F pgS T rR 20 0 7 sAS 23 00 H TLR4 747
114. TLR4 1 TLR4 1 2 2 652 839 1 1 WO 2007 114296 6 PCT JP2007 056962 3 2 7 OB BBEU E 747 2 4 1 4 1 1 TLR4
115. EGFp NF B lt IRF3 OS Oh LPS TLR4 lt BER
116. TLR WT TLR4 1CA TL OoooA R4 666 CO A 2CA TLR 706A C747A C831A P714H K653R 3CA TLR4 4CA TLR PH TLR 1KR TLR R92KR T LR 3KR TLR 4KR TLR SKR TLR 6KR TLR 7K R TLR 8KR TLRS KR TLR 3 TLR4 TLR4 OS MA B3 1 tb B 3 2 SMC 3 3 AMD 3 4 LPS NF WT TLR4 1CA TLR4 2CA TL Ro 3CA TLRA ACA TLR PH TLRs 1KR TLR 2KR TLR O 3KR TLR 4KR TLR SKR TLR 6KR TLR 7 KR TLR 8KR TLR SKR TLRS EV TLR4 TLR4 WO 2007 114296 8 PCT JP2007 056962 oo3 034 TLR4 1 N
117. KADNA RE DNA PCR DNA DNA DNA Molecular Cloning 2 BUR DNA Mutan super Express Km Mutan K opA LA pcr Gapped duplex e K unkel
118. 8 CH n CH n n 1 4 2 9 SO YAE R OR R TREN WO 2007 114296 oe 1 0 22 023 0024 00 5 0026 1610 A i le R 4 PCT JP2007 056962 R R NH Y NH A 1 2 ORS
119. TLR4 747 HS MB TE C OS D HAMA OSB TE C D 747 LR4 LR4 BELT
120. TLR4 1 7 1 5 WO 2007 114296 PCT JP2007 056962 1 A SFE 1 2 3 4 kDa K 37 25 2 5 WO 2007 114296 PCT JP2007 056962 2 WT 2CA 3CA 4CA WT PH IKR 2KR 3KR 4KR 5KR 6KR 7KR 8KR 9KR WT 1CA 30A 3 1 f LPS 100 ng mL gza LPS A 30 nmol L i O N r LPS A 100 nmol L mam PS A 300 nmol L aD NF kB FL RL Co O v oo ww UT ET _ 2CA 3CA ACA WT 1CA PH 120 NF kB FL RL 8 8 SASS A LTITIT ww TTT R CD a A R 5KR NF kB FL RL 8 88898 wl i 6KR 9KR EV 3 5 WO 2007 114296 PCT JP2007 056962 3 2 NF kB PL RL NF kB FL RL NF kB FL RL 200 150 100 50 0 LPS 100 ng mL LPS B 30 nmol L LPS
121. B 100 nmol L O LPS B 300 nmol L WT ICA 2CA 3CA 4CA PH WT IKR 2KR 3KR 4KR WT 6KR TKR 8KR OKR EV SKR 4 5 WO 2007 114296 PCT JP2007 056962 3 3 NF kB FL RLj NF kB FL RL NF KB FI RE 200 130 100 30 200 150 100 30 LPS 100 ng mL LPS C 30 nmol L LPS C 100 nmol L E LPS E HC 300 nmol L 7 F WT 1CA 2CA 3CA 4CA PH WT IKR 2KR 3KR 4KR 5KR e j E WO 2007 114296 3 4 NF kB FLRL NF kB TL RI NF kB FL RI 200 150 100 30 100 100 5 5 PCT JP2007 056962 MELUA O LPS 100 ng mL LPS D 30 nmol L LPS D 100 nmol L BLPS E AHD 300 nmol L SKR EV ERNATIONAL SEARCH RE International application No PCT JP2007 056962 GO07 33 50 A CLASSIFICATION OF SUBJECT MATTER C1201 02 2006 01 i C12N15 09 2006 01 i 2006 01 i GO1N33 15 2006 01 i According to International Patent Classification IPC or to both national classification and IPC B FIELDS SEARCHED Minimum documentation searched classification system followed by classification symbols C12 Q17 02 C12N1 5 09 GOIN3 3 15 GOIN3 3 50 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic d
122. polyacrylamiae gel electrophoresis PAGE FAsample buffer 1 00C Cs E E SpS PAGE 4 0 1 cgps L 1A rR2 0 0 sAS 23 00 LENE HTA WA lks ST TLR4 TIR cSr BAB cSr 1B 2 PCR TLs4 pNA pEFpo Stratasene o uikChange II Site directed Mutagen esis Kit
123. 3 0 N E 31 K WO 2007 114296 45 PCT JP2007 056962 0107 LATIN Gi iv 32 FER CC 33
124. FBS A SARE SESE FE 2004 01 08 amp wo 2003 084527 all 1 8 amp US 2006 0058288 Al amp EP 1495756 Al A AKIRA Ss et al Pathogen recognition and innate immunity Cell Feb 1 8 2006 vol 124 pp 783 801 E C TILER IA lt IT IX LY i
125. GB OR R 4 1 4 5 LS Ar b11 NCES 12 ins cH m 0 2 n 1 3 m n 4 Th NCH n X Y WO 2007 114296 5 PCT JP2007 056962 027 0028 0 9 NO TNF ca IL 1 IL 6
126. Thr Met WO 2007 114296 11 PCT JP2007 056962 oes 009 oo d Bowie Science 247 13 amp 1310 199 0 NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool 0 BLOSUM6 2 7 oFF Karlin Proc Natl Aca d Sci USA 9 0 5873 5877 1993 NBLAST xBLAST v ersion 2 0 Altschul Nucleic Acids Res 25 3389 340 19 97 Needleman J Mol Biol 48 444 453 1970
127. 0058288 Al amp EP 1495756 Al Special categories of cited documents document defining the general state of the art which is not considered to be of particular relevance cytokine of Pharmacol later document published after the international filing date or promty date and not in conflict with the application but cited to understand the pnciple or theory underlying the invention document of particular relevance the claimed invention cannot be considered novel or cannot be considered to involve an inventive step when the document is taken alone document of particular relevance the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents such combination being obvious to a person skilled in the art document member of the same patent family Date of mailing of the international search report 12 June 2007 12 06 07 A thonzed officer Telephone No INTERNATIONAL SEARCH RI EPORT C Continuation AKIRA s et al innate RT pp 783 801 Pathogen Cell Feb VERSTAK B signalling lie within pp 1 10 et al Toll like and the cl Inflamm Res 2 Form PCT ISA 210 continuation of second sheet April 2005 DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication where appropriate of the relevant passages recognition linical benefits International application No P
128. 1 21 22 25 26 30 71 72 75 54 UUM TATA A OAN 12 f 19 43 2007 10 11 11 10 2007 C12Q 1 02 2006 01 CI2N 15 09 2006 01 GOIN 33 15 2006 01 GOIN 33 50 2006 01 PCT JP2007 056962 2007 3 29 29 03 2007 2006 095936 2006 3 30 30 03 2006 JP lt HIROSHIMA UNIVERSITY PvP 7398S11 1 3 2 Hiroshima JP TAKEDA PHARMACEUTICAL COMPANY LIMITED JP JP 75410045 1 1 Osaka JP HAZEKI Osamu JP JP 77348551 1 2 3 Hiroshima JP 3 HAZEKI Kaoru JPAP T 7348551 1 2 3 Hiroshima JP FH II Masayuki JP US
129. 60015 1 RA Minois US X MATSUNAGA Naoko JP JP 75328686 Title SCREENING METHOD 54 74 81 84 10 WO 2007 114296 Al 17 85 Osaka JP TAKASHIMA Hajime 15410044 1 15 Osaka JP AE AG AL AM AT AU AZ BA BB BG BH BR BW BY BZ CA CH CN C CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD MG MK MN MW MX MY MZ NA NR NI No NZ oM PG PH PL PT RO RS RU SC SD SE SG SK SL SM SV SY TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
130. A pBR 322 pBR325 pUC12 pUC13 pUB110 pTPS pC194 pSH1g pSH15 pA1 11 pXT1 pRc CMV pRc RSV pcDNAILNeoO SR w SV40 L TR CMV HSV T CMV SR a trp 1lac recA AP lpp
131. BRE CAE 3 Rm fe TLR4 A
132. CT JP2007 056962 Relevant to claim No j uw 2006 vol ae receptor that 56 007 vol PCT JP2007 056962 r pc Tnt C1 C1201 02 2006 01 i C12N15 09 2006 01 i GO1N33 15 2006 01 i GO1N33 50 2006 01 i e r gc Tnt C1 1201 02 C12N15 09 G01N33 15 G01N33 50 BIOSIS MEDLINE WPIDS CAplus STN GenBank EMBL DDBJ GeneSeq UniProt Geneseq JMEDP lus JST7580 JSTP lus JDream2 c 9 A IIM et al A novel cyclohexene derivative ethyl 1 8 6R 6 N 2 Chloro 4 f 1uoropheny sulf amoy1 cyclohex l ene l carboxy late TAK 242 selectively inhibits toll like receptor 4 mediated cytokine production through suppression of intracellular signaling MoI Pharmacol Epub 2005 vol 69 pps 1288 1295 A JP 2004 002370 a pd
133. F a IL 1 IL 6 COREY TLR TLR4 TLR4 YOY ay SIRS CARS WO 2007 114296 35 PCT JP2007 056962 S28 GEE E AmA E DIC
134. a cell capable of expressing a TLR4 mutant N ws WO 2007 T R4 1 R4 2 T R4 1 T R4 WO 2007 114296 1 PCT JP2007 056962 0001 oor 0003 oom 0005 0006 BRK 1 1 CH Ai R
135. ata base consulted during the international search name of data base and where practicable search terms used BIOSIS MEDLINE WPIDS CAplus STN GenBank E MBL DDBJ GeneSeq UniProt Geneseq J MEDPlus JST7580 JSTPlus JDream2 C DOCUMENTS CONSIDERED TO BE RELEVANT Citation of document with indication where appropriate of the relevant passages et al A novel cyclohexene derivative 6R 6 N 2 Chloro 4 fluorophenyl Relevant to claim No nae l carboxy inhibits earlier application or patent but published on or after the international filing date document which may throw doubts on priority claim s or which is cited to establish the publication date of another citation or other special reason as specified document referring to an oral disclosure use exhibition or other means document published pror to the international filing date but later than the poty date claimed Date of the actual completion of the international search 29 May 2007 29 05 07 Name and mailing address of the ISA Japanese Patent Office Faosimile No Form PCT ISA 210 second sheet April 2005 sulfamoyl cyclohex 1 en late TAK 242 selectively i ibi toll like receptor 4 mediated j production through suppression intracellular signaling Mol Epub 2005 vol 69 pp 1288 1295 JP 2004 002370 A Takeda Chemical Industries Ltd 08 January 2004 08 01 04 amp WO 2003 084527 Al amp US 2006
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