Patent Abstract:
a peptide for inhibiting toll - like receptor 4 signalling comprising the amino acid sequence of seq id no . 4 , seq id no 55 , seq id no 68 , seq id no . 69 , seq id no 70 , seq id no 71 , seq id no 72 , seq id no 79 , seq id no 82 , seq id no 85 , seq id no 88 , seq id no 91 , seq id no 94 , seq id no 97 , seq id no 100 , seq id no 103 , seq id no 106 , seq id no 109 , seq id no 112 , or seq id no 115 . the peptide may comprise a delivery sequence such as a cationic peptide .

Detailed Description:
vaccinia virus proteins a52r and a46r have a distinct profile of inhibition against the tlr system , it may be possible to generate a repertoire of peptides with differential anti - inflammatory effects on the tlr system , whereby some peptides would specifically inhibit one distinct tlr signalling axis , while other peptides would block a number of pathways . this is particularly important to preserve anti - pathogen immunity while inhibiting an over active inflammatory response that causes pathogenesis . although exogenous proteins with intracellular targets are unlikely to be attractive therapeutics , peptides based on critical amino acid sequences from inhibitory proteins , and peptide derivatives such as peptidomimetics have more potential . in fact others have shown that a peptide containing 11 amino acids of the a52r sequence fused to a cell - penetrating peptide ( called p13 , seq id no . 61 ) can reduce in vivo bacterial - induced inflammation , and lps - induced inflammatory mediators in mice ( mccoy et al , 2005 ; tsung et al , 2007 ). we describe an 11 amino acid peptide derived from the a46 protein sequence that inhibits tlr4 responses when fused to a cell - penetrating peptide . this peptide has several potential advantages over p13 ( seq id no . 61 ): firstly , it is specific for one tlr , namely tlr4 . this is surprising given that full - length a46 protein can inhibit multiple tlrs . secondly it is active in human cells , which is not the case for p13 ( seq id no . 61 ) ( see fig4 ). thirdly , it inhibits tlr4 responses in vitro at a lower concentration than that reported for p13 ( seq id no . 61 , mccoy et al , 2005 ): fig1 shows significant inhibition of tlr4 by a464 ( seq id no . 20 ) at 1 μm , in both murine and human cells . furthermore , shorter forms of the peptide , namely sfklil ( seq id no . 55 ) and fklil ( seq id no . 62 ) are also shown to inhibit tlr4 when fused to a delivery peptide , making the development of a peptidomimetic more likely than would be the case with p13 ( seq id no . 61 ). peptides derived from mammalian tir protein bb loops have been shown to inhibit tlr signalling in vitro ( toshchakov et al , 2005 ; loiarro et al , 2005 ; toshchakov and vogel , 2007 ; toshchakov et al , 2007 ), which might suggest that a peptide from the a46 bb loop would also be inhibitory . however , the peptide discovered here , a464 ( seq id no . 4 and 20 ), is from a region of a46 upstream of the bb loop . in fact , peptides derived from the a46 bb loop fused to cell - penetrating peptides , such as a467 ( seq id no . 23 ), were found not to be inhibitory . interestingly , a464 ( seq id no . 20 ) has activity against tlr4 at a much lower concentration than peptides from mammalian bb loops ( toshchakov et al , 2007 ), and acts in a more specific manner since the only tlr it targets is tlr4 . the invention will be more clearly understood from the following examples . the empty vector pcdna3 . 1 and the pfr - luciferase reporter construct containing five yeast gal4 binding sites were purchased from stratagene . nfκb - luciferase reporter construct containing 5 × κb elements inserted into a pgl3 basic vector was obtained from r . hofmeister , university of rosenburg ( 93042 rosenburg , germany ). the tk - renilla luciferase reporter plasmid containing the herpes simplex virus ( hsv ) thymidine kinase promoter and the renilla reniformis luciferase gene in a prl vector was from promega . the plasmid encoding the fusion protein cd4 - tlr4 was a gift from r . medzhitov ( yale university , new haven , conn ., usa ). the plasmid encoding tlr3 was a gift from d . golenbock ( university of massachusetts medical school , worchester , mass ., usa ). the plasmids encoding flag - mal and flag - tram were gifts from k . fitzgerald ( university of massachusetts medical school , worchester , mass ., usa ). the human embryonic kidney cell line 293 ( hek293 ) and hek293 cells stably transfected with the interleukin - 1 receptor ( hek293_r1 ) were a gift from tularik inc ( san francisco , calif . 94080 , usa ). hek293 cells stably transfected with toll - like receptor ( tlr ) 2 , 3 , 4 and 8 ( hek293_tlr2 , hek293_tlr3 , hek293_tlr4 and hek293_tlr8 ) were a gift from k . fitzgerald ( university of massachusetts medical school , worcester , mass ., usa ). immortalised murine macrophages derived from bone marrow were a gift from d . golenbock ( university of massachusetts medical school , worcester , mass ., usa ). the mouse leukaemia monocyte - macrophage cell line raw264 . 7 , the human acute monocytic leukemia cell line thp - 1 and the human leukemic monocyte lymphoma cell line u937 were obtained from the european collection of animal cell cultures ( ecacc , salisbury , uk ). adherent cells were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) and suspension cells were grown in rpmi medium supplemented with 10 % ( v / v ) heat inactivated foetal calf serum ( fcs ), 2 mm l - glutamine and 100 μg / ml gentamycin ( referred to as complete medium ). cells were subcultured every two to three days when 60 - 80 % confluent . human peripheral blood mononuclear cells ( pbmc ) were isolated from the buffy coat of heparinized whole blood by density centrifugation on low - endotoxin ficoll - hypaque . isolated pbmcs were washed three times in sterile phosphate - buffered saline ( pbs : 137 mm nacl , 2 . 7 mm kcl , 10 mm na 2 hpo 4 , 2 mm nah 2 po 4 ), counted and seeded at a density of 1 × 10 6 cells / ml in complete rpmi medium . antibodies used were mouse anti - β - actin antibody ( sigma ), anti - flag monoclonal antibody ( sigma ), rabbit polyclonal anti - phosphospecific p38 antibody ( cell signalling ), anti - p38 antibody ( cell signalling ), rabbit polyclonal anti - phosphospecific jnk antibody ( biosource ), anti - jnk antibody ( biosource ), and mouse monoclonal anti - iκbα antibody ( a gift from ron hay , university of dundee , scotland ). ultra - pure lipopolysaccharide ( lps ) from eschericia coli (& gt ; 99 . 9 % pure in respect to protein , dna and tlr2 agonists contaminants ) was from alexis corporation , the synthetic double - stranded rna poly ( i : c ) was from amersham biosciences , il - 1α was from the national cancer institute ( frederick , wash ., usa ) and tnfα was a gift from zeneca pharmaceutics ( macclesfield , uk ). cpg dna and pma were a gift from k . mills ( trinity college dublin , dublin , ireland ). peptides were chemically synthesised commercially by genscript ( www . genscript . com ) and were at least 95 % pure . peptides were reconstituted with molecular biology grade sterile water to 10 mm and stored at − 80 ° c . working stocks of 1 mm and 200 μm were stored at 4 ° c . for a maximum of 2 weeks or else kept at − 20 ° c . cytokine and chemokine production from cells or mouse serum was measured by elisa using duoset kits from r & amp ; d systems , according to the manufacturer &# 39 ; s instructions . cell viability was measured by assessing mitochondrial function using an mtt ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ) assay . cells were seeded into 96 well plates and treated with peptides and agonists as described above in relation to the figs . wells containing medium only served as a blank control . to terminate treatment of cells , medium was removed and the cells were washed once with pbs . 200 μl per well of 1 mg / ml mtt in pbs was added directly to the cells and the plates were incubated at 37 ° c . for 14 - 16 hrs in the dark . mtt was then removed and 200 μl per well of dimethyl sulphoxide ( dmso ) was added and cells incubated for 20 min at 37 ° c . in the dark , prior to absorbance being read at 595 nm on a spectrophotometer . hek293 cells were transfected using genejuice ® a proprietary non - toxic transfection reagent . 200 μl / well cells at a density of 1 × 10 5 cells / ml were seeded the day before transfection . 0 . 8 μl of genejuice was mixed with 9 . 2 μl of serum - free dmem ( sfm ) per transfection and incubated for 5 min at room temperature before adding the appropriate amount of the plasmid dna . the genejuice / sfm / dna mixture was then incubated at room temperature for 15 min before adding to the cells . 10 μl per well of the genejuice / smf / dna mixture was added to the cells in triplicate and the cells were allowed to recover for 18 - 20 hours before stimulation . for the nfκb and isre reporter gene assays , 60 ng per well of either nfκb - luciferase or isre - luciferase reporter genes were used , together with 20 ng of the constitutively active internal control renilla - luciferase gene reporter . to measure irf3 activation , the stratagene pathdetect system ™ was used which employs a plasmid encoding a fusion protein of irf3 and the gal4 dna - binding domain ( dbd ), together with the pfr - luciferase reporter plasmid which contains five yeast gal4 binding sites that control expression of the firefly luciferase gene . the dbd of the fusion trans - activator protein binds to the reporter plasmid at the ga14 binding sites . phosphorylation of the irf3 transcription activation domain of the fusion trans - activator protein activates transcription of the luciferase gene from the reporter plasmid . therefore , activity of luciferase reflects the activation of irf3 . as before , renilla - luciferase gene reporter plasmid was used as an internal control . in all cases the total amount of dna per well was kept constant at 230 ng by the addition of pcdna3 . 1 . cells were harvested 6 hours post - stimulation and lysed for 20 min using 50 μl of passive lysis buffer ( promega , madison , wis ., usa ) with vigorous shaking . 20 μl of the lysates were placed in two different plates to analyse for firefly and renilla luciferase activity . the substrate for firefly - luciferase was 40 μl of luciferase assay mix ( 20 mm tricine , 1 . 07 mm ( mgco 3 ) 4 mg ( oh ) 2 5h2o , 2 . 67 mm mgso 4 , 0 . 1m edta , 33 . 3 mm dtt , 270 mm coenzyme a , 470 mm luciferin and 530 mm atp ), and the substrate for renilla luciferase was 40 μl of coelentrazine ( 2 μg / ml in pbs ). luciferase activity was analysed using a luminometer . raw264 . 7 cells were seeded at a density of 1 × 10 5 cells / ml in 6 well plates . after treatment with peptides and agonists , as described above in relation to the figs , the medium was removed and the cells were washed once with pbs before scraping them in 100 μl lysis buffer ( 100 mm nacl , 50 mm hepes ( ph 7 . 5 ), 1 mm edta , 0 . 5 % np - 40 , 10 % glycerol ) and incubating them on ice for 40 min . samples were spun at 15 , 000 × g for 10min and supernatants collected and stored at − 20 ° c . a bradford assay was used to determine the protein concentration in the samples . 1 μl of the sample was added to 480 μl quick start ® bradford dye reagent ( biorad laboratories inc , hercules , calif . 94547 , usa ) together with 19 μl deionised distilled water . the protein concentration was determined by monitoring the change in the solution absorbance at 280 nm using a spectrophotometer compared to protein standards . cell lysates with a known protein concentration were then diluted with water to obtain a concentration of 30 - 40 mg / ml protein in each sample . 20 μl of the diluted samples were mixed with 5 × sample buffer ( 62 . 5 mm tris ph 6 . 8 , 2 % ( w / v ) sds , 10 % ( v / v ) glycerol , 0 . 1 % ( w / v ) bromophenol blue , 50 mm dtt ) and boiled for 5 min at 99 ° c . cooled samples were resolved on a sodium dodecyl sulphate ( sds ) polyacrylamide gel , the resolved proteins were transferred to immobilontm polyvinylidene difluoride ( pvdf ) membrane , and membranes were blocked for 1 hour at room temperature or over night at 4 ° c . with 5 % ( w / v ) marvel ™ ( non - fat dried milk ) in 1 % ( v / v ) pbs - tween for iκbα , ink , β - actin and p38 and 1 % ( v / v ) tbs - tween for phospho - jnk and phospho - p38 . the membrane then was incubated in the relevant primary antibody diluted 1 : 1000 in either 5 % ( w / v ) marvel ™ in 1 % ( v / v ) pbs - tween for iκb , jnk , β - actin and p38 and in 3 % ( w / v ) bsa in 1 %( v / v ) tbs - tween for phospho - jnk and phospho - p38 , for 2 hours at room temperature or overnight at 4 ° c . thereafter , the membranes were washed , incubated in appropriate secondary antibody and then developed using standard enhanced chemi - luminescence ( ecl ) reagents . hek293_t cells were seeded in 6 well plates ( 1 . 5 × 10 5 cells / ml ) 24 hours before transfection . 8 μl of genejuice was mixed with 32 μl of serum - free dmem ( sfm ) per transfection and incubated for 5 min at room temperature before adding 1 μg per transfection of flag - mal or 2 μg of flag - tram encoding plasmid dna . genejuice / sfm / dna mixture was then incubated for 15 min at room temperature before adding to the cells . the total amount of plasmid dna transfected into the cells was made up to 2 μg by adding pcdna . 40 μl per well of the final mixture was added per well . 24 hours post - transfection supernatant was removed and cells were washed once with ice - cold 1 × pbs before lysing in 600 μl of 1 % np - 40 lysis buffer ( 100 mm nacl , 50 mm hepes ( ph 7 . 5 ), 1 mm edta , 1 % np - 40 , 10 % glycerol ) containing 20 mm imidazole on ice for 45 min . the lysates containing either flag - mal or flag - tram were pooled together to ensure equal amount of the protein in each sample . 550 μl of lysates were incubated with 25 μl of either his - a464 ( h - h - h - h - h - h - kysfklilaey — seq id no . 66 ) or his - a467 ( h - h - h - h - h - h - rntisgniysa — seq id no . 67 ) peptide and 40 μl of ni - agarose beads or beads only ( control ) for 2 hr at 4 ° c . rolling to avoid sedimentation of the beads . after incubation beads were washed 5 times in 1 % np - 40 lysis buffer with 20 mm imidazole . after the final wash the buffer was completely removed and 35 μl of 1 . 5 × sample buffer ( 62 . 5 mm tris ph 6 . 8 , 2 % ( w / v ) sds , 10 % ( v / v ) glycerol , 0 . 1 % ( w / v ) bromophenol blue , 50 mm dtt ) was added to each tube . samples were boiled for 5 min at 99 ° c . and then were resolved on 10 % sodium dodecyl sulphate ( sds ) polyacrylamide gel . the resolved proteins were transferred to immobilontm polyvinylidene difluoride ( pvdf ) membrane , and membranes were blocked for 1 hour at room temperature or over night at 4 ° c . in blocking buffer ( 5 % ( w / v ) marveltm ( non - fat dried milk ) in 1 % pbs - tween ). the membrane then was incubated in the anti - flag primary antibody diluted 1 : 10000 in the blocking buffer , for 2 hours at room temperature or overnight at 4 ° c . thereafter , the membranes were washed 6 times in 1 % ( v / v ) pbs - tween , and then incubated in anti - mouse secondary antibody diluted 1 : 10000 in blocking buffer . the signal was read using odyssey infrared imaging system , li - cor ® biosciences . as the full - length a46 protein can inhibit tlr signalling and tlr - dependent gene induction ( stack et al , 2005 ), peptides derived from the a46 protein were assayed for tlr inhibitory effects . secondary prediction programs suggest that a46 folds like a tir domain , consistent with its ability to interact with mammalian tir - containing proteins . a46 - derived peptides were designed based on two regions of the a46 sequence that represent conserved tir motifs . firstly , the bb loop of the tir domain is know to be important for signalling , and may mediate protein - protein interactions . further , peptides derived from mammalian tir protein bb loops have been shown to inhibit tlr signalling ( toshchakov et al , 2005 ; loiarro et al , 2005 ; toshchakov and vogel , 2007 ; toshchakov et al , 2007 ). secondly , another region thought to be important for tir - tir interactions is the cd / dd loop . truncated versions of a46 which retained the predicted cd / dd loop region are still inhibitory against tlrs . therefore this region was also considered of interest . the predicted position of these loops in a46 ( from western reserve strain of vacv , protein id : p26672 ) is shown below ( underlined ): the numbers 1 - 15 refer to the position of the first amino acid in 15 peptides synthesised , to cover the bb , cd and dd loop regions of a46 . each peptide contained 11 amino acids from a46 , and they overlap by approx 5 amino acids each . peptides 1 - 8 surround the predicted bb loop of the protein , and peptides 9 - 15 cover the cd / dd loop regions . peptides 1 to 15 have the following sequences : in order to deliver these peptides into cells , peptides 1 to 15 were linked to cell - penetrating peptides ( also called peptide / protein transduction domains ), which are short cationic peptides of normally 8 - 16 amino acids in length ( murriel & amp ; dowdy , 2006 ). cationic peptides are thought to allow delivery of their cargo into cells by a receptor - independent , fluid - phase macropinocytosis ( murriel & amp ; dowdy , 2006 ). once peptide - enclosed macropinosomes enter the cytoplasm , low ph stimulates endosomal release of the peptide - conjugated cargo ( murriel & amp ; dowdy , 2006 ). common cell - penetrating peptides employed include the amino acids 47 - 57 of hiv tat ( ygrkkrrqrrr ) ( seq id no . 34 ), amino acids 43 - 58 of the antennapedia homeotic transcription factor ( ant , rqikiwfqnrrmkwkk ) ( seq id no . 35 ) and synthetic peptide carriers such as polyarginine ( for example containing nine r residues , here termed 9r ) ( seq id no . 33 ) ( murriel & amp ; dowdy , 2006 ). as the rate of cellular uptake of the 9r peptide ( seq id no . 33 ) has been shown to be significantly faster than tat ( seq id no . 34 ) or ant ( seq id no . 35 ) for cells in vitro ( wender et al , 2000 ), the 9r peptide ( seq id no . 33 ) was selected to be fused to the a46 - derived peptides ( seq id no . 1 to 15 ) in order to test their ability to inhibit tlr signaling in vitro . wender et al ( 2000 ) showed that a polyarginine peptide containing only 6 , 7 or 8 rs is also taken up by cells , although less effectively than 9r ( seq id no . 33 ). 9r ( seq id no . 33 ) was fused to the c - terminus of the a46 - derived peptides ( seq id no . 1 to 15 ). the sequences of the peptides therefore were : ( the peptides were named as a46x where x represents the peptide number from the order in which it appears in seq id no . 16 ) these peptides were initially screened for effects on tlr - induced gene induction in vitro using the murine macrophage cell line raw264 . 7 . unmethylated cpg dinucleotides of bacterial dna sequences are detected by tlr9 , which leads to production of pro - inflammatory cytokines and chemokines , including tnfα via a myd88 - dependent pathway ( latz et al , 2004 ). raw264 . 7 cells showed very potent cytokine production when stimulated with 1 μg / ml cpg ( fig1 ). the effect of the peptides on two tlr9 - induced cytokines , namely tnfα and mip - 2 , was measured by elisa . tnfα is nfκb - dependent , and mip - 2 as well as nfκb also requires irfs for transcriptional activation ( de filippo et al , 2008 ). the cells were plated out at 1 × 10 5 cells / ml 24 hours before treatment . peptides were added at concentrations of 5 and 20 μm 1 hour before stimulation with cpg and supernatants were collected 24 hours post - cpg stimulation for mip - 2 and mtnfα measurement . this showed that only a463 ( seq id no . 19 ) inhibited cpg - induced mip - 2 secretion ( fig1 ), while only a469 ( seq id no . 25 ) inhibited tnfα production ( fig2 ). lps is a component of a cell wall of gram - negative bacteria which is recognised by a homodimer of tlr4 and signals to nfκb via both a myd88 - and trif - dependent pathway ( o &# 39 ; neill and bowie , 2007 ). in order to examine the effect of the peptides on tlr4 - dependent gene induction in human cells , the human monocytic lymphoma cell line u937 was used . u937 cells were differentiated into macrophages by treatment with 200 nm pma for 24 hours before any further manipulations . the a46 peptides ( seq id no . 17 to 31 ) were added to the cells as described for raw264 . 7 above and the cells were stimulated with 100 ng / ml lps for 24 hours . in this case , a464 ( seq id no . 20 ) was found to potently inhibit tlr4 - mediated tnfα production , while a4610 ( seq id no . 26 ) was also effective , but less so ( fig3 ). a more detailed investigation of a463 ( seq id no . 19 ), a464 ( seq id no . 20 ), a469 ( seq id no . 21 ) and a4610 ( seq id no . 22 ) was undertaken , and this showed that generally a463 ( seq id no . 19 ), a469 ( seq id no . 21 ) and a4610 ( seq id no . 22 ), as well as inhibiting tlrs , also reduced cell viability ( as assessed by the mtt assay , which measures activity of mitochondrial reductase , which can be related to the number of living cells ( domart - coulon et al , 2000 ; henneke et al , 2002 ). thus , future work focused on a464 ( seq id no . 20 ), which was found to be less toxic , and to be more potent than the other peptides at lower doses . a464 ( seq id no . 20 ) showed a very potent inhibitory effect on lps ( tlr4 )- induced tnfα production in u937 human cells , as shown in fig4 , and little or no effect on cpg ( tlr9 )- induced cytokine production ( fig1 a and fig2 a ). a52 is another vaccinia virus protein which can inhibit tlr signaling , and it has been shown that a peptide containing 11 amino acids of the a52 sequence together with a 9r delivery sequence ( termed p13 , seq id no . 61 ) can reduce in vivo bacterial - induced inflammation in a murine model ( mccoy et al , 2005 ). therefore the ability of a464 ( seq id no . 20 ) and p13 ( seq id no . 61 ) to inhibit tlr4 - induced cytokine production in human cells was compared . for this , the human acute monocytic leukemia cell line thp - 1 , which is widely used to study lps responses , was employed . thp - 1 cells were stimulated with 100 ng / ml lps . in this experiment the supernatants were collected 6 hours after stimulation , because tnfα production by these cells was shown to be highest after only 4 - 6 hours post - stimulation ( takashiba et al , 1999 ). in the majority of the further described elisa experiments assaying for tnfα , the supernatants were harvested 6 hours after stimulation , unless otherwise stated . here and for following experiments , a467 ( seq id no . 23 ) was used as a control peptide , since we found it to be inert towards tlrs in every cell type and tlr pathway tested . as before 20 μm a464 ( seq id no . 20 ) inhibited tlr4 - mediated tnfα production ( fig4 ). surprisingly however , p13 ( seq id no . 61 ), even at a high dose of 40 μm , was ineffective at inhibiting tlr4 ( fig4 ). in fact we consistently found that p13 ( seq id no . 61 ) was unable to block tlr responses in human cells , suggesting that any in vivo effects of p13 ( seq id no . 61 ) might be restricted to the murine system . in contrast , a464 ( seq id no . 20 ) was inhibitory against tlr4 in both human ( fig4 ) and murine cells . fig5 shows that a464 ( seq id no . 20 ) potently inhibited lps - induced murine tnfα production in raw264 . 7 cells at just 5 μm ( fig5 a ). a simultaneous mtt assay showed no toxicity at the concentrations of 5 and 20 μm in the presence of lps , and some cytotoxicity at 20 μm in the absence of lps ( fig5 b ). next , lower doses of a464 ( seq id no . 20 ) were tested for efficacy against tlr4 in murine cells , compared to the other a46 - derived peptides . as shown in fig6 , only a464 ( seq id no . 20 ), and not a461 ( seq id no . 17 ), a462 ( seq id no . 18 ), a463 ( seq id no . 19 ), a465 ( seq id no . 21 ), a466 ( seq id no . 22 ), a467 ( seq id no . 23 ), a468 ( seq id no . 24 ), a469 ( seq id no . 25 ), a4611 ( seq id no . 27 ), a4613 ( seq id no . 29 ) or a4614 ( seq id no . 30 ), inhibited lps - induced mtnfα at 1 and 5 μm . further , in human thp - 1 cells complete inhibition of tlr4 was achieved at 5 and 10 μm peptide with no cytotoxicity ( fig7 ). seeing such a strong inhibition of the tlr4 - induced cytokine production by the peptide , it was interesting to assay the potency of the peptide to inhibit the signal when the cells were stimulated before the peptide was added , and this showed that 5 and 10 μm a464 ( seq id no . 20 ) almost completely inhibited lps - induced htnfα production even when added 1 hour after the stimulation with lps ( fig8 ). thus far , a464 ( seq id no . 20 ) had been shown to inhibit both murine and human tlr4 , but not murine tlr9 . in order to determine whether a464 ( seq id no . 20 ) was specific for tlr4 , other tlr pathways were next assayed for sensitivity to a464 ( seq id no . 20 ). a scrambled version of a464 ( seq id no . 20 ) peptide was designed to use as a further control peptide , and this had the sequence : kalsifyekylrrrrrrrrr ( cona464 , seq id no . 32 ). the a464r ( seq id no . 20 ) and cona464 ( seq id no . 32 ) peptides were assayed for inhibition of tlr2 signalling in thp - 1 cells . pam3cysk4 ( s -( 2 , 3 - bis ( palmitoyloxy )-( 2 - rs )- propyl )- n - palmitoyl -( r )- cys -( s )- ser -( s )- lys ( 4 )) is a synthetic tripalmitoylated lipopeptide that is recognized by a tlr2 / tlr1 heterodimer and leads to activation of nfκb through a mal - and myd88 - dependent pathway . a464 ( seq id no . 20 ) and cona464 ( seq id no . 32 ) had only very minor inhibitory effects on htnfα secretion by 100 ng / ml pam3cysk4 ( fig9 a ), which were slightly heightened when 10 ng / ml pam3cysk4 was used instead ( fig9 b ). however , these effects were likely not significant since there was a similar level of toxicity upon peptide treatment ( fig9 c ). similar results were obtained in murine cells , in that tlr2 was unaffected by a464 ( seq id no . 20 ). next , the effect of a464 ( seq id no . 20 ) on raw264 . 7 cells was assayed . tlr3 , like tlr4 , uses trif to mediate both nfκb and irf3 activation ( o &# 39 ; neill and bowie , 2007 ). here some inhibition of mtnfα production in response to stimulation with 25 mg / ml poly ( i : c ) was seen but only at a concentration of 50 μm ( fig1 a ), where there was toxicity ( fig1 b ). tlr3 - mediated nfκb activation and isre induction was also assayed , and found to be insensitive to inhibition by either cona464 ( seq id no . 32 ) or a464 ( seq id no . 20 ) ( fig1 ). in fact a464 ( seq id no . 20 ) slightly enhanced activation of nfκb and induction of isre ( fig1 ). these luciferase reporter gene assays were performed in human hek293_tlr3 cells , which stably express tlr3 . the cells were transfected with either nfκb - or isre - luciferase and tk - renilla - luciferase plasmid constructs 24 hours before treatment with the peptide . as before , peptide was added to the cells at various concentrations and the cells were stimulated with poly ( lc ) for 6 hours . a464 ( seq id no . 20 ) was also tested for an effect on nfκb activation via tlr8 in hek293 cells . tlr8 - dependent nfκb activation proceeds via myd88 ( o &# 39 ; neill and bowie , 2007 ). cells were stimulated with 2 . 5 μg / ml cl075 , a thiazoloquinolone derivative which is a potent synthetic tlr8 agonist ( gorden et al , 2005 ). it was found that neither a467 ( seq id no . 23 ) nor a464 ( seq id no . 20 ) had any inhibitory effect on tlr8 - signalling , even at doses of 25 μm ( fig1 ). although murine tlr8 is non - functional , the closely related murine tlr7 is functional and this was also found to be insensitive to a464 ( seq id no . 20 ) inhibition . next , the a464 ( seq id no . 20 ) peptide was tested in primary human cells . peripheral blood mononuclear cells ( pbmcs ) were purified from uncoagulated whole blood using the ficol gradient method , and seeded in 96 well plates 24 hours before treatment . a464 ( seq id no . 20 ) and a467 ( seq id no . 23 ) were then added to the cells at concentrations of 1 , 5 and 25 μm 1 hour before stimulation with 10 ng / ml lps for 6 hours . as shown in fig1 a , 5 μm a464 ( seq id no . 20 ) peptide almost completely blocked htnfα production in response to lps , while a467 ( seq id no . 23 ) had no effect at any dose . the mtt assay showed that both peptides were not toxic for these cells at all concentrations used ( fig1 b ). thus , a464 ( seq id no . 20 ) is a potent inhibitor of human and murine tlr4 , but has little or no inhibitory effect against human tlr2 , tlr3 or tlr8 , or murine tlr2 , tlr3 , tlr7 or tlr9 . further , it is capable of inhibiting lps responses in primary human cells . effect of altered stereochemistry and delivery peptide on the inhibitory potential of a464 for further development of the peptide as a potential therapeutic agent it was of use to investigate a464 &# 39 ; s ( seq id no . 4 ) behaviour if the orientation of the amino acids were changed from the natural l -( lavarotatory )- form to d -( dextrarotatory )- form , since the d - form increases peptide stability and is therefore often used in vivo . thus , the d - form of a464 ( seq id no . 20 ) ( d - a464 ) was chemically synthesised and tested for inhibitory effects on lps - induced tnfα production in primary human pbmcs and immortalized murine macrophages . in pbmcs d - a464 still inhibited tnfα , although it was less potent than a464 ( seq id no . 20 ), since it only blocked tnfα secretion at 25 μm ( fig1 a ). however , in the murine cells d - a464 was as potent as a464 ( seq id no . 20 ), since both demonstrated complete inhibition at 1 ( fig1 b ). we also compared the tat delivery peptide ( seq id no . 34 ) to 9r ( seq id no . 33 ) since although here 9r ( seq id no . 33 ) proved to be very efficient in vitro , there are reports that tat ( seq id no . 34 ) is more efficient in vivo ( schwarze et al , 1999 ). bearing this in mind it was decided to test the tat delivery sequence ( seq id no . 34 ) for delivering the 464 ( seq id no . 4 ) peptide into primary cells in vitro . therefore d - a464 tat ( seq id no . 36 ) and d - a467 tat ( seq id no . 37 ) peptides with the tat delivery sequence ( seq id no . 34 ) at the c - terminus rather than 9r ( seq id no . 33 ) were synthesised and tested in primary human and murine cells . in pbmcs d - a464 - tat ( seq id no . 36 ) failed to inhibit lps - induced htnfα production at 1 - 25 μm ( fig1 a ), while in murine cells it still inhibited , but only at 5 - 25 μm ( fig1 b ). next we tested whether attaching the 9r delivery sequence ( seq id no . 33 ) to the n - terminus of the peptide instead of c - terminus would affect the inhibitory potential , since this might further protect the inhibitory amino acids from degradation . thus new 9rn - a464 ( seq id no . 38 ) and 9rn - a467 ( seq id no . 39 ) peptides with the 9r delivery sequence on n - termini were synthesised and tested in primary human and murine cells . it was found that 9rn - a464 ( seq id no . 38 ) peptide inhibited lps - induced mtnfα more potently than a464 ( seq id no . 20 ) in murine cells , since complete inhibition was observed at 1 μm peptide ( fig1 a ). however this modification did not improve inhibitory potential of the peptide in the human pbmcs , since inhibition was only observed from 5 - 25 μm ( fig1 b ). changing the peptide with the 9r at the n - terminus to the d - form did not improve but weakened its inhibitory potential . in the murine cells no inhibition at 1 μm was now observed ( fig1 a ), while in the human cells it inhibited tnfα only at concentration of 24 μm ( fig1 b ). these results demonstrate that the amino acid residues derived from a46 which are represented in 464 ( seq id no . 4 ) can still inhibit tlr4 responses when the stereochemistry of the peptide , or the orientation and nature of delivery peptide , are altered . however , in murine cells , attachment of the 9r delivery peptide ( seq id no . 33 ) to the n - terminus rather than the c - terminus improves the potency of inhibition of tlr4 . previously we showed that peptides derived from the regions of a46 surrounding the a464 sequence ( seq id no . 20 ), namely a463 ( seq id no . 19 ) and a465 ( seq id no . 21 ), did not inhibit tlr4 responses ( fig3 ). since these peptides partially overlapped with a464 ( seq id no . 4 ), it was possible that a shorter sequence within a464 ( seq id no . 4 ) might be critical for tlr4 inhibition , and that a shorter inhibitory peptide could be designed . thus four shorter peptides with amino acids removed from either end of the a46 - derived amino acids in a464 ( seq id no . 4 ) were designed and synthesized : a464n - 1 : ysfklilaey - 9r ( seq id no . 40 )— a peptide with a deletion of the first amino acid from the n - terminus ; a464n - 2 : sfklilaey - 9r ( seq id no . 41 )— a peptide with a deletion of the first two amino acids from the n - terminus ; a464c - 3 : kysfklil - 9r ( seq id no . 42 )— a peptide with a deletion of the last three amino acids from the c - terminus ; and a464c - 6 : kysfk - 9r ( seq id no . 43 )— a peptide with a deletion of the last six amino acids from the c - terminus . when these peptides were assayed in murine macrophages for the ability to inhibit tlr4 , it was found that only the peptide with the deletion of the 6 amino acids from the c - terminus ( a464c - 6 ) ( seq id no . 43 ) lost its ability to inhibit lps - induced tnfα production ( fig1 ). other deletions appeared to affect the inhibitory potential of the peptide to a minor extent , since they did not inhibit tnfα production at 1 μm but only at 5 μm inhibition ( fig1 ). importantly , the same results were obtained when primary human cells ( pbmcs ) were used ( fig1 ). a464n - 2 ( seq id no . 41 ) and a464c - 3 ( seq id no . 42 ) were then assayed for inhibition of lps - and poly ( i : c )- induced nfκb activation in hek293_tlr4 and hek293_tlr3 cells . both peptides inhibited lps - induced nfκb activation completely at 1 μm ; at the same time there was no inhibition of tlr3 signalling to nfκb up to 25 μm peptide ( fig2 ). based on these experiments , the amino acids sfklil ( seq id no . 55 ) were defined as the ones essential for inhibition by a464 ( seq id no . 20 ). next an alanine scan of a464 ( seq id no . 20 ) was performed , which involved synthesis of a series of a464 ( seq id no . 20 ) peptides with a substitution of each individual amino acid for alanine . thus , 10 new peptides were designed with the following sequences ( position 9 in a464 ( seq id no . 20 ) is already occupied by an alanine ): these peptides were assayed for lps - induced cytokine inhibition in primary human and murine cells , and compared to a464r ( seq id no . 20 ). the parental a464 peptide ( seq id no . 20 ) was used at the concentration of 5 μm as a positive control . the dashed line represents the level of inhibition by parental peptide ( seq id no . 20 ). in the human pbmcs peptides f4a ( seq id no . 47 ), k5a ( seq id no . 48 ), l6a ( seq id no . 49 ), i7a ( seq id no . 50 ) and e10a ( seq id no . 52 ) showed reduced inhibitory potential compared to the parental a464 ( seq id no . 20 ) ( fig2 a ). of note , substitution of the glutamic acid for alanine in e10a ( seq id no . 52 ) made the peptide insoluble , which was the likely reason for the reduced inhibitory effect . in the murine cells only l6a ( seq id no . 49 ) displayed reduced inhibition compared to a464r ( seq id no . 20 ) at 5 μm ( fig2 b ). these results largely correlate with the data obtained using truncated a464 peptides above ( seq id no . 44 to 53 ): namely that ( in human cells at least ), fkli ( seq id no . 54 ) is essential for optimal inhibition of tlr4 . based on the previous experiments with the deletions and alanine scanning a new shorter version of the a464 peptide ( seq id no . 20 ) was synthesised . this new peptide , called sa464 ( seq id no . 55 ), had the sequence sfklil , with the 9r delivery sequence attached to either c - or n - terminus , named accordingly sa464c9r ( seq id no . 56 ) or sa464n9r ( seq id no . 57 ). the control a467 peptide ( seq id no . 7 ) underwent similar modifications and its amino acid sequence became tisgni ( seq id no . 58 ) with the 9r delivery sequence attached to either n - or c - terminus . sa467c9r ( seq id no . 59 ) or sa467n9r ( seq id no . 60 ). the short a464 ( seq id no . 55 , 56 and 57 ) and a467 ( seq id no . 58 , 59 and 60 ) peptides were assayed in murine raw264 . 7 cells . the peptides were added to the cells at concentrations of 5 and 25 μm . a464 ( seq id no . 20 ) and a467 ( seq id no . 23 ) were used at concentrations of 5 μm as a positive and negative controls . it was found that sa464 ( seq id no . 55 ) was able to inhibit lps - induced tnfα , but only when the delivery sequence was at the n - terminus ( seq id no . 57 ) ( fig2 ). further sa464n9r ( seq id no . 57 ) inhibited lps - induced tnfα production as potently as a464 ( seq id no . 20 ) at 5 μm , demonstrating that the removal of the flanking amino acids did not affect the potency of the peptide to inhibit tlr4 at this concentration in murine cells ( fig2 ). next the effect of even shorter versions of a464 on tlr4 - induced murine tnfα in immortalised murine macrophages was tested . the peptide sa464n9r ( seq id no . 57 ) was further reduced in size with a deletion of one amino acid from either n - terminus ( 9r - fklil ) ( seq id no . 62 ) or c - terminus ( 9r - sfkli ) ( seq id no . 63 ) or both ( 9r - fkli ) ( seq id no . 64 ) and one with substitution of isoleucine ( i ) to phenylalanine ( f ) ( 9r - sfklfl ) ( seq id no . 65 ). this showed that 9r - fklil retained the ability to inhibit tlr4 , while 9r - sfkli , 9r - fkli , or 9r - sfklfl did not ( fig2 a ). none of these peptides were toxic to cells ( fig2 b ). thus the sequence fklil ( seq id no . 68 ), or a derivative of it such as a peptidomimetic , is a strong candidate for drug development of a specific tlr4 inhibitor with efficacy in human cells . a464 inhibits tlr4 - dependent signaling pathways ( transcription factors and map kinases ) and interacts with adaptor components of the tlr4 receptor complex activation of tlr4 by lps stimulates activation of the transcription factors nfκb and irfs , as well as mapks , such as p38 and jnk , all of which contribute to altered gene expression . a464 ( seq id no . 20 ) was shown to be capable of inhibiting lps - induced nfκb activation ( fig2 ). in contrast , nfκb activated by forced dimerisation of tlr4 tir domains in the absence of lps ( by overexpressing the fusion protein cd4 - tlr4 ) was insensitive to inhibition by a464 ( seq id no . 20 ) ( fig2 ). thus preformed dimers of tlr4 may be insensitive to a464 ( seq id no . 20 ) and therefore a464 ( seq id no . 20 ) may act to prevent normal tlr4 dimerisation after lps stimulation . to further assay activation of nfκb , the degradation of its inhibitor iκbα , an event required for the translocation of the nfκb into the nucleus , was measured . raw264 . 7 cells were seeded at 1 . 5 × 10 5 cells / ml in 12 well plates 24 hours before treatment . a467 ( seq id no . 23 ) and a464 ( seq id no . 20 ) were added to the cells at concentrations of 4 μm 1 hour before stimulating with 10 ng / ml lps for 5 , 15 , 30 or 60 min . cells were then harvested and lysed in 1 % ( v / v ) np - 40 lysis buffer ( see materials and methods ). the protein concentration in each sample was determined by bradford assay and samples were diluted accordingly to ensure equal protein loading onto a 12 % ( w / v ) sds - page gel . resolved proteins were transferred from the gel onto pvdf membrane by semi - dry transfer and immunoblotted for iκbα . as seen in fig2 a , iκbα was present in the control untreated samples and in the unstimulated samples treated with a464 ( seq id no . 20 ) or a467 ( seq id no . 23 ). stimulation with lps for 5 min did not significantly affect levels of iκbα in the samples , but after 15 min of lps treatment iκbα had completely disappeared in the sample treated with a467 ( seq id no . 23 ), and re - appeared again at 60 min treatment , due to rapid re - synthesis of iκb protein ( krappmann and scheidereit , 1997 ). however , a464 ( seq id no . 20 ) blocked the degradation of iκbα seen at 15 and 30 min ( fig2 a ). re - probing for β - actin protein confirmed that the gel was loaded evenly ( fig2 b ). as well as nfκb , a464 ( seq id no . 20 ) also completely inhibited lps - induced irf3 activation at 1 μm in hek293 cells ( fig2 ). next , the effect of a464 ( seq id no . 20 ) on mapk activation was measured by assaying the phosphorylation of p38 and jnk by western blot . to examine the effect of a464 ( seq id no . 20 ) on the phosphorylation of jnk , raw264 . 7 cells were treated with 10 ng / ml lps for 5 - 60 min . the membrane was probed with the antibody specific for phospho - jnk protein and the levels of jnk expression in the samples were checked by re - probing the blot for total jnk . as before the protein concentration in the samples was determined by bradford assay and the accuracy of the loading was confirmed by re - probing the blot for β - actin . as shown in fig2 there was no phosphorylated jnk detected in the unstimulated samples in the presence of a467 ( seq id no . 23 ) or a464 ( seq id no . 20 ), or in the untreated sample . when stimulated with 10 ng / ml for 5 min the phosphorylation of both jnk1 and jnk2 isoforms was detected as two faint bands in the sample treated with a467 ( seq id no . 23 ), which became much stronger at 15 min , and then diminished at 30 and 60 min treatment with lps . a464 ( seq id no . 20 ) completely inhibited phosphorylation of jnk at all times of the lps stimulation ( fig2 a ). the levels of the total jnk protein were equal throughout the all samples ( fig2 b ) and the loading was even ( fig2 c ). the effect of the a464 ( seq id no . 20 ) peptide on p38 mapk was also examined , and this was of particular interest since p38 has been shown to play an important role in the development of various autoimmune diseases ( kumar et al , 2003 ). phosphorylation of p38 was assayed in a manner similar to the experiment described for jnk above . cells were pretreated with no peptide , or with a467 ( seq id no . 23 ) or a464 ( seq id no . 20 ) prior to stimulation with 10 ng / ml lps for 5 , 30 or 60 min . as in the previous experiment , a464 ( seq id no . 20 ) showed very potent inhibition of lps - induced p38 phosphorylation at all the time points of lps stimulation ( fig2 a ). fig2 b shows that the expression of total p38 was equal throughout . consistent with tlr4 inhibition by a464 ( seq id no . 20 ) and not a467 ( seq id no . 23 ), a his - tagged version of 464 ( without the 9r delivery sequence , seq id no . 66 ) but not 467 ( seq id no . 67 ) was capable of pulling down overexpressed mal or tram from cell lysates ( fig2 ), demonstrating the ability of the peptide 464 to interact with critical adaptor components of the active tlr4 receptor complex . given that a464 ( seq id no . 20 ) was shown to be a specific tlr4 inhibitor in both human and murine cells , it was of interest to determine whether a related peptide could block lps responses in vivo . to do this we used a well characterised model of gram negative sepsis , where mice are injected with lps and serum cytokines are later analysed by elisa . for this , the effect of d - 9rn - a464 ( seq id no . 38 ) or l - 9rn - a464 ( seq id no . 38 ) was compared to l - 9rn - a467 ( seq id no . 34 ), since both of these forms of a464 ( seq id no . 38 ) were shown to inhibit tnf production in murine macrophages ( fig1 and 17 ). mice were injected i . v . with lps alone , or with peptide in the presence of lps . four hours later serum il - 12p40 concentrations were measured . fig3 shows that both d - 9rn - a464 ( seq id no . 38 ) or l - 9rn - a464 ( seq id no . 38 ) significantly inhibited lps - induced il - 12p40 to the extent that mice treated with these peptides displayed levels of il - 12p40 which were not significantly different from the pbs - treated animals . in contrast , although the control peptide l - 9rn - a467 ( seq id no . 39 ) did display some inhibition of lps - induced il - 12p40 , it failed to cause as dramatic a reduction as the a464 peptides ( fig3 ). thus peptides based on the 464 sequence ( seq id no . 4 ) have efficacy in vivo . thus peptides comprising the 464 sequence ( seq id no . 4 ) can inhibit lps induced proinflammatory il - 12p40 in vivo . we have shown that a464 ( seq id no . 20 ) inhibits tlr4 - induced map kinase activation , transcription factor activation and induction of gene expression leading to cytokine and interferon production . therefore biological responses , including but not limited to map kinase activation , transcription factor activation and gene induction , that are inhibited by administration of a464 ( seq id no . 20 ) would be expected to be tlr4 - dependent . in this way , in vitro treatment of cells in culture or in vivo treatment of a whole animal with a464 ( seq id no . 20 ), and measuring alterations in biological responses perturbed by the administration of a464 ( seq id no . 20 ) compared to control cells or an untreated animal , can be used as a method to implicate tlr4 in such a biological response . thus a pathogen - or host - derived substance which induces a biological response in cells or animals could be tested for its ability to activate tlr4 in the presence and absence of a464 ( seq id no . 20 ). in this way compounds or substances that activate tlr4 could be identified and such activators may have use as adjuvants and boosters of an immune response . lps signals via tlr4 in order to induce its proinflammatory effects . tlr4 has been implicated in many diseases in man . in autoimmune and inflammatory conditions , endogenous ligands for tlrs have been implicated in their pathogenesis . although lps is not be the causative agent of many of these diseases , it follows that potential endogenous ligands that signal via tlr4 could also be blocked using this approach , as 464 ( seq id no . 4 ) peptides block internally . tlr antagonists hold promise as therapeutics in immune and inflammatory diseases ( kanzler at al , 2007 ). further , tlr4 inhibitors warrant particular interest for use in the prophylaxis and / or treatment of severe sepsis , which kills more than 200 , 000 people in the us each year ( lolis and bucala , 2003 ). sepsis results from uncontrolled activation of lps / tlr4 - induced cytokine induction , and is also amplified by the endogenous tlr4 activators myeloid - related protein - 8 ( mrp8 ) and mrp14 ( vogl et al , 2007 ). mrp8 and mrp14 are cytosolic proteins in neutrophils and monocytes whose expression are strongly upregulated in inflammatory diseases such as sepsis , rheumatoid arthritis , inflammatory bowel disease and cancer . mrp8 and mrp14 are secreted by activated phagocytes and were shown to have a significant role in the pathogenesis of sepsis and to promote lethality in a murine septic shock model . mrp8 bound directly to the tlr4 - md2 complex to mediate its effects ( vogl et al , 2007 ). thus a specific tlr4 inhibitor would be expected to have a significant protective effect if administered to patients at risk of sepsis . traditionally , attempts to control sepsis have centred on blockage of pro - inflammatory cytokines such as tnfα , a presumed critical effector of lps / tlr4 toxicity . however tlr4 itself may be a much more effective target for intervention in sepsis , since cellular activation by mrp8 - tlr4 amplifies inflammation ( vogl et al , 2007 ). apart from sepsis , specific inhibition of tlr4 while leaving other pathogen detection pathways intact would have great therapeutic potential in a number of other diseases . sterile inflammation , which is caused by chemical insult and / or tissue damage rather than by pathogens , may also be mediated by tlr4 in certain contexts such as bleomycin - induced lung inflammation ( kanzler at al , 2007 ). tlr4 has also been implicated in kidney ischemia / reperfusion injury ( iri ), since in a murine model of iri , mice lacking tlr4 were protected against kidney dysfunction , tubular damage , neutrophil and macrophage accumulation in the kidney , and cytokine production after ischemia ( wu et al , 2007 ). further , tlr4 may also have a role in liver iri ( zhai et al , 2004 ), in ischemic brain injury ( tang et al , 2007 ) and in plaque development in atherosclerosis - prone apolipoprotein e - deficient mice ( michelsen et al , 2004 ). acute lung injury ( ali ), for which treatment options are currently limited and which is a leading cause of death in human h5n1 avian influenza infections , is also tlr4 dependent . ali is triggered by oxidized phospholipids in the lung which stimulate tlr4 signalling through the trif - dependent pathway , leading to cytokine production ( imai et al , 2008 ). given the important role of tlr4 in disease pathogenesis , the development of specific tlr4 inhibitors is important . viral immunosuppressive proteins have been finely - tuned and honed by evolution to target the host immune system with maximal effectiveness . this is analogous to a ‘ naturally occurring drug development programme ’, whereby the protein has already undergone cycles of modification due to natural selection , leading to enhanced inhibitory function . vaccinia virus has developed effective ways of inhibiting tlrs . thus peptides derived from such proteins may more potently and specifically target tlr - mediated inflammation compared to current non - specific therapeutic strategies . the a46 protein , when expressed in human or murine cells , has been shown to inhibit activation of nfκb ( which is a critical transcription factor in mediating inflammation ) in response to il - 1 ( bowie et al , 2000 ), to tlr agonists including tlr4 ( stack et al , 2005 ; aravalli et al , 2007 ), and in murine cells to hsv infection ( aravalli et al , 2007 ). furthermore , a46 can also block il - 1 and tlr - mediated map kinase and irf activation in human cells ( stack et al , 2005 ). a46 works by binding to tir domains in host proteins , and has been shown to be able to associate with tlr4 , il - 1racp , myd88 , mal , trif and tram , which explains how a46 can inhibit multiple tlr pathways ( stack et al , 2005 ). in contrast , the effects of the a464 peptide ( seq id no . 20 ) are specific to inhibition of tlr4 signalling . a46 protein inhibits tlrs by interacting with tir domains , it is likely that the a464 peptide ( seq id no . 20 ) acts in a similar manner to prevent critical tir - tir interactions in tlr4 signalling . tlr4 signalling involves five distinct tir - domain containing proteins : tlr4 , mal , myd88 , tram and trif . given that a464 ( seq id no . 20 ) did not inhibit tlrs which use trif ( tlr3 ) or myd88 ( tlr9 ) it is unlikely that the peptide targets either of these two adaptors directly . rather it is likely that a464 ( seq id no . 20 ) targets tlr4 , mal or tram in order to disrupt tlr4 - tlr4 and / or tlr4 - tram and / or tlr4 - mal tir interactions . consistent with this , his - tagged a464 ( seq id no . 66 ) was capable of interacting with overexpressed mal or tram when added to cell lysates . it is likely that a464 is binding to a novel site on mal or tram that is essential for tlr4 function . therefore the a464 or related peptides may be useful in screening for novel inhibitors of tlr4 or mal or tram . assuming that a464 binds to a site on mal or tram essential for tlr4 function , a screen could be established to assay for small molecules that would bind to this site and thus displace a464 . for example , fitc - labelled a464 displacement from recombinant tram or recombinant mal could be measured as an assay of small molecule binding to said site . the invention is not limited to the embodiment hereinbefore described , with reference to the accompanying drawings , which may be varied in construction and detail . akira s , uematsu s , takeuchi o . 2006 . pathogen recognition and innate immunity . cell 124 , 783 - 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