Patent Application: US-10236008-A

Abstract:
cyclooxygenase and inducible nitric oxide synthase are two major inflammatory mediators . inducible nos specifically binds to cox2 and s - nitrosylates it , enhancing cox2 catalytic activity . selectively disrupting inos - cox2 binding prevents no - mediated activation of cox2 . the synergistic molecular interaction between two inflammatory systems permits assays for developing anti - inflammatory drugs .

Description:
the inventors have found that inos binds directly to cox2 without a scaffold protein . moreover , we found that inos binding to cox2 leads to s - nitrosylation and activation of cox2 . we further found that the binding of inos to cox2 leads to augmented formation of prostaglandins by cox2 . based on these findings , one can screen and develop novel anti - inflammatory drugs by monitoring the ability of candidate drugs to inhibit the binding of inos to cox 2 . peptide agents that mimic the portions of the two binding partners that interact with each other , can be used to block the activation of cox2 by inos . similarly antibodies which bind to the interacting portions of the binding partners can be use to inhibit inflammation responses . for screening assays , any molecules comprising the binding domains of cox2 and inos can be used . these include full - length cox2 and inos polypeptides , fragments comprising the binding domains of the two full - length polypeptides , fusion proteins comprising the binding domains of the two full length polypeptides . as defined experimentally , the binding domains are within the n - terminal 10 % of inos and the c - terminal 20 % of cox2 , as these residues have been found to be sufficient to mediate binding . fragments contain less than the full - length proteins , typically less than 50 %, more typically less than 25 %. fragments of cox2 do not contain the membrane binding domain while fragments of inos do not contain the reductase or cam ( calmodulin ) domains . any of these forms of inos protein can be used to bind to any form of the cox2 protein . binding can be determined by measuring one or both of the binding partners , either in the bound or the free form . increase in the bound form or decrease in the free form indicates binding of the two partners . conversely , added test compounds which inhibit binding can be characterized by the decrease in bound form or increase in free form of one or both of the binding partners . for screening assays , the test compound is preferably a small molecule that binds to and occupies , for example , the binding site of the cox2 or inos polypeptide , such that normal binding of the two binding partners is prevented . examples of such small molecules include , but are not limited to , small peptides or peptide - like molecules . any small organic molecule can be used . libraries of natural or synthetic compounds can be screened . small molecules which mimic three dimensional structure of peptides can be designed . in screening assays , either the cox2 or inos polypeptide can comprise a detectable label , such as a fluorescent , radioisotopic , chemiluminescent , or enzymatic label , such as horseradish peroxidase , alkaline phosphatase , or luciferase . detection of bound cox2 or inos polypeptide can then be accomplished , for example , by direct counting of radioemmission , by scintillation counting , or by determining conversion of an appropriate substrate to a detectable product . alternatively , inhibition of binding of a human cox2 to an inos polypeptide by a test compound can be determined without labeling the interactants . for example , a microphysiometer can be used to detect binding of a human cox2 and inos polypeptide . a microphysiometer ( e . g ., cytosensor ™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light - addressable potentiometric sensor ( laps ). changes in this acidification rate can be used as an indicator of the interaction between a human cox2 or inos polypeptide ( mcconnell et al ., science 257 , 1906 - 1912 , 1992 ). determining the ability of a mammalian cox2 and an inos polypeptide to bind also can be accomplished using a technology such as real - time bimolecular interaction analysis ( bia ) ( sjolander & amp ; urbaniczky , anal . chem . 63 , 2338 - 2345 , 1991 , and szabo et al ., curr . opin . struct . biol 5 , 699 - 705 , 1995 ). bia is a technology for studying biospecific interactions in real time , without labeling any of the interactants ( e . g ., biacore ™). changes in the optical phenomenon surface plasmon resonance ( spr ) can be used as an indication of real - time reactions between biological molecules . in yet another aspect of the invention , a mammalian cox2 or inos polypeptide can be used in a two - hybrid assay or three - hybrid assay ( see , e . g ., u . s . pat . no . 5 , 283 , 317 ; zervos et al ., cell 72 , 223 - 232 , 1993 ; madura et al ., j . biol . chem . 268 , 12046 - 12054 , 1993 ; bartel et al ., biotechniques 14 , 920 - 924 , 1993 ; twabuchi et al ., oncogene 8 , 1693 - 1696 , 1993 ; and brent wo94 / 10300 ), to identify agents which inhibit binding of the cox2 and inos polypeptides and thereby modulate cox2 activity . the two - hybrid system is based on the modular nature of most transcription factors , which consist of separable dna - binding and activation domains . briefly , the assay utilizes two different dna constructs . for example , in one construct , polynucleotide encoding a mammalian cox2 or inos polypeptide can be fused to a polynucleotide encoding the dna binding domain of a known transcription factor ( e . g ., gal - 4 , particularly yeast gal - 4 ). in the other construct a dna sequence that encodes the other of cox2 or inos can be fused to a polynucleotide that codes for the activation domain of the known transcription factor . when the fusion proteins are able to interact in vivo to form a protein - dependent complex , the dna - binding and activation domains of the transcription factor are brought into close proximity . this proximity allows transcription of a reporter gene ( e . g ., lacz , particularly e . coli lacz ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor . contacting such cells ( typically but not limited to yeast cells ) with test substances will permit an assay for binding inhibition . expression or inhibition of expression of the reporter gene can be detected , thus identifying an agent which inhibits the interaction of cox2 and inos polypeptide . it may be desirable to immobilize either the cox2 or inos polypeptide or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants , as well as to accommodate automation of the assay . thus , either the cox2 or inos polypeptide or the test compound can be bound to a solid support . suitable solid supports include , but are not limited to , glass or plastic slides , tissue culture plates , microtiter wells , tubes , silicon chips , or particles such as beads ( including , but not limited to , latex , polystyrene , or glass beads ). any method known in the art can be used to attach the polypeptide or test compound to a solid support , including use of covalent and non - covalent linkages , passive absorption , or pairs of binding moieties attached respectively to the polypeptide or test compound and the solid support . test compounds can be bound to the solid support in an array , so that the location of individual test compounds can be tracked . binding of a test compound to a mammalian cox2 or inos polypeptide can be accomplished in any vessel suitable for containing the reactants . examples of such vessels include microtiter plates , test tubes , and microcentrifuge tubes . in one embodiment , the inos polypeptide is a fusion protein comprising a domain that allows the inos polypeptide to be bound to a solid support . for example , glutathione - s - transferase fusion proteins can be adsorbed onto glutathione sepharose beads ( sigma chemical , st . louis , mo .) or glutathione derivatized microtiter plates , which are then combined with the test compound or the test compound and the non - adsorbed cox2 polypeptide ; the mixture is then incubated under conditions conducive to complex formation ( e . g ., at physiological conditions for salt and ph ). following incubation , the beads or microtiter plate wells are washed to remove any unbound components . binding of the interactants can be determined either directly or indirectly , as described above . alternatively , the complexes can be dissociated from the solid support before binding is determined . in another alternative , the cox2 polypeptide is a fusion protein . other techniques for immobilizing proteins on a solid support also can be used in the screening assays of the invention . for example , either an inos or cox2 polypeptide or a test compound can be immobilized utilizing conjugation of biotin and streptavidin . biotinylated inos or cox2 polypeptides test compounds can be prepared from biotin - nhs ( n - hydroxysuccinimide ) using techniques well known in the art ( e . g ., biotinylation kit , pierce chemicals , rockford , ill .) and immobilized in the wells of streptavidin - coated 96 well plates ( pierce chemical ). alternatively , antibodies which specifically bind to an inos or cox2 polypeptide , or a test compound , but which do not interfere with a desired binding site , can be derivatized to the wells of the plate . unbound target or protein can be trapped in the wells by antibody conjugation . methods for detecting such complexes , in addition to those described above for the gst - immobilized complexes , include immunodetection of complexes using antibodies which specifically bind to the inos or cox2 polypeptide or test compound , enzyme - linked assays which rely on detecting an activity of the inos or cox2 polypeptide , and sds gel electrophoresis under non - reducing conditions . screening for test compounds which bind to an inos or cox2 polypeptide also can be carried out in an intact cell , in whole animals , or cell - free systems . any cell which comprises an inos or cox2 polypeptide can be used in a cell - based assay system . an inos or cox2 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above . binding of the test compound to an inos or cox2 polypeptide is determined as described above . once a test compound has been identified that inhibits the binding of inos to cox2 , whether in cells , in whole animals , or in a cell - free system , it can be further tested to determine its effect on inflammation . for example , it can be tested for its effect on prostaglandin synthesis or for its effect in any of several animal models of inflammation known in the art . exemplary animal models include the following mouse strains , without limitation . the mouse strain cd1 - tg ( gadd45β - luc )- xen ( gadd45β - luc lpta ®) animal model is useful in studying apoptosis , gadd45β gene regulation , map kinase - and nf - κb - mediated signaling pathways , and the treatment of inflammatory diseases and cancer . the mouse strain fvb / n - tg ( inos - luc ) xen ( inos - luc lptar ®) animal model is useful in studying sepsis , arthritis , and anti - inflammatory compounds . the mouse strain fvb / n - tg ( epx - luc )- xen ( epx - luc lpta ®) animal model is useful in studying changes in eosinophil production resulting from parasite infection and asthma , and may be used as donor animals for studying bone marrow transplantation . the mouse strain balb / c - tg ( saa1 - luc )- xen ( saa1 - luc lpta ®) animal model is useful in studying sepsis , arthritis , amyloidosis , and a - saa - mediated disorders . the mouse strain cd1 - tg ( il2 - luc )- xen ( il2 - luc lpta ®) animal model is useful in studying il - 2 gene regulation , inflammatory diseases , and cancer . the mouse strain balb / c - tg ( tnfα - luc )- xen ( tnfα - luc lpta ®) animal model is useful in studying sepsis , arthritis , inflammatory bowel disease , apoptosis , tnfα gene regulation , and the treatment of tnfα - mediated inflammatory diseases . the mouse strain balb / c - tg ( nfκb - re - luc )- xen ( nfκb - re - luc lpta ®) animal model is useful in studying sepsis , arthritis , inflammatory bowel disease , apoptosis , tumor development , nfκb gene regulation , and the treatment of inflammatory diseases and cancer . the mouse strain dba / 1 , balb / c - tg ( nfkb - re - luc [ oslo ])- xen ( nfκb - luc ( oslo ) lpta ®) animal model is useful in studying sepsis , arthritis , inflammatory bowel disease , apoptosis , tumor development , nfκb gene regulation , and the treatment of inflammatory diseases and cancer . the mouse strain balb / c - tg ( ikba - luc )- xen ( iκba - luc lpta ®) animal model is useful in studying sepsis , arthritis , inflammatory bowel disease , apoptosis , tumor development , transcriptional regulation of iκbα gene and other genes responsive to nfκb , and the treatment of inflammatory diseases and cancer . any animal model of inflammation can be used . cox2 and inos for use in any of the methods or compositions of the invention can be from any species , particularly from mammals , such as rat , mouse , rabbit , dog , cat , and most particularly from humans . these proteins are well known in the art and a version of these proteins from any individual can be used even though the version of the protein from that individual may vary slightly from that which is documented in a protein sequence database . exemplary sequences which can be used are those which are documented at genbank as p35228 and p35354 . other cox2 sequences which can be used include those from p . troglodytes , c . familiaris , m . musculus , r . norvegicus , and g . gallus . exemplary sequences include xp - 520238 , np — 001003023 , np — 032995 , xp — 579388 , and xp — 425326 , respectively . other inos sequences which can be used include those from c . familiaris , m . musculus , r . norvegicus , and g . gallus . exemplary sequences include np — 001003186 , np — 035057 , and np — 990292 , respectively . all referenced protein and / or gene sequences are incorporated by reference herein as they exist on apr . 26 , 2006 . treatment modalities based on the discovery of binding of inos and cox2 include administration of a polypeptide , administration of a nucleic acid encoding a polypeptide , and administration of an antibody . the nucleic acid can be a naturally occurring genomic or cdna sequence or can be any sequence which encodes the desired protein . each of these is designed to inhibit binding of the two binding partners . both the polypeptides and the antibodies bind to the portion of the binding partners that interact , thereby inhibiting the interaction . inhibition of the interaction inhibits the inos - mediated s - nitrosylation of cox2 which activates cox2 . polypeptides delivered directly or indirectly via nucleic acids can be fragments of fusion proteins comprising a binding domain or sufficient amounts of a binding domain to bind and interferen with cox2 / inos binding . inflammation which can be treated according to the present invention includes any which is exacerbated by prostaglandin synthesis . these include , without limitation , chronic inflammatory diseases , such as rheumatoid arthritis , inflammatory bowel disease , systemic lupus erythematosus , multiple sclerosis , type idiabetes , rheumatoid arthritis , chronic obstructive pulmonary disease ( copd ), asthma , allergic rhinitis , cardiovascular disease , psoriasis . inflammatory diseases of the brain include abscess , meningitis , encephalitis and vasculitis . any of these diseases can be treated according to the methods of the present invention . antibodies for use in the present invention can be monoclonal or polyclonal . they can gain their specificity by purification or by limitation of the inducing immunogen . the antibodies will bind to the n - terminal 10 % of inos or to the c - terminal 20 % of cox2 . administration of antibodies can be by any means known in the art , but typically the antibodies are administered by injection , such as intrathecal , intraventricular , intravascular ( intravenous or intraarterial ), subcutaneous , intramuscular , intraperitoneal , intrapleural , by perfusion through a regional catheter , or by direct intralesional injection . when administering antibodies by injection , the administration may be by continuous infusion or by single or multiple boluses . polypeptides and polynucleotides can be administered by similar means . in general , the dosage of administered antibodies will vary depending upon such factors as the patient &# 39 ; s age , weight , height , sex , general medical condition , and previous medical history . preferably , a saturating dose of antibody is administered to a patient . antibodies can be administered as whole igg , f ( ab ′) 2 , f ( ab ) 2 , fab ′, or fab . typically , it is desirable to provide the recipient with a dosage of antibody that is in the range of from about 50 to 500 milligrams of antibody , although a lower or higher dosage also may be administered as circumstances dictate . effective in vivo dosages of an antibody are in the range of about 5 mg to about 50 mg / kg , about 50 mg to about 5 mg / kg , about 100 mg to about 500 mg / kg of patient body weight , and about 200 to about 250 mg / kg of patient body weight . high doses of antibody may cause anaphylaxis due to complement activation with endogenous antibodies . this side effect , however , can be prevented by administration of oligosaccharides that bind with endogenous antibodies , as detailed below . the antibodies of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions , whereby antibodies are combined in a mixture with a pharmaceutically acceptable carrier . a composition is said to be a “ pharmaceutically acceptable carrier ” if its administration can be tolerated by a recipient patient . sterile phosphate - buffered saline is one example of a pharmaceutically acceptable carrier . other suitable carriers are well - known to those in the art . see , for example , remington &# 39 ; s pharmaceutical sciences , 19th ed . ( mack publishing co . 1995 ), and gilman &# 39 ; s the pharmacological basis of therapeutics , 7th ed . ( macmillan publishing co . 1985 ). for purposes of therapy , an antibody and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount . a combination of an antibody and a pharmaceutically acceptable carrier is said to be administered in a “ therapeutically effective amount ” if the amount administered is physiologically significant . an agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient . in the present context , an agent is physiologically significant if its presence results in the modulation of an immune response or malignant t cell malignancy growth . polynucleotides can be administered in any vector , whether viral or non - viral designed for delivery and expression of inserted nucleic acid sequences . polynucleotides and / or proteins can be further formulated in liposomes or cationic vesicles or particles for added stability . viral vectors include adenovirus vectors , adeno - associated virus vectors , retrovirus vectors , lentivirus vectors . non - viral vectors include plasmid vectors . exemplary types of viruses include hsv ( herpes simplex virus ), aav ( adeno associated virus ), hiv ( human immunodeficiency virus ), biv ( bovine immunodeficiency virus ), and mlv ( murine leukemia virus ). nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels , including in virus particles , in liposomes , in nanoparticles , and complexed to polymers . inducible nos and cox2 physiologically bind , bringing no in proximity with cox2 , facilitating its s - nitrosylation and activation . earlier findings that nos inhibition decreases prostaglandin formation suggested a relationship between the two systems ( 10 , 26 ), but might have reflected a generally diminished stressful stimulus to the cox2 system rather than a direct intermolecular linkage . because no is a labile molecule which can be rapidly inactivated within cells by the high physiologic concentrations of glutathione or other reducing agents , it is possible that many of no &# 39 ; s physiologic actions will require delivery of no to its targets ( 15 ). other instances of no delivery have primarily involved nnos acting via scaffold proteins . thus , a scaffold protein capon links nnos to dexrasl and provides no to s - nitrosylate dexrasl and act as its guanine nucleotide exchange factor ( 27 ). similarly the scaffold protein psd95 links nnos to n - methyl - d - aspartate ( nmda ) receptors where no s - nitrosylates and inactivates nmda receptors ( 28 ). in contrast to these examples , inos binds directly to cox2 with no intervening scaffold protein . a similar direct delivery of a regulatory metabolite via protein - protein interactions involves the binding of glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ) to inositol 1 , 4 , 5 - trisphosphate ( ip3 ) receptors with nadh formed by gapdh selectively augmenting calcium release activity of ip3 receptors ( 29 ). the molecular synergism between inos and cox2 may represent a major mechanism of inflammatory responses . inhibitors of inos do relieve fever and pain , classically associated with prostaglandin production which may reflect the inos - cox2 interaction , though such actions are sufficiently indirect that one cannot draw strong conclusions ( 30 , 31 ). our findings have therapeutic relevance . thus drugs which block the inos - cox2 interaction might have anti - inflammatory action . moreover , such agents might synergize with cox2 inhibitor drugs permitting lower doses with less side - effects . while it has been speculated that adverse cardiovascular effects of cox2 inhibitors reflect inhibition of pge 2 formation , this has not been directly established so that other actions of the drugs might be involved ( 32 ). the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . cells . hek293t , cos1 and raw 264 . 7 murine macrophages were obtained from the american type culture collection ( manassas , va .). they were grown in a humid atmosphere of 95 % air and 5 % co2 at 37 ° c . in dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fetal bovine serum , l - glutamine ( 300 ug / ml ), penicillin ( 100 u / ml ), and streptomycin ( 100 μg / ml ). preparation of peritoneal macrophages . peritoneal macrophages were obtained following intraperitoneal injection of 1 . 5 ml of 3 % sterile thioglycolate medium . after 4 days mice were sacrificed and macrophages were harvested as described ( 1 ). plasmid constructions . full length human cyclooxygenase2 - encoding gene ( gene bank sequence nm — 000963 ) was purchased from atcc in pcmv - sport6 vector . the sequence was amplified by pcr using primers harboring sal1 / not1 restriction sites and cloned into the pcmv - myc vector ( clontech , palo alto , calif .). the murine inducible nitric oxide synthase ( accession number nm — 010927 ) cloned into the pcdna3 . 1 + vector ( invitrogen , carlsbad , calif .). immunoprecipitation . for co - immunoprecipitation experiments , 1 × 10 6 hek293t cells were plated in 10 cm 2 culture dishes ( invitrogen , carlsbad , calif .). cells were transfected with 4 μg pcmv - myc - cox2 , 2 ug pcdna3 . 1 - inos , or empty pcmv - myc vector using lipofectamine plus ( invitrogen , carlsbad , calif .) according to the manufacturer &# 39 ; s protocol . after 48 h , cells were harvested and lysed in ice - cold lysis buffer ( 100 mm tris ph 7 . 4 , 150 mm nacl , 10 % glycerol , 1 % triton x - 100 , and complete protease inhibitors ). the supernatants ( 800 ul ) from the hekt cell extracts were pre - cleaned for non - specific binding with 50 ul protein a - sepharose , then mixed overnight at 4 ° c . with 2 ug / ml anti - myc antibody ( roche , alameda , calif .). after the addition of 80 μl of protein a - sepharose , the immunoprecipitates were mixed for another 1 h at 4 ° c . the mixture was washed three times with the buffer described above and the pellet boiled in 10 ul sds - loading dye , which was subjected to 6 % sds page gel run in mops buffer . the proteins were then transferred to nitrocellulose membrane . the bands were visualized by ecl reagent ( pierce , milwaukee , wis .) as described by the manufacturer . site directed mutagenesis . the quickchange site - directed mutagenesis system ( stratagene , la jolla , calif .) was employed per manufacturer &# 39 ; s instructions to alter the thirteen cysteine residues in cox2 to serine . each mutant was verified via automated sequencing by the hopkins core facility . s - nitrosylation assay . cells were homogenized by 26g needle in hen ( 250 mm hepes - naoh ph 7 . 7 , 1 mm edta , 0 . 1 mm neocuproine ) buffer and then centrifuged at 1000 g for 10 min at 4 ° c . cells lysates ( 240 μg ) was added 4 vol of blocking buffer ( 9 vol of hen buffer plus 1 vol 25 % sds , adjusted to 20 mm mmts with a 2 m stock prepared in dimethylformamide ( dmf )) at 50 ° c . for 20 min with frequent vortexing . the mmts was then removed by desalting three times with the microbiospin6 column ( bio - rad , hercules , calif .) pre - equilibrated in hen buffer . to the eluate was added biotin - hpdp prepared fresh as a 4 mm stock in dmso from a 50 mm stock suspension in dmf . sodium ascorbate was added to a final concentration of 1 mm . after incubation for 1 h at 25 ° c ., biotinylated proteins were precipitated by streptavidin - agarose beads . the streptavidin - agarose was then pelleted and washed 5 times using hens buffer . the biotinylated proteins were eluted by sds - page sample buffer and subjected to western - blot analysis . fluorometric detection of s - nitrosothiols . the methods were modified according to cook et al . ( 2 ) raw264 . 7 cells were treated with lps / ifnγ for overnight and cox2 was immunoprecipitated by donkey anti - rat cox2 antibodies . after that the samples were reacted with 100 μm 2 , 3 - diaminonaphthalene ( dan ) in the presence of 100 μm of hgcl2 and incubated in darkness for 30 min at room temperature . the generated fluorescent compound 2 , 3 - napththyltrazole ( nat ) was then measured at an excitation wavelength of 375 nm and an emission wavelength of 450 nm . cox2 enzymatic assay . recombinant human cox2 was obtained from cayman biochemical inc . cox2 was treated with no donor , snp for 30 min and was then passed through a spin column ( bio - rad , hercules , calif .) to remove excess no . cox2 enzymatic activity was measured with a cox assay kit ( cayman biochemical inc , ann arbor , mich .) according to the manufacturer &# 39 ; s instructions . measurement of pge 2 . cells were washed with worm pbs ( x2 ) and incubated in the phenol free dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 20 μm arachidonic acid and 3 % albumin for 20 min . media was collected and pge2 production was measured with a pge 2 elisa kit from assay design ( ann arbor , mich .) according to the manufacturer &# 39 ; s instructions . immunohistochemistry . raw264 . 7 cells were transfected with deletion constructs of cox2 . cells were fixed in 4 % paraformaldehyde in pbs for 5 min , permeablized in 0 . 1 % triton x - 100 for 10 s , and then rinsed twice in pbs . coverslips were then blocked in 10 % goat serum at room temperature for 1 h and incubated with pge 2 ( cayman biochemical inc , ann arbor , mich .) and myc antibodies for 24 hr at 4 ° c . rhodamine or fluorescein - conjugated secondary antibodies ( jackson immunochemicals , west grove , pa .) were then added at 10 μg / ml for 1 hr at room temperature as indicated . coverslips were mounted in prolong ( molecular probes , eugene , oreg .). confocal microscopy , in which the immunofluorescent staining is superimposed on phase contrast images , employed a noran oz ( noran instruments , middleton , wis .) confocal laser - scanning system , fitted to an olympus ix - 50 fluorescence microscope . viscosity studies . viscosity experiments were performed with sucrose ( 0 - 31 % w / w ) as described previously ( 3 , 4 ). 1 . nunoshiba , t ., derojas - walker , t ., wishnok , j . s ., tannenbaum , s . r . & amp ; demple , b . activation by nitric oxide of an oxidative - stress response that defends escherichia coli against activated macrophages . proc natl acad sci usa 90 , 9993 - 7 ( 1993 ). 2 . cook , j . a ., kim , s . y ., teague , d ., krishna , m . c ., pacelli , r ., mitchell , j . b ., vodovotz , y ., nims , r . w ., christodoulou , d ., miles , a . m ., grisham , m . b ., & amp ; wink , d . a . convenient colorimetric and fluorometric assays for s - nitrosothiols . anal biochem 238 , 150 - 158 ( 1996 ). 3 . cole , p . a ., burn , p ., takacs , b ., walsh , c . t . evaluation of the catalytic mechanism of recombinant human csk ( c - terminal src kinase ) using nucleotide analogs and viscosity effects . j bio chem 269 , 30880 - 30887 ( 1994 ). 4 . brouwer , a . c ., and kirsch , j . f . investigation of diffusion - limited rates of chymotrypsin reactions by viscosity variation . biochem 21 , 1302 - 1307 ( 1982 ) for initial studies we employed raw264 . 7 cells , a macrophage cell line in which lps and ifnγ massively activate both inos and cox2 . in lps / ifnγ treated cells immunoprecipitation with cox2 antibodies leads to co - precipitation of inos ( fig1 a ). coprecipitation of cox2 and inos is also evident in hek293t cells transfected with both proteins ( fig5 a ). to examine interactions between the two enzymes in intact organisms , we injected mice with thioglycollate , an inflammatory stimulus which typically produces peritonitis or pleuritis , and observed coprecipitation of inos and cox2 ( fig5 b ). to determine whether catalytic activity of the enzymes influences their binding interactions , we co - precipitated the two proteins with inos antibodies or cox2 antibodies and examined the effect of the inos - selective inhibitor 1400w ( fig1 b ) or the cox2 selective inhibitor sc58125 ( fig1 c ). co - precipitation of inos and cox2 is unaffected by either inos or cox2 inhibitors . the binding of inos and cox2 is selective , as we do not detect immunoprecipitation of cox1 with inos ( data not shown ). to map the binding sites , we employed selective deletions of inos ( fig1 d - f ) and cox2 ( fig1 g and h ) sequences . amino acids 1 - 114 appear to be the minimal portion of inos mediating binding , while amino acids 484 - 604 of cox2 are required . the binding area of inos occurs within the oxygenase domain , while the c - terminal portion of cox2 that mediates binding includes a cox2 domain which does not exist in cox1 . the two major mechanisms whereby no influences its targets are stimulation of guanylyl cyclase by direct binding of no to iron in heme at the active site of guanylyl cyclase ( 11 , 12 ) or s - nitrosylation of protein targets on appropriate cysteines ( 13 - 15 ). since cox2 possesses heme at its active site ( 16 ), this would be a potential target . however , no binding to heme in cox does not alter its activity ( 17 ). cox2 also contains 13 cysteines whose roles are not fully understood ( 18 , 19 ). hence , we explored the possibility of s - nitrosylation of cox - 2 by no , examining multiple no donors including nitrosoglutathione ( gsno ) ( fig2 a ), sodium nitroprusside ( snp ), spermine - no and deta - nonoate ( fig7 a ). utilizing the biotin switch method ( 20 ), we demonstrate that all four no donors elicit s - nitrosylation of cox2 in hek293t cells transfected with cox2 - myc ( fig2 a ). we wondered whether physiological induction of no formation leads to s - nitrosylation of cox2 . in raw264 . 7 cells treated with lps / ifnγ we observe s - nitrosylation of cox2 which is prevented by the inos specific inhibitor 1400w using the biotin switch assay ( fig2 b ) as well as the fluorometric method ( fig7 b ). to ensure specificity of the biotin switch method , we have observed that h 2 o 2 does not elicit s - nitrosylation ( fig8 a ). we ruled out the possibility that sulfenic acid modification is detected by the biotin switch assay by demonstrating that arsenite , which reverses sulfenic acid modifications but not s - nitrosylation , fails to provide the biotin switch signal afforded by ascorbate utilizing gsno with purified cox2 or lps / ifnγ treatment of raw 264 . 7 cells ( fig8 b ). as no is freely diffusible , in some instances there may be no need to deliver no directly to targets , as some actions of no are prevented by hemoglobin , which can sequester freely diffusible no ( 21 ). we examined the effects of hemoglobin on s - nitrosylation of cox2 under varying conditions . in hek293t cells transfected with cox2 , hemoglobin prevents the s - nitrosylation elicited by gsno ( fig9 a ) whereas it fails to alter s - nitrosylation of cox2 in raw264 . 7 cells activated by lps / ifnγ ( fig9 b ). thus in the more physiologic macrophage cell line , the s - nitrosylation of cox2 generated by an inflammatory stimulus does not appear to be elicited by freely diffusible no . to determine whether s - nitrosylation of cox2 alters enzyme activity , we examined cox2 enzyme activity in hek293t cells transfected with cox2 - myc . the no donor snp , added to cell lysates , elicits a substantial augmentation of cox2 activity , which reflects s - nitrosylation , as ascorbic acid , which reverses s - nitrosylation ( 20 , 22 ), prevents the increase ( fig2 c and d ). the reversal by ascorbate of cox2 activation by no donors is not merely a reflection of ascorbate influences on enzymes substrates or intermediate products , as ascorbate fails to affect cox2 activity in preparations not treated by snp . a relationship of s - nitrosylation and cox2 activation is further supported by the closely similar concentration - response relationship between the effects of the no donor gsno on s - nitrosylation and on cox2 activity ( fig2 e ). no activates cox2 by increasing its apparent vmax without changing its km ( fig2 e ). the higher concentration of snp required to activate cox2 in vitro compared to intact cells accords with earlier studies showing greater potency of no donors in intact cells ( 23 , 24 ). to ascertain the kinetic basis for no activation of cox2 , we conducted enzyme assays with increasing concentrations of sucrose to augment viscosity and slow down enzyme kinetics ( fig2 g ). as expected , with increasing viscosity , the ratio of control enzyme activity to the activity in more viscous solutions increases . this increase is diminished in snp samples consistent with snp accelerating the release of product from the enzyme . we wondered which of the 13 cysteines of cox2 are critical for the augmentation of cox2 activity elicited by s - nitrosylation . in raw 264 . 7 cells transfected with the n - terminal 483 amino acids or the c - terminal 120 amino acids of cox2 , lps / ifnγ treatment leads to s - nitrosylation of the c - terminal fragment ( which contains 3 cysteines ) but not the n - terminal fragment ( fig1 a ). to ascertain which of these 3 cysteines is responsible for augmented cox2 activity we mutated each of them to serine . the c526s mutation prevents activation of cox2 activity by the no donor snp , while the c561s mutation does not ( fig1 b ). the c555s mutation abolishes enzyme activity so the effects of no stimulation cannot be assessed . individual mutation of the 13 cysteines in cox2 does not detectably diminish total s - nitrosylation of the enzyme ( data not shown ), which suggests that multiple cysteines can be s - nitrosylated but only c526 is responsible for enzyme activation by no . to clarify the influence of no on pge 2 formation in a more physiologic preparation , we employed raw264 . 7 cells . the formation of pge 2 in response to lps / ifnγ is inhibited by the inos inhibitor 1400w with 50 % reduction of pge 2 formation at drug concentrations which provide 50 % inhibition of inos activity ( fig3 a ). specificity of the no association is evident by inhibition of pge 2 formation with the active l - isomer of the nos inhibitor nitro - argininemethylester ( l - name ) but not by d - name ; the effects of l - name are reversed by added l - arginine ( fig3 b ). thus , about 50 % of induced cox2 activity is determined by s - nitrosylation . as raw264 . 7 cells are a continuous macrophage cell line which may not behave the same as macrophages in intact organisms , we also tested peritoneal macrophages obtained from inos knockout mice . pge 2 formation from macrophages of lps / ifnγ - treated mice is profoundly reduced in the inos knockout mice in parallel with a similar reduction in nitrite formation by the macrophages ( fig3 c ) and a decrease in s - nitrosylated cox2 ( fig3 d ). these observations concur with findings of decreased urinary pge 2 in inos knockout mice ( 25 ). fragment of cox2 ( amino acids 484 - 604 ) abolishes the co - precipitation of inos and cox2 we hypothesized that the augmentation of pge 2 formation by inos activation reflects binding of inos to cox2 to deliver no in appropriate proximity for s - nitrosylation . to explore this possibility we utilized dominant - negative constructs to block inos - cox2 binding using the fragment of cox2 , amino acids 484 - 604 , which binds inos ( fig4 a ). transfection of 484 - 604 into raw264 . 7 cells abolishes the co - precipitation of inos and cox2 and is associated with precipitation of 484 - 604 together with inos ( fig4 a ). moreover , this interference of binding between cox2 and inos by 484 - 604 decreases s - nitrosylation of cox2 in raw264 . 7 cells ( fig4 b ). the dominant - negative transfection also reduces pge 2 formation by more than 50 %, whereas transfection of a fragment comprising amino acids 1 - 483 , which does not bind inos , fails to influence pge2 formation ( fig4 c and d ). 1 . m . e . turini , r . n . dubois , annu . rev . med . 53 , 35 ( 2002 ). 2 . s . moncada , j . r . soc . med . 92 , 164 ( 1999 ). 3 . r . j . flower , nat . rev . drug discov . 2 , 179 ( 2003 ). 4 . d . mukherjee , s . e . nissen , e . j . topol , jama 286 , 954 ( 2001 ). 5 . e . j . topol , jama 293 , 366 ( 2005 ). 6 . t . p . misko , j . l . trotter , a . h . cross , j . neuroimmunol . 61 , 195 ( 1995 ). 7 . i . appleton , a . tomlinson , d . a . willoughby , adv . pharmacol . 35 , 27 ( 1996 ). 8 . d . salvemini et al ., j . clin . invest 93 , 1940 ( 1994 ). 9 . j . r . vane , y . s . bakhle , r . m . botting , annu . rev . pharmacol . toxicol . 38 , 97 ( 1998 ). 10 . d . salvemini et al ., proc . natl . acad . sci . u . s . a 90 , 7240 ( 1993 ). 11 . j . m . braughler , c . k . mittal , f . murad , j . biol . chem . 254 , 12450 ( 1979 ). 12 . j . c . edwards et al ., biochem . pharmacol . 30 , 2531 ( 1981 ). 13 . j . s . stamler , d . j . singel , j . loscalzo , science 258 , 1898 ( 1992 ). 14 . s . r . jaffrey , h . erdjument - 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