Patent Application: US-22221605-A

Abstract:
the present invention is directed to conopeptides termed conkunitzins and their use for blocking the flow of potassium ions through voltage - gated potassium channels . in view of the kunitz domain in the conkunitzins , they are also useful for inhibiting platelet aggregation and as protease inhibitors .

Description:
the present invention is directed conkunitzins and their uses as described above . potassium channels comprise a large and diverse group of proteins that , through maintenance of the cellular membrane potential , are fundamental in normal biological function . the therapeutic applications for compounds that regulate the flow of potassium ions through k + channels are far - reaching and include treatments of a wide range of disease and injury states . disorders which can be treated using these conopeptides include multiple sclerosis , other demyelinating diseases ( such as acute dissenmiated encephalomyelitis , optic neuromyelitis , adrenoleukodystrophy , acute transverse myelitis , progressive multifocal leukoencephalopathy ), sub - acute sclerosing panencephalomyelitis ( sspe ), metachromatic leukodystrophy , pelizaeus - merzbacher disease , spinal cord injury , botulinum toxin poisoning , huntington &# 39 ; s chorea , compression and entrapment neurophathies ( such as carpal tunnel syndrome , ulnar nerve palsy ), cardiovascular disorders ( such as cardiac arrhythmias , congestive heart failure ), reactive gliosis , hyperglycemia , immunosuppression , cocaine addiction , cancer , cognitive dysfunction and disorders resulting from defects in neurotransmitter release ( such as eaton - lambert syndrome ). in addition , these conkunitzins can provide reversal of the actions of curare and other neuromuscular blocking drugs . the conkunitzins of the present invention are identified by isolation from conus venom or by using recombinant dna techniques by screening cdna libraries of various conus species using conventional techniques , such as the use of reverse - transcriptase polymerase chain reaction ( rt - pcr ) or the use of degenerate probes . clones which hybridize to degenerate probes are analyzed to identify those which meet minimal size requirements , i . e ., clones having approximately 400 nucleotides ( for a propeptide ), as determined using pcr primers which flank the cdna cloning sites for the specific cdna library being examined . these minimal - sized clones and the clones produced by rt - pcr are then sequenced . the sequences are then examined for the presence of a peptide having the characteristics noted above for conkunitzins . the biological activity of the peptides identified by this method is tested as described herein or conventionally in the art . these peptides are sufficiently small to be chemically synthesized . general chemical syntheses for preparing the foregoing conopeptides are described hereinafter , along with specific chemical synthesis of conopeptides and indications of biological activities of these synthetic products . various ones of these conopeptides can also be obtained by isolation and purification from specific conus species using the techniques described in u . s . pat . no . 4 , 447 , 356 ( olivera et al ., 1984 ), u . s . pat . no . 5 , 514 , 774 ( olivera et al ., 1996 ) and u . s . pat . no . 5 , 591 , 821 ( olivera et al ., 1997 ), the disclosures of which are incorporated herein by reference . although the conopeptides of the present invention can be obtained by purification from cone snails , because the amounts of conopeptides obtainable from individual snails are very small , the desired substantially pure conopeptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid - phase strategy . for example , the yield from a single cone snail may be about 10 micrograms or less of conopeptide . by “ substantially pure ” is meant that the peptide is present in the substantial absence of other biological molecules of the same type ; it is preferably present in an amount of at least about 85 % purity and preferably at least about 95 % purity . chemical synthesis of biologically active conopeptides depends of course upon correct determination of the amino acid sequence . thus , the conopeptides of the present invention may be isolated , synthesized and / or substantially pure . the conopeptides can also be produced by recombinant dna techniques well known in the art . such techniques are described by sambrook et al . ( 1989 ). the peptides produced in this manner are isolated , reduced if necessary , and oxidized to form the correct disulfide bonds , if present in the final molecule . one method of forming disulfide bonds in the conopeptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature . this procedure results in the creation of a substantial amount of the bioactive , disulfide - linked peptides . the oxidized peptides are fractionated using reverse - phase high performance liquid chromatography ( hplc ) or the like , to separate peptides having different linked configurations . thereafter , either by comparing these fractions with the elution of the native material or by using a simple assay , the particular fraction having the correct linkage for maximum biological potency is easily determined . it is also found that the linear peptide , or the oxidized product having more than one fraction , can sometimes be used for in vivo administration because the cross - linking and / or rearrangement which occurs in vivo has been found to create the biologically potent conopeptide molecule . however , because of the dilution resulting from the presence of other fractions of less biopotency , a somewhat higher dosage may be required . the peptides are synthesized by a suitable method , such as by exclusively solid - phase techniques , by partial solid - phase techniques , by fragment condensation or by classical solution couplings . in conventional solution phase peptide synthesis , the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence . use of various coupling reagents , e . g ., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole , various active esters , e . g ., esters of n - hydroxyphthalimide or n - hydroxy - succinimide , and the various cleavage reagents , to carry out reaction in solution , with subsequent isolation and purification of intermediates , is well known classical peptide methodology . classical solution synthesis is described in detail in the treatise , “ methoden der organischen chemie ( houben - weyl ): synthese von peptiden ,” ( 1974 ). techniques of exclusively solid - phase synthesis are set forth in the textbook , “ solid - phase peptide synthesis ,” ( stewart and young , 1969 ), and are exemplified by the disclosure of u . s . pat . no . 4 , 105 , 603 ( vale et al ., 1978 ). the fragment condensation method of synthesis is exemplified in u . s . pat . no . 3 , 972 , 859 ( 1976 ). other available syntheses are exemplified by u . s . pat . no . 3 , 842 , 067 ( 1974 ) and u . s . pat . no . 3 , 862 , 925 ( 1975 ). the synthesis of peptides containing γ - carboxyglutamic acid residues is exemplified by rivier et al . ( 1987 ), nishiuchi et al . ( 1993 ) and zhou et al . ( 1996 ). synthesis of conopeptides have been described in u . s . pat . no . 4 , 447 , 356 ( olivera et al ., 1984 ), u . s . pat . no . 5 , 514 , 774 ( olivera et al ., 1996 ) and u . s . pat . no . 5 , 591 , 821 ( olivera et al ., 1997 ). common to such chemical syntheses is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed . usually also common is the protection of an α - amino group on an amino acid or a fragment while that entity reacts at the carboxyl group , followed by the selective removal of the α - amino protecting group to allow subsequent reaction to take place at that location . accordingly , it is common that , as a step in such a synthesis , an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side - chain protecting groups linked to various ones of the residues having labile side chains . as far as the selection of a side chain amino protecting group is concerned , generally one is chosen which is not removed during deprotection of the α - amino groups during the synthesis . however , for some amino acids , e . g ., his , protection is not generally necessary . in selecting a particular side chain protecting group to be used in the synthesis of the peptides , the following general rules are followed : ( a ) the protecting group preferably retains its protecting properties and is not split off under coupling conditions , ( b ) the protecting group should be stable under the reaction conditions selected for removing the α - amino protecting group at each step of the synthesis , and ( c ) the side chain protecting group must be removable , upon the completion of the synthesis containing the desired amino acid sequence , under reaction conditions that will not undesirably alter the peptide chain . it should be possible to prepare many , or even all , of these peptides using recombinant dna technology . however , when peptides are not so prepared , they are preferably prepared using the merrifield solid - phase synthesis , although other equivalent chemical syntheses known in the art can also be used as previously mentioned . solid - phase synthesis is commenced from the c - terminus of the peptide by coupling a protected α - amino acid to a suitable resin . such a starting material can be prepared by attaching an α - amino - protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin , or by an amide bond to a benzhydrylamine ( bha ) resin or paramethylbenzhydrylamine ( mbha ) resin . preparation of the hydroxymethyl resin is described by bodansky et al . ( 1966 ). chloromethylated resins are commercially available from bio rad laboratories ( richmond , calif .) and from lab . systems , inc . the preparation of such a resin is described by stewart and young ( 1969 ). bha and mbha resin supports are commercially available , and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the c - terminus . thus , solid resin supports may be any of those known in the art , such as one having the formulae — o — ch 2 - resin support , — nh bha resin support , or — nh - mbha resin support . when the unsubstituted amide is desired , use of a bha or mbha resin is preferred , because cleavage directly gives the amide . in case the n - methyl amide is desired , it can be generated from an n - methyl bha resin . should other substituted amides be desired , the teaching of u . s . pat . no . 4 , 569 , 967 ( kornreich et al ., 1986 ) can be used , or should still other groups than the free acid be desired at the c - terminus , it may be preferable to synthesize the peptide using classical methods as set forth in the houben - weyl text ( 1974 ). the c - terminal amino acid , protected by boc or fmoc and by a side - chain protecting group , if appropriate , can be first coupled to a chloromethylated resin according to the procedure set forth in horiki et al . ( 1978 ), using kf in dmf at about 60 ° c . for 24 hours with stirring , when a peptide having free acid at the c - terminus is to be synthesized . following the coupling of the boc - protected amino acid to the resin support , the α - amino protecting group is removed , as by using trifluoroacetic acid ( tfa ) in methylene chloride or tfa alone . the deprotection is carried out at a temperature between about 0 ° c . and room temperature . other standard cleaving reagents , such as hcl in dioxane , and conditions for removal of specific α - amino protecting groups may be used as described in schroder and lubke ( 1965 ). after removal of the a - amino - protecting group , the remaining α - amino - and side chain - protected amino acids are coupled step - wise in the desired order to obtain the intermediate compound defined hereinbefore , or as an alternative to adding each amino acid separately in the synthesis , some of them may be coupled to one another prior to addition to the solid phase reactor . selection of an appropriate coupling reagent is within the skill of the art . particularly suitable as a coupling reagent is n , n ′- dicyclohexylcarbodiimide ( dcc , dic , hbtu , hatu , tbtu in the presence of hobt or hoat ). the activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art . examples of suitable activating reagents are carbodiimides , such as n , n ′- diisopropylcarbodiimide and n - ethyl - n ′-( 3 - dimethylaminopropyl ) carbodiimide . other activating reagents and their use in peptide coupling are described by schroder and lubke ( 1965 ) and kapoor ( 1970 ). each protected amino acid or amino acid sequence is introduced into the solid - phase reactor in about a twofold or more excess , and the coupling may be carried out in a medium of dimethylformamide ( dmf ): ch 2 cl 2 ( 1 : 1 ) or in dmf or ch 2 cl 2 alone . in cases where intermediate coupling occurs , the coupling procedure is repeated before removal of the α - amino protecting group prior to the coupling of the next amino acid . the success of the coupling reaction at each stage of the synthesis , if performed manually , is preferably monitored by the ninhydrin reaction , as described by kaiser et al . ( 1970 ). coupling reactions can be performed automatically , as on a beckman 990 automatic synthesizer , using a program such as that reported in rivier et al . ( 1978 ). after the desired amino acid sequence has been completed , the intermediate peptide can be removed from the resin support by treatment with a reagent , such as liquid hydrogen fluoride or tfa ( if using fmoc chemistry ), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the α - amino protecting group at the n - terminus if it was not previously removed to obtain the peptide in the form of the free acid . if met is present in the sequence , the boc protecting group is preferably first removed using trifluoroacetic acid ( tfa )/ ethanedithiol prior to cleaving the peptide from the resin with hf to eliminate potential s - alkylation . when using hydrogen fluoride or tfa for cleaving , one or more scavengers such as anisole , cresol , dimethyl sulfide and methylethyl sulfide are included in the reaction vessel . cyclization of the linear peptide is preferably affected , as opposed to cyclizing the peptide while a part of the peptido - resin , to create bonds between cys residues . to effect such a disulfide cyclizing linkage , fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis , as is well known in the art , to yield the fully protected amide intermediate , which is thereafter suitably cyclized and deprotected . alternatively , deprotection , as well as cleavage of the peptide from the above resins or a benzhydrylamine ( bha ) resin or a methylbenzhydrylamine ( mbha ), can take place at 0 ° c . with hydrofluoric acid ( hf ) or tfa , followed by oxidation as described above . a suitable method for cyclization is the method described by cartier et al . ( 1996 ). muteins , analogs or active fragments , of the foregoing conkunitzins are also contemplated here . see , e . g ., hammerland et al ( 1992 ). derivative muteins , analogs or active fragments of the conkunitzins may be synthesized according to known techniques , including conservative amino acid substitutions , such as outlined in u . s . pat . no . 5 , 545 , 723 ( see particularly col . 2 , line 50 to col . 3 , line 8 ); u . s . pat . no . 5 , 534 , 615 ( see particularly col . 19 , line 45 to col . 22 , line 33 ); and u . s . pat . no . 5 , 364 , 769 ( see particularly col . 4 , line 55 to col . 7 , line 26 ), each incorporated herein by reference . pharmaceutical compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques . see , for example , remington &# 39 ; s pharmaceutical sciences , 18th ed . ( 1990 , mack publishing co ., easton , pa .). typically , an antagonistic amount of the active ingredient will be admixed with a pharmaceutically acceptable carrier . the carrier may take a wide variety of forms depending on the form of preparation desired for administration , e . g ., intravenous , oral or parenteral . for examples of delivery methods , see u . s . pat . no . 5 , 844 , 077 , incorporated herein by reference . for oral administration , the compounds can be formulated into solid or liquid preparations such as capsules , pills , tablets , lozenges , melts , powders , suspensions or emulsions . in preparing the compositions in oral dosage form , any of the usual pharmaceutical media may be employed , such as , for example , water , glycols , oils , alcohols , flavoring agents , preservatives , coloring agents , suspending agents and the like in the case of oral liquid preparations ( such as , for example , suspensions , elixirs and solutions ); or carriers such as starches , sugars , diluents , granulating agents , lubricants , binders , disintegrating agents and the like in the case of oral solid preparations ( such as , for example , powders , capsules and tablets ). because of their ease in administration , tablets and capsules represent the most advantageous oral dosage unit form , in which case solid pharmaceutical carriers are obviously employed . if desired , tablets may be sugar - coated or enteric - coated by standard techniques . the active agent can be encapsulated to make it stable for passage through the gastrointestinal tract , while at the same time allowing for passage across the blood brain barrier . see for example , wo 96 / 11698 . for parenteral administration , the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension . illustrative of suitable carriers are water , saline , dextrose solutions , fructose solutions , ethanol , or oils of animal , vegetative or synthetic origin . the carrier may also contain other ingredients , for example , preservatives , suspending agents , solubilizing agents , buffers and the like . when the compounds are being administered intrathecally , they may also be dissolved in cerebrospinal fluid . the active agent is preferably administered in a therapeutically effective amount . the actual amount administered , and the rate and time - course of administration , will depend on the nature and severity of the condition being treated . prescription of treatment , e . g . decisions on dosage , timing , etc ., is within the responsibility of general practitioners or specialists , and typically takes into account the disorder to be treated , the condition of the individual patient , the site of delivery , the method of administration and other factors known to practitioners . examples of techniques and protocols can be found in remington &# 39 ; s pharmaceutical sciences . typically , the active agents of the present invention exhibit their effect at a dosage range of from about 0 . 001 mg / kg to about 250 mg / kg , preferably from about 0 . 01 mg / kg to about 100 mg / kg , of the active ingredient and more preferably , from about 0 . 05 mg / kg to about 75 mg / kg . a suitable dose can be administered in multiple sub - doses per day . typically , a dose or sub - dose may contain from about 0 . 1 mg to about 500 mg of the active ingredient per unit dosage form . a more preferred dosage will contain from about 0 . 5 mg to about 100 mg of active ingredient per unit dosage form . dosages are generally initiated at lower levels and increased until desired effects are achieved . alternatively , targeting therapies may be used to deliver the active agent more specifically to certain types of cells , by the use of targeting systems such as antibodies or cell - specific ligands . targeting may be desirable for a variety of reasons , e . g . if the agent is unacceptably toxic , if it would otherwise require too high a dosage , or if it would not otherwise be able to enter target cells . the active agents , which are peptides , can also be administered in a cell - based delivery system in which a dna sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient , especially in the spinal cord region . suitable delivery systems are described in u . s . pat . no . 5 , 550 , 050 and in published pct applications no . wo 92 / 19195 , wo 94 / 25503 , wo 95 / 01203 , wo 95 / 05452 , wo 96 / 02286 , wo 96 / 02646 , wo 96 / 40871 , wo 96 / 40959 and wo 97 / 12635 . suitable dna sequences can be prepared synthetically for each active agent on the basis of developed sequences and the known genetic code . the present invention is described by reference to the following examples , which are offered by way of illustration and are not intended to limit the invention in any manner . standard techniques well known in the art or the techniques specifically described below were utilized . voltage - gated ion channels determine the membrane excitability of cells . although many conus peptides that interact with voltage - gated na + and ca ++ channels have been characterized , relatively few have been identified that interact with k + channels . we describe novel conus peptides that interact with the shaker k + channel , the conkunitzins . the peptide was chemically synthesized . abbreviations : fmoc , n -( 9 - fluorenyl ) methoxycarboxyl ; hplc ; high performance liquid chromatography ; i . c . v ., intracerbrovascular ; i . p ., intraperitoneal ; i . t , intrathecal ; tfa , trifluoroacetic acid . synthesis : the peptides were synthesized on solid support using standard fmoc chemistry and a peptide synthesizer symphony / multiplex ™ ( protein technologies inc .). coupling time with hbtu and diea was 1 . 5 h . peptide 32 - 60 cleavage / deprotection was accomplished with reagent k ( 82 . 5 % tfa : 5 % phenol : 5 % water : 5 % thioanisole : 2 . 5 % edt ) for 4 hours at room temperature . soluble crude peptide product was precipitated with cold mtbe , washed with mbte and then dissolved in 25 % aqueous acetonitrile , 1 % tfa and lyophilized . peptide was purified on a semi - preparative rp - hplc column to 99 % of purity . for synthesis of the thioester peptide , peptide 1 - 31 , the first amino acid was attached by mixing 4 - sulfamylbutyryl am resin ( 0 . 2 mmol ), ch 2 cl 2 ( 5 ml ), diea ( 342 μl , 2 mmol ), and the fmoc - gln ( trt )- oh ( 1 mmol ) ( backes and ellman , 1999 ). the reaction mixture was stirred for 20 min , followed by cooling to − 20 ° c . then 420 mg of benzotriazole - 1 - yl - oxy - tris - pyrrolidino - phosphonium hexafluorophosphate ( pybop ) was added as a solid , and the reaction mixture was stirred for 12 h at − 20 ° c ., filtered and washed with ch 2 cl 2 ( 5 × 5 ml ). the extent of incorporation was quantitated by the fmoc release uv assay ( atherton and sheppard , 1989 ). coupling was repeated when loading & lt ; 70 %. the last lys residue was introduced manually as a boc - lys ( boc )- oh derivative . twenty mg of the resin - bound protein ( 1 . 0 mmol / g ) was prepared for activation by initial addition 3 ml dried under solid na tetrahydrofuran ( thf ) and after 15 min , 3 ml of 2 m tms - chn 2 ( 2 m solution in hexane ) was added . after stirring on a rotary plated for 6 hours , the resin was washed with thf ( 5 × 5 ml ) and dmf ( 5 × 5 ml ) and used in the displacement reaction . the activated resin was swollen in dmf and drained . dmf ( 875 μl ), ethyl - 3 - mercaptopropionate ( 130 μl , 1 mmol , 50 equiv ) and sodium thiophenate ( 1 . 5 mg , 10 μmol , 0 . 5 equiv ) was then added . the mixture was stirred on a rotary plated for 24 hours and the resin was filtered and washed with dmf ( 3 × 2 ml ). the combined filtrate and washes were collected in a round - bottom flask and rotary evaporated , and then the oil was dried over koh in vacuo for a minimum of 5 h . the side - chain - protecting groups on the crude peptide were removed with 2 ml of reagent b ( 88 % tfa : 5 % phenol : 2 % triisopropylsilane : 5 % water ) for 3 h at room temperature ( sole and barany , 1992 ). the tfa solution was added dropwise to screw - cap centrifuge tubes containing mtbe with a tfa / mtbe ratio of 1 / 20 . after centrifugation at 3200 g ( 30 min ), the ether was removed and the peptide precipitate resuspended in mtbe . the process was repeated twice . the final precipitate was dissolved in 25 % aqueous acetonitrile and lyophilized . peptide was purified on a semi - preparative rp - hplc column to 99 % of purity . to initiate native chemical ligation , peptide 1 - 31 ( 1 . 2 mg , 0 . 33 mol ) and peptide 32 - 60 ( 1 . 75 mg , 0 . 5 μmol ) were dissolved in 160 μl of 0 . 1 sodium phosphate buffer containing 6m guhcl , ph 7 . 5 to which 4 % thiopheno was added ( 1 . 6 μl ) ( shin et al ., 1999 ). the reaction was incubated for 10 h and monitored by analytical c18 reversed - phase hplc separations in the linear gradient of acetonitrile from 10 % to 60 % buffer b in 45 minutes . the flow rate was 1 ml / min and the elution was monitored by uv detection at 220 nm . the final ligation product was purified on c18 reversed - phase hplc column using semi - preparative column vydac c - 18 column ( 5 μm . 1 cm × 25 cm ) and a flow rated of 5 ml / min . the concentration of linear form of the peptide was determined spectrophotometrically using the molar absorbance coefficient at 274 . 5 nm , ε = 1420 m − 1 × cm − 1 ( litchfield and wilcoxon , 1949 ). oxidative folding : folding reactions were carried out in buffered solution ( 0 . 1 m tris - hcl , 1 mm edta , ph 8 . 7 ) containing appropriate concentration of the linear peptide and a mixture of 0 . 5 mm reduced and 2 . 5 mm oxidized glutathione . the analytical folding reactions were initiated by adding 10 μl of the linear peptide , ( resuspended in 0 . 01 % tfa ) to 40 μl folding mixture . the final peptide concentration was 20 μm . after appropriate time , the reaction was quenched by acidification with 5 μl of formic acid . the reaction mixtures was analyzed by analytical rp - hplc ( linear 40 min gradient from 21 % to 25 % buffer b ) at 45 ° c . peaks were collected and analyzed by maldi - tof on a bruker daltoncis omniflex mass spectrometer using α - cyano - 4 - hydroxy cinnamic acid as matrix . disulfide mapping : qualitative disulfide mapping of the peptide was performed with immobilized pepsin ( pierce biotechnology ). immobilized pepsin , 100 μl was resuspended in 200 μl digest buffer ( 20 mm sodium acetate , ph 3 . 5 ). peptide ( 10 nmoles ) was dissolved in 200 μl digest buffer and 40 μl with pepsin was then added . the solution was incubated at 37 ° c . after 1 h , sample was stirred ( 2 min , 10 , 000 rpm ) and 200 μl supernatant was applied to analytical rp - hplc ( linear 40 min gradient from 2 % to 40 % buffer b ). peaks were collected and analyzed by maldi - tof - ms . peptide corresponding to peak 5 , about 8 nmoles , was dissolved in 160 μl digest buffer and 32 μl digest buffer with pepsin was added . the solution was incubated at 37 ° c . after 1 h , sample was stirred ( 2 min , 10 , 000 rpm ) and 150 μl supernatant was applied to analytical rp - hplc ( linear 40 min gradient from 2 % to 40 % buffer b ). peaks were collected and analyzed by maldi - tof - ms . assessment of proconvulsive activity : male cf - 1 mice ( 26 - 35 g ; charles river laboratories ) were housed in a temperature controlled ( 23 °± 2 ° c .) room with a 12 hour light - dark cycle with free access to food and water . conkunitzin shk str - 1 was administered to mice either by intraperitoneal injection ( ip ) or by freehand intracerebroventricular ( icv ) injection (%: 1 volume ) using a 10 : 1 hamilton syringe . five doses ( 0 . 1 , 0 . 3 , 1 , 2 and 3 nmol / mouse ) of conkunitzin shk str - 1 with 3 - 5 mice per dose group were used for ed 50 determination . the percent of mice having seizures was calculated for each group , and the ed 50 and 95 % confidence limits were calculated using probit analysis ( litchfield and wilcoxon , 1949 ). electrophysiological methods : the xenopus expression system was used for investigating the potential effects of the conkunitizins on voltage - gated na + and k + channels . oocytes from xenopus laevis were prepared as described previously ( methfessel et al ., 1986 ; stühmer , 1992 ). frogs were anaesthetized with 0 . 2 % tricaine in ice water for surgery . following crna injection , the oocytes were incubated 1 - 5 days to allow expression of the protein . prior to the electrophysiological measurements , the vitelline membranes of the oocytes were removed mechanically with fine forceps . crnas encoding various cloned na + and k + channels to be tested were prepared by standard techniques . whole cell currents were recorded under two - electrode voltage clamp control using a turbo - tec amplifier ( npi electronic , tamm germany ). the intracellular electrodes were filled with 2 m kcl and had a resistance between 0 . 6 and 1 mω . current records were low - pass filtered at 1 khz ( k + channels ) or 3 khz ( na + channels ) (− 3 db ) and sampled at 4 or 10 khz , respectively . leak and capacitive currents were corrected online by using a p / n method . the bath solution was normal frog ringer &# 39 ; s ( nfr ) containing ( in mm ): 115 nacl , 2 . 5 kcl , 1 . 8 cacl 2 , 10 hepes ph 7 . 2 ( naoh ). lyophilized conkunitzin was dissolved in nfr , diluted to the final concentration and added to the bath chamber . all electrophysiological experiments were performed at room temperature ( 19 - 22 ° c .). protein isolation : conkunitzin shk str - 1 and shk str - 2 were obtained by isolation and purification from conus striatus using the techniques described in u . s . pat . no . 4 , 447 , 356 ( olivera et al ., 1984 ), u . s . pat . no . 5 , 514 , 774 ( olivera et al ., 1996 ) and u . s . pat . no . 5 , 591 , 821 ( olivera et al ., 1997 ), molecular biology : the cdna library from c . striatus venom ducts was prepared as previously described ( colledge et al ., 1992 ; jacobsen et al ., 1998 ). two conkunitzins were isolated from conus striatus and sequenced . one additional conkunitzin was isolated from conus magus and sequenced . conkunitzin shk str - 1 was found to have the following sequence : the lys at position 1 may be replaced by an arg . conkunitzin shk str - 2 was found to have the following partial sequence : to access synthetic conkunitzin by native chemical ligation strategy , two separate peptides were synthesized : lys 1 - gln 31 - α - thiosester ( peptide 1 - 31 ) and cys 32 - thr 60 ( peptide 32 - 60 ). both peptides were prepared in a stepwise solid - phase method using fmoc chemistry , purified by preparative rp - hplc and characterized by maldi mass spectrometry . peptide lys 1 - gln 31 - α - thioester was synthesized as described above . ligation reaction was observed on rp - hplc and all details are described above . after oxidative folding conkunitizin shk str - 1 , the observed molecular mass decreased from 6934 . 43 da ( calculated mass of mh + = 6934 . 66 da , average ) to 6930 . 41 da ( calculated mass of mh + = 6930 . 66 da , average ), reflecting the loss of four protons because of the formation of two disulfide bonds . the 60 - residue polypeptide chain of conkunitzin shk str - 1 contains four cysteines that form two disulfide bonds . the peptide was folded in buffered solution as described above . the yield of correct disulfide linkages was very high , with six intermediates observed on analytical rp - hplc . each intermediate was collected and analyzed by maldi - tof - ms . in the mass spectrum of intermediates i a , i b , i c and i d , a succession of peaks was observed at 6932 . 17 , 6932 . 07 , 6932 . 53 and 6932 . 31 da ( calculated mass of mh + = 6932 . 66 da , average ), reflecting loss of two protons and indicating the presence of one disulfide bridge . in the mass spectrum of intermediates ii a and ii b , a succession of peaks was observed at 6930 . 80 and 6930 . 12 da ( calculated mass of mh + = 6930 . 66 da , average ), reflecting loss of four protons and indicating the presence of two disulfide bridge . enzymatic cleavage with pepsin in acidic conditions to reduce the potential for disulfide bond interchange was performed to generate individual disulfide - linkage peptides . pepsin digestion of conkunitzin shk str - 1 was performed in two steps as described above . at the first step , conkunitzin shk str - 1 was cleaved into several linear fragments . one fragment was interpreted as being three linear peptides , kdrpslcdlpadsgsgtka ( amino acids 1 - 19 of seq id no : 1 ), nsarkqclrf ( amino acids 26 - 35 of seq id no : 1 ) and rrtydcqrtcl ( amino acids 48 - 58 of seq id no : 1 ), connected by two disulfide bonds and having the molecular mass of 4549 . 74 da ( calculated mass of mh + = 4549 . 23 da , monosiotopic ). this peptide fragment was separated by analytical rp - hplc and used in the second pepsin digest step . mass spectrometric analyses of four additional peaks identified three ions corresponding to peptides linked by a single disulfide bond . the rp - hplc peak 6 at 2161 . 46 da ( calculated mass of mh + = 2161 . 06 da , monoisotopic ), represents peptide nsarkqclrf ( cys2 - cys3 ) rrtydcq ( amino acids 26 - 35 of seq id no : 1 ( cys2 - cys3 ) amino acids 48 - 54 of seq id no : 1 ). peak 7 at 1960 . 31 da ( calculated mass of mh + = 1599 . 99 da , monoisotopic ) represents peptide arkqclrf ( cys2 - cys3 ) rrtydcq ( amino acids 28 - 35 of seq id no : 1 ( cys2 - cys3 ) amino acids 48 - 54 of seq id no : 1 ). peak 8 at 2407 . 67 da ( calculated mass of mh += 2407 . 18 da , monoisotopic ) represents peptide kdrpslcdlpadsgsgtka ( cys1 - cys4 ) rtcl ( amino acids 1 - 19 of seq id no : 1 ( cys1 - cys4 ) amino acids 55 - 58 of seq id no : 1 ). in summary , the linkage of the two disulfide bonds in conkunitzin shk str - 1 is identified as cys1 - cys4 and cys2 - cys3 , by pepsin digestion followed by mass mapping . based upon the sequences of the mature peptides isolated from conus magus and conus striatus , degenerate primers were designed and used to isolate full - length cdna clones for three conkunitzin peptides via 3 ′ and 5 + race techniques . the cdna for each peptide encoded a precursor sequence containing a hydrophobic signal sequence n - terminal to the mature peptide . primers were then designed to target the conserved 3 ′ and 5 ′ utr regions of conkunitzins and were used to screen various conus venoms . the dna sequences of the clones , the sequences of the encoded propeptides , and the sequences of the mature peptides are shown in table 1 . one of these , conkunitzin s1 , has been expressed in insect cell lines . most of the clones consist of a single kunitz domain . however , a few conkunitzins contain 2 or 3 kunitz domains in tandem . the conkunitzins can be grouped into four classes as shown in tables 2 - 5 . the groupings are based upon cysteine patterns ( 6 cysteines vs . 4 cysteines ) and upon the number of tandem kunitz domains found in the peptide . icv administration of conkunitzin shk str - 1 resulted in a dose - dependent increase in the percentage of animals displaying seizures . in general , icv administration of conkunitzin shk str - 1 produced spastic running followed by tonic extension seizures . the ed 50 for icv conkunitzin shk str - 1 was determined to be 0 . 96 nmol / mouse ( 95 % confidence limits : 0 . 29 to 2 . 00 nmol ). in contrast , ip administration of conkunitzin shk str - 1 ( 3 nmol / mouse ) was without effect . conkunitzin shk str - 1 has been tested on the shaker k + channel and an inhibition of channel conductance was observed . it will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . atherton , e . and sheppard , r . c . 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