CN1125402A

CN1125402A - Potassium channel blocking compounds and their use

Info

Publication number: CN1125402A
Application number: CN94192030A
Authority: CN
Inventors: M.C.桑金内蒂; A.L.米勒
Original assignee: NPS Pharmaceuticals Inc
Current assignee: Shire NPS Pharmaceuticals Inc
Priority date: 1993-03-18
Filing date: 1994-03-14
Publication date: 1996-06-26
Also published as: CA2158051A1; AU6407594A; WO1994021278A1; EP0689450A1; EP0689450A4; JPH08508250A

Abstract

Specific potent transient outward potassium channel inhibitors, and polypeptides derived from spider venom, and uses thereof.

Description

Potassium channel blocking compounds and uses thereof

Invention field

The present invention relates to the inhibitor of instantaneous export-oriented potassium-channel and other potassium-channel.

Background of invention

Below be to description of related art, wherein whichever can not be thought the prior art of claim.

Potassium ion (K ⁺) passage is transmembrane protein, it can be along with the variation of transmembrane potential, perhaps along with the activation of cation and/or ligand optionally K ⁺Move into or emigrated cells.K ⁺The main effect of passage is to keep resting membrane electric potential: another effect is to make action potential multipole in the sensitive cells.Potassium-channel has been represented dissimilar ionophorous proteins, and to have illustrated several toxin now mainly be by blocking one or more special K ⁺Passage work (if not being unique effect) (25Neuroscience 729,1988 for Rudy, ＂ Diversity and Ubiquityof K Channels ＂).Heart cell is with K ⁺The remarkable multiformity of passage different subtype is a feature.In action potential process, can open the K of several types because of the depolarization of film ⁺Passage, ion flow (currents) summation of these different passage deliveries can cause the multipole of film and reach resting potential.In case the film depolarization, the instantaneous export-oriented K of one of these channel type ⁺Passage can produce one ion flow (I of (at the 1-10 millisecond) startup fast ₁₀), (10-200 millisecond) decline (inactivation) rapidly then.I ₁₀Initial multipole to heart action potential plays the most significant effect, and can be by several non-specific medicinal compounds such as aminopyridines and tedisamil, a kind of three class anti-arrhythmic agents block (Dukes etc., " Tedisamil Blocks the Transient and Delayed Rectifier K ⁺Currents in Mammal ian Cardiac and Glial Cells " 254J.Pharmacd.Exp.Ther.560,1990).Retardance I ₁₀Cause the prolongation of heart action potential.From the human heart cell, noted I ₁₀, at " Two Types ofTransient Outward Currents inAdult Human Atrial Cells ", 252Am.J.Physiol H142 is described in 1987 as Escande etc.Allegedly prolonging heart action potential (and so vacuum response) is a kind of mechanism (Lynch etc. that suppress again activeness atrium and ventricular arrhythmia, " Therapeutic Potential of ModulatingPotassium Currents in the Diseased Myocardium " 6 FASEB J.2952,1992).Present available III class anti-arrhythmic agents, wherein majority can be called delayed-type correction K by retardance ⁺A kind of independent K of passage ⁺The passage hypotype works, it can cause the excessive prolongation of heart action potential, can cause the appearance of ventricular arrhythmia like this, be called torsades de pointes (Sanguinetti, " Modulation of Potassium Channels by Antiarrhythmicand Antihypertensive Drugs ", 19 Hypertension 228,1992).

Voltage-dependent K ⁺Opening of passage also is the mechanism that occurs the cell membrane multipole in the very short action potential characteristic procedure of axoneuron.In this process, instantaneous export-oriented K ⁺Channel ions stream (is called I in neuron _A) play an important role.Dendrotoxin (dendrotoxin), a kind of toxin that comes from the Serpentis class, the non-inactivation K of slow type in the selectivity retardance neuroganglion neuron ⁺Ion flow (Penner etc., " Dendrotoxin:ASelective Blocker of a Non-inactivating PotassiumCurrent in Guinea-Pig Porsal Root Ganglion Neurones; 407 Pfluqers Arch.365; 1986), and in the section of hippocampus, also can block instantaneous export-oriented K ⁺Ion flow (I _A) (Halliwell etc., " CentralAction of Dendrotoxin:Selective Reduction of aTransient K Conductance in Hippocampus and Binding toLocalized Acceptors ", 83Proc.Natl.Acad.sci.USA493,1986).Increase (the Harvey and Anderson that can cause neurotransmitter release by the prolongation of action potential duration in the caused neuron of dendrotoxin, " Dendrotoxins:Snake Toxins That Block Potassium Channels andFacilitate Neurotransmitter Release ", 31 Pharmac.Ther.33,1985).Someone proposed in this field further research susceptible of proof this will become a kind of pharmacology approach (Lavretskyand Jarvik of treatment cognitive-ability disease such as Alzheimer's disease, " A Group of Potassium-Channel Blockers-Acetylcholine Releasers:Nev Potentials for AlzheimerDisease? A Review ", 12J.Clinical Psychopharm.110,1992).Summary of the invention

The present invention relates generally to that instantaneous export-oriented potassium-channel (for example, is called I respectively in heart cell and neuron ₁₀Or the ion flow of IA) new specificity and effective inhibitors or blocker.The present invention also relates to isolated novel polypeptide or its equivalent from spider venom, they have activity to one or more potassium-channels such as instantaneous export-oriented potassium-channel.

Specifically, provide the new active example of polypeptide separated toxin from the venom of Aranea heteropoda venatoria and Olios fasciculatus.These peptides (it being abbreviated as

chemical compound

1,2 and 3 here) are the instantaneous export-oriented K of voltage-dependent ⁺The effective blocker of the specificity of passage, the corresponding whole cell plasma stream (I of their retardance heart cells ₁₀).These medicaments itself, its fragment or use the chemical compound that these toxin or its equivalent are found with a kind of ligand binding assay (or its equivalent processes), or its equivalent, can be used for treating arrhythmia, and can be used for treating obstacle (as Alzheimer), Parkinson's disease, multiple sclerosis, schizophrenia, epilepsy, apoplexy and the muscle spasm disease of learning and memory.

In general, from the venom of Aranea heteropoda venatoria and Olios fascicul atus, can be separated to the useful K of described herein and claimed type ⁺The channel blocking polypeptide.What also can be separated to other retardance potassium-channel has similar or homologous amino acid (or other chemical compound or monomer) polypeptide of sequence (or its equivalent).The applicant believes that the present invention has measured this K in the toxin that comes from spider venom first ⁺The active existence of channel blocking shows that thus screening to other this class polypeptide in the spider venom is useful with producible.The invention still further relates to and use these polypeptide to screen the method that can act on other medicaments in common site (that is instantaneous export-oriented potassium-channel) as activating agent.

The applicant believes can optionally block I in the axoneuron _APassage causes that also neurotransmitter discharges the chemical agent that increases and can be used for treating Alzheimer and other sacred disease, and similarly, the applicant also believes and can block I in the heart cell by selectivity ₁₀The medicament of passage can be used for treating arrhythmia.Therefore, can be used for the treatment of arrhythmia to these polypeptide with relevant chemical compound or medicament, Alzheimer, parkinson, multiple sclerosis disease, schizophrenia, epilepsy, apoplexy and muscle spasm disease.

So first aspect present invention is set forth the effective instantaneous export-oriented potassium-channel inhibitor of specificity, blocker or antagonist.

Term " instantaneous export-oriented potassium-channel " is a term of knowing, and this term definition is the potassium-channel of specific hypotype in the variety classes cell, as, in heart cell and neurocyte respectively with above-mentioned dated ion flow I ₁₀And I _AK for feature ⁺Passage.

Term " inhibitor " is to be used to refer to the medicament of minimizing by the ion flow that instantaneous export-oriented potassium-channel produced.In the art, term is to exchange with term " inhibitor " to use as " blocker " and " antagonist ".

" specificity " is meant the half maximum (IC to instantaneous export-oriented potassium channel blocking ₅₀) be 100nM or be lower than 100nM, and be higher than instantaneous export-oriented K at least ⁺Passage IC ₅₀When being worth 10 times concentration to other K ⁺Passage (is proofreaied and correct K as slow type ⁺Passage, interior to proofreading and correct K ⁺Passage, the K that acetylcholine is activated or ATP suppresses ⁺Passage), Na ⁺Or Ca ²⁺Passage is all less than effect.

" effectively " is meant concentration at inhibitor less than 100nM, preferably can suppress to specify 50% of instantaneous export-oriented K+ passage less than 10nM with when being more preferably less than 1nM.

In preferred embodiments, these medicaments are polypeptide or come from polypeptide in the spider venom.Although this paper provides the instantiation of this peptide species, but these examples are not determinate in the present invention, and those of ordinary skills should recognize in various spider venoms can identify other polypeptide easily, comprises but being not limited to polypeptide as herein described.In addition, those of ordinary skills should recognize with standard method and can synthesize the polypeptide that is equal to.Can identify the given activity position of the selected instantaneous export-oriented potassium-channel of these peptide retardances, inhibition or antagonism at an easy rate with the screening technique of routine.For example, can synthesize or from complete polypeptide, generate the particular peptide fragment, and order these fragments that record in assay method as mentioned below have and suppress active with various peptidases.Having active these fragments as inhibitor in the present invention can be used in various screening tests and the treatment application.

The analog or the mutain that can synthesize in addition, this peptide species at an easy rate.Modify the zone that does not influence original polypeptide inhibition or blocking activity in the aminoacid sequence these synthetic comprising.In many conservative regions of this inhibitor, can substituted amino acid and significantly do not change the activity of this inhibitor, for example, use less aminoacid such as glycine to replace other less aminoacid such as valine, perhaps with the aminoacid of the similar electric charge of aminoacid replacement of positively charged or negative charge.

Term " is derived ", and expression can be according to the polypeptide formula synthetic compound of being identified in the spider venom simply.Can carry out this deriving with method known in the art, this specific examples above is provided prevailingly.Term " is derived " and is preferably included aforesaid analog, mutain and fragment, and they have the required adjusting activity of polypeptide toxin as indicated above.

The polypeptide of finding in the venom with Aranea heteropoda venatoria and Olios fasciculatus illustrates foregoing invention, comprise heteropoda venatoria peptide compounds 1 (SEQ.ID.NO.1) according to three kinds of specific polypeptide of the present invention and level part of containing this polypeptide, heteropoda venatoria peptide compounds 2 (SEQ.ID.NO.2) and Ol ios fasciculatus peptide compounds 3 (SEQ.ID.NO.3).In an example, in polypeptide of the present invention retardance heart and the neurocyte instantaneous outer, to K ⁺Passage.The present invention includes and have aminoacid sequence substantially the same and substantially the same K with the polypeptide of heteropoda venatoria peptide compounds 1 and chemical compound 2 and Olios fascicul atus peptide compounds 3 ⁺The polypeptide of ion flow blocking activity.

Second aspect present invention is set forth a kind of method of screening instantaneous export-oriented potassium-channel activating agent, it comprise the instantaneous export-oriented potassium-channel inhibitor of a kind of instantaneous export-oriented potassium-channel and a kind of known specificity (as above-mentioned those) and a kind of possible instantaneous export-oriented potassium-channel activator contact, measure the bonded inhibitory action of possible activator then to known inhibitor.Show it is a kind of useful instantaneous export-oriented potassium-channel activator in conjunction with inhibitory action.Can filter out this class activator at an easy rate and measure its specificity.

" activating agent " is meant increase (if it is as a kind of agonist) or reduces (if it is as a kind of inhibitor) a kind of chemical compound by instantaneous export-oriented potassium-channel ion flow.

The one side that the present invention is correlated with is set forth a kind of a kind of useful K that screens from spider venom ⁺The method of channel activator is as illustrating of methods described herein.Screening this venom measures and contains required active level part.

" K ⁺The channel activity agent " be meant the K that increases or suppress any other type ⁺Passage (is proofreaied and correct K as slow type ⁺Passage, interior to proofreading and correct K ⁺Channel C a ²⁺The K of-activation or ATP-responsive type ⁺Passage) chemical compound.

In preferred embodiments, this screening technique comprises the instantaneous export-oriented potassium-channel that uses heart or nervous tissue.

The scope of the invention also comprise a kind of differentiate can with the method for the bonded chemical compound of instantaneous export-oriented potassium-channel, described chemical compound preferably with heteropoda venatoria peptide compounds 1 and chemical compound 2, and combination on the Olios fasciculatus peptide compounds 3 bonded same locis.

Third aspect present invention is set forth a kind of method for the treatment of disease or symptom, wherein by using a kind of instantaneous export-oriented potassium-channel inhibitor of a species specificity or a kind of polypeptide (or its equivalent) that comes from the spider venom for the treatment of effective dose to organism, regulate the activity of instantaneous export-oriented potassium-channel, reach the treatment useful results.Comprised (but not limiting) above-mentioned listed those by the special disease of being treated.

" adjusting " is meant and reduces instantaneous K ⁺The activity of passage.For example, by blocking the pore of this passage, perhaps by changing the voltage-dependent of passage gate.

Treating the step that comprises is, at first suffers from patient's (people or non-human) of disease or symptom with conventional clinical method diagnosis, provides a kind of treatment the effective present composition for then this class patient.

" treatment effectively " is meant a kind of consumption of one or more remissions (to a certain degree) of the described disease that can make the patient or state.In addition, " treatment effectively " also refers to make physiology relevant with this disease or state or this disease or the state cause of disease or biochemical parameter to return to normal a kind of consumption some or all ofly.Usually this consumption is between about 1nmol/kg and 1 μ mol/kg molecule, and this depends on its EC ₅₀And patient's age, body weight and disease.

The present invention sets forth a kind of Pharmaceutical composition in addition on the one hand, and it comprises instantaneous export-oriented potassium-channel inhibitor of a species specificity or spider venom polypeptide (or its equivalent).

The present invention also sets forth an obtainable peptide species (or its analog) from a kind of spider venom on the one hand, and it is the active a kind of inhibitor of potassium-channel.The present invention also provides the unique fragment of this peptide species.As hereinbefore defined, this analog is not a resulting polypeptide itself from venom, but deutero-or obtain by screening other venom by analyzing a kind of polypeptide separated (illustrational as this paper) institute.

Term " unique fragment " is meant the part of not finding same equivalent in the application's applying date known array.The data base of a parsing polypeptide that exists during with the applying date measures equivalent and can discern these fragments at an easy rate.

" pharmaceutically acceptable compositions " is meant and contains a kind of of the present invention a kind of chemical compound for the treatment of effective dose in a kind of pharmaceutically acceptable carrier, promptly, a kind of preparation wherein can add this chemical compound wherein dissolving or make this chemical compound be easy to administration with method for distinguishing.The pharmaceutically acceptable carrier example comprises water, normal saline and physiological buffer saline.This pharmaceutical composition provides with suitable dosage.These compositionss generally are meant those compositionss of being ratified to be used for the treatment of a kind of specified disease by the same mechanism of the FDA or the non-U.S..

" disease or state " is meant above-mentioned those listed diseases and the relevant disease that relates to heart or neurocyte.

This chemical compound is also included within the scope of the present invention as insecticide.The applicant believes that chemical compound as herein described has significant insecticidal action, perhaps is by the instantaneous export-oriented K of retardance with cardiac muscle of mammal ⁺Passage similar on the channel design works.We know that the venom that Aranea produces contains the multiple toxin (Quistad with effective insecticidal activity, G.B, Deng " Insecticidal activity of Spider (Araneae); centipede (Chilopoda); scorpion (Scorpionidae); and snake (Serpentes) venoms 85 Journal Economic Entomology 33, l992).These toxin are under the effect of release pressure and emit, can be effectively and kill or benumb the toxin (Jackson that catches insecticide rapidly thereby produce, H and P.N.R.Usherwood, " Spider toxins as tools for dissectingelements of excitary amino acid transmission ", 11Trends in Neurosciences 278,1988).

Other features and advantages of the present invention will be conspicuous in following described preferred embodiment and claim.

The description of preferred embodiment

Brief description accompanying drawing accompanying drawing at first

Fig. 1 (carries out the chromatography figure of isolating heteropoda venatoria venom (120 μ l) at, Vydac C18 reversed-phase HPLC post equilibrated with 80%A/20%B on 10 * 250mm).(A=0.1%TFA (moisture), B=0.1%TFA (is dissolved in CH to the linear gradient elution of 65%A/35%B with 76%A/24%B in 44 minutes ₃Among the CN)).Inject this venom and begin this gradient elution after 5 minutes, and in the time of 39 minutes, stop eluting and post is washed with 50%B with the last peak of eluting with 3 fens clock times.Trap in the monitoring eluate of 220nm place.Numeric indicia level part (#1-8 and last level part) with the absworption peak bottom.

Fig. 2 is the chromatography figure that carries out isolating

peptide compounds

1 and 2 on cation exchange column.(a figure) for peptide compounds 1, launches this post with the linear gradient of the 50mM sodium acetate pH4.0 of 0～0.32M NaCl in 32 minutes, launches this post with the linear gradient of the 50mM sodium acetate pH4.0 of 0.32～1M NaCl in 5 minutes then.(b figure) uses the linear gradient of the 50mM sodium acetate pH4.0 of 0-0.3M NaCl to launch this post in 3 minutes for peptide compounds 2, launches this post with the linear gradient of the 50mM sodium acetate pH4.0 that contains 0.3-1M NaCl in 35 minutes then.With 1ml/ minute flow velocity eluting, and at 280nm place monitoring effluent.Shown in chromatogram, collect level part.

Fig. 3 is that (300 carry out the chromatogram of isolating Olios fasciculatus venom (108ul) on 10 * 250mm) at Vydac C-18 reversed-phase column.Behind the sample injection 5 minutes, in 75 minutes, launch this post with 20-45% acetonitrile/0.1%TFA linear gradient.In the time of 50 minutes, flow through pillar 7 minutes with 100% acetonitrile/0.1%TFA.Flow velocity was at 3.0ml/ minute, and monitoring is at 320nm place effluent.Shown in chromatogram, collect level part.

Fig. 4 is a chromatogram of analyzing the peptide compounds 3 of purification with cation-exchange chromatography.Behind the sample injection 5 minutes, use the linear gradient of the 50mM sodium-acetate buffer pH4.0 of 0.25-lM NaCl in 75 minutes, to launch this post.With 1ml/ minute flow velocity eluting and at 280nm place monitoring effluent.Shown in this chromatogram, collect level part.

Below describe in detail to find and use the useful compounds for treating of the present invention such as the rhythm of the heart is not normal and the method and the experiment of memory, learning disorder.A kind of main method is can filter out chemical compound (synthetic and natural product) rapidly with radioactivity ligand combination technology (or its equivalent technologies), discerns and can revise combined thing 1,

chemical compound

2 or 3 bonded instantaneous export-oriented K of chemical compound ⁺Bonded those chemical compounds in site on the passage.Can carry out from spider venom, screening K with a kind of similarity method ⁺Channel blocker.In addition, also can screen useful derivant or the analog or the mutein of this peptide species at an easy rate with this technology.

Other experiments comprise in record heart and the neurocyte whole cell plasma stream determine those can be as instantaneous export-oriented potassium current activity specific medicament and with radiolabeled chemical compound 1, chemical compound 2 or chemical compound 3 competitive combinations, and the passage of other type not have the remarkable medicament that acts on.

The ideal characterisitics of the medicament that the method for the invention is differentiated comprises:

1) blocks a kind of potassium-channel specifically and effectively, as heart or neural instantaneous export-oriented K ⁺Passage.Specificity retardance be meant this medicament in vivo during specific concentrations to other ion channel or not significantly effect of receptor, said specific concentrations is meant with to arrhythmia or study, memory imbalance or the effective dosage retardance of above listed other treatment of diseases I ₁₀Or I _AThe time bulk concentration.

2) non-evident effect when therapeutic dose comprises the Electrocardiographic QT of excessive prolongation at interval, bradycardia, fever or stroke.The separation of spider venom toxin

Below be a kind of infinite method example, can separate inhibitor spider venom of the present invention by this method.Those skilled in the art will be contemplated to equivalent processes can separate and discern other polypeptide (or its equivalent) with useful activity of the present invention.Being equal to chemical compound is analog as referred to herein, mutain and derivant, and be having one or more K of discerning of available method as herein described ⁺Passage activated analog, mutain and derivant.In case those skilled in the art will be contemplated to identification and a kind of useful K that checks order out ⁺The channel activity agent, just can chemosynthesis its all or fragment, and as described below, can modify this sequence.In addition, this class fragment or polypeptide itself can be used for screening its active other activating agent that is equal to former polypeptide.

According to method well-known to those skilled in the art is obtained venom with electricity irritation secretion method from Aranea heteropoda venatoria and 0lios fasciculatus.Used method should guarantee that preferably whole venom are not polluted by anti-flow liquid of abdominal part or hemolymph.This method is known those skilled in the art.About-78 ℃, deposit whole venom of such acquisition up to being used for as described below making with extra care with freezing state.In various preparations with partly prepare post such as C-4 and C-18 Vydac post (Rainin Instrument Co.Inc., Mack Road; Woburn, Massachusetts 01801) go up with reversed phase high-performance liquid chromatography and realize refining component from whole venom.Carry out monochromatic peak value measurement at 220nm.Measuring instrument (the Millipore Corporation that arranges with Waters990 diode for example, WatersChromatography Division, 34Maple Street, Milford, Massachusetts 01757) the polychrome U V data of collecting of measuring further analyze level part.Collect level part in the post with known method as using level part to collect instrument and an ISCO2159 peak value measurement instrument (ISCO, 4700Superior, Lincoln, Nebraska, 68504).Container such as aseptic polyethylene labware with suitable size are collected this grade part.Use the lyophilizing eluent then, lyophilizing moisture content is finished concentrating level part again.The dissimilar pillar of used system is measured the purity of resulting level part when using with final refining level part by chromatogram analysis method.

The order-checking of peptide

Can the polypeptide of invention be checked order as the polypeptide that this paper discerned according to known method.The conventional method of measuring main structure comprises as following step: 1) cysteine residues that disulfide bond is connected reduces and carries out the S-pyridineization and improve the sensitivity of substrate to the enzyme effect; 2) control the cracking of this peptide by enzymolysis single or repeatedly step; 3) fragment of usefulness reversed-phase high-performance liquid chromatography chromatography (HPLC) separation and purified peptide; 4) make the fragments of peptides characterization through N-end sequencing and ion injection mass spectral analysis.

For example in solution, carry out the S-pyridine ethyl research of this polypeptide cysteine residue, then carry out the amino acid sequencing of this polypeptide.The step that can finish the S-pyridine ethylization as described below.

The polypeptide of about 1 to 10 μ g is dissolved in or be diluted in maximum 5 μ l-kind of buffer in, sort buffer liquid is to mix 1 part of Tris hydrochloric acid and 3 parts of 8M guanidine hydrochloride that contain 4mM EDTA pH8.5 to make.Add 2.5 μ l10%2-mercaptoethanol aqueous solutions and in the dark in the argon under the room temperature mixture insulation two hours, after the insulation, add 2 μ l 4-vinylpridines (the fresh reagent of depositing in 20 ℃ of argon), and mixture in the dark is incubated two hours under the room temperature in the argon again.Remove the salinity in the mixture then, preferably on short reversed-phase column, carry out with chromatography.At last according to the alkylation sequencing polypeptides of known method to reclaiming.

On the other hand, as Kruft etc., after described reduction in position of 193Aral.Biochem.306 (1991) and the S-pyridine ethylization to this sequencing polypeptides.

At present, with regard to from the peptide compounds in the heteropoda venatoria venom 1 and chemical compound 2 with from regard to the peptide compounds 3 in the olios fasciculatus venom, herein disclosed is specific advantage, use can not obtain other peptide by the method for the whole venom of separation/purification yet.Use recombinant DNA technology can produce polypeptide of the present invention by a kind of coded sequence of cloning described polypeptide or its part.For example, use the hybridization probe that has utilized present known this polypeptid acid sequence information to clone the coded sequence of whole polypeptide according to method well-known to those skilled in the art.Be used in combination recombinant DNA technology and external albumen synthetic technology also can be produced polypeptide of the present invention.This external albumen synthetic method comprises, but be not limited to, use an ABI430A solid-phase peptide synthesizer (Applied Biosystems, Ins., 850LincolnCentre Drive, Foster City California94404) uses conventional Merrifield chemical method well-known to those skilled in the art or other solid state chemistry method.The peptide that is equal to

As everyone knows, in polypeptide, can carry out the replacement of specific amino acids and do not influence or do not influence basically the function of said polypeptide.This feasibility to be substituted in the practical operation difference because of polypeptide different.According to method well known to those skilled in the art admissible replacement is measured.Therefore, has substantially the same aminoacid sequence and substantially the same active all polypeptide of K+ channel blocking all should belong within the scope of the present invention.Biological activity

This peptide species and fragment thereof can be used for treating arrhythmia or memory, study is lacked of proper care as Alzheimer and above-mentioned other disease.When being used for this class indication, this polypeptide and fragment thereof according to conventional formulation method well known in the art as at Remington ' sPharmaceutical Sciences (latest edition, Mack Pnblishing Company, Easton, PA) disclosed those methods are prepared into preparation in.The performance of said preparation will depend on route of administration and required dosage.The conventional Techniques of Optimum of using peptide medicament to use always can realize the dosage optimization to specific adaptations disease.

In general, preferred intravenous, intramuscular, subcutaneous or peritoneal injection administration.Concerning injection, at fluid matrix such as ringer's solution, this peptide of preparation or fragment in the normal saline of Hank solution or other form.Preparation also comprises lyophilized formulations, and it can be prepared when administration again.The another kind of method that reactive compound of the present invention is offered receptor comprises that wherein said preparation contains penetration enhancer through mucosa or transdermal administration, as detergent, and other excipient.Suitably the oral administration of preparation is also included within the scope of the

invention.Use peptides

1,2 and 3 to carry out Screening test

In addition, this peptide class and bioactive fragment thereof can be used for screening test with measure micromolecule or other accurate medicine suppress

chemical compound

1,2 or 3 with heart or the bonded ability of the instantaneous export-oriented potassium-channel of nerve.Described below this paper the bonded suitable mensuration of competitiveness, wherein used

chemical compound

1,2 of the present invention and 3.When being used for this mensuration, generally providing

polypeptide compounds

1,2 or 3 (or a kind of active fragment), and estimate the ability that institute's chemical compound of surveying and radio-labelled compound are competed with radiolabeled form.

Following examples are to be used for describing in detail rather than limiting of the present invention.

Embodiment 1: venom level part of heteropoda venatoria

By the rough venom of 75-120 μ l equal portions being diluted to 1ml with A, and on the part that will dilute sample to the equilibrated Vydae C18 post of 20%B (10 * 250mm), the heteropoda venatoria venom of about 900 μ l is carried out fractionated.(A=0.1%TFA (moisture); B=0.1%TFA (is dissolved in CH ₃CN)).After 3 minutes, gradient is become 24%B and the 5th minute the time, begin linear gradient to be become 35%B by 24% with 44 fens clock times with 1 fen clock time.Flow velocity is 3.5ml/ minute, and measures effluent (Fig. 1) at 220nm.Collect following level part: level part 1 (peak of eluting between 5 and 16 minutes), level part 2 (peaks of eluting between 16 and 19 minutes), level part 3 (peaks of eluting between 19 and 23.5 minutes), level part 4 (peaks of eluting between 23.5 and 26.5 minutes), level part 5 (peaks of eluting between 26.5 and 29.5 minutes), level part 6 (peaks of eluting between 29.5 and 33 minutes), level part 7 (peaks of eluting between 33 and 37 minutes), level part 8 (at the peaks of eluting between 37 and 39 minutes) and last level part (peak of eluting between 39 and 46 minutes).In the time of 39 minutes, most of venom component behind the eluting, was crossed post 3 minutes with 50%B.When not having the peak value eluting to go out again (～7 minutes), post is returned to 20%B and the balance pillar is used for chromatographic isolation next time with 4 fens clock times.Merging and lyophilizing are from 8 effusive similar level parts of chromatographic isolation.As described in dividing in addition in following

enforcement

2 and 4, level parts 6 is consistent with

chemical compound

1 and 2 with 7 main peaks.

Embodiment 2 Heteropoda peptide compounds 1

The rough venom of heteropoda venatoria (～50 μ l) is gone up sample to reversed-phase HPLC post (Vydac, C-18300A, 22 * 250nm), and in 60 minutes, use and carry out eluting (A=0.1% trifluoracetic acid (TFA) to 65%A and 35%B two-phase linear gradient from 80%A and 20%B, the B=acetonitrile), measure at the 220nm place, flow velocity is 15ml/ minute.Collect needed 43 to 44 minutes level part.With the concentrated component of freeze-drying by each time collection in service.

Measure and determine the structure of peptide compounds 1 with following method.Use Waters Pico-Tag system that three parts of identical 1-10nmol samples are carried out the PTC amino acid analysis.Upward natural and reductive/pyridine ethyl peptide is carried out the N-end sequencing at a pulse-liquid-phase sequencing instrument (ABI).Spray mass spectrograph from a SCI-EXAPI III ion and obtain mass spectral analysis.

According to Kruft etc., the method produced in situ of 193 Anal.Biochem.306 (1991) is suitable for the pyridine ethyl derivant of the chemical compound 1 of N-end sequencing.

These data acknowledgements of Huo Deing simultaneously the structure of peptide compounds 1 as follows.3030 residues of SEo.ID.No.1:Asp Asp Cys Gly Lys Leu Phe Ser Gly Cys Asp Thr Ash Ala1 5 10Asp Cys Cys Glu Gly Tyr Val Cys Arg Leu Trp Cys Lys Leu15 20 25Asp Trp, 6 cysteine, 3 disulfide bond.Quality=3412.86 (amide) of calculating.The quality that records=3412.70 (ion injection mass spectrum).The p1=3.76 of estimation.

Embodiment 3:Heteropoda peptide compounds 1

At HEMA-IEC BIO SB post (10 μ m, a 4.6 * 150cm; Alltech Associates, Deerfield, IL 60015) upward be further purified peptide compounds 1 with the ion exchange chromatography partition method.The lyophilized products that contains peptide compounds 1 that obtains from the reversed phase chromatography method is dissolved in 3ml50mM sodium acetate (pH4.0), and followingly carries out chromatography with trisection.Sample on the 1ml sample is being used on the equilibrated HEMA-IEC BIO of 50mM acetic acid steel (pH4.0) the SB pillar.After 5 minutes, the linear gradient with the 50mM sodium acetate solution (pH4.0) of 0-0.32M NaCl in 32 minutes is launched this post, and (Fig. 2 a) then to launch this post with the linear gradient of the 50mM sodium acetate solution (pH4.0) of 0.32-1M NaCl in 5 minutes.After 10 minutes, in 5 minutes, post is returned to initial conditions, and this post of balance is to be used for next step chromatography.With 1ml/ minute eluting, and in 280nm monitoring eluate.Shown on the chromatogram, collect level part.Other 2ml crude compound 1 is carried out chromatographic isolation as mentioned above and merge level part similar in the separation of tertiary colo(u)r(s) spectrum.

At Vydac C-18 reversed-phase column (a 10 * 250mm, 300 ) make in the main absworption peak desalination that eluting goes out from the ion exchange column between 26.5 and 29 minutes, the level that merges part (～10ml) be loaded into on the equilibrated reversed-phase column of 20% acetonitrile/0.1%TFA.After 10 minutes, in 30 minutes, launch this post with 3.5ml/ minute flow velocity with the linear gradient of acetonitrile/0.1%TFA of 20-35%, and in 220nm place monitoring eluate.The level part lyophilization that eluting between 35.5 and 38 minutes goes out, obtain the purified peptide chemical compound 1 of 641 μ g.Recording this peptide quality is 3412.72 (electrospray ionization massspectrum methods).

Embodiment 4Heteropoda peptide compounds 2

The rough venom of heteropoda venatoria (～50 μ l) is gone up sample to a reversed-phase HPLC post (Vydac, C-18,300A, on 22 * 250mm), and in 60 minutes, use and carry out eluting (A=0.1 trifluoracetic acid (TFA) to the two-phase linear gradient of 65%A and 35%B by 80%A and 20%B, the B diacetonitrile), measure at 220nm, flow velocity is 15ml/ minute.Between 46 to 48.5 minutes, collect desired level part.With the concentrated level part of freeze-drying from each time collection in service.

Sample to a reversed-phase HPLC post (Vydac on the material of the above-mentioned level part that comes from the rough venom of 50 μ l, C-18,300A, on 22 * 250mm), and use 75%A and 25%B+ (A=0.1%TFA, the B=acetonitrile) equivalent elution program carries out eluting, measures at the 220nm place, and operates with 3.5ml/ minute flow velocity.Between 55 to 68 minutes, collect required level part.With the concentrated level part of freeze-drying from each time collection in service.

Measure and determine the structure of peptide compounds 2 with following method.With Waters Pico-Tag system three parts of identical 1-10nmol samples are carried out the PTC amino acid analysis.With a pulse-liquid-phase sequencing instrument (ABI) natural and reductive/pyridine ethyl peptide is carried out the N-end sequencing, spray mass spectrograph from a SCI-EXAPI III ion and obtain mass spectral analysis.

According to Kruft etc., the method produced in situ of 193Anal Biochem 306 (1991) is fit to the pyridine ethyl derivant of the chemical compound of N-end sequencing.

These data acknowledgements of Huo Deing simultaneously the structure of peptide compounds 2 as follows.30 31 residues of SEQ.ID.No.2:Glu Cys Gly Thr Leu phe ser Gly Cys Ser Thr His Ala Asp1 5 10Cys Cys Giu Gly Phe Ile Cys Lys Leu Trp Cys Arg Tyr Glu15 20 25Arg Thr Trp, 6 cysteine, 3 disulfide bond.The pl=5.41 of quality=3599.38 (the ion injection mass spectrum) estimation that quality=3599.05 (amide) of calculating record.

Embodiment 5:Heteropoda peptide compounds 2

(10 μ m, 4.6 * 150cm) go up with cation exchange chromatography purified peptide chemical compound 2 at a HEMA-IEC BI0 SB post.The lyophilized products that contains peptide compounds 2 that obtains from initial reversed phase chromatography is dissolved in the 3ml50mM sodium acetate (pH4.0), and following three equal parts is carried out chromatography.Sample on the 1ml sample is extremely being used on the equilibrated HEMA-IEC BIO of 50mM sodium acetate (pH4.0) the SB post.After 5 minutes, the linear gradient with the 50mM sodium acetate solution (pH4.0) of 0-0.3M NaCl in 3 minutes is launched this pillar, and then 50mM sodium acetate solution (pH4.0) linear gradient with 0.3-1M NaCl is launched this post (Fig. 2 b) in 35 minutes.After 5 minutes, in 10 minutes, this post is returned to initial conditions and carry out balance to be used for next step chromatography.With 1ml/ minute flow velocity eluting, and in 280nm monitoring eluate.Shown on the chromatograph, collect level part.As mentioned above other two milliliters of crude compound 2 are carried out chromatography.And merge same stages part from three chromatographies.

A Vydac c-18 reversed-phase column (10 * 250mm, 300 ) go up to divide two parts slough between 30 and 34 minutes from the cation exchange column the salinity of the main absworption peak of eluting.This post balance in 25% acetonitrile/0.1%TFA, and with initial solvent elution 10 minutes then in 20 minutes with the flow velocity eluting of 25-35% acetonitrile/0.1%TFA linear gradient with 3.5ml/ minute.Monitor eluate at the 220nm place, and went out peptide compounds 2 with unimodal form eluting at 26.5 to 29 minutes.Then other eluate that obtains is sloughed salinity from cation exchange column, and merge identical level part.These effluent of lyophilization obtain the peptide compounds 2 of 1.88mg purification.The quality that records this peptide is 3599.52 (electrospray ionization massspectrum methods).

The fractionated of embodiment 6:Olios fasciculatus venom

Dilute with 20% acetonitrile/0.1%TFA of 1.5ml about 108 μ l Oliosfasci culatus venom and with sample on the sample (300 , 10 * 250mm) carry out fractionated with whole venom to a VydacC-18 post of crossing with the same buffer balance.After the injected sample 5 minutes, in 75 minutes, launch this post (Fig. 3) with the linear gradient of 20-45% acetonitrile/0.1%TFA.In the time of 50 minutes, after most of venom compositions elute, pillar is washed with 100% acetonitrile/0.1%TFA with 7 fens clock times.Flow velocity is 3.0ml/ minute, and in 220nm monitoring eluate.Shown on the chromatogram, collect level part.Eluting goes out between 40 and 42 minutes contains the level part (#21) of peptide compounds 3 in lyophilization, and residue is dissolved in 2ml 50nM sodium acetate, in the 0.25M NaCl solution (pH4.0).

Embodiment 7:Olios fasciculatus chemical compound 3

(from Alltech Associates, Deerfield IL60015) upward is further purified peptide compounds 3 with cation exchange chromatography for 10 μ m, 4.6 * 150cm at a HEMA-IEC BIO SB post.The solution (1.5ml) of the peptide containing compound 3 that obtains from reversed phase chromatography is loaded on the HEMA-IEC BIO SB post of crossing with the same buffer balance.After 5 minutes, the linear gradient with the 50mM sodium-acetate buffer (pH4.0) of 0.25-1M NaCl in 75 minutes is launched this post (Fig. 4).With 1ml/ minute flow velocity eluting, and in 280nm place monitoring eluate.Shown on this chromatogram, collect level part.

Go up material (the level part #4) desalination of the main peak value that between 27 and 32 minutes, elutes from cation exchange column at a Vydac C-18 reversed-phase column (10 * 250mm, 300 ).The level part (～4.5ml) be loaded on the reversed-phase column of crossing with the 0.1%TFA balance.After 3 minutes, in 3 minutes, launch this post, then in 5 minutes, launch this post with the linear gradient of 15-30% isopropyl alcohol/0.1%TFA with the linear gradient of 0-15% isopropyl alcohol/0.1%TFA.With 1.0ml/ minute flow velocity eluting, and in 220nm place monitoring eluate.Lyophilization level part that eluting goes out between 43 and 45 minutes obtains the peptide compounds 3 of 70 μ g purification.The quality that records this peptide is 3786.64 (electrospray ionization massspectrum methods).

Obtained the N-terminal sequence analysis of reduction, deutero-peptide compounds 3.Sequence is as follows

SEQ.ID.No.3：

Asp?Asp?Cys?Ala?Gly?Trp?Met?Glu?Ser?Cys?Ser?Ser?Lys

1 5 10

Pro?Cys?Cys?Ala?Gly?Arg?Lys?Cys?Phe?Ser?Glu?Trp?Tyr

15 20 25

Cys?Lys?Leu?Val?Val?Asp?Gln?Asn

3034 residues, 6 cysteine, 3 disulfide bond.It is lower to the discriminating credibility of aminoacid 33 and 34 to record quality=3786.64 (ion injection mass spectrum).Embodiment 8: the K of heteropoda venatoria and Olils fasCiculatus peptide level part and

chemical compound

1,2 and 3 ⁺The channel blocking activity

Confirm peptide level part of the present invention and

chemical compound

1,2 and the instantaneous export-oriented K of 3 retardances with following method ⁺The ability of passage.

According to foregoing method (Kamp etc., " Voltage-and Use-dependent Modulation of Cardiac Calcium Channels bythe Dihydropyridine (+)-202-791 ", 64 Circ.Res.3381989) separate the myocardial cell of Mus ventricle.This method comprises that thereby the rat heart of using solution retroperfusion to a cutting-out that contains collagenase and protease comes the whole heart of enzymolysis to separate and is applicable to that the standard electric potential difference applies the single cardiac myocyte of test.With other (Hamill etc. described in detail, " Improved Patch Clamp Techniqnes for High-resolutionCurrent Recording from Cells and Cell-Free MembranePatches ", 391 Pflugers Arch.85,1981) the potential difference technology that applies writes down the ion flow of whole cells from isolating myocyte.Cell is put into a 0.5ml recording room, and soak with the buffer of following compositions, this compositions is (in mM): NaCl 132:MgCl ₂, 1.2:CaCl ₂, 1.8; KCl, 4; HEPES, 10; Glucose, 10:pH are 7.4.At record K ⁺In the great majority test of ion flow, remove CaCl ₂And in this solution, add 1mM Co ²⁺Block Ca ²⁺Ion flow.Apply a potential difference for this myocyte with commercial commercially available folder amplifier (Axon Instruments Axopatch ID), and with personal computer Collection and analysis data.Apply-voltage of 60mV to cell.Test voltage (500 ms interval) scope of using is-40 to+30mV.Utilize these technology can write down several K in these cells ⁺Ion flow comprises interior to proofreading and correct K ⁺Ion flow; The slow type of activation, non-inactivation is proofreaied and correct K rapidly ⁺Ion flow; Instantaneous export-oriented K with voltage-dependent ⁺Ion flow (I ₁₀).Respectively the dried level part residue of the venom level part 1-8 that makes described in the embodiment 1 and last level part is dissolved in the 1ml water.Each sample that dilutes 10 μ l with the buffer of 3ml is tested heart K then ⁺The effect of ion flow.Under these conditions, peptide level part 2-9 blocks I in a kind of mode of voltage-dependent ₁₀Block fully when the test current potential of-10mV, retardance is reduced to 30 to 70% of control value when+30mV test current potential.As above-mentioned embodiment 3 with separate described in 5 and the main peptide of purification level part 6 (chemical compound 1) and level part 7 (chemical compounds 2).Chemical compound 1 blocks I in the voltage-dependent mode ₁₀, when less depolarization test current potential, have bigger retardance to occur.Suppress 50% I by measuring ₁₀Required concentration (IC ₅₀) come this retardance of quantificational expression with the method for the maximum retardance of this ion flow when three kinds of different test current potentials.At-10mV ,+20mV and+I during 50mV test current potential ₁₀Maximum retardance be respectively 100%, 79% and 69%, retardance I ₁₀IC ₅₀-10mV is 16nM ,+20mV be 35nM and+50mV is 138nM (n=4-6).With retardance I ₁₀The same, when 90% multipole, record, chemical compound 1 (30nM) has prolonged the action potential duration of 34 ± 5% isolating Mus ventricular muscle cells.Optionally over reach potential time is the activity (VaughanWilliams of the 3rd class anti-arrhythmic, " Delayed ventricular repolarization as anantiarrhythmic principle ", 6 Eur.Heart J.145,1985).Measure chemical compound 1 or 2 other heart ion flow is used for estimating its specificity.As use the full cell potential of standard to apply technology and test, chemical compound 1 or chemical compound 2 do not influence the ion flow of following heart when 0.2-1.0 μ m concentration:---the slow type of hypervelocity is proofreaied and correct K in the Mus ventricular muscle cell ⁺Ion flow (I _Kur)---slow slow type is proofreaied and correct K among the myocyte of guinea-pig ventricular ⁺Ion flow (I _Ks)---interior among Mus and the myocyte of guinea-pig ventricular to proofreading and correct K ⁺Ion flow (I _Ki)---the sodium ion stream (I of Mus ventricular muscle cell _Na)---L type Ca among Mus and the myocyte of guinea-pig ventricular ²⁺Ion flow (I _Ca-L)

In isolating people's ventricle myocyte, chemical compound 1 (0.2 μ M) also blocks I ₁₀But do not block I _Kw, be similar to the discovery in the Mus ventricular muscle cell.

Chemical compound 3 also has similar activity, blocks I when testing current potential＜0mV at 1 μ M fully ₁₀, simultaneously to slow type correction or interior to proofreading and correct K ⁺Ion flow is influence not.In case lost toxin, the effect of chemical compound 1 and chemical compound 2 (1 μ M) is opposite basically.

Chemical compound

1 or 2 pairs have also been tested by many other K that isolating non-heart cell write down ⁺The activity of passage.At 0.2-1.0 μ M,

chemical compound

1 or 2 pairs of following no effects:---Mus maincenter cell (hand over for Pu Kenyeshi neuron, cerebellar myeloid by hippocampal pyramidal cell

Feel neural ganglionic cell), GH ₃Pituicyte or rabbit osteoclast slow rapidly

Type is proofreaied and correct K ⁺Ion flow (I _k)---the instantaneous outward current (I of Mus cerebellar myeloid or orthosympathetic ganglionic cell _n)---expressed a kind of clone's passage in Xenopus oocytes.

Therefore show one type passage (a kind of instantaneous export-oriented K of voltage-activated in the chemical compound 1 and the 2 pairs of cardiac myocytes ⁺Ion flow) is the instantaneous export-oriented K of inhibition (in the neurocyte) that has very much the unique report of specificity ⁺Other toxin of ion flow is dendrotoxin (Halinell etc. " Central action of dendrotoxin:Selective veduction of a transient K conductance in hippocampus and binding to localized acceptors " 83 Proc.Natl.Acad.Sci.USA493,1986).But we have proved that dendrotoxin (2 μ M) is to rat heart I ₁₀Not

effect.So chemical compound

1,2 and 3 is to describe first to block heart I by specificity ₁₀Toxin.

Embodiment 9:

chemical compound

1 and 2 effects on neural system

Electric physiological action with The compounds of this

invention

1 and 2 pairs of hippocampal slices confirms that it influences the active ability of nervus centralis.

Put to death male Sprague-Dawley Mus (100-2009) with decapitation.Take out brain from intracranial, put into (the 95%O of cold (4-6 ℃) oxygenation at once ₂/ 5%CO ₂) in the artificial cerebrospinal fluid (aCSF), this artificial cerebrospinal fluid is by NaCl, 126; KCl, 2.5, NaH ₂PO ₄, 1.24; MgSO ₄, 1.3; CaCl ₂, 2.4; NaHCO ₃, 26; Glucose 11 (mM of unit) is formed, as front (Mueller etc., " Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmission in rat hippocampalslices ", 9synapse244,1991) described preparation hippocampal slices.Under the room temperature section is kept in the 200ml container of aCSF of oxygenation.After 1 hour convalescent period, a section is transferred in the recording room of low capacity (～300 μ l).In this section, place the stability that lighter platinum increases record.Cover this section with aCSF, and keep with a kind of cooling system of crossing.The aCSF flow velocity of fresh oxygenation is 2ml/ minute.Keep this section to be immersed among this mobile aCSF, drop to minimum so that medicine enters the potential problem of this section.During the outer electrogram of pair cell, the temperature of recording room is remained on 33 ℃.Be placed near under the radiating layer at CA1-CA2 edge at following bipolar coaxial stimulating electrode of perusal.Be to cause synaptic response, the unidirectional pulse that discharged 50 microseconds of 3-50V to each section in per 30 seconds is measured this reaction simultaneously, till obtaining the amplitude peak of current potential from specific record position.Voltage is set then causes the maximum reaction of half.Carry out record with the 2-3MW glass microelectrode that is filled with 0～9%NaCl, this electrode is also placed under the meat limit is observed.Record derives from the synaptic response of CA1 pyramidal layer (overall peak) or radiating layer (excitatory postsynaptic potential (EPSP) of field and input volley (AV)), and with its digitized, imports in a kind of data acquisition and storage system based on the IBMPC machine.With phosphate buffer (PBS:NaCl, 140mM; KCl, 2.5mM; KH ₂PO ₄, 1.5mM; Na ₂HPO ₄, 8.1mM; PH7.4) 100-1000 with required ultimate density doubly prepares toxin, changes over to then in the deposit syringe, so that reach required ultimate density by dilution and mixing.Whole medicines and toxin are used the general balance of response amplitude this moment after supercool 30 minutes.All wave mode datumization and deposits.Calculate the extracellular of medication during preceding 5 minutes after (contrast is before the medication) and the medication during 5 minutes (after the medication 25-30 minute) and write down the response amplitude meansigma methods.

A lasting growth appears in chemical compound 1 on the amplitude at overall peak, this growth does not restore to the original state during with fresh aCSF flush compound 1.Average response to 500nM chemical compound 1 is to increase by 35 ± 9% (meansigma methods ± S.E.M., n=5 section) in two sections of these sections, the medicated cap degree of the field excitatory postsynaptic potential that writes down simultaneously increases with 16% meansigma methods, and the input volley does not change.

Use the chemical compound 2 of purification can obtain analog result, when the final concentration with 1 μ M used chemical compound 2, it can produce the growth of 25 ± 7% (meansigma methods ± S.E.M., n=9 sections) on the amplitude at overall peak.In two sections of these sections, the amplitude of the field excitatory postsynaptic potential of record increases with 12% meansigma methods simultaneously, and the input volley does not change.

In sum, these results confirm

chemical compound

1 and 2 can increase for a long time the Schaffer pleurapophysis-synapse transmission in the synapse of CA1 pyramidal cell.These data can not be distinguished presynaptic and postsynaptic site of action, can not clearly illustrate that the mechanism of effect.This growth on synapse is transmitted may be because of due to the retardance of voltage sensitivity potassium-channel.

When intravenous injection (i.v.) maybe when when the intracerebral ventricle injection administration (i.c.v), many K ⁺Channel blocker can cause epilepsy.For example, when passing through intracerebral ventricle injection with 0.008 μ g/g, be equivalent to about 0.24 a μ g Mus, dendrotoxin can cause that Mus twitches and dead (Schweitz, H. etc., ＂ Purification andpharmacological Characterization of peptide toxins fromthe black mamba (Dendroaspis polylepis) venom ＂, 28Toxicon 847,1990).By comparison, give Mus that the AGS tendency is arranged with the chemical compound 2 of 1 μ g (n=3) or 2 μ g (n=1) during by intracerebral ventricle injection, chemical compound 2 can not cause tic or epilepsy.Behind 10,15 or 24 μ g (the each dosage of n=1) dosage intravenous injection chemical compound 2, it can cause the instantaneous ataxia of Mus, but does not observe tic.

Embodiment 10: other K ⁺The channel blocking toxin

Chemical compound

1,2 and 3 be meant from spider venom, separate can block specificity K ⁺The toxin example of the reported first of passage.Also contain incoherent toxin on the structure (peptide class and non-peptide class) in the spider venom that is not heteropoda venatoria and Oliosfasciculatus kind, these toxin can block the I in the mammalian cell effectively ₁₀The perhaps K of other type ⁺Passage.

Now known the characteristic of isolating several other toxin from spinal animals and non-spinal animal venom.For example, from Apis Apis mellifera venom, isolate two kinds of toxin and can block K ⁺Passage.Apamin can block low electric conductance Ca ²⁺Activated K ⁺Passage, and MCD (mastocyte threshing) Toplink blocks the slow type correction of non-inactivation K ⁺Passage (Strong, " Potassium Channel Toxins ", 46Pharmac.Ther.137,1990).From the venom of Scorpio and Serpentis generation, isolated other K ⁺The specificity retardance toxin of passage.For example, contain at least two kinds of toxin in the venom of Leiurus quinquestriatus Scorpio, charybdotoxin and leiurotoxin, they can block high electric conductance and low electric conductance Ca respectively ²⁺Activity K ⁺Passage (Strong, " potassiumChannel Toxins ", 46Pharm.Ther.127,1990).Now from the venom of mamba, isolated and to have blocked the slow type correction of neuronic non-inactivation K ⁺The toxin of passage (Harvey and Anderson, " Dendrotoxins:SnakeToxins That Block Potassium Channels and FacilitateNeurotransmitter Release ", 31Pharmac.Ther.33,1985).All have and β-Bungarus fasciatus toxin obvious sequence homology from the dendrotoxin of green mamba (Dendroaspis angusticeps) with from the toxin 1 of black mamba (D.Polylepis), wherein β-Bungarus fasciatus toxin be from the venom of Bungarusmulticinctus, separate also can block same type K ⁺Neurotoxin (Moczydlowski etc., " An EmergingPharmacology of Peptide Toxins Targeted Against PotassiumChannels ", 105J.Membrane Biol95,1988) before a kind of inhibitory synapse of passage.It is reported that the toxin of above indication has the slow type correction of the non-inactivation of the retardance of removing K ⁺Effect outside the passage.For example, β-Bungarus fasciatus toxin also has phospholipase A ₂Active (Moczydlowski etc.), and dendrotoxin also can block the sodium ion stream of hippocampal neuron and a kind of instantaneous K of slow inactivation ⁺Ion flow (Li and McArdle " Dendrotoxin Inhibits Sodium andTransient Potassium Cuments in Murine HippocampalNeurons ", 64.Biophys.J.A198,1993).

Above-mentioned toxin has been used for defining the specificity K of Normocellular physiology and diseased tissue cell ⁺The effect of passage.But have been found that several K ⁺Passage is not also found than high specific and effective regulator.For finding the new passage ligand of this class, spider venom is a kind of resource that can excavate.

By using the full cell voltage recording technique of exerting pressure to measure whole venom, by standard HPLC method isolating venom level part and isolating toxin to isolating mammalian heart and neurocyte K ⁺Ion flow be used for detecting K in the spider venom ⁺The existence of passage specificity toxin.

Embodiment 11: sieving and washing can concentrating one instantaneous export-oriented K in tissue ⁺Access site and chemical compound

The method of 1/ chemical compound, 2/chemical compound, 3 bonded chemical compounds

Use with method as known in the art (lactoperoxidase, Bolton-Hunter, toluene-sodium-sulfonchloramide etc.) ¹²⁵I labelled

compound

1,2 or 3 or related peptides.Use following method to measure the candidate compound that acts on chemical compound 1/ chemical compound 2/ chemical compound 3 binding sites, replace usefulness by measuring it ¹²⁵The I labelling [ ¹²⁵I] chemical compound 1, [ ¹²⁵I] chemical compound 2, or [ ¹²⁵I] the bonded ability of specificity of chemical compound 3 or related peptides estimates described candidate compound.

Following method of testing as a kind of large-duty method of testing, screen product library (as the chemical compound of, natural product storehouse and the application of main drugmaker) thus be identified in I ₁₀Chemical compound 1/ chemical compound 2/ chemical compound 3 binding sites on the passage have an active new compounds.Use this class noval chemical compound conduct with neural I then ₁₀ Chemical compound 1/ chemical compound 2/ chemical compound 3 binding sites are the chemical main structure of the drug development project of target on the passage.Imbalance provides a kind of new treatment approach as Alzheimer and above-mentioned other listed disease to the chemical compound of discerning with this method of testing to learning and memory.If in quantitative test, used a kind of peptide, guarantee that the biological activity that this peptide is keeping it is very important.Just block heart I ₁₀, iodinating ( ¹²⁵I) chemical compound 1, chemical compound 2 and chemical compound 3 are keeping their normal activity.For example, ¹²⁵I-chemical compound 1 blocks the I of Mus ventricular muscle cell in a kind of voltage-dependent mode ₁₀, estimate IC ₅₀-10mV is 25nM ,+20mV is 70nM, and+50mV is 150nM.

According to (" Effects of Polyamines on theBinding of[such as Williams ³H] MK-801 to the NNDA Receptor:Pharmacological Evidence for the Existence of aPolyamine Recognition Site " 36Molec.Pharmacol.575; 1989) method prepare the Mus meninges, as follows: is weight that the male Sprague-Dawley mice (Simonsen Laboratories) of 100-200g is put to death with decapitation.With one table glass/Teflon refiner in containing the 300ml0.32M sucrose solution (pH7.0) of 5mMK-EDTA 20 mouse brain (removing cerebellum and brain stem) homogenate.Homogenate centrifugalize 10 minutes, remove supernatant at 1000xg, again 30,000xg centrifugalize 30 minutes.Resulting precipitate is suspended in 250ml 5mM K-EDTA (pH7.0), and stirred 15 minutes on ice.Then 30,000xg centrifugalize 30 minutes.This precipitate is suspended in 90ml 5mM K-EDTA (pH7.0), and the layering in the discontinuous sucrose gradient of 0.9M and 1.2M sucrose solution (each 10ml) of 15-ml aliquot.95,000xg is collected in the synapse serous coat (SPM) at 0.9M/1.2M sucrose solution interface to this gradient solution centrifugalize 90 minutes.Suspension with 500ml5mM K-EDTA (pH7.0) washes this film, and 32 ℃ of insulations 30 minutes, again 100,000xg centrifugalize 30 minutes.This rinsing step of triplicate comprises 30 minutes insulation, and last precipitate resuspending in 60ml 5mMK-EDTA (pH7.0), and is deposited with aliquot at-80 ℃.For carry out with [ ¹²⁵I] chemical compound 1,2 or 3 in conjunction with test, synapse serous coat (SPMs) is melted, 32 ℃ of incubations 30 minutes, flushing once, then with 100,000xg centrifugalize 30 minutes.The SPMs resuspending in buffer A (20mM K-HEPES, 1mM K-EDTA, (pH7.0).In this reactant mixture, add [ ¹²⁵I] chemical compound 1,2 or 3.In the polypropylene test tube, carry out the combination test.Last incubation volume is 200 μ l.There is mensuration non-specific binding down at the nonradioactive chemical compound 1,2 of 100 μ m or 3.Three parts of same sample 32 ℃ of incubations 2 hours.Add the ice-cold buffer A of 10ml and finish test, filter (Schl eicher﹠amp with glass fiber filter paper then; Schuell No.30).Wash filter paper with other 10ml buffer A, and measure with the γ calculating instrument ¹²⁵The radioactivity of I.

In order to confirm above-mentioned test, also carried out following test.

(a) 200 μ l are contained 100nM[ ¹²⁵I]

chemical compound

1,2 or 3 buffer A by this glass fiber filter paper measure with this filter paper non-specific binding [ ¹²⁵I]

chemical compound

1,2 or 3 amount.Wash this filter paper with other 10ml buffer A, and measure and the bonded radioactivity of this filter paper with scintillation counting technique.If a large amount of non-specific binding [ ¹²⁵I]

chemical compound

1,2 or 3, then wash this filter paper in advance with the

chemical compound

1,2 or 3 that does not identify and limit this combination.If higher non-specific binding do not occur, then finish this test with centrifuging rather than Filtration, measure the amount of radioactivity in the precipitate with scintillation counting technique.

(b) with the SPMs resuspending in buffer A, make a saturation curve.Assay buffer contains the protein of 75 μ g.Use [ ¹²⁵I]

chemical compound

1,2 or 9 concentration of 3, scope from 10nM to 100 μ M (half log unit).Make a saturation curve by data, and measure approximate K with Scatchard analytic process (Scatchard, " The Attractionsof Proteins for Small Molecules and Ions ", 51Ann.N.Y.Aead.Sci.660,1949) _DValue and Bmax.Drawing (Hill with the Hill curve, " A New Mathematical Treatmentof Changesof Ionic Concentrations in Musele and Nerve Under theAction of Electric Currents; With a Theory to TheirMode of Excitation " 40 J.Physiol.190,1910) measure [ ¹²⁵I]

chemical compound

1,2 or 3 in conjunction with concertedness.

(c) SPMs being suspended in buffer A measures in conjunction with the dependency to albumen (receptor) concentration.This test buffer (200 μ l) contains and its K _DThe value equal concentrations [ ¹²⁵I]

chemical compound

1,2 or 3 and can increase the albumen of concentration.[ ¹²⁵I]

chemical compound

1,2 or 3 specificity be in conjunction with should be linear with the content of albumen (receptor).

(d) SPMs is suspended in the time course of measuring ligand-receptors bind in the buffer A.The buffer of this test usefulness (300 μ l) contains and its K _DThe value equal concentrations [ ¹²⁵I]

chemical compound

1,2 or 3 and 100 μ g albumen.In different time length in three parts of identical samples of 32 ℃ of incubations; Mensuration reaches balance time, and uses this time point usually in all tests of carrying out subsequently.

(e) can analyze the pharmacology of binding site with competitive trials.In this test, keep [ ¹²⁵I]

chemical compound

1,2 or 3 concentration and protein content be constant, changes test (competition) drug concentrations simultaneously.This test can be measured the IC of competition medicine ₅₀With approximate K _D(Cheng and Prusoff, " Relationship Between theInhibition Constant (K ₁) and the Concentration ofInhibitor Which Causes 50 Percent Inhibition (IC ₅₀) ofan Enzymatic Reaction ", 22J.Biochem.Pharmacol.3099,1973).With Hill tracing analysis method determine this competition medicine in conjunction with concertedness.

[ ¹²⁵I]

chemical compound

1,2 or 3 specificity in conjunction be meant can and I ₁₀A new position combination on the passage.According to like this, should compete in competitive mode with

chemical compound

1,2 or 3 relevant peptides [ ¹²⁵I]

chemical compound

1,2 or 3 combination, and in this test they effect should with at retardance I ₁₀(as, in isolated nerve or heart cell to I ₁₀Inhibition) functional trial in their inhibition effect relevant.On the contrary, at I ₁₀Other position on the passage have active chemical compound can not replace in competitive mode [ ¹²⁵I]

chemical compound

1,2 or 3 combination.On the contrary, may occur to [ ¹²⁵I]

chemical compound

1,2 or 3 bonded complicated allosterics adjustings, this shows it is noncompetitive interaction.

(f) [ ¹²⁵I]

chemical compound

1,2 or 3 combination reach balance (seeing above-mentioned (d)) back and measure their combination, and the competitive drug that adds more excessive no radioactivity in reactant mixture is assessed the dynamic research of dissociating.Then the different time measuring space [ ¹²⁵I]

chemical compound

1,2 or 3 combination.Use this method of testing, measure [ ¹²⁵I]

chemical compound

1,2 or 3 bonded combinations and dissociate than (Titeler, " Multiple Dopamine Receptors:Receptor Binding Studiesin Dopamine Pharmacology ", Marcel Dekker, Inc., NewYork, 1983).Test in addition comprises that changing reaction temperature (20 ℃ to 37 ℃) does these parameters of research to dependence on temperature.

Embodiment 12: the instantaneous export-oriented K that can be bonded to heart tissue ⁺The screening technique of the chemical compound of chemical compound 1/ chemical compound 2/ chemical compound 3 binding sites on the passage

Use by known method in this area (lactoperoxidase, Bolton-Hunter toluene-sodium-sulfonchloramide etc.) ¹²⁵I labelled compound 1, chemical compound 2, chemical compound 3 and relevant peptide.With the candidate compound that following described technical measurement acts on chemical compound 1/ chemical compound 2/ chemical compound 3 binding sites replace [ ¹²⁵I] chemical compound 1, [ ¹²⁵I] chemical compound 2, [ ¹²⁵I] chemical compound 3 or usefulness ¹²⁵The bonded ability of the specificity of the related peptides of I labelling is assessed them.

Following method of testing is used as a kind of large-duty test method makes and be used for screening product library (as the chemical compound of, natural product storehouse and the application of most drugmaker), thereby be identified in heart I ₁₀Chemical compound 1/ chemical compound 2/ chemical compound 3 bound fractions have an active new compounds on the passage.Use this class noval chemical compound conduct with heart I then ₁₀The binding site of chemical compound 1/ chemical compound 2/ chemical compound 3 is the chemical main structure of the drug development project of target on the passage.The chemical compound that identifies with this method of testing is given and is treated on the activeness chamber and the arrhythmia of ventricle provides a new treatment approach again.

Method (" Saxitoxin binding and " Fast " SodiumChannel Inhibition in Sheep Heart Plasma Membrane " 249Am.J.Physiol H328,1985) according to Doyle etc.And the method for Jones and Besch (" Isolation of Canine Cardiac Sarcolemmal Vesicles ", 5Methods in Pharmacology 1,1984), method (" Voltage-dependent Nitrendipine Binding toCardiac Sarcolemmal Vesicles " as Kamp and Miller improvement, 32Mol.Pharmacol.278,1987) and preparation heart sarcolemma vesicle.Prepare the fresh cattle or the heart sarcolemma vesicle of other suitable mammalian heart tissue down at 0-4 ℃.Heart is cut into 1cm ³Fritter, and make pasty state with a meat grinder.(being buffered to pH7.4 with 30mMN-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid (HEPES)-15mMTris) sticks with paste homogenate to this meat in the 0.75M choline chlorine of 4 times of meat paste volumes.Carrying out twice homogenizes of 30 seconds with a kind of Tekmar T185 axle in the 300ml polypropylene centrifuge tube stirs.This and all other buffer all comprises protease inhibitor: 0.2mM phenyl methanesulfonamide acyl fluorides, 1mM EGTA and 1mM dithiothreitol, DTT (dithiothreitol).With 27,000xg was centrifugal 20 minutes of resulting homogenate in the GSA of Sorvall centrifuge rotor.Remove supernatant and the precipitate resuspending in 10mM HEPES-5mM Tris (pH7.4), and as above-mentioned carry out again centrifugal.Current centrifugal sediment resuspending in 10mM HEPES-Tris, and was carried out three homogenizes respectively 30 seconds with the T185 axle of Tekmar with 5 grades of settings.With 14,000xg was centrifugal 20 minutes of homogenate in the GSA rotor.Then in the GSA rotor with 27,500xg was centrifugal 70 minutes of supernatant.

After preliminary centrifugal, this film is suspended in 50% sucrose, and 150mM KCl is in 100mMTris Cl and the 5mM tetrasodium pyrophosphate.The concentration that these vesicles is filled into other step of bottom of four step discontinuous gradients is 30%, 21.5% and 9.5% sucrose.With 193,000xg was centrifugal 1.5 hours of this gradient solution in a Beckman50.2Ti rotor.Thin film at 9.5%-21.5% sucrose solution interface is enriched on the surperficial sarcolemma.Collect this thin film and it is released and contain 150mM KCl, 0.8mM NgSO ₄In the buffer (pH=7.4@22 ℃) of 10mM Tris HCl, centrifugal 35 minutes then at 193.000 * g., and once centrifugal again resulting precipitate resuspending in the filler buffer.Is last precipitate resuspending 2mg albumen/ml in the filler buffer to final protein concentration, and liquid nitrogen freezing also stores use at-70 ℃.

With 150mM KCl filling film vesicle (20-40 μ g albumen) and dilute 50 times and make the binding buffer liquid that 1ml contains 150mM KCl.This vesicle 37 ℃ of pre-incubations 5 minutes, then do not have concentration [ ¹²⁵I]

chemical compound

1,2 or 3 (1nM-1 μ m) exists down once more incubation to reach the required time of equilibrium condition (recording correct time by initial test).Add the ice-cold binding buffer liquid of 4ml and stop association reaction, filter rapidly on Whatman GF/C filter paper then, the ice-cold binding buffer liquid with other three parts of 4ml washes subsequently.γ counting technology with routine is measured and the bonded radioactivity of this filter paper.[ ¹²⁵I] there is the combination that records down in

chemical compound

1,2 or 3 specificity in conjunction with being defined as to deduct at the

cold chemical compound

1,2 or 3 of 1-10 μ m with total binding.

The method that is outline among (a)-(f) figure with embodiment 8 confirms above-mentioned test.

Embodiment 13: recombinant receptor is in conjunction with test

Be the test example of screening the useful chemical compound of the present invention rapidly below.In this test, can obtain the I that encodes from suitable organism as going into conventional method ₁₀The cDNA or the gene clone of passage binding site (receptor).Cloned this receptoroid, and be known in the art.The isolated fragment of expressing this clone in a kind of suitable expression vector is produced obtainable minimum polypeptide from this receptor, and this peptide species has kept the ability of binding

compounds

1,2 or 3.Can identify the polypeptide that contains new chemical compound 1/ chemical compound 2/ chemical compound 3 receptors in this way.Use and express I ₁₀The mammal cell line of the stable transfection of passage (as, the HEK293 cell) can make this test become easy.

On the other hand, chemical compound 1/ chemical compound 2/ chemical compound 3 receptors can with the chemical compound 1 of chemical modification, 2 or 3 carry out chemical reaction with a kind of like this method, promptly modify with the chemical compound of selecting and contact the amino acid residue of the chemical compound 1/ chemical compound 2/ chemical compound 3 peptide receptors of (or adjacent), and can obtain identification thus.As mentioned above, use the fragment that the standard expression vector can recombinant expressed chemical compound 1/ chemical compound 2/ chemical compound 3 receptors, this fragment contain after measured can with

chemical compound

1,2 or 3 in conjunction with and be enough to can with bonded those aminoacid of said molecule.

Can combine recombinant polypeptide with a kind of solid support with standard chemical process with required binding characteristic.Then this solid phase or affinity substrate and

chemical compound

1,2 or 3 contacts are confirmed that these chemical compounds can combine with this pillar, and determine the condition of removing this chemical compound from these solid phases.Then with the chemical compound among the big data base repeat this step measure can with bonded those chemical compounds of this affinity substrate, use the similar manner with

chemical compound

1,2 or 3 that it is discharged then.However, also can use other combination and release conditions with obtain can be different from chemical compound 1/ chemical compound 2/ chemical compound 3 peptides in conjunction with used condition (as, especially under pathological state, can better simulate the condition of physiological status) bonded chemical compound down.Therefore filtering out in can the very a large amount of chemical compound from be present in fluid matrix or extract really can bonded those chemical compound.

Can combine the bonded chemical compound of polypeptide with above-claimed cpd 1/ chemical compound 2/ chemical compound 3 in case identify, can easily measure those chemical compounds with above-mentioned various tests, determine whether they or its simple derivant are the useful chemical compounds that can be used for treating heart and sacred disease.

In another method,

native compound

1,2 or 3 receptors can be combined with a kind of post or other solid support.Can identify then not by those chemical compounds of the bonded reagent place competition at other position of this receptor.This compounds has defined binding site new on this receptor.The chemical compound of being competed by other known compound can combine with known point, perhaps combines with the new position that known point overlaps.In any case, this class medicine structurally is different from known compound.Therefore, can be used for defining the analeptic or the antagonist of the new chemical classes of useful as therapeutics.Preparation and administration

Confirm that as this paper the useful chemical compound of the present invention can be used for treating nervous system disease or imbalance.Although these chemical compounds are usually used in treating human patients, they also can be used to treat other vertebrates such as other primate, agricultural animal such as pig, and cattle and poultry, and physical culture is with animal with enjoy with animal such as horse, Canis familiaris L. and the similar or same disease of retouching.

In treatment and/or diagnosis are used, The compounds of this invention is mixed with the preparation of different modes of administration, these administering modes comprise whole body and surgery or topical.At Remington ' sPharmaceutical Sciences, Mack Publishing Co. generally can find preparation technique and dosage form among the EastonPA.

Concerning systemic administration, the preferred oral medication.On the other hand, also can use injection, as intramuscular, intravenous, intraperitoneal and subcutaneous injection.Concerning injection, The compounds of this invention liquid solution, preferred physiologically acceptable buffer such as HankShi solution or Ringer's mixture are made into preparation.On the other hand, also can be made into preparation to chemical compound of the present invention with one or more excipient (as, Polyethylene Glycol), this excipient generally is acceptable with the safety of USP prescribed by standard.In addition, can be made into solid form to this chemical compound, and dissolving or suspension more rapidly before use.Also comprise lyophilized form.

Also can the whole body administration with seeing through mucosa or transdermal method, perhaps oral this compound administration.Concerning seeing through mucosa or administration through skin, in said preparation, use the penetrating agent that is fit to this obstacle of infiltration.These penetrating agent generally all are known in the prior art.For example, concerning seeing through mucosa delivery, this penetrating agent comprises bile salts and fusidic acid derivatives.In addition, use detergent can help infiltration.See through mucosa delivery and can pass through portion's nose spray delivery, for example, perhaps use the suppository administration.Concerning oral administration, this chemical compound is made into conventional peroral dosage form such as capsule, tablet and health-care medicament.

Concerning topical, as is known in the art, can be made into oil preparation to The compounds of this invention, ointment, gel or cream.

Can determine the necessary dosage of the different chemical compounds of the present invention with conventional method.The treatment consumption generally is for the weight of animals that per kilogram is treated, and dosage is 1 to 50mg.

Other embodiment is included in the following claim.

" sequence table " (1) physical data: (i) applicant: Michael.C.Sangui netti Alan Mueller is denomination of invention (ii): potassium channel blocking compounds and uses thereof is sequence number (iii): 3 (iv) mailing addresses:

(A) address: Lyon﹠amp; Lyon

(B) street: 6l1 West Sixth Street

(C) city: Los Angeles

(D) state: California

(E) country: the U.S.

(F) postcode: 90017 (v) computer-reader form:

(A) media type: 3.5 cun dishes, 1.44Mb capacity

(B) computer: IBM compatible

(C) operating system: IBM MS-DOS (5.0 editions)

(D) software: WordPerfeet (5.1 editions) (vi) current request for data:

(A) application number:

(B) applying date:

(C) classification: (vii) request for data formerly:

All, comprise application what follows: 1 in first to file

(A) application number: 08/033,388

(B) application: 03/18/93 (viii) lawyer/agent's data:

(A) name: WARBURG, RICHARD J.

(B) registration number: 32,327

(C) reference/case number: 206/093 (ix) communications data:

(A) phone: (213) 489-1600

(B) fax: (213) 955-0440

(C) data of fax: 67-3510 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 30

(B) type: aminoacid

(C) chain: sub-thread

(D) topological structure: linear (xi) sequence description: the data of SEQ ID NO:1:Asp Asp Cys Gly Lys Leu Phe Ser Gly Cys Asp Thr Asn Ala1 5 10Asp Cys Cys Glu Gly Tyr Val Cys Arg Ler Trp Cys Lys Leu15 20 25 Asp Trp 30 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 31

(B) type: aminoacid

(C) chain: sub-thread

(D) topological structure: linearity, (xi) sequence description: SEQ ID NO:2:Glu Cys Gly Thr Leu Phe Ser Gly Cys Ser Thr His Ala Asp 15 10 Cys Cys Glu Gly Phe Ile Cys Lys Leu Trp Cys Arg Tyr Glu 15 20 25 Arg Thr Trp 30, (3) data of SEQ ID NO:3:, (i) sequence signature:

(A) length: 34

(B) type: aminoacid

(C) chain: sub-thread

(D) topological structure: linear (xi) sequence description: SEQ ID NO:3:Asp Asp Cys Ala Gly Trp Met Glu Ser Cys Ser Ser Lys Pro 15 10 Cys Cys Ala Gly Arg Lys Cys Phe Ser Glu Trp Tyr Cys Lys 15 20 25 Leu Val Val Asp Gln Asn 30

Claims

1. the export-oriented potassium-channel inhibitor of specificity significant instant.

2. screen the method for instantaneous export-oriented potassium-channel activating agent, the step that comprises is:

A kind of described instantaneous export-oriented potassium-channel contacted with a kind of possible instantaneous export-oriented potassium-channel activating agent with the instantaneous export-oriented potassium-channel inhibitor of a kind of known specificity and

Measure described possible instantaneous export-oriented potassium-channel activating agent to the bonded inhibitory action of the instantaneous export-oriented potassium-channel inhibitor of described known specificity, wherein bonded inhibition is shown it is a kind of useful instantaneous export-oriented potassium-channel activating agent.

3. treat the method for a kind of disease or symptom, wherein regulating instantaneous export-oriented potassium-channel activity is useful to described disease in treatment, and this method comprises the following steps: the instantaneous export-oriented potassium-channel inhibitor of the effective specificity of a kind of treatment of administration.

4. treat the method for a kind of disease or symptom, wherein regulating instantaneous export-oriented potassium-channel activity is useful to described disease in treatment, this method comprises the following steps: the effective potassium-channel inhibitor of a kind of treatment of administration, and described inhibitor is corresponding to the inhibitor that is present in the spider venom.

5. the method for claim 4, wherein said passage is a kind of instantaneous export-oriented potassium-channel.

6. the analog of the unique fragment of obtainable inhibitor or this polypeptide or polypeptide from spider venom, it has active to regulating a kind of potassium-channel.

7. screen a kind of method of potassium-channel activating agent, comprise the following steps:

A kind of described potassium-channel is contacted with a kind of possible potassium-channel activating agent with a kind of known potassium-channel inhibitor that is derived from spider venom, and

Measure described possible activating agent to the bonded inhibitory action of described known inhibitor, wherein bonded inhibition is shown it is a kind of useful potassium-channel activating agent.

8. the method for claim 2, wherein said export-oriented potassium-channel inhibitor is selected from chemical compound 1, chemical compound 2 and chemical compound 3.

9. the method for claim 3, wherein said instantaneous export-oriented channel inhibitor is selected from chemical compound 1, chemical compound 2 and chemical compound 3.

10. the method for claim 2, wherein said instantaneous export-oriented potassium-channel comes from heart or nervous tissue.

11. compositions that contains chemical compound 3 or its pharmaceutically acceptable salt.

12. a pharmaceutical composition contains a kind of chemical compound that is selected from chemical compound 1, chemical compound 2 and the chemical compound 3.

13. a kind of inhibitor of claim 1 is selected from chemical compound 1, chemical compound 2 and chemical compound 3.

14. a kind of potassium-channel inhibitor that use is separated from spider venom as the method for insecticide, comprises the following steps: to use a kind of inhibitor that is present in the spider venom to insecticide or its environment.