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WO1998016185A2 - Composes bloquants a canal potassium et leur utilisation - Google Patents

Composes bloquants a canal potassium et leur utilisation Download PDF

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Publication number
WO1998016185A2
WO1998016185A2 PCT/US1997/018710 US9718710W WO9816185A2 WO 1998016185 A2 WO1998016185 A2 WO 1998016185A2 US 9718710 W US9718710 W US 9718710W WO 9816185 A2 WO9816185 A2 WO 9816185A2
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WIPO (PCT)
Prior art keywords
compound
potassium channel
seq
transient outward
inhibitor
Prior art date
Application number
PCT/US1997/018710
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English (en)
Inventor
Michael C. Sanguinetti
Alan L. Mueller
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Nps Pharmaceuticals, Inc.
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Publication date
Application filed by Nps Pharmaceuticals, Inc. filed Critical Nps Pharmaceuticals, Inc.
Priority to AU47589/97A priority Critical patent/AU4758997A/en
Publication of WO1998016185A2 publication Critical patent/WO1998016185A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to inhibitors of the transient outward potassium channel and other potassium channels.
  • Potassium (K*) channels are membrane-spanning proteins that allow the selective movement of K * into or out of cells in response to changes in membrane potential, or in response to activation by cations and/or ligands.
  • the primary role of K" channels is maintenance of the resting membrane potential; another role concerns their contribution to repolarization of action potentials in excitable cells.
  • Potassium channels represent a diverse group of ion channel proteins, and several toxins have been described that act primarily (if not exclusively) by blocking one or more specific K + channels (Rudy, "Diversity and Ubiquity of K Channels", Neuroscience 25:729, 1988).
  • K + channel subtypes Cardiac cells are characterized by a remarkable variety of different K + channel subtypes.
  • K* channel types are opened in response to depolarization of the membrane during an action potential, and the currents carried by these different channels sum to cause repolarization of the membrane to the resting potential.
  • One of these channel types, the transient outward K + channel conducts a current (I to ) which activates very rapidly (within 1-10 milliseconds) upon membrane depolarization and then decays (inactivates) rapidly (10-200 milliseconds) .
  • the opening of voltage -dependent K* channels is also the mechanism by which ' repolarization of the cell membrane occurs during the very short action potential characteristic of central neurons.
  • Transient outward K* currents (referred to as I A in neurons) play a role in this process.
  • Dendrotoxin a toxin derived from snakes, selectively blocks the delayed non-inactivating K* current in dorsal root ganglion neurons (Penner et al . , "Dendrotoxin: A Selective Blocker of a Non-inactivating Potassium Current in Guinea-pig Dorsal Root Ganglion Neurones", Pflugers Arch .
  • Alzheimer's disease Lavretsky and Jarvik, "A Group of Potassium-channel Blockers- Acetylcholine Releasers: New Potentials for Alzheimer Disease? A Review", J. Clinical Psychopharm. 12:110, 1992) .
  • This invention generally concerns specific and potent novel inhibitors or blocking agents of the transient outward potassium channel, for example, currents referred to as I co or I A in cardiac cells and in neurons, respectively, or of a subset of types of transient outward potassium channels.
  • the invention also features novel polypeptides isolated from spider venom, or their equivalent, which are active at one or more potassium channels, e . g. , the transient outward potassium channel.
  • Examples are provided of the novel activity of polypeptide toxins isolated from the venom of the spiders Heteropoda vena toria , and Olios fasciculatus
  • These peptides referred to herein simply as Compounds 1, 2, 3, and 4 are examples of specific and potent blockers of voltage-dependent transient outward K* channels, which block the corresponding whole-cell current (I to ) of cardiac cells.
  • these toxins, fragments thereof, or compounds discovered using a ligand binding assay (or its equivalent) employing these toxins, fragments, or their equivalent are useful in the treatment of cardiac arrhythmias and have utility in the treatment of disorders of learning and memory, (such as Alzheimer's disease), Parkinson's disease, multiple sclerosis, schizophrenia, epilepsy, stroke and muscle spasticity. Further, such agents are useful as reagents in analyzing the distribution and function of particular types and subtypes of potassium channels.
  • useful K* channel inhibiting poly- peptides of the type described and claimed herein can be isolated from the venom of spiders, for example, of the spiders Heteropoda venatoria and Olios fasciculatus .
  • Other polypeptides (or their equivalent) with similar or homologous amino acid (or other compound or monomer) sequences that block potassium channels can also be isolated.
  • the determination of the existence of such K + channel blocking activity in toxins derived from spider venom thus demonstrates that it is useful and productive to screen for other such polypeptides in spider venom.
  • This invention also concerns methods of using these polypeptides to screen for other agents acting at a common site (i.e., the transient outward potassium channel) as active agents.
  • the invention features specific potent transient outward potassium channel inhibitors, blockers or antagonists.
  • transient outward potassium channel is a well recognized phrase which defines a specific sub- type of potassium channels within a variety of cells, e.g., as characterized by the currents l to and I A noted above, in cardiac and neural cells, respectively.
  • Subtypes of transient outward potassium channels have been cloned, isolated, and identified (e.g., Kvl.4, Kv4.2, and Kv4.3) .
  • inhibitor is used to mean agents which reduce current conducted by transient outward potassium channels.
  • terms such as “blocker” and “antagonist” have been used interchangeably with the term “inhibitor”.
  • transient outward K* channels are half-maximal (IC 50 )at or below 100 nM, and that no effect on currents of other K * channels (such as delayed rectifier, inward rectifier, acetylcholine-activated or ATP-inhibited K + channels) , Na * or Ca 2 * channels occurs at concentrations at least 10-fold greater than the IC 50 for transient outward K* channels.
  • K * channels such as delayed rectifier, inward rectifier, acetylcholine-activated or ATP-inhibited K + channels
  • potent is meant that a given transient outward K* channel is blocked by 50% at a concentration of an inhibitor less than 100 nM, more preferably less than 10 nM, and even more preferably less than 1 nM.
  • these agents are polypeptides or are derived from polypeptides present in spider venom. While specific examples of such polypep- tides are provided herein, these examples are not limiting in this invention and those of ordinary skill in the art will recognize that other polypeptides can be readily identified within various spider venoms, including but not limited to those described herein. In addition, those of ordinary skill in the art will recognize that equivalent polypeptides can be synthetically formed using standard procedures. Specific portions of those peptides which are active in blocking, inhibiting, or antagonizing a selected transient outward potassium channel can be readily iden- tified using standard screening procedures.
  • specific peptide fragments can be synthesized, or produced from intact polypeptides using various peptidases, and those fragments assayed for inhibitory activity in assays, as described below. Those fragments which are active as inhibitors are useful in this invention in various screening assays, and in therapeutic applications.
  • analogues or muteins of such polypeptides can be readily synthesized. These may contain modifications in the amino acid sequence in regions which do not affect the inhibitory or blocking activity of the original polypeptide. In more conserved regions of the inhibitor, amino acids may be substituted such that the activity of the inhibitor is not significantly altered, for example, by substitution of small amino acids, such as glycine, for other small amino acids, such as valine, or positively or negatively charged amino acids for similarly charged amino acids.
  • derived indicates that compounds can be synthesized based upon the general structure of polypeptides identified in spider venom. Such deriva- tization is performed by methods well recognized in the art, specific examples of which are generally provided above.
  • the term “derived” includes analogues, uteins and fragments, as described above, which have a desired modulatory activity of a polypeptide toxin described herein.
  • the above inventions are exemplified by polypeptides found in the venom of the spiders Heteropoda venatoria and Olios fasciculatus .
  • Four specific poly- peptides of this invention and the fractions in which they are present according to this invention include Heteropoda venatoria peptide Compound 1 (SEQ. ID. NO. 1) , Heteropoda venatoria peptide Compound 2 (SEQ. ID. NO. 2) , Olios fasciculatus peptide Compound 3 (SEQ. ID NO. 3), and Heteropoda venatoria peptide Compound 4 (SEQ. ID. NO. 4).
  • polypeptides of this invention block transient outward K* channels in cardiac and neural cells.
  • This invention includes polypeptides which have substantially the same amino acid sequence, and substantially the same K* current blocking activity as the polypeptides, Heteropoda venatoria peptides Compound 1
  • Heteropoda venatoria peptide Compound 4 (SEQ ID NO. 4) , and.
  • the invention features a method for screening for a transient outward potassium channel active agent by contacting a transient outward potassium channel with a known specific transient outward potassium channel inhibitor (such as those described above) and a potential transient outward potassium channel active agent, and detecting inhibition of binding of the known inhibitor by the potential active agent. Inhibition of binding is an indication of a useful transient outward potassium channel active agent.
  • a known specific transient outward potassium channel inhibitor is active on a subset of types of transient outward potassium channels.
  • an agent may be active on a Kv4.2 channel, but not on a Kvl.4 channel, or an agent may be active on a Kvl.4 channel, but not on a Kv4.2 channel.
  • selectivity also applies to other types of transient outward potassium channels which may be identified.
  • An "active agent” is a compound that either increases (if it acts as an agonist) or decreases (if it acts as an inhibitor) current through a transient outward potassium channel.
  • references to a "subset" of types of transient outward potassium channels in this context indicates that an agent is active on one or more of the types of transient outward potassium channels (e.g., Kvl.4, Kv4.2, and Kv4.3), but not on all such channels. Thus, the agent is selectively active on these different channels.
  • this invention provides a method for screening for agents active on the Kv4.2 or Kv4.3 potassium channels . This method involves detecting competition for binding of a labeled compound known to be active on these channels by a potential agent active on such channels.
  • the Kv4.2 or Kv4.3 channels are expressed in oocytes, e . g. , Xenopus oocytes .
  • the compound known to be active on the Kv4.2 or Kv4.3 channels is not active on the Kvl.4 channels.
  • examples of such compounds include Compounds 1 and 2.
  • the invention features a method for screening spider venom for a useful
  • K* channel active agent as exemplified by methods described herein. Such venom is screened to determine fractions which contain the desired activity.
  • K * channel active agent is meant a compound that either increases or inhibits any other type of K* channel such as delayed rectifier, inward rectifier, Ca 2 *- activated or ATP-sensitive K + channels.
  • the screening method involves use of transient outward potassium channels from cardiac or neural tissue.
  • Also within the scope of this invention is a method for identifying compounds that bind to the tran- sient outward K + channel, preferably at the same site as that bound by one of the Heteropoda venatoria peptides Compound 1 (SEQ ID NO. 1) , Compound 2 (SEQ ID NO. 2) , or Compound 4 (SEQ ID NO. 4) , or Olios fasciculatus peptide Compound 3 (SEQ ID NO. 3) .
  • Compounds 1, 2, 3, and 4 may bind to the same site, different sites, or overlapping sites on a transient outward K* channel, however it is likely that the differences in binding sites are small.
  • binding refers to an interaction between a compound (such as one of Compounds 1, 2, 3, or 4) and a site on a K + channel (such as a transient outward potassium channel) .
  • a binding site generally is formed by a number of amino acid residues of the channel, which together form a set of contact points and/or a space or pocket for locating and/or interacting with the bound compound .
  • the invention features a method for treatment of a disease or condition in which a therapeutically useful result is achieved by modulating a transient outward potassium channel activity, by adminis- tering to the organism a therapeutically effective amount of a specific transient outward potassium channel inhibitor, or a polypeptide (or its equivalent) from spider venom.
  • Specific diseases to be treated include (but are not limited to) those listed above.
  • the treatment involves modulating the activity of Kv4.2 or Kv4.3 or Kvl.4 potassium channels by administering a therapeutically effective amount of a compound active on such a channel .
  • modulating is meant a decrease of transient
  • K + channel activity for example, by blocking the pore of the channel, or by changing the voltage dependance of channel gating.
  • Treatment involves the steps of first iden- tifying a patient (human or non-human) that suffers from a disease or condition by standard clinical methodology and then providing such a patient with a therapeutically effective composition of the present invention.
  • terapéuticaally effective is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by “therapeutically effective” is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of the disease or condition. Generally, it is an amount between about 1 nmole/kg and 1 ⁇ mole/kg of the molecule, dependent on its EC S0 and on the age, size, and disease associated with the patient.
  • the invention features a pharmaceutically acceptable composition including a specific transient outward potassium channel inhibitor or spider venom polypeptide (or its equivalent) .
  • the invention features a polypeptide (or analogues thereof) obtainable from a spider venom, that is an inhibitor of potassium channel activity.
  • the invention also features unique fragments of such a polypeptide.
  • Such analogues as defined above, are not themselves obtained from the venom but are derived by analysis of an isolated polypeptide (as exemplified herein) or can be obtained by screening other venoms.
  • fragments refers to portions that find no identical counterpart in known sequences as of the date of filing this application. These fragments can be identified easily by an analysis of polypeptide data bases existing as of the date of filing to detect counterparts .
  • composition a therapeutically effective amount of a compound of the present invention in a pharmaceutically acceptable carrier, i.e., a formulation to which the compound can be added to dissolve or otherwise facilitate administration of the compound.
  • pharmaceutically acceptable carriers include water, saline, and physiologically buffered saline.
  • Such a pharmaceutical composition is pro- vided in a suitable dose.
  • Such compositions are generally those which are approved for use in treatment of a specified disorder by the FDA or its equivalent in non-U.S. countries .
  • disease or condition is meant those diseases listed above and related diseases concerning cardiac or neural cells.
  • a further aspect within the scope of the invention is the use of such compounds as insecticidal agents.
  • the compounds described herein are believed to possess significant insecticidal action, perhaps through block of a channel that is structurally similar to the transient outward K + channel of mammalian heart muscle.
  • Spiders are known to produce venoms that contain a variety of toxins with potent insecticidal activities (Quistad, G.B., et al . "Insecticidal activity of spider (Araneae) , centipede (Chilopoda) , scorpion (Scorpionidae) , and snake (Serpentes) venoms", Journal Economic En tomology 85:33, 1992) .
  • Fraction 5 contained Compound 4
  • Fraction 6 contained Compound 1 (SEQ ID NO. 1)
  • Fraction 7 contained Compound 2 (SEQ ID NO. 2) .
  • Figs. 2 and 3 are chromatograms of peptide
  • Fig. 2 For peptide Compound 1 the column was developed with a linear gradient from 0-0.32 M NaCl in 50 mM sodium acetate, pH 4.0 in 32 min followed by a linear gradient from 0.32-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 5 min.
  • Fig. 3 For peptide Compound 2 the column was developed with a linear gradient from 0-0.3 M NaCl in 50 mM sodium acetate, pH 4.0 in 3 min followed by a linear gradient from 0.3-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 35 min.
  • Fig. 4 is a chromatogram of Olios fasciculatus venom (108 ⁇ l) fractionated on a Vydac C-18 reversed phase column (300 A, 10 x 250 mm) . Five minutes after injection of the sample, the column was developed with a linear gradient from 20-45% acetonitrile/0.1% TFA in 75 minutes. At 50 minutes, the column was taken to 100% acetonitrile/0.1% TFA over 7 min. The flow rate was 3.0 ml/min and the effluent was monitored at 220 nm. Fractions were collected as noted on the chromatogram.
  • Fig. 5 is a chromatogram of peptide Compound 3 purified by cation-exchange chromatography. Five minutes after injection of the sample, the column was developed with a linear gradient from 0.25-1 M NaCl in 50 mM sodium acetate buffer, pH 4.0 in 75 min. Elution was at 1 ml/minute and the effluent was monitored . at 280 nm. Fractions were collected as noted on the chromatogram.
  • Figs. 6 and 7 are graphs showing the fractional voltage dependent block of the potassium channel Kv4.2 expressed in Xenopus oocytes.
  • Fig. 6 shows the block by Compound 2 (SEQ ID NO.2 ) and Compound 4 (SEQ ID NO. 4)
  • Fig. 7 shows the block by Compound 1 (SEQ ID NO. 1) .
  • Additional testing can include whole-cell recording of ionic currents in cardiac and neural cells to confirm that agents which compete with radiolabeled Compound 1, Compound 2, Compound 3, or Compound 4 binding act as specific active agents of transient outward K* currents, and are without significant effect on other channel types.
  • the desired properties of agents identified by means outlined in this invention include:
  • Specific and potent block of a potassium channel e.g., cardiac or neural transient outward K* channels.
  • Specific block implies that the agent will not have demonstrable effects on other ionic channels or receptors at concentrations in vi tro that block I to or I A at dosages that prove to be of therapeutic utility in the treatment of cardiac arrhythmias or disorders of learning and memory, or the other diseases listed above.
  • inhibitory spider toxins of this invention may be isolated.
  • equivalent methods can be used to isolate and identify other polypeptides (or their equivalent) having useful activity in this invention.
  • Equivalent compounds are those referred to herein as analogues, muteins and derivatives which can be identified by methods described herein as having useful activity on one or more K* channels.
  • K* channel active agent once one useful K* channel active agent is identified and sequenced it can be chemically synthesized in totality or as fragments, and modifications in the sequence made, as described below.
  • fragments or the polypeptide itself may be used to screen for other such active agents which are equivalent in their activity to the original polypeptide.
  • Venom is obtained from the spiders Heteropoda venatoria and Olios fasciculatus through the process of milking by electrical stimulation according to standard methods well known to those skilled in the art. It is preferred that the method employed is one which safeguards against contamination of the whole venom by abdominal regurgitant or hemolymph. Such methods are well known to those skilled in the art .
  • the whole venom so obtained is stored in a frozen state at about -78°C until used for purification as described below. Purification of the constituents from the whole venom is accomplished by reversed-phase high performance liquid chromatography (HPLC) on a variety of preparative and semi-preparative columns such as C-4 and C-18 Vydac columns (Rainin Instrument Co.
  • HPLC reversed-phase high performance liquid chromatography
  • Peak detection is carried out monochromatically at 220 nm. Further analysis of the fractions can be accomplished with, for example, polychrome UV data collected with a Waters 990 diode array detector (Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, Massachusetts 01757) .
  • the fractions from the columns are collected by known methods such as through the use of a fraction collector and an ISCO 2159 peak detector (ISCO, 4700 Superior, Lincoln, Kansas, 68504) .
  • the fractions are collected in appropriately sized vessels such as sterile polyethylene laboratory ware. Concentra- tion of the fractions is then accomplished by lyophilization from the eluant followed by lyophilization from water. Purity of the resulting constituent fractions then can be determined by chromatographic analysis using a different type of column than the system used in the final purification of the fractions.
  • polypeptides of the invention can be sequenced according to known methods .
  • a general strategy for determining the primary structure includes, for example, the following steps: 1) Reduction and S-pyridylation of disulfide- bridged cysteine residues to enhance substrate susceptibility to enzymatic attack; 2) Controlled cleavage of the peptide through single or multi-step enzymatic digestion;
  • S-pyridylethylation of cysteine residues of the polypeptides under study can be performed, for example, in solution followed by amino acid sequencing of the polypeptides.
  • One such procedure for S-pyridylethylation can be accomplished as described below.
  • About 1 to 10 ⁇ g of polypeptide is dissolved or diluted in up to 50 ⁇ l of a buffer prepared by mixing 1 part TrisHCl, pH 8.5, containing 4 mM EDTA and 3 parts 8M guanidine HCI .
  • 2.5 ⁇ l of 10% aqueous 2-mercaptoethanol is added and the mixture is incubated at room temperature in the dark under argon for two hours.
  • polypeptide can be sequenced after in si tu reduction and S-pyridylethylation as described in Kruft et al . , Anal. Biochem. 193:306 (1991).
  • polypeptides of this invention can be produced using recombinant DNA techniques through the cloning of a coding sequence for said polypeptides or portions thereof.
  • hybridization probes which take advantage of the now known amino acid sequence information of said polypeptides can be employed according to methods well known to those skilled in the art to clone a coding sequence for the entire polypeptide.
  • a combination of recombinant DNA techniques and in vi tro protein synthesis can also be employed to produce the polypeptides of this invention.
  • Such in vi tro protein synthesis methods include, but are not limited to, use of an ABI 430A solid phase peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, California 94404) employing standard Merrifield chemistry or other solid phase chemistries well known ' to those skilled in the art.
  • polypeptides or fragments thereof are useful in the treatment of cardiac arrhythmias or in disorders of memory and learning such as Alzheimer's disease and those other diseases noted above.
  • the peptides and fragments are formulated according to standard formulation methods known in the art, such as those disclosed in Remington's Pharmaceutical Sciences (latest edition, Mack Publishing Company, Easton, PA) .
  • the nature of the formulation will depend on the route of administration and the dosage required. Optimization of the dosage for a particular indication can be accomplished using standard optimization techniques as is generally practiced for peptide medicaments.
  • administration by injection is preferred, either intravenous, intramuscular, subcutaneous or intraperitoneal .
  • the peptide or fragments are formulated in liquid medium, such as Ringer's solu- tion, Hank's solution, or other forms of physiological saline.
  • Formulations may also involve lyophilized preparations which can be reconstituted for administration.
  • Alternative means of providing the active compounds of the invention to the subject include transmucosal and trans- dermal administration, wherein the formulation includes a permeation enhancer, such as a detergent, as well as additional excipients.
  • a permeation enhancer such as a detergent
  • oral administration is also within the scope of the invention.
  • Kvl.4 Shaker subfamily
  • Kv4.2 Shal subfamily
  • Kv4.2 While the presence of Kv4.2 was demonstrated in rat myocytes, recent results suggest that the highly homologous Kv4.3 potassium channel is present in human heart tissue, while Kv4.2 appears to be present in human brain. (Dixon et al . , "The Role of the Kv4.3 Channel in Cardiac Myocyte Function” , Biophys, J. 70:A307, 1996.)
  • the peptides and biologically active fragments thereof are useful in screening assays to assess the ability of small molecules or other candidate drugs to inhibit the binding of Compounds 1, 2, 3, or 4 (SEQ ID NO. 1, 2, 3, and 4) to cardiac or neural transient outward K* channels. Described herein below is a suitable assay for competitive binding in which the Compound 1, 2, 3 or 4 of the invention are useful.
  • the polypeptides Compound 1, 2, 3, or 4 (or an active fragment) is supplied in radiolabeled form and the ability of the candidate compound to compete with radiolabeled Compound 1, 2, 3, or 4 for binding to the potassium channel is assessed.
  • fraction 1 peaks eluting between 5 and 16 minutes
  • fraction 2 peaks eluting between 16 and 19 minutes
  • fraction 3 peaks eluting between 19 and 23.5 minutes
  • fraction 4 peaks eluting between 23.5 and 26.5 minutes
  • fraction 5 peaks eluting between 26.5 and 29.5 minutes
  • fraction 6 peaks eluting between 29.5 and 33 minutes
  • fraction 7 peaks eluting between 33 and 37 minutes
  • fraction 8 peaks eluting between 37 and 39 minutes
  • the end fraction peaks eluting between 39 and 46 minutes
  • PTC amino acid analysis was carried out on 1-10 nmols in triplicate using the Waters Pico-Tag system.
  • N-terminal sequencing was carried out on a pulse-liquid sequenator (ABI) on both native and reduced/pyridylethylated peptide.
  • Mass spectral analysis was obtained from a SCI -EX API III ion spray mass spectrometer.
  • the major absorbance peak which eluted from the cation-exchange column between 26.5 and 29 min, was desalted on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A) .
  • the pooled fraction (10 ml) was loaded onto the reversed-phase column equilibrated in 20% acetonitrile/0.1% TFA. After 10 min, the column was developed with a linear gradient from 20-35% acetonitrile/0.1% TFA in 30 min at a flow rate of 3.5 ml/min and the effluent was monitored at 220 nm.
  • the fraction eluting between 35.5 and 38 min was lyophilized to give 641 ⁇ g of purified peptide Compound 1.
  • the observed mass of this peptide was 3412.72 (electrospray ionization) .
  • the desired fraction was collected from 55 to 68 min. Pooled like fractions from individual runs "were concentrated by lyophilization .
  • the structure of peptide Compound 2 was determined and verified by the following methods. PTC amino acid analysis was carried out on 1-10 nmols in triplicate using the Waters Pico-Tag system.
  • N-terminal sequencing was carried out on a pulse-liquid sequenator (ABI) on both native and reduced/pyridylethylated peptide.
  • ABSI pulse-liquid sequenator
  • Mass spectral analysis was obtained from a SCI-EX API III ion spray mass spectrometer.
  • Cys Cys Glu Gly Phe lie Cys Lys Leu Trp Cys Arg Tyr Glu 15 20 25 Arg Thr Trp 30
  • Peptide Compound 2 was also purified by cation- exchange chromatography on a HEMA-IEC BIO SB column (10 ⁇ m, 4.6 x 150 cm).
  • the lyophilized material containing peptide Compound 2 from the initial reversed-phase chromatography was dissolved in three ml of 50 mM sodium acetate, pH 4.0 and chromatographed in three equal portions as follows. One ml was loaded onto the HEMA-IEC BIO SB column equilibrated in 50 mM sodium acetate, pH 4.0.
  • the column was equilibrated in 25% acetonitrile/0.1% TFA and eluted with the starting solvent for 10 min, followed by a linear gradient from 25-35% acetonitrile/0.1% TFA in 20 min at a flow rate of 3.5 ml/min.
  • the effluent was monitored at 220 nm and peptide Compound 2 eluted as a single peak from 26.5 to 29 min.
  • the remaining pool from the cation-exchange column was then desalted and like fractions were combined. This pool was lyophilized to give 1.88 mg of purified peptide Compound 2.
  • the observed mass of this peptide was 3599.52 (electrospray ionization) .
  • Peptide Compound 4 was also purified by cation- exchange chromatography on a HEMA-IEC BIO SB column (10 ⁇ m, 4.6 x 150 cm) .
  • the lyophilized material containing peptide Compound 4 from the initial reversed-phase chromatography was dissolved in 50 mM sodium acetate, pH 4.0 and chromatographed as follows. An aliquot was loaded onto a HEMA-IEC BIO SB column equilibrated in 50 mM sodium acetate, pH 4.0. The column was developed with a linear gradient from 0.1-1.0 M NaCl in 50 mM sodium acetate, pH 4.0 over 45 min. Elution was at 1 ml/min and the effluent was monitored at 280 nm.
  • the major absorbance peak was desalted on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A) .
  • the column was equilibrated in 0.1% TFA, developed with a 10 min linear gradient from 0 to 25 % Solution B, and eluted with a linear gradient from 25-35% Solution B in 20 min at a flow rate of 3.5 ml/min.
  • the effluent was monitored at 220 nm and peptide Compound 4 eluted as a single peak from 24 to 25.5 min.
  • the observed mass of this peptide was 3910.57 (ion spray mass spectrometer, see Example 7 below) .
  • Portions of the cation exchange purified Compound 4 prepared as described above were used for N- terminal sequencing and mass spectral analysis.
  • the mass of Compound 4 was determined using a triple quadrupole mass spectrometer with an ionspray interface (Perkin-Elmer SCIEX API III system) .
  • the sample was dissolved in 10% acetic acid and the sample solution
  • PTC amino acid analysis was performed on 1-10 nmols of the peptide in triplicate using a Waters Pico-Tag system.
  • N- terminal sequencing was carried out on a pulse- liquid sequenator (ABI) on both native and S- pyridylethylated derivatives.
  • S-pyridylethylated peptides were generated in si tu according to the method of Kruft et al., Anal. Bioche . 193:306-309, 1991.
  • Olios fascicula tus venom was fractionated by diluting the whole venom with 1.5 ml of 20% acetonitrile/0.1% TFA and loading the sample on to a Vydac C-18 column (300 A, 10 X 250 mm) equilibrated in the same buffer. Five minutes after injection of the sample, the column was developed with a linear gradient from 20-45% acetonitrile/0.1% TFA in 75 min (Fig. 4). At 50 min, after the majority of the venom components had eluted, the column was taken to 100% acetonitrile/0.1% TFA over 7 min.
  • the solution containing peptide Compound 3 (1.5 ml) from the reversed-phase chromatography was loaded onto the HEMA-IEC BIO SB column equilibrated in the same buffer. After 5 minutes, the column was developed with a linear gradient from 0.25-1 M NaCl in 50 mM sodium acetate buffer, pH 4.0 in 75 min (Fig 5) . Elution was at 1 ml/min and the effluent was monitored at 280 nm. Fractions were collected as noted on the chromatogram.
  • fraction #4 The major peak of material (fraction #4) , which eluted from the cation-exchange column between 27 and 32 min, was desalted on a Vydac C-18 reversed-phase column (10 x 250 mm, 300 A). The fraction (-4.5 ml) was loaded onto the reversed-phase column equilibrated in 0.1%TFA. After 3 min, the column was developed with a linear gradient from 0-15% iso-propanol/0.1% TFA in 3 min followed by a linear gradient from 15-30% iso- propanol/0.1% TFA in 30 min and from 30-50% iso- propanol/0.1% TFA in 5 min.
  • N-terminal sequence analysis was obtained for reduced, derivatized peptide Compound 3.
  • the sequence is as follows:
  • Rat ventricular myocytes were isolated according to the procedure described previously (Kamp et al . , "Voltage- and Use-dependent Modulation of Cardiac Calcium Channels by the Dihydropyridine (+) -202-791" , Circ . Res . 64:338, 1989).
  • the method involves retrograde perfusion of an excised rat heart with a solution containing collagenase and protease to enzymatically digest the entire heart so as to isolate individual cardiac myocytes suitable for use in standard voltage clamp experiments.
  • K* currents were recorded, Ca 2+ current was blocked by omission of CaCl 2 and addition of 1 mM Co 2* to this solution.
  • the myocytes are voltage clamped using a commercially available patch clamp amplifier (Axon Instruments Axopatch ID) , and data acquisition and analysis is performed using a personal computer. Cells were clamped at a potential of -60 mV.
  • Test potentials 500 msec duration were applied to potentials ranging from -40 to +30 mV. Using these techniques several K* currents can be recorded in these cells, including an inward rectifier K* current; a rapidly activating, non-inactivating delayed rectifier K* current; and a voltage-dependent transient outward K* current (I to ) .
  • the dried fraction residues of venom fractions 1-8 and the end fraction prepared as described in Example 1 were each dissolved in 1 ml of water. A 10 ⁇ l sample of each was then diluted with 3 ml of the buffered solution to test for effects on cardiac K* currents. Under these conditions, peptide fractions 2-9 blocked I -0 in a voltage- dependent manner.
  • Block was complete at a test potential of -10 mV, with block reduced to 30 to 70% of control values at a test potential of +30 mV.
  • the predominant peptides of fractions 6 (Compound 1 (SEQ ID NO. 1)) and 7 (Compound 2) were isolated and purified as described in Examples 3 and 5 above.
  • Compound 1 blocked I to in a voltage-dependent manner, with greater block occuring at less depolarized test potentials. This was quantified by determining the concentration required to inhibit I- 0 by 50% (IC 50 ) and the maximum block of this current at three different test potentials.
  • the maximum block of I. 0 at test potentials of -10 mV, +20 mV and +50 mV was 100%, 79% and 69%, respectively.
  • Compound 1 or 2 did not affect the following cardiac currents as measured using standard whole cell-voltage clamp techniques:
  • Compound 1 (0.2 ⁇ M) also blocked I to but not I Kur , similar to the findings in rat ventricular myocytes. I to in human myocytes is believed to be be due to Kv4.3 channels.
  • Compound 3 had similar activity on rat ventricular myocytes, blocking I co completely at test potentials ⁇ 0 V at 1 ⁇ M, while having no effect on either delayed rectifier or inward rectifier K* currents.
  • the effects of Compound 1 and Compound 2 (1 ⁇ M) were substantially reversed upon washout of the toxins.
  • Compound 1 or 2 was also tested for activity on a number of other K* channels recorded from isolated non- cardiac cells. At 0.2 - 1.0 ⁇ M, Compound 1 or 2 had no effect on:
  • K* currents I of rat neural cells (Purkinje neurons, cerebellar granule cells, hippocampal pyramidal cells, sympathetic ganglion cells) , GH 3 pituitary cells, or rabbit osteoclasts.
  • Example 11 Neural effects of Compound 1. 2. and 4 The ability of Compounds 1, 2, and 4 (SEQ ID NO.
  • the temperature in the recording chamber was held at 33°C for extracellular field potential recording.
  • Bipolar concentric stimulating electrodes were placed under visual guidance in the stratum radiatum near the border of CA1-CA2.
  • monophasic 50 ⁇ sec pulses of 3-50 V were delivered to the slice every 30 sec while testing the response until potentials of maximal amplitude were obtained from a particular recording site. The voltage was then set so as to evoke a half-maximal response. Recording was done with 2-3 MW glass microelectrodes filled with 0.9% NaCl, which were also placed under visual guidance. Synaptic responses were recorded from the CAl pyramidal cell layer
  • Toxins were made up in phosphate-buffered saline (PBS: NaCl, 140 mM; KC1, 2.5 mM; KH 2 P0 4 , 1.5 mM; NajHPO*, 8.1 mM; pH 7.4) at 100-1000 times the desired final concentration, and then transferred to the reservoir syringe so as to achieve, via dilution and mixing, the desired final concentration.
  • PBS phosphate-buffered saline
  • Compound 1 produced a sustained increase in the population spike amplitude which did not recover during washout with fresh aCSF.
  • the amplitude of the simultaneously recorded field EPSP was increased by an average of 16%, while the afferent volley was unchanged.
  • the amplitude of the simultaneously recorded field EPSP was increased by an average of 12%, while the afferent volley was unchanged.
  • K* channel blocking agents can cause seizures when injected intravenously (i.v.), or when administered by intracerebroventricular (i.e. v.) injection.
  • dendrotoxin causes convulsions and death in mice when injected i.e. v. at 0.008 ⁇ g/g, equivalent to about 0.24 ⁇ g/mouse (Schweitz, H. et al . , "Purification and pharmacological characterization of peptide toxins from the black mamba (Dendroaspis polylepis) venom", Toxicon 28:847, 1990).
  • Compound 2 did not cause convulsions or seizures in audiogenic seizure-prone mice injected i.e. v.
  • Compounds 1, 2, 3 and 4 represent the first reported examples of toxins isolated from spider venoms that block specific K* channels.
  • Venoms from species of spiders other than Heteropoda vena toria and Olios fasciculatus may contain structurally unrelated toxins (peptides and nonpeptides) that potently block I to or other types of K* channels in mammalian cells.
  • toxins isolated from venoms of invertebrate and vertebrate venoms have been well characterized.
  • two toxins have been isolated from venom of the bee Apis mellifera that block K* channels.
  • Apamin blocks a low conductance Ca 2 *-activated K + channel
  • MCD (mast cell degranulating) peptide blocks a non- inactivating delayed rectifier K* channel (Strong, "Potassium Channel Toxins", Pharmac. Ther. 46: 137, 1990) .
  • K* channel specific blocking toxins have been isolated from venoms produced by scorpions and snakes.
  • venom from the scorpion Leiurus quinquestriatus contains at least two toxins, charybdo- toxin and leiurotoxin that block high conductance, and low conductance Ca 2* -activated K * channels, respectively (Strong, "Potassium Channel Toxins", Pharm . Ther . 46:137, 1990) .
  • Toxins that block non-inactivating delayed rectifier K * channels of neurons have also been isolated from the venoms of mamba snakes (Harvey and Anderson, "Dendrotoxins: Snake Toxins That Block Potassium Channels and Facilitate Neurotransmitter Release", Pharmac . Ther. 31:33, 1985).
  • the above noted toxins are reported to have effects beyond block of non- inactivating delayed rectifier K + channels.
  • ⁇ -bungarotoxin also exhibits phospholipase A2 activity (Moczydlowski et al . ) and dendrotoxin also blocks sodium current and a slow inactivating transient K* current in hippocampal neurons (Li and McArdle, "Dendrotoxin Inhibits Sodium and Transient Potassium Currents in Murine Hippocampal Neurons", Biophys . J. 64:A198, 1993).
  • the above-noted toxins have been useful in defining the role of specific K + channels in the physiology of normal cells and cells of diseased tissues. However, there are several K + channels known for which no highly specific and potent modulators have been discovered. This invention demonstrates that spider venoms represent an untapped source for the discovery of such novel channel ligands .
  • K* channel-specific toxins in spider venoms is examined by testing the effects of whole venoms, venom fractions separated by standard HPLC methodology, and isolated toxins on K* currents measured using standard whole-cell voltage clamp recording techniques on isolated mammalian cardiac and neural cells as described in Example 10 above.
  • Example 13 Method for Screening Compounds that Bind to a Compound 1 /Compound 2 /Compound 3 /Compound 4 Site on the Transient Outward K* Channel in Neural Tissue Compound 1, 2, 3, or 4 (SEQ ID NO. 1, 2, 3, and
  • the following assay can be utilized as a high throughput assay to screen product libraries (e.g., natural product libraries and compound files at major pharmaceutical companies) to identify new classes of compounds with activity at the Compound 1/Compound 2 /Compound 3/Compound 4 binding site on the I to channel. These new classes of compounds are then utilized as chemical lead structures for a drug development program targeting the Compound 1/Compound 2/Compound 3/Compound 4 binding site on the neural I to channel.
  • the compounds identified by this assay offer a novel therapeutic approach to disorders of learning and memory such as Alzheimer's disease, and those other diseases listed above. It is important to demonstrate that a peptide retains its biological activity if it is to be used in a quantitative binding assay.
  • Iodinated ( ⁇ r7 I) Compound 1 Compound 1, Compound 2, Compound 3, and Compound 4 retain their normal activity with regard to block of cardiac I co .
  • 125 I-Compound 1 blocked I co of rat ventricular myocytes in a voltag -dependent manner, with approximate IC 50 's of 25 nM at -10 mV, 70 nM at +20 mV, and 150 nM at +50 mV.
  • the IC S0 compares favorably with the IC 50 for non- iodinated samples of Compound 1.
  • Rat brain membranes are prepared according to the method of Williams et al . ("Effects of Polyamines on the Binding of [ 3 H]MK-801 to the NMDA Receptor: Pharmacological Evidence for the Existence of a Polyamine Recognition Site", Molec . Pharmacol . 36:575, 1989) as follows: Male Sprague-Dawley rats (Simonsen Laboratories) weighing 100-200 g are sacrificed by decapitation. The brains from 20 rats (minus cerebellum and brainstem) are homogenized at 4°C with a glass/Teflon homogenizer in 300 ml 0.32 M sucrose containing 5 mM K-EDTA (pH 7.0).
  • the homogenate is centrifuged for 10 minutes at 1,000 x g and the supernatant removed and centrifuged at 30,000 x g for 30 minutes.
  • the resulting pellet is resuspended in 250 ml 5 mM K-EDTA (pH 7.0) stirred on ice for 15 minutes, and then centrifuged at 30,000 x g for 30 minutes.
  • the pellet is resuspended in 90 ml 5 mM K-EDTA (pH 7.0), and 15-ml aliquots are layered over discontinuous sucrose gradients of 0.9 M and 1.2 M sucrose (10 ml each).
  • the gradients are centrifuged at 95,000 x g for 90 minutes, and the synaptic plasma membrane (SPM) fraction at the 0.9 M/1.2 M sucrose interface collected.
  • SPM synaptic plasma membrane
  • Membranes are washed by resuspension in 500 ml 5 mM K-EDTA (pH 7.0), incubated at 32°C for 30 minutes, and centrifuged at 100,000 x g for 30 minutes. The wash procedure, including the 30 minutes incubation, is repeated three times. The final pellet is resuspended in 60 ml 5 mM K-EDTA (pH 7.0) and stored in aliquots at -80°C.
  • the amount of nonspecific binding of the [ 125 I] Compound 1, 2, 3, or 4 to the filters is determined by passing 200 ⁇ l of buffer A containing 100 nM [ 12S I] Compound 1, 2, 3, or 4 through the glass-fiber filters. The filters are washed with another 10 ml of buffer A, and radioactivity bound to the filters is determined by scintillation counting. If a significant amount of nonspecific binding of the [ 125 I] Compound 1, 2, 3, or 4 occurs, then filters are prewashed with unlabeled Compound 1, 2, 3, or 4 to limit this binding. If high nonspecific binding remains a problem, assays will be terminated by centrifugation rather than by filtration, and the amount of radioactivity in the pellet will be determined by scintillation counting.
  • a saturation curve is constructed by resuspending SPMs in buffer A.
  • the assay buffer 200 ⁇ l contains 75 ⁇ g of protein.
  • a saturation curve is constructed from the data, and an apparent K D value and B-, ax value determined by Scatchard analysis (Scatchard, "The Attractions of Proteins for Small Molecules and Ions", Ann. N. Y. Acad . Sci . 51:660, 1949).
  • the cooperativity of binding of the [ 125 1] Compound 1, 2, 3, or 4 is determined by the construction of a Hill plot (Hill, "A New Mathematical Treatment of Changes of Ionic Concentrations in Muscle and Nerve Under the Action of Electric Currents, With a Theory to Their Mode of Excitation", J. Physiol . 40:190, 1910) .
  • the dependence of binding on protein (receptor) concentration is determined by resuspending SPMs in buffer A.
  • the assay buffer 200 ⁇ l contains a concentration of [ 12S I] Compound 1, 2, 3, or 4 equal to its K D value and increasing concentrations of protein.
  • the specific binding of [ 125 1] Compound 1, 2, 3, or 4 should be linearly related to the amount of protein (receptor) present.
  • the time course of ligand-receptor binding is determined by resuspending SPMs in buffer A.
  • the assay buffer 300 ⁇ l contains a concentration of [ 12S I] Compound 1, 2, 3, or 4 equal to its K D value and 100 ⁇ g of protein.
  • Triplicate samples are incubated at 32°C for varying lengths of time; the time at which equilibrium is reached is determined, and this time point is routinely used in all subsequent assays.
  • the pharmacology of the binding site can be analyzed by competition experiments. In such experiments, the concentration of [ 125 I] Compound 1, 2, 3, or 4 and the amount of protein are kept constant, while the concentration of test (competing) drug is varied.
  • This assay allows for the determination of an IC 50 and an apparent K D for the competing drug (Cheng and Prusoff, "Relationship Between the Inhibition Constant (K and the Concentration of Inhibitor Which Causes 50 Percent Inhibition (IC 50 ) of an Enzymatic Reaction", J. Biochem . Pharmacol . 22:3099, 1973) .
  • the cooperativity of binding of the competing drug is determined by Hill plot analysis.
  • Specific binding of the [ 125 1] Compound 1, 2, 3, or 4 represents binding to a novel site on the I to channel .
  • peptides related to Compound 1, 2, 3, or 4 should compete with the binding of [ 125 I] Compound 1, 2, 3, or 4 in a competitive fashion, and their potencies in this assay should correlate with their inhibitory potencies in a functional assay of I to block (e.g., inhibition of I co in isolated neural or cardiac cells) .
  • compounds which have activity at the other sites on the I to channel should not displace [ 12S I] Compound 1, 2, 3, or 4 binding in a competitive manner. Rather, complex allosteric modulation of [ 12S I] Compound 1, 2, 3, or 4 binding, indicative of noncompetitive interactions, might be expected to occur.
  • Studies to estimate the dissociation kinetics are performed by measuring the binding of
  • [ 125 I] Compound 1, 2, 3, or 4 after it is allowed to come to equilibrium (see (d) above) , and a large excess of nonradioactive competing drug is added to the reaction mixture. Binding of the [ 12S I] Compound 1, 2, 3, or 4 is then assayed at various time intervals. With this assay, the association and dissociation rates of binding of the [ 12S I] Compound 1, 2, 3, or 4 are determined (Titeler, "Mul tiple Dopa ine Receptors : Receptor Binding Studies in Dopamine Pharmacology", Marcel Dekker, Inc., New York, 1983) . Additional experiments involve varying the reaction temperature (20°C to 37°C) in order to understand the temperature dependence of these parameters .
  • Candidate compounds acting at the Compound 1/Compound 2/Compound 3/Compound 4 binding site are assessed by determining their ability to displace specific binding of [ 125 I] Compound 1, [ 125 1] Compound 2, [ 125 1] Compound 3, [ 125 I] Compound 4 or related peptides labeled with 125 I or other detectable label using techniques known to those skilled in the art, for example, as described below.
  • the following assay can be utilized as a high throughput assay to screen product libraries (e . g. , natural product libraries and compound files at major pharmaceutical companies) to identify new classes of compounds with activity at the Compound l/Compound 2/ Compound 3/Compound 4 binding site on the cardiac I to channel.
  • Cardiac sarcolemmal vesicles are prepared according to the method of Doyle et al . ("Saxitoxin binding and "Fast” Sodium Channel Inhibition in Sheep
  • Cardiac sarcolemmal vesicles are prepared from fresh bovine or other suitable mammalian heart tissue at 0-4°C. The heart is cut into 1 cm 3 pieces, and converted into a paste with a meat grinder.
  • the paste was homogenized in 4 -times its volume of 0.75 M choline Cl buffered to pH 7.4 with 30 mM -2-hydroxyethyl- piperazine-lV' -2- ethanesulfonic acid (HEPES) -15 mM Tris.
  • the homogenization was carried out twice for 30 seconds in 300 ml polypropylene centrifuge jars with a Tekmar T185 shaft. This and all other buffers include the proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, and 1 mM dithiothreitol .
  • the resultant homogenate is centrifuged for 20 minutes at 27,000 x g in the GSA rotor of a Sorvall centrifuge. The supernatant is discarded, and the pellet is resuspended in 10 mM HEPES-5 mM Tris, pH 7.4 and recentrifuged as before. The pellet from this centrifugation is resuspended in 10 mM HEPES-Tris and homogenized three times for 30 seconds each with the T185 shaft of the Tekmar at a setting of 5. The resulting homogenate is centrifuged for 20 minutes in the
  • the membranes are suspended in 50% sucrose, 150 mM KC1, 100 mM TrisCl, and 5 mM Na pyrophosphate . These vesicles are loaded onto the bottom of a four step discontinuous gradient with additional steps at 30%, 21.5%, and 9.5% sucrose. This gradient is centrifuged for 1.5 hour at 193,000 x g in a Beckman 50.2Ti rotor. The pellicle at the 9.5%-21.5% sucrose interface is enriched in surface sarcolemma.
  • Membrane vesicles (20-40 ⁇ g protein) are loaded with 150 mM KCl and diluted 50-fold into 1 ml of binding buffer containing 150 mM KCl. The vesicles are preincubated 5 minutes at 37°C and then incubated for an additional time required for the attainment of equilibration conditions (exact time determined by preliminary experiments) in the presence of varying concentrations of [ 125 I] Compound 1, 2 , 3, or 4 (1 nM-1 ⁇ M) . The binding reaction is terminated by addition of 4 ml of ice cold binding buffer and then rapid filtration over Whatman GF/C filters followed by three additional 4 ml washes with ice cold binding buffer. The radioactivity associated with the filters is determined using standard gamma counting techniques. Specific [ 12S I] Compound 1, 2, 3, or 4 binding is defined as total binding minus binding measured in the presence of 1-10 ⁇ M cold Compound 1, 2, 3, or 4.
  • Example 15 Recombinant Receptor Binding Assay The following is one example of a rapid screening assay for useful compounds of this invention.
  • a cDNA or gene clone encoding the I to channel binding site (receptor) from a suitable organism such as a human is obtained using standard procedures.
  • receptors have been cloned and are known in the art.
  • Distinct fragments of the clone are expressed in an appropriate expression vector to produce the smallest polypeptide (s) obtainable from the receptor which retain the ability to bind Compound 1, 2, 3, or 4.
  • the polypeptide (s) which includes the novel Compound 1/ Compound 2/Compound 3/Compound 4 receptor for these compounds can be identified.
  • Such experiments can be facilitated by utilizing a stably- ransfected mammalian cell line ( e . g. , HEK 293 cells) expressing the I co channel.
  • a stably- ransfected mammalian cell line e . g. , HEK 293 cells
  • Compound 3/Compound 4 receptor can be chemically reacted with chemically modified Compound 1, 2, 3, or 4 in such a way that amino acid residues of the Compound l/Compound 2/Compound 3/Compound 4 peptide receptor which contact (or are adjacent to) the selected compound are modified and thereby identifiable.
  • the fragment (s) of the Compound 1/Compound 2/Compound 3/Compound 4 receptor containing those amino acids which are determined to interact with Compound 1, 2, 3, or 4 and are sufficient for binding to said molecules can then be recombinantly expressed, as described above, using a standard expression vector (s) .
  • the recombinant polypeptide (s) having the desired binding properties can be bound to a solid phase support using standard chemical procedures.
  • This solid phase, or affinity matrix may then be contacted with Compound 1, 2, 3, or 4 to demonstrate that those compounds can bind to the column, and to identify conditions by which the compounds may be removed from the solid phase.
  • This procedure may then be repeated using a large library of compounds to determine those compounds which are able to bind to the affinity matrix, and then can be released in a manner similar to Compound 1, 2, 3, or 4.
  • alternative binding and release conditions may be utilized in order to obtain compounds capable of binding under conditions distinct from those used for Compound 1/ Compound 2/Compound 3/Compound 4 peptide binding (e.g., conditions which better mimic physiological conditions encountered, especially in pathological states) .
  • Those compounds which do bind can thus be selected from a very large collection of compounds present in a liquid medium or extract .
  • native Compound 1, 2, 3, or 4 receptor can be bound to a column or other solid phase support. Those compounds which are not competed off by reagents which bind other sites on the receptor can then be identified. Such compounds define novel binding sites on the receptor. Compounds which are competed off by other known compounds, thus bind to known sites, or bind to novel sites which overlap known binding sites. Regardless, such compounds may be structurally distinct from known compounds and thus may define novel chemical classes of agonists or antagonist which may be useful as therapeutics.
  • Xenopus frogs were anesthetized by immersion in
  • Barth's solution contained (in mM) : 88 NaCl, 1 KCl, 0.4 CaCl 2 , 0.33 Ca(N0 3 ) 2 , 1 MgS0 4 , 2.4 NaHC0 3 , 10 HEPES; pH 7.4.
  • Plasmids containing Kvl.4 and Kv4.2 cDNA were used, as described in Blair et al . , 1991, FEBS Letters 295:211-213 and Po et al . , 1992, Circ . Res . 71:732-736.
  • Templates for cRNA synthesis from channel DNA were prepared as described by Po et al . , 1992, Circ . Res . 71:732-736.
  • Two-microelectrode voltage clamp of oocytes Oocytes were bathed in ND96 solution. This solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES; pH 7.6. Currents were recorded at room temperature (21-23°C) using standard two-microelectrode voltage clamp techniques. Glass microelectrodes were filled with 3 M KCl and their tips broken to obtain tip resistances of 1-3 M ⁇ for the voltage-recording electrode and 0.6-1 M ⁇ for the current-passing electrode. Oocytes were voltage-clamped with an Axoclamp 2A amplifier. Voltage commands were generated using pCLAMP software (ver.
  • the voltage dependence of Kv4.2 inactivation was determined using 8 sec conditioning test pulses, applied once every 30 sec. Each conditioning pulse was applied to a potential ranging from -120 mV to 0 mV, and was followed by a pulse to +40 or +75 mV to monitor the extent of channel inactivation.
  • the voltage- dependence of block of Kv4.2 was determined using a concentration of toxin that decreased current by 50% at a test potential near 0 mV (100 nM for Compound 4 and Compound 1, and 67 nM for Compound 2) (See Figs. 6 and 7) .
  • the voltage-dependence of block was linear for 100 nM Compound 4 and 67 nM Compound 2 (Fig. 6) , but was curvilinear for 100 nM Compound 1 (Fig. 7) .
  • the data for Compound 1 was well described with the sum of a linear component and a sigmoidal component fit with a Boltzmann function. From these data, the voltage at which Kv4.2 was blocked by 50% was determined to be +4 mV for all three compounds .
  • useful compounds of this invention and their pharmaceutically acceptable salts may be used to treat neurological disorders or diseases. While these compounds will typically be used in therapy for human patients, they may also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sports animals and pets such as horses, dogs and cats. In therapeutic and/or diagnostic applications, the compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington ' s
  • salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, calcium edetate, camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide , hydrochloride , hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, aleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate) , pantothenate, phosphate/disphosphate, polygalacturonate, salicylate, stea
  • Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate pamoate (embonate), phosphate, salicylate, succinate, sulfate, or tartrate .
  • the useful compounds of this invention may also be in the form of pharmaceutically- acceptable complexes.
  • Pharmaceutically acceptable complexes are known to those of ordinary skill in the art and include, by way of example but not limitation, 8-chlorotheophyllinate (teoclate) .
  • the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical responses were not adequate (precluding toxicity) .
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • Such agents may be formulated into liquid or solid dosage forms and administered systemically or locally.
  • Techniques for formulation and administration may be found in Remington ' s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. Suitable routes may include oral, buccal, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives.
  • compositions of the present invention in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspension.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid ester, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose (CMC) , and/or polyvinylpyrrolidone (PVP: povidone) .
  • CMC carboxymethylcellulose
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG) , and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs) .
  • PEGs liquid polyethylene glycols
  • stabilizers may be added.
  • the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the amount of various compounds of this invention which should be administered can be determined by standard procedures .

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  • Insects & Arthropods (AREA)
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Abstract

L'invention porte sur de puissants inhibiteurs spécifiques de canal potassium de passage vers l'extérieur ainsi que sur des polypeptides dérivés de venin d'araignée. Elle a également trait à des techniques de criblage d'agents ayant une activité spécifique sur ces canaux potassium ou sur d'autres types de canaux potassium, à des méthodes thérapeutiques à l'encontre de maladies ou d'états pathologiques, consistant à appliquer un agent active sur le canal potassium de passage vers l'extérieur ou d'autres agents actifs sur d'autres canaux potassium, ainsi qu'à des procédés d'utilisation d'un inhibiteur de canal potassium dérivé du venin d'araignée en tant qu'agent insecticide.
PCT/US1997/018710 1996-10-17 1997-10-17 Composes bloquants a canal potassium et leur utilisation WO1998016185A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47589/97A AU4758997A (en) 1996-10-17 1997-10-17 Potassium channel blocking compounds and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73313196A 1996-10-17 1996-10-17
US08/733,131 1996-10-17

Publications (1)

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WO1998016185A2 true WO1998016185A2 (fr) 1998-04-23

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031133A3 (fr) * 1998-11-20 2000-10-05 Millennium Pharm Inc Proteines d'interaction de canaux potassiques (pcip) et procedes de leur utilisation
WO2001079455A1 (fr) * 2000-04-14 2001-10-25 Icagen, Inc. Kv10, un nouveau canal potassique commande par tension a partir de cerveau humain
US6361971B1 (en) 1998-11-20 2002-03-26 Millennium Pharmaceuticals, Inc. Nucleic acid molecules encoding potassium channel interactors and uses therefor
US6369197B1 (en) 1998-11-20 2002-04-09 Millennium Pharmaceuticals, Inc. Potassium channel interactors and uses therefor
WO2002026984A3 (fr) * 2000-09-27 2003-03-13 Millennium Pharm Inc Proteines d'interaction de canaux potassiques (pcip) et leurs procedes d'utilisation
US7078481B1 (en) 1998-11-20 2006-07-18 Wyeth Potassium channel interactors and uses therefor
US7115381B1 (en) 1998-11-20 2006-10-03 Kenneth Rhodes Methods for treating cardiovascular disorders
US7439029B2 (en) 1998-11-20 2008-10-21 Wyeth Human 9q polypeptides method
WO2010052276A2 (fr) * 2008-11-06 2010-05-14 Basf Se Test de criblage ea pour insecticides

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7439029B2 (en) 1998-11-20 2008-10-21 Wyeth Human 9q polypeptides method
US7078481B1 (en) 1998-11-20 2006-07-18 Wyeth Potassium channel interactors and uses therefor
US6361971B1 (en) 1998-11-20 2002-03-26 Millennium Pharmaceuticals, Inc. Nucleic acid molecules encoding potassium channel interactors and uses therefor
US6369197B1 (en) 1998-11-20 2002-04-09 Millennium Pharmaceuticals, Inc. Potassium channel interactors and uses therefor
WO2000031133A3 (fr) * 1998-11-20 2000-10-05 Millennium Pharm Inc Proteines d'interaction de canaux potassiques (pcip) et procedes de leur utilisation
US6689581B1 (en) 1998-11-20 2004-02-10 Wyeth Potassium channel interactors and uses therefor
US7556938B1 (en) 1998-11-20 2009-07-07 Millennium Pharmaceuticals, Inc. Nucleic acids encoding potassium channel interactors
US7041795B2 (en) 1998-11-20 2006-05-09 Millennium Pharmaceuticals, Inc Potassium channel interacting polypeptides and uses thereof
US7115381B1 (en) 1998-11-20 2006-10-03 Kenneth Rhodes Methods for treating cardiovascular disorders
WO2001079455A1 (fr) * 2000-04-14 2001-10-25 Icagen, Inc. Kv10, un nouveau canal potassique commande par tension a partir de cerveau humain
US6727353B2 (en) 2000-04-14 2004-04-27 Icagen, Inc. Nucleic acid encoding Kv10.1, a voltage-gated potassium channel from human brain
US7528231B2 (en) 2000-04-14 2009-05-05 Icagen, Inc. Kv10.1, a novel voltage-gated potassium channel from human brain
WO2002026984A3 (fr) * 2000-09-27 2003-03-13 Millennium Pharm Inc Proteines d'interaction de canaux potassiques (pcip) et leurs procedes d'utilisation
WO2010052276A2 (fr) * 2008-11-06 2010-05-14 Basf Se Test de criblage ea pour insecticides
WO2010052276A3 (fr) * 2008-11-06 2010-07-08 Basf Se Test de criblage ea pour insecticides
JP2012507273A (ja) * 2008-11-06 2012-03-29 ビーエーエスエフ ソシエタス・ヨーロピア 殺虫剤のスクリーニングアッセイ
AU2009312751B2 (en) * 2008-11-06 2015-03-19 Basf Se A screening assay for insecticides
AU2009312751A8 (en) * 2008-11-06 2015-04-09 Basf Se A screening assay for insecticides
AU2009312751B8 (en) * 2008-11-06 2015-04-09 Basf Se A screening assay for insecticides
US9029080B2 (en) 2008-11-06 2015-05-12 Basf Se Screening assay for insecticides

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