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WO1996033206A1 - Peptides de conotoxine - Google Patents

Peptides de conotoxine Download PDF

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Publication number
WO1996033206A1
WO1996033206A1 PCT/US1996/005262 US9605262W WO9633206A1 WO 1996033206 A1 WO1996033206 A1 WO 1996033206A1 US 9605262 W US9605262 W US 9605262W WO 9633206 A1 WO9633206 A1 WO 9633206A1
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WO
WIPO (PCT)
Prior art keywords
cys
conotoxin
peptide
xaa
gly
Prior art date
Application number
PCT/US1996/005262
Other languages
English (en)
Inventor
Ki-Joon Shon
Michelle M. Grilley
Baldomero M. Olivera
Doju Yoshikami
Maren Marsh
Lourdes J. Cruz
David R. Hillyard
Original Assignee
University Of Utah Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/599,556 external-priority patent/US5670622A/en
Priority claimed from US08/624,123 external-priority patent/US5739276A/en
Application filed by University Of Utah Research Foundation filed Critical University Of Utah Research Foundation
Priority to JP8531855A priority Critical patent/JPH11503909A/ja
Priority to AU55502/96A priority patent/AU700592B2/en
Priority to EP96912813A priority patent/EP0821694A4/fr
Publication of WO1996033206A1 publication Critical patent/WO1996033206A1/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
    • 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 relatively short peptides, and more particularly to peptides between about 22 and about 35 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which include three cyclizing disulfide linkages.
  • Mollusks of the genus Conus produce a highly toxic venom which enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom which is injected by means of a highly specialized venom apparatus, a disposable hollow tooth which functions both in the manner of a harpoon and a hypodermic needle.
  • Venom may be used as a primary weapon to capture prey or as a defense mechanism. These venoms disrupt essential organ systems in the envenomated animal, and many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
  • the predatory cone snails have developed a unique biological strategy.
  • Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid- encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
  • the cone snails that produce these toxic peptides which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species.
  • the major paralytic peptides in these fish-hunting cone venoms were the first to be identified and characterized.
  • C geographus venom three classes of disulfide-rich peptides were found: the ⁇ -conotoxin peptides (which target and block the nicotinic acetylcholine receptors); the ⁇ - conotoxin peptides (which target and block the skeletal muscle Na + channels); and the ⁇ -conotoxin peptides (which target and block the presynaptic neuronal Ca "+ channels).
  • ⁇ -conotoxin peptides which target and block the nicotinic acetylcholine receptors
  • the ⁇ - conotoxin peptides which target and block the skeletal muscle Na + channels
  • the ⁇ -conotoxin peptides which target and block the presynaptic neuronal Ca "+ channels.
  • there are multiple homologs in each toxin class for example,
  • ⁇ -Conotoxin peptides because of their ability to preferentially block muscle but not axonal Na channels, are convenient tools for immobilizing skeletal muscle without affecting axonal or synaptic events, ⁇ - Conotoxin peptides have become standard pharmacological reagents for investigating voltage- sensitive Ca 2+ channels and are used to block presynaptic termini and neurotransmitter release.
  • conotoxin peptides have also found utility in screening newly isolated conotoxin peptides or analogs for medical purposes (Miljanich et al., 1993).
  • Site I is the classical binding site for channel blockers. notably the guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX); this site is generally postulated to be at the extracellular end of the channel pore. Only one family of polypeptide toxins, the ⁇ -conotoxins, has been shown to act at this site and functionally affect voltage-gated sodium currents. These were originally isolated from the venom of the marine snail Conus geographus (Stone and Gray, 1982; Sato et al.. 1983; Cruz et al., 1985; and Olivera et al., 1985).
  • the C-terminus may be free or amidated. This conotoxin is vertebrate-specific and targets voltage- sensitive Na channels.
  • a second aspect of the present invention describes a new member of the ⁇ -conotoxin peptide family - ⁇ -conotoxin PI ⁇ A from Conus purpurascens, an Eastern Pacific fish-hunting species.
  • the new ⁇ -conotoxin shows considerable sequence divergence from the ⁇ -conotoxins of Conus geographus.
  • electrophysiological and binding data which demonstrate that ⁇ -conotoxin P ⁇ IA is a powerful pharmacological tool for distinguishing among different tetrodotoxin-sensitive Na + channel subtypes.
  • the tetrodotoxin-sensitive sodium channels can now be resolved into three categories: 1 ) sensitive to ⁇ -PniA and ⁇ -conotoxin GI ⁇ A; 2) sensitive to ⁇ -PniA but not to ⁇ -Gni ⁇ ; and 3) sensitive to neither ⁇ -conotoxin (examples are skeletal muscle, rat brain Type II and motor axon subtypes, respectively).
  • ⁇ -conotoxin PIIIA is a key tool for distinguishing among different sodium channel subtypes.
  • One aspect of the present invention is directed to conotoxin peptides having 25-35 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines. respectively.
  • the invention is directed to ⁇ -conotoxin PVIA having the formula Glu-Ala-Cys-Tyr-Ala-Xaa 1 -Gly-Thr-Phe-Cys-Gly-Ile-Lys-Xaa 2 -Gly-Leu-Cys-Cys- Ser-Glu-Phe-Cys-Leu-Pro-Gly-Val-Cys-Pro-Gly (Xaa ! or Xaa 2 is Pro or 4-trans- hydroxyproline) (SEQ ID NO: 1).
  • the C-terminus may be free or amidated.
  • This conotoxin is vertebrate-specific and targets voltage-sensitive Na channels.
  • a second aspect of the present invention is directed to conotoxin peptides having 22 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines. respectively.
  • the invention is directed to ⁇ -conotoxin PIIIA having the formula Xaa r Arg-Leu-Cys-Cys-Gly-Phe-Xaa 2 -Lys-Ser-Cys-Arg-Ser-Arg-Gln-Cys-Lys-Xaa 2 - His-Arg-Cys-Cys (SEQ ID NO:2) where Xaa] represents pyroglutamate or glutamine and Xaa 2 represents 4-trans-hydroxyproline or proline.
  • This peptide targets sodium channels.
  • Figure 1 shows the nucleic acid sequence and predicted amino acid sequence of a cDNA clone encoding the lockjaw peptide precursor and a comparison of the lockjaw peptide to two other related Conus peptide precursor sequences.
  • the sequences labeled 1, 2 and 3 in this figure are precursors of ⁇ -conotoxin GVIA, ⁇ -conotoxin TxVIa and ⁇ -conotoxin PVIA respectively.
  • Figure 2A shows the nucleic acid sequence derived by analyzing cDNA clones from a Conus purpurascens venom duct library.
  • the sequence encoding the inferred C-terminal end of the open reading frame is shown; mature conotoxins are always encoded at the C-terminus of the precursor sequence.
  • the pattern of Cys residues suggested that the encoded C-terminal peptide might be a ⁇ - conotoxin.
  • the arrow indicates the predicted site of proteolytic cleavage to generate the mature toxin.
  • a -Lys-Arg- sequence is the most common motif for proteolytic cleavage of conotoxin precursors.
  • Figure 2B shows the predicted sequence of the post-translationally processed peptide.
  • the amino acid sequence shown in Figure 2A would be predicted to be post-translationally processed at the four indicated sites as follows: proline would be hydroxylated to 4-trans-hydroxyproline (sites)
  • the C-terminal -Cys-Gly-Arg- sequence would be processed by an exopeptidase and amidation enzymes to a -Cys-NH 2 moiety (site 4); and after proteolysis the encoded glutamine residue would be converted to pyroglutamate (site 1).
  • FIG 3A is a sketch of electrophysiological recording chamber for testing toxin on frog cutaneus pectoris muscle's response to direct electrical stimulation.
  • a rectangular Sylgard trough was partitioned into four compartments (A through D) by three Mylar sheets (1-3). Mylar sheets were inserted into slots in the wall of the trough after the muscle had been pinned to the floor of the trough.
  • the cutaneus end of the muscle was in A and the episternum (cartilage) was in D.
  • Stimulating electrodes were in A and B (i.e., stimulation was across partition 1).
  • a ground electrode was in B.
  • Recording electrodes were in C and D (with electrode in D leading to the "+ " input of the preamp). Compartment D served as the test chamber - only it was exposed to toxin.
  • Figure 3C shows the time course of block of directly-evoked action potentials. Maximum amplitudes of the positive phase (open circles) and negative phase (closed circles) of the response are plotted as a function of time. Solution in compartment D was replaced with 1 ⁇ M PIIIA at time 0 (downward arrow), and the toxin was washed out 23 minutes later (upward arrow).
  • Figure 4 shows the results of binding competition experiments with [ 3 H]saxitoxin (SXT). Specific binding was determined by subtracting nonspecific binding of [ HJsaxitoxin from total binding; the nonspecific binding was measured by using 12 ⁇ M tetrodotoxin (TTX) to displace [ H]saxitoxin binding. Open circles, ⁇ -PuiA displacement for eel electroplax sites; squares. ⁇ -PniA displacement for rat brain sites: triangles, ⁇ -GniA displacement for rat brain sites
  • Figures 5A, 5B and 5C show that ⁇ -PniA blocks rat type II Na + channel expressed in Xenopus oocytes.
  • Figure 5 A shows whole cell current recorded from an oocyte expressing rat type II Na + channels. Voltage steps ranging from -80 mV to +60 mV, in 10 mV increments, were generated from a holding potential of -100 mV.
  • Figure 5B shows results (profound block of the currents) when 2 mM PIIIA was added to the bath solution.
  • Figure 5C shows the results following a wash with Normal Frog Ringers solution.
  • the present invention is directed to conotoxin peptides having 22-35 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines. respectively, and to the precursors of these peptides.
  • One aspect of the invention is directed to ⁇ -conotoxin PVIA having the formula Glu-Ala-Cys-Tyr-Ala-Xaa,-Gly- Thr-Phe-Cys-Gly-Ile-Lys-Xaa 2 -Gly-Leu-Cys-Cys-Ser-Glu-Phe-Cys-Leu-Pro-Gly-Val-Cys-Pro- Gly (Xaa, or Xaa 2 is Pro or 4-tr ⁇ HS-hydroxyproline) (SEQ ID NO:l).
  • the C-terminus may be free or amidated.
  • This conotoxin is vertebrate-specific and targets voltage-sensitive Na channels and is useful as an active agent for muscle contraction in instances where lack of muscle contraction is problematic, such as for treating urinary or fecal incontinence.
  • This conotoxin is also useful for tagging tumors since it is able, as are other conotoxins, to detect antibodies which form against tumors. Since conotoxins bind to cell surface receptors, ion channels, they are capable of inhibiting tumor growth and are useful as anti-neoplastic agents.
  • ⁇ -conotoxins inhibit voltage-gated Ca channels, distinguishing various subtypes.
  • the ⁇ -conotoxins are without effect on Ca ⁇ + channels; the results shown below demonstrate that that they do not compete for binding with ⁇ -conotoxin GVIA and do not induce the shaking syndrome in mice characteristic of the ⁇ -conotoxins.
  • Biologically active ⁇ -conotoxin GmVIA has been chemically synthesized, demonstrating that the biological activity is not due to contaminants.
  • a different family of Conus peptides, the ⁇ - conotoxins is known which also affects Na + channels.
  • these have a different disulfide framework, are channel blockers specific for the muscle subtype, and, like the ⁇ -conotoxins, are highly basic molecules. Given the very different chemical character of ⁇ -conotoxins, it is likely that their site of action on the Na + channel is quite distinct.
  • ⁇ -Conotoxin PVIA (sometimes called herein the lockjaw peptide), present in the venom of the fish hunting snail C purpurascens, is the first biochemically characterized toxin shown to underlie such symptoms, as well as to increase excitability at the vertebrate neuromuscular junction.
  • lockjaw peptide is a vertebrate- targeted ⁇ -conotoxin.
  • the C purpurascens lockjaw peptide was inactive in the molluscan test system at doses 100-fold higher than required to potently affect both fish and mice.
  • ⁇ -conotoxin TxVIA which potently potentiates molluscan Na channels showed no biological activity in any assays involving vertebrate systems.
  • ⁇ -conotoxin TxVIA and the lockjaw peptide competed for the same binding site in rat brain membranes.
  • ⁇ -Conotoxin TxVIA binds specifically and with high affinity to voltage-sensitive sodium channels in the mammalian central nervous system, even though it has no inhibitory effect (Fainzilber et al., 1994).
  • the primary structure of the lockjaw peptide, the predicted amino acid sequence of the precursor, the electrophysiological results using the frog neuromuscular junction, the binding data, and the in vivo symptoms induced by the peptide are consistent with the conclusion that the lockjaw peptide is a vertebrate-targeted ⁇ -conotoxin.
  • ⁇ - conotoxin PVIA had clear effects on this mammalian channel.
  • the toxin inhibited channel inactivation.
  • a persistent conductance was observed in the presence of this peptide. That is, when voltage is brought from a resting potential to 0 mV and held there for 18 mseconds, virtually no conductance remains in the control, whereas a residual Na conductance remains in the presence of ⁇ -conotoxin PVIA.
  • the cloning data described below demonstrate that the toxin precursor must have an amidated C-terminus.
  • the amidated lockjaw peptide is designated as ⁇ -conotoxin PVIA and the form of the peptide with the free carboxyl terminus as [deamido]- ⁇ -conotoxin PVIA.
  • Conus striatus another fish-hunting Conus species, Conus striatus, has a toxin which apparently acts by the same physiological mechanism.
  • a second aspect of the invention is directed to ⁇ -conotoxin PIIIA having the formula Xaa,- Arg-Leu-Cys-Cys-Gly-Phe-Xaa 2 -Lys-Ser-Cys-Arg-Ser-Arg-Gln-Cys-Lys-Xaa 2 -His-Arg-Cys-Cys (SEQ ID NO:2) where Xaa ( represents pyroglutamate or glutamine and Xaa 2 represents 4-trans- hydroxyproline or proline and is also directed to precursors of these peptides and nucleic acids encoding these peptides.
  • This peptide is a sodium channel blocker and is useful as an active agent for muscle contraction in instances where lack of muscle contraction is problematic, such as for treating urinary or fecal incontinence. It is also useful as an active agent for anti-seizures, e.g.. an anti-epileptic. Further, this conotoxin may also be useful for tagging tumors since they are able, as are other conotoxins, to detect antibodies which form against tumors. Since conotoxins bind to cell surface receptors, ion channels, they are capable of inhibiting tumor growth and are useful as anti-neoplastic agents.
  • the conotoxin peptides of the present invention can be obtained by purification from cone snails, because the amounts of conotoxin peptides obtainable from individual snails are very small, the desired substantially pure conotoxin peptides are best practically obtained in commercially valuable amounts by chemical synthesis.
  • the yield from a single cone snail may be about 10 micrograms or less of conotoxin peptide.
  • substantially pure is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% by weight and preferably at least about 95% of such biological molecules of the same type which are present (i.e., water, buffers and innocuous small molecules may be present).
  • Chemical synthesis of biologically active conotoxin peptides depends of course upon correct determination of the amino acid sequence.
  • conotoxin peptides can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1979). The peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds.
  • One method of forming disulfide bonds in the conotoxin peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides.
  • the oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined.
  • HPLC reverse-phase high performance liquid chromatography
  • a second method of forming the disulfide bonds in the conotoxin peptides of the present invention involves the use of acetamidomethyl (Acm) as protection agent on the second and fifth cysteines during the synthesis of the conotoxin peptides.
  • the peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
  • a suitable method such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
  • the employment of recently developed recombinant DNA techniques may be used to prepare these peptides, particularly the longer ones containing only natural amino acid residues which do not require post-translational processing steps.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • N-protecting groups e.g., dicyclohexylcarbodiimide or carbonyldimidazole
  • active esters e.g., esters of N- hydroxyphthalimide or N-hydroxy-succinimide
  • cleavage reagents to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology.
  • Classical solution synthesis is described in detail in the treatise.
  • the protecting group preferably retains its protecting properties and is not split off under coupling conditions
  • the protecting group should be stable under the reaction conditions selected for removing the ⁇ -amino protecting group at each step of the synthesis
  • the side chin protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
  • Solid-phase synthesis is commenced from the C- terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin.
  • a suitable resin can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine
  • BHA resin paramethylbenzhydrylamine (MBHA) resin
  • MBHA resin supports Preparation of the hydroxymethyl resin is described by Bodansky et al., (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, CA) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart et al. (1969). BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus.
  • solid resin supports may be any of those known in the art, such as one having the formulae -O-CH 2 -resin support. -NH BHA resin support, or -NH- MBHA resin support.
  • the C-terminal amino acid, protected by Boc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in K. Horiki et al. (1978), using KF in DMF at about 60°C for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized.
  • the ⁇ -amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.
  • TFA trifluoroacetic acid
  • the deprotection is carried out at a temperature between about 0°C and room temperature.
  • Other standard cleaving reagents, such as HCI in dioxane, and conditions for removal of specific ⁇ -amino protecting groups may be used as described in Schroder & Lubke (1965).
  • the remaining ⁇ -amino- and side chain- protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor.
  • activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
  • suitable activating reagents are carbodiimides, such as N,N'- diisopropylcarbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
  • Other activating reagents and their use in peptide coupling are described by Schroder & Lubke (1965) and Kapoor (1970).
  • Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH 2 C1 2 ( 1 : 1 ) or in DMF or CH 2 C1 2 alone.
  • DMF dimethylformamide
  • the coupling procedure is repeated before removal of the ⁇ -amino protecting group prior to the coupling of the next amino acid.
  • the success of the coupling reaction at each stage of the synthesis if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970).
  • Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. ( 1978).
  • the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the ⁇ -amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid.
  • a reagent such as liquid hydrogen fluoride
  • the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation.
  • one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel. Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptidoresin, to create bonds between Cys residues.
  • fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected.
  • deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0°C with hydrofluoric acid (HF). followed by oxidation as described above.
  • HF hydrofluoric acid
  • ⁇ -Conotoxin PIIIA appears to target a wider spectrum of mammalian voltage-gated sodium channel subtypes than does ⁇ -GniA.
  • ⁇ -Conotoxin PIIIA was able to displace a larger fraction of specific [ HJSTX binding to high affinity rat brain sites than could ⁇ -GniA.
  • voltage-gated sodium channels are primarily distinguished in situ by their tetrodotoxin sensitivity or insensitivity.
  • the discovery and characterization of ⁇ -conotoxin PIIIA described above provides the basis for dividing tetrodotoxin-sensitive sodium channels into three categories distinguishable by their sensitivity to ⁇ -conotoxins:
  • This category would be the voltage-gated sodium channels present in motor axons. which are resistant to both ⁇ -conotoxins.
  • the binding data indicate that a major fraction of the total CNS channels may fall into this category.
  • the results indicate that the subtype of voltage-gated sodium channels present in motor axons must be distinct from the Type II sodium channels present in the mammalian central nervous system.
  • ⁇ -conotoxin PIIIA The discovery of ⁇ -conotoxin PIIIA is also indicative that the ⁇ -conotoxin peptide family may be broadly distributed in Conus species. Different ⁇ -conotoxin sequence variants found in the about 500 species of Conus may be expected to exhibit different affinities for the various voltage- gated sodium channel subtypes. The situation is somewhat analogous to that found for the ⁇ - conotoxins, where the subtype specificity of different ⁇ -conotoxin peptides has been used to advantage to investigate the functional roles of different Ca 2+ channel subtypes. Similarly, the ⁇ - conotoxins should prove useful for dissecting the role of an individual Na + channel subtype in a neuron, circuit or event slice preparation, particularly in those situations when multiple molecular forms of voltage-gated Na channels are present.
  • Milked Venom Extraction C purpurascens specimens were collected from the Gulf of California, and venom was collected by a milking procedure. Milked venom (0.5 ml) was pooled from 50 Eppendorf tubes stored in a -70°C deep freezer. The pooled venom was kept over ice and diluted with 10 mL of 0.1% TFA in water. The solution was spun for a few minutes using a bench-top microfuge, and the supernatant was immediately subjected to purification. There were several prominent components of the venom; when these were tested for biological activity, several of the major peaks caused flaccid paralysis if fish.
  • the peptides were eluted with a linear gradient of 1 % buffer B increase per minute.
  • the C ) g analytical column was also used for purifying alkylated peptides for amino acid sequence analysis. The major components which elicit these symptoms were purified to apparent homogeneity.
  • the protected peptide resin was built using standard fmoc chemistry, couplings being carried out with equimolar amounts of amino acid derivative, DCC, and HOBT. All amino acids were purchased from Bachem (Torrence, CA), and the side chains were protected as follows: Hyp (t-Bu), Lys (boc). Ser (t-Bu), Tyr (t-Bu), Glu (t-Bu), and Thr (t-Bu). Cys residues 3, 17, 18 and 27 were protected by trt, while Cys residues 10 and 22 were protected by acm.
  • the terminal fmoc group was removed by standard treatment with piperidine NMP (20% by volume).
  • Peptide was removed from the resin by treatment for 2 h at 20°C with TFA/H 2 O/ethanedithiol/phenol/thioanisole (90/5/2.5/7.5/5 by volume), and the whole mixture was filtered rapidly into tert-butyl methyl ether at -10°C.
  • the three isomers were purified using a reversed-phase HPLC preparative column with a gradient of acetonitrile (27-50%) in 0.1% TFA and a flow rate of 20 mL/min.
  • One of the three isomers gave native-like material after oxidation with 1 mM I 2 in 10% TFA and acetonitrile (5 min at room temperature, followed by a quench with 30 mM ascorbic acid).
  • both the free and amidated forms of the peptide were synthesized and folded as described herein. Both synthetic peptides provided to be biologically active, the same in vivo symptomatology was induced by the synthetic forms and the native peptide purified from venom. The earlier eluting native lockjaw peptide peak exhibits the same retention time as the synthetic amidated peptide.
  • a cDNA clone encoding the lockjaw peptide was purified from a library of C. purpurascens ⁇ - and ⁇ -conotoxin cDNAs using the lockjaw-specific oligonucleotide DHOG 538 (5' GARGCNTGYTAYGCNCC 3'; SEQ ID NO:3) as probe (Colledge et al., 1992).
  • the library was constructed in the pUC vector ptzl 8u by cloning PCR amplicons generated using reversed- transcribed C. purpurascens venom duct RNA (AMV reverse transcriptase. Boehringer Mannheim) and an oligonucleotide A corresponding to the signal sequence of ⁇ - and ⁇ - conotoxins and oligonucleotide poly(dT) as primers. Putative clones were sequenced using the
  • the nucleotide sequence of the clone and the inferred amino acid sequence for ⁇ - conotoxin PVIA (lockjaw peptide) as shown in Figure 1 are set forth in SEQ ID NO:4 and SEQ ID NO:5.
  • the open reading frame encodes a typical signal sequence, with the prepropeptide organization found in other conotoxins (Olivera et al., 1990).
  • the precursor sequence has a typical conotoxin proteolytic cleavage site (Colledge et al., 1992; Woodward et al., 1990).
  • the predicted mature toxin sequence is identical to the peptide purified from venom, with a C- terminal amide group.
  • the C-terminal -Cys-Phe-Gly-Gly-OH sequence (SEQ ID NO:6) of the precursor would be processed to a -Cys-Phe-Gly-NH 2 sequence by peptidylglycine ⁇ -amidating monooxygenase (Bradbury et al., 1982; Murthy et al., 1987).
  • Radiolabeling of Conotoxins Iodination of ⁇ -conotoxin TxVIA was carried out using the water-soluble reagent chloramine T. Two nanomoles of ⁇ -conotoxin TxVIA dissolved in 50% acetonitrile in water was incubated for 10 min at room temperature with 2 nmol Na "" 1 ( 1.1 mCi/nmol) and 10 nmol of chloramine T in 200 mM Tris (pH 8.6). The reaction was quenched with 50 ⁇ L of 500 mM ascorbic acid and 50 ⁇ L of 200 mM methionine, and the solution was gently extracted twice with 500 ⁇ L of diethyl ether.
  • the monoiodinated TxVIA eluted shortly after the unmodified ⁇ -conotoxin TxVIA at approximately 56% acetonitrile on a linear gradient of acetonitrile (36-63%).
  • the label was stored as a HPLC effluent at -20°C with 57 mM methionine and centrifuged before use in binding assays.
  • ⁇ -Conotoxin GVIA was labeled by resuspending 10 nmol of peptide in 0.1% TFA, adding an equal volume of 0.25 M Tris-HCl, pH 7.0, and incubating with an equivalent amount of chloramine T and 4 nmol of Na 5 I (2.2 mCi/nmol) for 10 min at room temperature.
  • the [ D I] ⁇ -GVIA was purified by HPLC as previously described (Cruz and Olivera, 1986; Cruz et al., 1987).
  • purpurascens peptide exhibits an even greater similarity to the precursor sequence of a previously characterized peptide from a snail-hunting Conus venom, ⁇ -conotoxin TxVIA (Woodward et al.. 1990).
  • the ⁇ -conotoxins were previously shown to bind specifically to voltage sensitive Na channels (Fainzilber et al., 1994), causing a delay in channel inactivation resulting in an increase in Na conductance (Hasson et al., 1993; Shon et al., 1994).
  • the sequence homology in Figure 1 strongly suggested that the lockjaw peptide might be a member of the ⁇ -conotoxin family.
  • the peptide is a potent excitotoxin in mammals, a result consistent with Na channel-targeted ligand, which increases conductance, rather than a calcium channel blocker of the ⁇ -conotoxin class.
  • the peptide elicited spurts of rapid swimming, with twisted motions, quivering fins, and the lockjaw extended mouth syndrome. Rigid paralysis and death were observed if 0.5- 5.0 nmol was injected.
  • the method involves preparing cDNA libraries and screening these with mixed oligonucleotides in a manner similar to that described by Hillyard et al. (1992).
  • a cDNA library was prepared using mRNA from the venom duct of C purpurascens as previously described (Woodward et al., 1990;
  • the predicted 22-residue peptide, including post-translational modifications modeled on other related peptides was chemically synthesized.
  • the post-translational modifications include changing Glnl to pyroglutamate, prolines to hydroxyproline, the C-terminal Cys-Cys-Gly-Arg (SEQ ID NO: 7) to Cys-Cys-NH 2 .
  • the resulting peptide, with the disulfide bonding indicated, is referred to as ⁇ -conotoxin PIIIA ( ⁇ -P ⁇ iA) based on the physiological evidence detailed below.
  • the structure is:
  • the peptide was built in two stages based on the linear sequence predicted from the cDNA isolate.
  • the protected peptide resin minus the N-terminal pyroglutamate was built by standard Fmoc chemistry on an ABI model 477A peptide synthesizer. Pyroglutamate was then added manually to some of the resin to produce the complete peptide.
  • the linear peptides [1-22] and [2-22 J) were purified by preparative reversed phase HPLC. Disulfide bridges were allowed to form in the presence of a glutathione redox buffer, and the products were again fractionated by preparative HPLC. The major oxidation products in each case were obtained in highly purified form. Peptide bond coupling was carried out with equimolar amounts of amino acid derivative.
  • the side chain Fmoc-protected amino acids were purchased from Bachem (Torrance, CA): these are Hyp (t-Bu), Lys (Boc), Ser (t-Bu), Arg (pmc), Gin (trt), His (tit) and Cys (trt).
  • pyroglutamic acid was manually coupled to the peptide resin. Pyroglutamic acid (0.25 mmol; Sigma) was activated in 1 ml solution of 1 M DICC/1 M HOBT in
  • the pellet was dissolved in 60% acetonitrile containing 0.1% TFA, and purified by reversed phase HPLC. Several runs were required on both preparative and semi-preparative columns to obtain pure linear peptide.
  • the glutathione oxidation protocol previously described was used to oxidize the linear peptide.
  • the major peak from overnight oxidation was repurified on both preparative and semi-preparative columns.
  • the peptide solution (5-10 nmol) in sodium phosphate buffer (0.25 M, pH 7.5) (about 0.4 ml) was incubated with an equal volume of 2 mM I 2 dissolved in methanol, for 10 minutes at ambient temperature.
  • the reaction mixture was quenched with ascorbic acid, and then subjected to reversed phase HPLC. With this incubation time, most of the product was the di-iodinated peptide.
  • Disulfide bridge analysis was carried out on two analogs (the peptide without an N-terminal pyroglutamate, and the same peptide with the His residue di-iodinated).
  • the disulfide connectivity of ⁇ -P ⁇ iA[2-22] was analyzed by the partial reduction strategy of Gray (Gray, 1993), and found to be the same as that of the known ⁇ -conotoxins from C. geographus.
  • Gray Gray
  • Partial reduction with TCEP gave intermediates that were not well resolved from fully reduced or fully oxidized peptides. and only one suitable product could be isolated.
  • FIG. 3A A sketch of the recording chamber is shown in Figure 3A. Current was injected into the muscle across partition 1 ; the recording electrodes monitored the potential across partition 3. Partition 2 served to electrically isolate the recording from the stimulating electrodes. Toxin was added only into the compartment D.
  • the cutaneus pectoris muscle from ⁇ 7 cm Rana pipiens frogs was used.
  • the muscle was trimmed longitudinally so that only the lateral one-quarter of muscle remained (cf. Yoshikami et al., 1989).
  • the trimmed muscle was pinned flat on the floor of a shallow trough ( ⁇ 4 mm x 16 mm x 1 mm deep) fabricated from Sylgard (a silicone elastomer, Dow Chemical Co.).
  • Sylgard a silicone elastomer, Dow Chemical Co.
  • the trough had four transverse slits cut into its wall by a razor blade (see Figure 3A).
  • the trough could be partitioned into four compartments by inserting a 0.1 mm thick Mylar sheet into each slot.
  • the stimulating electrodes were connected to a stimulus isolation unit, and supramaximal, 1 ms-long rectangular pulses were used to directly elicit action potentials in the muscle. Stimuli were applied at a frequency of 1 /minute or less.
  • a positive response was recorded by the preamplifier, and the further propagation of the action potential into chamber D was registered by the preamplifier as a negative response.
  • the extracellularly recorded action potential from the population of fibers in the muscle was recorded as a biphasic response, with the phases separated from each other by only a few milliseconds (see Figure 3B).
  • the plain Ringer ' s solution in chamber D was replaced by one containing toxin.
  • FIG. 3B A control response before toxin addition is shown in Figure 3B.
  • the progression of the action potential between segments C and D is readily apparent; the biphasic waveform generated represents propagation of the action potential from C to D.
  • the peaks of the responses as a function of time before, during, and after toxin addition are shown in Figure 3C.
  • the action potential clearly propagated into segment C, causing the voltage change characteristic of the first half of the biphasic waveform in Figure 3B; however, the negative phase was completely abolished, indicating that although a normal action potential was generated, transmission in segment D of the muscle was abolished.
  • the initial rising phase of the positive phase is also slightly delayed following exposure to toxin; this is thought to be due to leakage of the toxin into compartment C with an attendant decrease in the propagation velocity of the action potential in that compartment. Leak of toxin into compartment C is also thought to be responsible for the decrement in the amplitude of the positive phase as well as delayed time to peak observed in the response taken >4.5 hours later.
  • Nerve-muscle preparations were also examined; when the motor nerve was electrically stimulated, a muscle action potential was recorded and a muscle twitch observed. When the entire nerve-muscle preparation was exposed to toxin, muscle twitching and action potentials were completely abolished; however, excitatory post-synaptic responses were still recorded (results not shown). Thus, propagation of action potentials in the motor axon is not blocked, unlike action potential propagation in muscle.
  • homogenizing buffer used was 10 mM HEPES-Tris, 10 mM EDTA, 10 mM EGTA, 1 mM PMSF, 1 ⁇ M leupeptin and pepstatin, pH 7.0.
  • ⁇ -conotoxin PIIIA displaces [ 3 H]saxitoxin binding to electric organ membranes. which contain a high density of the skeletal muscle subtype of voltage-gated sodium channels.
  • ⁇ -conotoxin PIIIA has a high affinity (K D - 3 x 10 "9 M) for the saxitoxin binding site in the electric organ.
  • the vitelline membranes of the oocytes were removed mechanically with fine forceps and currents were recorded 2-6 days after injection under two-electrode voltage clamp control with a Turbo-Tec amplifier (NPI Elekronik, Tamm, Germany) driven by the Pulse+PulseFit software package (HEKA Electronik, Lambrecht, Germany).
  • Intracellular electrodes were filled with 2 M KC1 and had a resistance between 0.6 and 0.8 M ⁇ . Current records were low-pass filtered at 3 kHz and sampled at 10 kHz.
  • the bath solution was normal frogs Ringer's (NFR) containing (in mM): 115 NaCl, 2.5 KC1, 1.8 CaCl 2 , 10 Hepes pH 7.2 (NaOH). Leak and capacitive currents were corrected on-line by using a P/n method. Toxin solution was prepared in NFR added to the bath chamber.
  • Type II voltage-gated sodium channels were previously shown to be TTX-sensitive but resistant to 1-2 ⁇ M of ⁇ -conotoxin GIIIA (Terlau et al., 1996; Noda et al., 1986).
  • ⁇ -PniA blocked type II Na + channels from rat expressed in Xenopus oocytes (Noda et al., 1986); the presence of ⁇ - PIIIA (2 ⁇ M) in the bath solution abolished nearly all Na + currents (Figure 5B), but in a reversible manner (Figure 5C).
  • rat brain Type II Na + channels apparently belong to the TTX- and ⁇ - PiiiA-sensitive, but ⁇ -GniA resistant class of Na + channels in the mammalian CNS.
  • ADDRESSEE Venable, Baetjer, Howard & Civiletti, LLP
  • NAME Saxe, Stephen A.
  • ORGANISM Conus purpurascens ( ix ) FEATURE :
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO

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Abstract

Peptides de conotoxine, en particulier de δ-conotoxine PVIA et de ν-conotoxine PIIIA. La δ-conotoxine PVIA se trouve dans une espèce de mollusque conidé vivant dans le Pacifique est, chassant le poisson, appelée Conus purpurascens. Cette substance est composée de 29 restes d'acides aminés de la séquence Glu-Ala-Cys-Tyr-Ala-Xaa1-Gly-Thr-Phe-Cys-Gly-Ile-Lys-Xaa2-Gly-Leu-Cys-Cys-Ser-Glu-Phe-Cys-Leu-Pro-Gly-Val-Cys-Pro-Gly (SEQ ID No. 1), où Xaa1 ou Xaa2 sont Pro ou 4-trans-hydroxyproline. La terminaison C peut être libre ou munie d'un amide. La δ-conotoxine PVIA est spécifique aux vertégrés et cible des canaux Na+ sensibles à la tension. La ν-conotoxine PIIIA se trouve également dans le Conus purpurascens. Elle consiste en 22 restes d'acides aminés de la séquence Xaa¿1?-Arg-Leu-Cys-Cys-Gly-Phe-Xaa2-Lys-Ser-Cys-Arg-Ser-Arg-Gln-Cys-Lys-Xaa2-His-Arg-Cys-Cys (SEQ ID No. 2) où Xaa1 représente pyroglutamate ou glutamine et Xaa2 représente 4-trans-hydroxyproline ou proline. Ce dernier peptide cible les canaux de sodium.
PCT/US1996/005262 1995-04-17 1996-04-17 Peptides de conotoxine WO1996033206A1 (fr)

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EP96912813A EP0821694A4 (fr) 1995-04-17 1996-04-17 Peptides de conotoxine

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US42356195A 1995-04-17 1995-04-17
US08/599,556 US5670622A (en) 1996-02-15 1996-02-15 Conotoxin peptide PIIIA
US08/599,556 1996-03-29
US08/624,123 1996-03-29
US08/423,561 1996-03-29
US08/624,123 US5739276A (en) 1994-10-07 1996-03-29 Conotoxin peptides

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Cited By (8)

* Cited by examiner, † Cited by third party
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EP1129106A4 (fr) * 1998-09-14 2002-07-03 Univ Queensland Peptides de conotoxines cyclises
EP0948526A4 (fr) * 1996-11-08 2003-01-02 Univ Utah Res Found Peptides contryphanes
WO2004066990A3 (fr) * 2003-01-30 2004-11-04 Dynogen Pharmaceuticals Inc Methodes destinees a traiter les troubles du tractus urinaire inferieur au moyen de modulateurs du canal sodique
US7084116B2 (en) 2003-03-10 2006-08-01 Dynogen Pharmaceuticals, Inc. Methods for treating lower urinary tract disorders and the related disorders vulvodynia and vulvar vestibulitis using Cav2.2 subunit calcium channel modulators
US7125848B2 (en) 2003-06-13 2006-10-24 Dynogen Pharmaceuticals, Inc. Methods of treating non-inflammatory gastrointestinal tract disorders using Cav2.2 subunit calcium channel modulators
US7223754B2 (en) 2003-03-10 2007-05-29 Dynogen Pharmaceuticals, Inc. Thiazolidinone, oxazolidinone, and imidazolone derivatives for treating lower urinary tract and related disorders
US8226537B2 (en) 2008-09-12 2012-07-24 Terumo Bct, Inc. Blood processing apparatus with cell separation chamber with baffles
US8722859B2 (en) 2000-04-11 2014-05-13 Genentech, Inc. Multivalent antibodies and uses therefor

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AU5474200A (en) * 1999-06-10 2001-01-02 Cognetix, Inc. Muo-conopeptides and their use as local anesthetics

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US5432155A (en) * 1993-06-29 1995-07-11 The Salk Institute For Biological Studies Conotoxins I

Patent Citations (1)

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US5432155A (en) * 1993-06-29 1995-07-11 The Salk Institute For Biological Studies Conotoxins I

Non-Patent Citations (5)

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Title
BIOCHEMISTRY, 18 April 1995, Volume 34, Number 15, SHON et al., "Purification, Characterization, Synthesis and Cloning of the Lockjaw Peptide from Conus Purpurascens Venom", pages 4913-4918. *
BIOCHEMISTRY, 1994, Volume 33, Number 38, SHON et al., "Delta-Conotoxin GmVIA, a Novel Peptide from the Venom of Conus Gloriamaris", pages 11420-11425. *
CHEMICAL REVIEWS, 1993, Volume 93, Number 5, MYERS et al., "Conus Peptides as Chemical Probes for Receptors and Ion Channels", pages 1923-1936. *
See also references of EP0821694A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 20 January 1995, Volume 270, Number 3, FAINZILBER et al., "A New Conotoxin Affecting Sodium Current Inactivation Interacts with the delta-Conotoxin Receptor Site", pages 1123-1129. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0948526A4 (fr) * 1996-11-08 2003-01-02 Univ Utah Res Found Peptides contryphanes
EP1129106A4 (fr) * 1998-09-14 2002-07-03 Univ Queensland Peptides de conotoxines cyclises
US7001883B1 (en) 1998-09-14 2006-02-21 The University Of Queensland Cyclized conotoxin peptides
US7312195B2 (en) 1998-09-14 2007-12-25 The University Of Queensland Cyclised conotoxin peptides
US8722859B2 (en) 2000-04-11 2014-05-13 Genentech, Inc. Multivalent antibodies and uses therefor
US9493579B2 (en) 2000-04-11 2016-11-15 Genentech, Inc. Multivalent antibodies and uses therefor
WO2004066990A3 (fr) * 2003-01-30 2004-11-04 Dynogen Pharmaceuticals Inc Methodes destinees a traiter les troubles du tractus urinaire inferieur au moyen de modulateurs du canal sodique
US7084116B2 (en) 2003-03-10 2006-08-01 Dynogen Pharmaceuticals, Inc. Methods for treating lower urinary tract disorders and the related disorders vulvodynia and vulvar vestibulitis using Cav2.2 subunit calcium channel modulators
US7223754B2 (en) 2003-03-10 2007-05-29 Dynogen Pharmaceuticals, Inc. Thiazolidinone, oxazolidinone, and imidazolone derivatives for treating lower urinary tract and related disorders
US7459430B2 (en) 2003-03-10 2008-12-02 Dynogen Pharmaceuticals, Inc. Methods of using ziconotide to treat overactive bladder
US7125848B2 (en) 2003-06-13 2006-10-24 Dynogen Pharmaceuticals, Inc. Methods of treating non-inflammatory gastrointestinal tract disorders using Cav2.2 subunit calcium channel modulators
US8226537B2 (en) 2008-09-12 2012-07-24 Terumo Bct, Inc. Blood processing apparatus with cell separation chamber with baffles

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EP0821694A1 (fr) 1998-02-04
JPH11503909A (ja) 1999-04-06

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