WO2009021289A1 - Potassium channel inhibitors - Google Patents

Potassium channel inhibitors Download PDF

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Publication number
WO2009021289A1
WO2009021289A1 PCT/AU2008/001181 AU2008001181W WO2009021289A1 WO 2009021289 A1 WO2009021289 A1 WO 2009021289A1 AU 2008001181 W AU2008001181 W AU 2008001181W WO 2009021289 A1 WO2009021289 A1 WO 2009021289A1
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seq
xaa
amino acid
peptide according
acid residue
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PCT/AU2008/001181
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French (fr)
Inventor
Raymond Stanley Norton
Brian John Smith
Michael Pennington
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The Walter And Eliza Hall Institute Of Medical Research
Bachem Biosciences, Inc.
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Publication of WO2009021289A1 publication Critical patent/WO2009021289A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides which are related to the polypeptide toxin ShK, compositions containing them and their use in blocking KvI.3 potassium channels. More particularly, the invention relates to polypeptides related to ShK in which all of the amino acid residues are in the D-configuration. The invention also relates to the use of the peptides in the treatment or prevention of autoimmune diseases.
  • K + ) channels All human T lymphocytes express predominantly two types of potassium (K + ) channels, Kv 1.3 and KCa3.1, which play crucial roles in human T-cell activation (1-3).
  • Kv 1.3 channels dominate in terminally-differentiated effector memory (T EM ) cells, and Kv 1.3 blockers inhibit the activation of these cells, while KCa3.1 blockers are ineffective (4, 5).
  • Naive and central-memory (T CM ) cells are less sensitive to Kv 1.3 blockade because they rapidly up-regulate KCa3.1 channels upon activation (4).
  • Sea anemones contain a family of polypeptide toxins that block potassium channels, the first representative of which to be isolated and characterized was ShK, from Stichodactyla helianthus (6, 7). Its solution structure, determined by NMR spectroscopy (8, 9), consists of two short ⁇ -helices encompassing residues 14-19 and 21-24, and an N-terminus with an extended conformation up to residue 8 followed by a pair of interlocking turns that resembles a 3 10 -helix.
  • scorpion K + channel blockers such as charybdotoxin (ChTX) (10) and margatoxin (MgTX) (11), but is similar to that of BgK toxin (12).
  • ShK blocks K + channels by binding to a shallow vestibule at the outer entrance to the ion conduction pathway and occluding the entrance to the pore.
  • ShK blocks not only Kv 1.3 (K d 11 pM) but also KvLl (K d 16 pM), KvI.6 (K d 165 pM) (14) and Kv3.2 (17, 18).
  • ShK-Dap22 in which the critical Lys22 was replaced by the shorter, positively charged, non-natural residue 1,3-diaminopropionic acid (Dap) (13), ShK-FoCA, a fluorescein-labeled analogue of ShK (14), and ShK(L5), in which a pTyr residue is attached through a hydrophilic linker to Argl (15).
  • Kv 1.3 blockers constitute a valuable source of new therapeutics for the treatment of autoimmune diseases, particularly those mediated by T EM cells, such as multiple sclerosis (MS), rheumatoid arthritis, and type 1 diabetes mellitus (4, 5).
  • autoimmune diseases particularly those mediated by T EM cells, such as multiple sclerosis (MS), rheumatoid arthritis, and type 1 diabetes mellitus (4, 5).
  • MS multiple sclerosis
  • ShK and its analogue ShK(L5) potently inhibit proliferation and cytokine production by disease- associated autoreactive T cells from patients with autoimmune diseases (4, 5).
  • ShK, ShK- Dap22 (16), and ShK(L5) (17, 5) have been shown to prevent and treat adoptive transfer experimental autoimmune encephalomyelitis (EAE) in rats, an animal model for MS, treat pristane-induced arthritis in rats, a model for rheumatoid arthritis, and suppress delayed- type hypersensitivity caused by skin-homing T EM cells.
  • EAE adoptive transfer experimental autoimmune encephalomyelitis
  • ShK has a short half-life in vivo ( ⁇ 30 min) (16) as a result of proteolytic degradation and/or rapid renal clearance.
  • Kv 1.3 channel blockers that have selectivity and improved in vivo stability.
  • the present invention is predicated in part on the surprising discovery that polypeptides related to ShK in which all of the amino acid residues are in the D-configuration, maintain the ability to bind and block the KvI.3 potassium channel and are completely resistant to proteolysis and are non-antigenic.
  • a peptide or a pharmaceutically acceptable salt thereof comprising amino acid sequence 1 : R -xaa 2 9xaa 2 8Cixaa 1 xaa 2 xaa3xaa4xaa5xaa 6 xaa 7 xaa8C 2 xaa9xaai 0 xaa 1 ixaa 12 c 3 xaa 13 xaa 14 xaai 5 xaai 6 xaai 7 xaa 18 xaa!9xaa 2 oxaa 2 ixaa 22 c 4 xaa 2 3xaa 24 xaa 25 C5xaa 26 xaa 27 C6-R 2
  • xaa 17 is a positively charged D-amino acid residue
  • xaa 18 is an aromatic D-amino acid residue
  • xaa ! to xaa 16 xaaig to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue;
  • R 1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of Ci and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C 6 through a disulfide, diseleno or sulfide-selenium bond; or both C 1 and C 6 are D-amino acid residues having a side chain double bond and the side chains of C 1 and C 6 are linked to form a dicarba bond; and each of C 2 to C 5 are independently selected from D-cysteine and D-selenocysteine and C 2 is linked to C 4 and C 3 is linked to C 5 through disulfide, diseleno or sulfide-selenium bonds.
  • amino acid refers to compounds having an amino group and a carboxylic acid group.
  • the amino acids incorporated into the amino acid sequences of the present invention are D-amino acids and may be the D-form of naturally occurring proteogenic amino acids, or may be D-forms of non-naturally occurring amino acids.
  • Amino acids incorporated into oligopeptides at the N- or C-terminus (R 1 or R 2 ) may be D-amino acids or naturally or non-naturally occurring L-amino acids.
  • L-amino acids are referred to using capital letters or initial capital letters whereas D-amino acids are referred to using lower case letters.
  • glycine may be referred to using codes designating D or L amino acids (GIy, gly, G, or g).
  • Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal- 5-phosphate followed by reduction with NaBH 4 .
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulfhydryl groups that may occur in oligopeptides at the N- or C-terminus (R 1 or R 2 ) or are other than those present in ci to C 6 may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid and 2-thienyl alanine.
  • Examples of suitable non-proteogenic or non-naturally occurring amino acids contemplated herein is shown in Table 2.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-pipecolic acid D-homo Dpip D-N-methylhomophenylalanine Nmhphe proline) N-(N-(3,3-diphenylpropyl) Nnbhe
  • non-naturally occurring amino acids listed in Table 2 are preferably in the D-conformation, although amino acids having corresponding L-conformations may be incorporated as the amino acid residue of R 1 or R 2 or into oligopeptides at the N- or C-terminus (R 1 and R 2 ).
  • Suitable ⁇ -amino acids include, but are not limited to, D- ⁇ -homoalanine, D- ⁇ -homoarginine, D- ⁇ -homoasparagine, D- ⁇ -homoaspartic acid, D- ⁇ -homoglutamic acid, D- ⁇ -homoglutamine, D- ⁇ -homoisoleucine, D- ⁇ -homoleucine, D- ⁇ -homolysine,
  • D- ⁇ -homomethionine D- ⁇ -homophenylalanine, D- ⁇ -homoproline, D- ⁇ -homoserine,
  • xaa ⁇ is a positively charged D-amino acid residue.
  • This is an amino acid having a side chain capable of bearing a positive charge.
  • suitable amino acid residues include, but are not limited to, D-lysine, D- 1,3-diaminopropionic acid, D-arginine, D-histidine, 5-hydroxy-D-lysine, D-norlysine, D-homolysine and D-ornithine, especially D-lysine, D-l,3-diaminopropionic acid, 5-hydroxy-D-lysine, D-norlysine and D-homolysine, more especially D-lysine and D-l,3-diaminopropionic acid.
  • xaais is an aromatic D-amino acid residue.
  • Suitable aromatic D-amino acid residues include, but are not limited to, D-tyrosine, D-phenylalanine, D-tryptophan, D-homotyrosine, D-naphthylalanine and D-phosphotyrosine, especially D-tyrosine, D-phenylalanine, D-tryptophan and D-homotyrosine, more especially D-tyrosine.
  • R 1 is selected from hydrogen, an amino acid residue, an N-terminal capping group and an oligopeptide optionally capped with an N-terminal capping group.
  • the amino acid residue or sequence of amino acid residues in the oligopeptide may include one or more of D- and L-amino acid residues, naturally occurring amino acid residues, non-naturally occurring amino acid residues, proteogenic amino acid residues and non-proteogenic amino acid residues.
  • the N-terminal capping group is a group that caps the N-terminal amino group without affecting the tertiary structure of the peptide.
  • the N-terminal capping group is an acyl group, such as acetyl or succinyl, or the N-terminal capping group may be a label such as a fluorescent biological label attached directly, or through a linker, to Xaa 2 9 or Xaa 28 or in their absence to ci.
  • a suitable fluorescent label is fluoroscein and suitable biological labels include biotin and streptavidin.
  • the N-terminal capping group is an antibody or a molecule that recognises and targets a T EM cell.
  • Linkers that link an N-terminal capping group to xaa 29 , xaa 28 or cj may be any divalent linker that provides the required length and does not inhibit or prevent the binding of the peptide to the KvI .3 channel.
  • Suitable linkers include, but are not limited to, alkylene groups -(CH 2 ) p - where p is an integer from 1-20, preferably 1-10 and alkylene groups in which one or more -CH 2 - group has been replaced by a heteroatom, selected from the oxygen, sulfur or nitrogen.
  • Each linker may also include substituents suitable for forming an attachment with the peptide and the label or other group such as an antibody.
  • the linker may contain a terminal carboxyl group that is able to form an amide bond with the amino terminus of xaa 2 g, xaa 28 or ci or with the label or a biological molecule, or the linker may include a terminal amino group that is able to link to a label or biological molecule or to a reactive group in the side chain of xaa 2 g or xaa 28 .
  • Suitable compounds for use as linkers include, but are not limited to, amino-ethyloxy-ethyloxy acetic acid, 5-amino-3-oxapentanoic acid, 6-aminohexanoic acid, 5-aminopentanoic acid and 8-aminooctanoic acid.
  • R 2 is selected from hydrogen, an amino acid residue, a C-terminal capping group and an oligopeptide optionally capped with a C-terminal capping group.
  • the amino acid residue or oligopeptide may be any amino acid residue or sequence of amino acid residues including both D- or L-amino acid residues, naturally occurring amino acid residues, non-naturally occurring amino acid residues, proteogenic amino acid residues and non-proteogenic amino acid residues.
  • the C-terminal capping group is a group that caps the C-terminal carboxy group without affecting the tertiary structure of the peptide.
  • the C-terminal capping group is an amide group, such as NH 2
  • the C-terminal capping group may be a label, such as a fluorescent or biological label attached directly, or through a linker, to C 6 .
  • a suitable fluorescent label is fluorescein and suitable biological labels include biotin and streptavidin.
  • the C-terminal capping group is an antibody or a molecule that recognises and targets a T EM cell.
  • Suitable linkers for linking R 2 to C 6 are similar to those that link R 1 to xaa 28 , xaa 29 or C 1 and may include suitable groups that can form an amide bond with the C-terminus, such as a terminal amino group and a group suitable for linking the linker to the biological molecule, such as a second terminal amino group or a terminal carboxylic acid.
  • suitable compounds for use as linkers include, but are not limited to, amino-ethyloxy-ethyloxy acetic acid,
  • suitable amino acid residues include, but are not limited to, D-allylglycine, D-but-3- enylglycine, 2-methylbut-3-enylglycine and the like, especially D-allylglycine.
  • C 1 and C 6 are linked by a dicarba bond
  • ci and C 6 are both D-allylglycine residues and the dicarba bond is a double bond formed by ring closing metathesis between the double bonds of the allyl groups.
  • the amino acid sequence has a minimum of 33 amino acids in the sequence. In some embodiments, the amino acid sequence is 33 to 45 amino acids in length.
  • the compounds of the invention may be in the form of pharmaceutically acceptable salts. It will be appreciated however that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium,
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • C 1 to C 6 are independently selected from D-cysteine or D-selenocysteine and C 1 is linked to C 6 , C 2 is linked to C 4 and C 3 is linked to C 5 through disulfide, diseleno or sulfide-selenium bonds.
  • C 1 is D-cysteine, D-homocysteine or D-penicillamine, especially D-cysteine.
  • xaaj is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-isoleucine.
  • NPaa non-polar D-amino acid
  • xaa 2 is a negatively charged D-amino acid (Naa), especially D-aspartic acid or D-glutamic acid, more especially D-aspartic acid;
  • xaa 3 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine.
  • PUaa polar, uncharged D-amino acid
  • xa ⁇ is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-isoleucine.
  • NPaa non-polar D-amino acid
  • NPaa non-polar D-amino acid
  • NPaa non-polar D-amino acid
  • xaa ⁇ is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine.
  • xaa 7 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine.
  • xaag is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine.
  • C 2 is D-cysteine.
  • xaa 9 is polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine,
  • xaaio is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan or glycine, more especially D-alanine, D-valine, D-leucine, D-isoleucine or glycine, most especially D-alanine.
  • NPaa non-polar D-amino acid
  • xaa ⁇ is a non-polar or aromatic D-amino acid, especially a non-polar D-amino acid (NPaa); especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine, D-tryptophan or D-tyrosine, especially a non-polar and aromatic D-amino acid such as D-phenylalanine or D-tryptophan, most especially D-phenylalanine.
  • NPaa non-polar D-amino acid
  • xaai 2 is polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-glutamine or D-asparagine, most especially D-glutamine.
  • PUaa uncharged D-amino acid
  • C 3 is D-cysteine.
  • xaa 13 is a positively charged amino acid (Paa), especially D-lysine, D-arginine,
  • D-histidine or D-l,3-diaminopropionic acid more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine.
  • xaai 4 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-histidine.
  • xaa 15 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine.
  • PUaa polar, uncharged D-amino acid
  • xaai 6 is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-methionine, D-alanine, D-valine, D-leucine or D-isoleucine, most especially D-methionine.
  • NPaa non-polar D-amino acid
  • xaa 17 is selected from D-lysine, D-l,3-diaminopropionic acid, D-arginine, D-histidine, 5-hydroxy-D-lysine, D-norlysine, D-homolysine and D-ornithine, especially D-lysine, D-l,3-diaminopropionic acid, 5-hydroxy-D-lysine, D-norlysine, D-homolysine, more especially D-lysine or D-l,3-diaminopropionic acid.
  • xaa 18 is selected from D-tyrosine, D-phenylalanine, D-tryptophan, D-homotyrosine, D-naphthylalanine and D-phosphotyrosine, especially D-tyrosine, D-phenylalanine, D-tryptophan or D-homotyrosine, more especially D-tyrosine.
  • Xaa ⁇ is a positively charged amino acid (Paa), especially D-lysine, D-arginine,
  • xaa 20 is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-leucine.
  • xaa 21 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine,
  • xaa 22 is a non-polar or aromatic D-amino acid, especially a non-polar D-amino acid
  • NPaa especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine, D-tryptophan or D-tyrosine, especially a non-polar and aromatic
  • D-amino acid such as D-phenylalanine and D-tryptophan, most especially
  • C 4 is D-cysteine.
  • xaa 23 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine.
  • xaa 24 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine.
  • xaa 25 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine.
  • PUaa polar, uncharged D-amino acid
  • C 5 is D-cysteine.
  • xaa 26 is glycine or a small non-polar D-amino acid (SNPaa), especially glycine, D-alanine, D-valine, D-leucine or D-isoleucine, more especially glycine or D-alanine, most especially glycine.
  • xaa 27 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine.
  • PUaa polar, uncharged D-amino acid
  • C 6 is D-cysteine, D-homocysteine or D-penicillamine, especially D-cysteine.
  • C 1 and C 6 are both D-allylglycine and they are linked by a dicarba double bond.
  • xaa 28 is absent or is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine.
  • xaa 29 is absent or is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine.
  • R 2 is hydrogen or NH 2 .
  • R 1 is hydrogen, acyl, a phosphorylated amino acid residue, or a fluorescent or biological label optionally linked to C 1 , xaa 2 g or xaa 28 through a linker, especially hydrogen, acetyl, phosphotyrosine or a fluoroscein or biotin label optionally linked to ci, xaa 29 or xaa 28 through a linker, more especially hydrogen, phosphotyrosine or a fluoroscein or biotin label linked to ci, xaa 29 or xaa 28 through a linker, even more especially hydrogen, D-phosphotyrosine or a fluoroscein label linked to xaa 29 through an amino-ethyloxy- ethyloxy-acetyl linker.
  • the peptide of SEQ ID NO: 1 has one of the following sequences: rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:2 rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO: 3 ptyr-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID N0:4 ptyr-rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO: 5 fluoroscein-aminoethyloxyethyloxyacetyl-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:6 fluoroscein-a
  • the compounds of the present invention may be prepared using techniques known in the art.
  • peptides can be synthesized using various solid phase techniques (45) or using an automated synthesis, for example, using a Pioneer peptide synthesizer and standard F-moc chemistry (46).
  • D-amino acids may be substituted for L-amino acids in such synthesis.
  • the peptides of the present invention may be substantially purified by preparative FIPLC.
  • the composition of the synthetic peptides can be confirmed by amino acid analysis or by sequencing (using the Edman degradation procedure).
  • Suitable protecting and deprotecting methods for reactive functional groups such as carboxylic acids and amines are known in the art, for example, in Protective Groups in Organic Synthesis, T.W. Green & P. Wutz, John Wiley & Son, 3 rd Ed, 1999.
  • the compounds of the present invention in which C 1 and C 6 are linked by a dicarba bond can also be prepared by methods known in the art.
  • a linear peptide may be prepared using Fmoc-solid phase synthesis where the residues at ci and c 6 both include a double bond in their side chains, for example, both C 1 and C 6 are D-allylglycine.
  • the peptide is subjected to ring closing metathesis conditions to form a dicarba double bond between a carbon atom of the double bond of ci side chain and a carbon atom of the double bond of C 6 side chain.
  • the ring closing metathesis is carried out in the presence of 5-10% second generation Grubbs catalyst in dichloromethane containing 10% 0.4M lithium chloride in dimethylfbrmamide.
  • the metathesis reaction may be carried out under standard conditions or may be carried out using microwave irradiation at 2.45 GHz for 1-2 hours thereby maintaining a temperature of about 100°C.
  • the peptide may be deprotected and cleaved from the resin.
  • the disulfide, diseleno or sulphide-selenium bonds can be formed between C 2 and C 4 and C 3 and C 5 before or after cleavage from the resin. If required, selective protection and deprotection at C 2 and C 4 and/or C 3 and C 5 may be used to ensure correct connectivity of disulfide bonds.
  • the peptides of the invention may be tested for their ability to selectively bind to Kv 1.3 channels using assays known in the art. For example, cells stably expressing mKvl.l, rKvl.2, mKvl .3 and hKvl.5 channels may be exposed to the peptides of the invention and the channel currents measured by the patch clamp method as described in Grissmer et al. (28). Alternatively, if a fluorescent label is present on the peptide, fluorescence detection may be possible as described in Beeton et al, (14).
  • a method of blocking a Kv 1.3 potassium channel comprising exposing a cell expressing the Kv 1.3 potassium channel to a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
  • R 1 -xaa 29 xaa 28 Cixaaixaa 2 xaa 3 xaa 4 xaa 5 xaa 6 xaa 7 xaa 8 c 2 xaa 9 xaai 0 xaai 1 xaa 12 c 3 xaai 3 xaai 4 xaai 5 xaai 6 xaa 17 xaa 18 xaa 19 xaa 2 oxaa 21 xaa 22 C 4 xaa 23 xaa 24 xaa 25 c 5 xaa 26 xaa 27 c 6 -R 2 SEQ ID NO:!
  • xaa ⁇ is a positively charged D-amino acid residue
  • xaajs is an aromatic D-amino acid residue
  • xaaj to xaai 6 , xaai 9 to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue;
  • R is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group
  • each of Ci and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C 1 is linked to C 6 through a disulfide, diseleno or sulfide-selenium bond
  • both C 1 and C 6 are D-amino acid residues having a side chain double bond and the side chains of C 1 and C 6 are linked to form a dicarba bond
  • each of C 2 to C 5 are independently selected from D-cysteine and D-selenocysteine and C 2 is linked to C 4 and C 3 is linked to C 5 through disulfide, diseleno or
  • a method of inhibiting activation of T-cells comprising exposing the T-cells to a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
  • xaai 7 is a positively charged D-amino acid residue
  • xaai 8 is an aromatic D-amino acid residue
  • xaai to xaa 16 , xaaig to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue
  • R 1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group
  • each of cj and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C 6 through a disulfide, diseleno or
  • the cell expressing a KvI.3 potassium channel or T-cells which are treated according to a method of the present invention may be located ex vivo or in vivo.
  • ex vivo is meant that the cell has been removed from the body of a subject wherein the modulation of its activity will be initiated in vitro.
  • the cell may be a cell which is to be used as a model for studying any one or more aspects of the pathogenesis of conditions which are characterised by aberrant activation of T-cells.
  • the subject cell is located in vivo.
  • a method of treatment and/or prophylaxis of a disease or disorder characterized by aberrant or abnormal activation of T-cells in a subject comprising administering to the subject an effective amount of a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
  • xaa ⁇ is a positively charged D-amino acid residue
  • xaais is an aromatic D-amino acid residue
  • xaai to xaai 6 , xaajg to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue
  • R 1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of C 1 and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C 1 is linked to C 6 through a disulfide, diseleno or sulfide-selenium bond; or both C 1 and C 6 are D-amino acid residues having a side chain double bond and the side chains of C 1 and C 6 are linked to form a dicarba bond; and each of C 2 to C 5 are independently selected from D-cysteine and D-selenocysteine and C 2 is linked to C 4 and C 3 is linked to c 5 through disulfide, diseleno or sulfide-selenium bonds.
  • the T-cells are human effector memory T-cells (T EM cells).
  • a method of treatment and/or prophylaxis of an autoimmune disease or organ or tissue transplant rejection in a subject comprising administering to the subject an effective amount of a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
  • xaai 7 is a positively charged D-amino acid residue
  • xaais is an aromatic D-amino acid residue
  • xaai to xaai 6 , xaa ⁇ to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue
  • R 1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of C 1 and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C 6 through a disulfide, diseleno or sulfide-selenium bond; or both ci and C 6 are D-amino acid residues having a side chain double bond and the side chains of cj and C 6 are linked to form a dicarba bond; and each of C 2 to C 5 are independently selected from D-cysteine and D-selenocysteine and c 2 is linked to C 4 and c 3 is linked to c 5 through disulfide, diseleno or sulfide-selenium bonds.
  • subject refers to an organism that has cells that express KvI .3 receptors, especially T-cells that express KvI.3 receptors.
  • the subject is a mammal.
  • mammal includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer).
  • the mammal is human or a laboratory test animal, especially a human.
  • autoimmune diseases refers to diseases in which the subjects own immune system malfunctions and immune cells such as T-cells become activated resulting in an immune response against the subject's own cells and/or tissues.
  • Autoimmune diseases that may be treated by the present invention include multiple sclerosis, rheumatoid arthritis, Type-I diabetes (IDDM), psoriasis, Hashimoto's disease, Sjogren's syndrome, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anaemia, autoimmune hepatitis, autoimmune oophoritis, Coeliac disease, Crohn's disease, gestational pemphigoid, Goodpasture's syndrome, Grave's disease, Guillian-Barre syndrome, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, polyarthritis (in dogs
  • T-cells Aberrant or abnormal activation of T-cells may also occur after organ or tissue transplants resulting in rejection of the transplanted organ or tissue.
  • the present invention also relates to methods of treating or preventing rejection of organ or tissue transplants. Suitable transplants include organs and tissues such as heart, lungs, kidney, liver, pancreas, intestine and skin.
  • an "effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated.
  • the amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • An effective amount in relation to a human patient may lie in the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage is preferably in the range of l ⁇ g to 1 g per kg of body weight per dosage, such as is in the range of lmg to Ig per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 ⁇ g to 1 mg per kg of body weight per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • treatment does not necessarily imply that a subject is treated until total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylaxis” may be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
  • the present invention further contemplates a combination of therapies, such as the administration of the peptides of the invention or pharmaceutically acceptable salts thereof together with the subjection of the subject to other agents or procedures which are useful in the treatment of diseases and disorders characterized by the aberrant or abnormal activation of T-cells or autoimmune diseases.
  • the compounds of the present invention may be administered in combination with, in the same composition or in separate compositions simultaneously or sequentially, other chemotherapeutic drugs used to treat the cause of or symptoms of the disease or disorder.
  • the peptides of the present invention may be combined with immunosuppressants, such as glucocorticoid immunosuppressants, cytostatic immunosuppressants, antibody immunosuppressants, immunosuppressants that act on immunophilins and other immunosuppressants.
  • immunosuppressants such as glucocorticoid immunosuppressants, cytostatic immunosuppressants, antibody immunosuppressants, immunosuppressants that act on immunophilins and other immunosuppressants.
  • Immunosuppressants that are useful in combination therapies with the peptides of the present invention include, but are not limited to, Cortisol, hydrocortisol, dexamethasone, cyclophosphamide, nitrosoureas, folic acid analogues such as methotrexate, purine analogues such as azathioprine and mercaptopurine, pyrimidine analogues, protein synthesis inhibitors, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, Atgam (R), Thymoglobuline (R), OKT3 (R), basilixmab, daclizumab, cyclosporine, tacrolimus, sirolimus, interferons, infliximab, etanercept, adalimumab, mycophenolic acid and FTY720.
  • Cortisol Cortisol, hydrocortisol, dex
  • a peptide of the invention or a pharmaceutically acceptable salt thereof may be administered as a neat chemical, it is preferable to present the active ingredient as a pharmaceutical composition.
  • composition comprising a peptide or pharmaceutically acceptable salt thereof comprising amino acid sequence 1 :
  • xaa 17 is a positively charged D-amino acid residue
  • xaa 18 is an aromatic D-amino acid residue
  • xaai to xaai 6 , xaajg to xaa 27 are each independently selected from any D-amino acid residue
  • xaa 28 and xaa 29 are independently absent or are selected from any D-amino acid residue;
  • R 1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
  • R 2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of cj and C 6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C 1 is linked to C 6 through a disulfide, diseleno or sulfide-selenium bond; or both C 1 and C 6 are D-amino acid residues having a side chain double bond and the side chains of cj and C 6 are linked to form a dicarba bond; and each of C 2 to C 5 are independently selected from D-cysteine and D-selenocysteine and C 2 is linked to C 4 and C 3 is linked to C 5 through disulfide, diseleno or sulfide-selenium bonds; and a pharmaceutically acceptable carrier.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • compositions and unit dosages thereof may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
  • Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to two hundred (200) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
  • the compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt or derivative of the compound of the invention.
  • pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from five or ten to about seventy percent of the active compound.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • preparation is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
  • a low melting wax such as admixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
  • parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
  • the peptides according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-i ⁇ lled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents, as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
  • Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
  • the formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.
  • the compounds according to the invention may be encapsulated with cyclodextrins, or formulated with their agents expected to enhance delivery and retention in the nasal mucosa.
  • Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofiuoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofiuoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • CFC chlorofluorocarbon
  • the aerosol may conveniently also contain a surfactant such as lecithin.
  • the dose of drug may be controlled by provision of a metered valve.
  • the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • a powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • the powder carrier will form a gel in the nasal cavity.
  • the powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
  • the compound In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 1 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
  • formulations adapted to give sustained release of the active ingredient may be employed.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • B Dose-dependent inhibition of Kv 1.3 (open symbols) and Kv 1.1 (closed symbols) by ShK (A), ShK-amide (T) and D-allo-ShK ( ⁇ ). Each data point is the mean ⁇ SD of three independent determinations.
  • C Selectivity of D-allo-ShK for K channels.
  • FIG. 1 Characterization of the effects of ShK and D-alloShK on Kv 1.3 currents, ShK blocks the closed state of the channel, but D-allo-ShK does not.
  • a Kv 1.3 control current was elicited by a 200ms depolarizing pulse to 40 mV.
  • D-allo-ShK preferentially suppresses the proliferation of T EM cells.
  • Fmoc-D-amino acids (Bachem Feinchemikalien) included D-Arg(Pmc), D-Asp(OtBu), D- Cys(Trt), D-GIn(Tn), D-His(Trt), D-Lys(Boc), D-Ser(tBu) and D-Thr(tBu). All other non- side chain protected derivatives used for synthesis were also in the D-configuration except for GIy which is achiral. Stepwise assembly was carried out on an Applied Biosystems 43 IA peptide synthesizer at the 0.25 mmol scale using Fmoc-Ramage Tm -amide-resin. Residues 34 through 22 were single coupled.
  • the peptide was collected on a fine filter funnel, washed with ice-cold ether and finally extracted with 20% AcOH in H 2 O.
  • the peptide extract was subsequently diluted into 2 L of H 2 O, the pH adjusted to 8.0 with NH 4 OH and allowed to air oxidize at room temperature for 36 h.
  • the peptide solution was acidified to pH 2.5 and pumped onto a Rainin Dynamax Ci 8 column (5.0 x 30 cm). The sample was eluted with a linear gradient from 5-30% acetonitrile into H 2 O containing 0.1% trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • Peptide Analysis Purified peptide was analysed by reverse phase HPLC (Vydac C18-silica, 5 ⁇ , 120 angstrom, 0.46 x 250 mm column) using gradient conditions of 5 to 50% B in 20 minutes at 1.5 mL/min (A buffer is 0.1% trifluoroacetic acid (TFA) in water and B is acetonitrile containing 0.1% TFA). The peptide was detected at 215 nm. The correctly folded peptide eluted at 10.5 minutes. Synthetic peptide samples were hydrolyzed in 6 N HCl at 110°C for 22 h in vacuo. Peptide and amino acid analysis was performed on a Beckman 126AA System Gold amino acid analyzer.
  • MALDI-ToF mass spectroscopic analysis was performed on a Kratos Kompact mass spectrometer using CCA as a matrix.
  • the theoretical mass of the peptide is 4054. ID, the observed mass [M+H] was 4055.2D.
  • Amino acid analysis of the purified D-allo-ShK showed the following average amino acid ratios: Asx (1) 0.97, Thr (4) 3.89, Ser (4) 4.04, GIx (1) 0.98, Pro (1) 0.93, GIy (1) 1.02, Ala (1) 1.00, Met (1) 0.88, He (2) 1.78, Leu (1) 1.01, Tyr (1) 0.99, Phe (2) 2.00, Lys (4) 3.99, His (1) 0.95, Arg (4) 3.89, and Cys (6) 5.26.
  • NMR Spectroscopy Spectra were recorded on a sample of D-allo-ShK in 95% H 2 O/5% 2 H 2 O at pH 4.9.
  • Two-dimensional homonuclear total correlation (TOCSY) spectra with a spin-lock time of 60 ms and double quantum filtered correlation (DQF-COSY) NMR spectra were acquired at 500 MHz on a Bruker AMX-500 spectrometer.
  • a two-dimensional nuclear Overhauser enhancement (NOESY) spectrum with a mixing time of 200 ms was also acquired on a Bruker AMX-500 spectrometer.
  • NOESY spectra for ShK were acquired at 500 MHz and 600 MHz as described previously (8, 9); the 600 MHz NOESY was used in the comparison with D-allo-ShK. Water was suppressed using the WATERGATE pulse sequence (20). All spectra were collected at 20 °C unless otherwise stated and were referenced to an impurity peak at 0.15 ppm or to the water resonance.
  • Diffusion measurements were performed using a pulsed field gradient longitudinal eddy- current delay pulse sequence (21, 22) as implemented by Yao et al. (23).
  • Ser20 NH could be indicative of a slight drift in pH and/or a difference in salt content, which is supported by the differences in chemical shift of the His 19 C(2)H and C(4)H protons, which were 8.42 ppm and 6.54 ppm in the D-allo-ShK spectrum and 8.46 ppm and 6.57 ppm in the ShK spectrum (the pKa of His 19 in ShK is 6.1) (4).
  • the 0.04 ppm difference in NH chemical shift for Ile4 may have arisen from the stereochemistry of its asymmetric side chain, which is not an exact mirror image of ShK.
  • D-allo-ShK The stability of D-allo-ShK to proteolytic digestion was investigated under the same conditions used to determine the disulfide bridges of ShK toxin (26).
  • D-allo-ShK (15 ⁇ g) was dissolved in 0.05 M HEPES, pH 6.5, containing 10 mM CaCl 2 (30 ⁇ L) and trypsin, chymotrypsin or a mixture of trypsin and chymotrypsin (enzyme:substratel :50, w/w, 30 0 C, 6 h).
  • the digestion was terminated by acidification with 10% aqueous TFA (3 ⁇ l), the solution centrifuged (13,000 g, 5 min), and the supernatant analyzed directly by RP-HPLC.
  • L929, B82 and MEL cells stably expressing mKvl.l, rKvl.2, mKvl.3, and hKvl.5 have been described previously (28) and were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, and 500 ⁇ g/ml G418 (Calbiochem).
  • LTK cells expressing hKvl.4 were obtained from M.
  • Tamkun (University of Colorado, Boulder), CHL cells expressing mKvl.7 from Vertex Pharmaceutical Inc (San Diego, CA), and HEK293 cells stably expressing hKCa3.1 were a kind gift from Dr. Khaled Houamed (Chicago, IL).
  • PAS T cells a MHC class II-restricted myelin basic protein (MBP)-specific encephalitogenic CD4 + rat T cell line (29), were a kind gift from Dr. Evelyne Beraud (Marseille, France).
  • Mononuclear cells were isolated from Lewis rat (Harlan-Sprague Dawley, Indianapolis, IN) spleens using Histopaque-1083TM gradients (Sigma).
  • KvI.1, KvI .2, KvI .3, KvI.4, KvI.5, and KvI .7 currents were recorded in normal Ringer solution with a calcium-free pipette solution containing (in mM): 145 KF, 10 HEPES, 10 EGTA, 2 MgCl 2 , pH 7.2, 300 mOsm, as described previously (28).
  • KCa3.1 currents were recorded as described previously (30). ⁇ s were determined from dose-response curves shown using Microcal Origin software.
  • Fig. IA shows the effects of ShK and D-allo-ShK on Kvl .3 (left) and KvI.1 (right) currents elicited by 200 ms depolarizing pulses from a holding potential of -80 mV to 40 mV. Both peptides reversibly blocked Kvl.3 and Kv 1.1 in a dose-dependent manner with Hill coefficients of 1 (Fig. IB).
  • D-allo-ShK blocked Kvl.3 with a 2,800-fold lower affinity (K d 36 ⁇ 3 nM) than ShK but displayed the same two-fold selectivity for Kvl.3 over KvI.1 as ShK (K d on KvI.1 83 ⁇ 9 nM) (Fig. 1).
  • D-allo-ShK had no effect on Kvl .2, Kvl .4, Kvl.5, Kvl .7, or KCa3.1 at concentrations up to 1 ⁇ M (Fig. 1C). Although D-allo-ShK had a lower affinity for Kvl.3 than native ShK, its selectivity profile was maintained.
  • ShK blocked 60% of the Kv 1.3 current at the first pulse after peptide incubation, and this blockade did not increase further after applying a further 10 depolarizing pulses, indicating that ShK binds to the closed state of the KvI.3 channel.
  • D-allo-ShK had no effect on the current at the first pulse after peptide incubation, and steady-state block was only reached after several depolarizing pulses, a phenomenon termed "use- dependent block", indicating that D-allo-ShK binds to an open or inactivated conformation of the channel.
  • D-allo-ShK was created by inverting the structure of ShK derived by NMR (8) (PDB access code IROO, structure 1), and correcting the side-chains of threonine and isoleucine residues for the appropriate stereochemistry.
  • D-allo model and NMR-derived structures were subjected to molecular dynamics (MD) simulation using the GROMACS (v3.3.1) package of programs (33). All simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed and MD continued for a further 10 ns.
  • the particle-mesh Ewald method was used to account for long-range electrostatics, applying a grid width of 1.2 A, and a fourth-order spline interpolation. Bond lengths were constrained using the LINCS algorithm (36). All simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed and MD continued for a further 10 ns.
  • Comparative models of the trans-membrane region (only) of the murine Kv 1.3 channel were constructed using the X-ray structure of the K + channel from Streptomyces lividans (KcsA, PDB accession code 1BL8) as a template.
  • the MODELLER (6v2) program (37) was used to create nine models based on the sequence alignment shown in Table S2.
  • mKvl .3 has good sequence similarity with KcsA over the entire pore domain (32% identity), whereas there is 91% sequence identity with Kv 1.2.
  • KcsA was chosen as the template for model building because the structure of the two loops comprising the extracellular face of KcsA, the site of toxin binding, has been well characterized (38), whereas those in the more closely related channel, Kv 1.2, are disordered in the electron density (39), and are therefore less suitable for model building.
  • ZDOCK program (40). This program uses a fast Fourier transform to explore all possible binding modes of the two proteins; docking was restricted to residues on the extracellular surface of the channel to ensure the exclusion of unphysical binding predictions. The interaction is evaluated using shape complementarity, desolvation energy and electrostatics.
  • Models of each form of the toxin were extracted at 1 ns intervals during the MD simulation. Including the initial model, we considered eleven models of each form of the toxin. Each model of the toxin was docked with one of the nine models of the channel; thus, we considered all 99 possible combinations of toxin with channel for both D-allo and
  • the Asp380 A and Ser379 A that contact Argi l are on a different channel monomer from the His404 ⁇ also close to Argl 1.
  • Mutant-cycle analysis also suggested that Ser379 and His404 of the channel were close to Arg29 of ShK. In the current model the separations are 8.3 A (between Ser379 ⁇ O ⁇ and Arg29 N ⁇ ) and 4.4 A (between His404 c N ⁇ and Arg29 N ⁇ ); these residues on the channel are on different monomers from those that interact with Argl 1.
  • the mutant-cycle analysis indicated cooperativity between Asp386 of the channel and Arg29 of ShK. In the model, these residues are ⁇ 9 A apart.
  • Buried surface areas were calculated from the difference in surface areas of channel and toxin from the complex. Surface areas were calculated using the NACCESS program (42). Table 4 - Summary of internuclear distances for the present models of ShK complexed with Kv 1.3, and a comparison with the earlier model of Lanigan et al. (32)
  • Channel subunits (A-D) are indicated by subscripts. X is the centroid of the aromatic ring. Distances are reported in Angstrom.
  • Phe27 packs alongside Lys22, in the space created by two Gly401 residues of the ion-selectivity filter from neighbouring channel monomers, and the side chains of Asp402 and His404.
  • the side chain of Met21 occupies the equivalent pocket in the space diametrically opposite to the pocket filled by Phe27.
  • the overlay of the two complexes illustrated the approximate mirror symmetry of the binding of the two diastereomers to the channel. Residues of the toxins that contact the channel and lie along this plane share common binding interactions in the two diastereomers. Additionally, residues of the toxins that contact the channel along the channel monomer interface perpendicular to this plane also share common binding modes in the two diastereomers.
  • PAS T cells (2x10 4 cells per well) were stimulated in the presence of 2x10 6 irradiated
  • a patch-clamp bioassay was used to determine the circulating half-life of D-allo-ShK.
  • rat serum did not exhibit detectable blocking activity, indicating an absence of endogenous channel blockers.
  • the spiked serum samples blocked KvI.3 currents in a dose-dependent fashion (K d 38 ⁇ 4 nM), similar to the effect of D-allo-ShK observed in the absence of serum (not shown).
  • Levels of D-allo-ShK in rats following a single subcutaneous injection of 1 mg/kg were determined by comparison with the standard curve. D-allo-ShK was detectable in serum 25 min after injection (Fig. 3A). Peak levels (950 nM) were reached within 50 min and the level then fell to 15 nM within 24 h (Fig. 3A). The disappearance of D-allo-ShK from the serum could be fitted by a single exponential (Fig. 3B) and its circulating half-life was estimated to be 40 min.
  • DTH delayed-type hypersensitivity
  • Ear swelling was measured 24 h later using a spring-loaded micrometer (Mitutoyo, Spokane, WA). All vehicle-treated control rats developed ear swelling 24 h after ovalbumin challenge in the ear, but the DTH reaction was significantly milder in animals treated with 1 mg/kg D-allo-ShK at the time of challenge in the ear (Fig. 3C). Thus, D-allo-ShK inhibited the T EM -mediated DTH response.
  • D-allo-ShK folded in good yield and gave a high-resolution NMR spectrum essentially identical to the native (all-L) polypeptide.
  • the most significant NMR spectral changes could be attributed to the presence of a C-terminal amide on D-allo-ShK, compared with a free C-terminal carboxylate on naturally occurring ShK.
  • D-allo-ShK is not a perfect mirror image of ShK because the stereochemistry of the side chains was not changed in concert with the change in the backbone, but this is only significant for the two He and four Thr side chains, and the NMR data show that the local structure in the vicinity of these residues is essentially identical to that of ShK.
  • D-allo-ShK blocked the Kv 1.3 channel with nM affinity, making it a weaker blocker than the native toxin but still a potent inhibitor. D-allo-ShK was also able to block T-cell proliferation. It is highly unusual for an all-D analogue of a folded polypeptide or protein that acts at a specific binding site on a target protein to retain activity.
  • ShK has a circulating half-life of - 30 min (16). Such a rapid clearance from the blood could be due to renal elimination and/or proteolysis.
  • the finding that D-allo-ShK has a similar half-life ( ⁇ 40 min) implies that the disappearance of ShK and D-allo-ShK is due to rapid renal clearance since endogenous proteases can only cleave L-forms of polypeptide chains and are therefore unable to proteolyse D-allo-ShK.
  • the kidney allows peptides up to 5 kDa to pass through without filtration and both forms of ShK are smaller than this cutoff.
  • D-allo-ShK is expected not to be recognized by the immune system as it should be resistant to proteolytic processing by T-cells for presentation on MHC complexes, and therefore is not likely to be antigenic in vivo.

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Abstract

The present invention relates to a peptide or a pharmaceutically acceptable salt thereof comprising amino acid sequence 1: R1 xaa29xaa28c1xaa1xaa2xaa3xaa4xaa5xaa6xaa7xaa8c2xaa9xaa10xaa11xaa12c3xaa13xaa14xaa15 xaa16xaa17xaa18xaa19xaa20xaa21xaa22c4xaa23xaa24xaa25c5xaa26xaa27c6-R2 SEQ ID NO:1 wherein xaa17 is a positively charged D amino acid residue; xaa18 is an aromatic D amino acid residue; xaa1 to xaa16, xaa19 to xaa27 are each independently selected from any D amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D amino acid residue; R1 is hydrogen or is an amino acid residue, an N- terminal capping group or an oligopeptide optionally capped with an N- terminal capping group; R2 is hydrogen or is an amino acid residue, a C -terminal capping group or an oligopeptide optionally capped with a C -terminal capping group; wherein each of c1 and c6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to c6 through a disulfide, diseleno or sulfide-selenium bond; or both c1 and c6 are D-amino acid residues having a side chain double bond and the side chains of ci and c6 are linked to form a dicarba bond; and each of c2 to c5 are independently selected from D-cysteine and D-selenocysteine and c2 is linked to c4 and c3 is linked to c5 through disulfide, diseleno or sulfide-selenium bonds.

Description

POTASSIUM CHANNEL INHIBITORS
Field of the Invention
The present invention relates to peptides which are related to the polypeptide toxin ShK, compositions containing them and their use in blocking KvI.3 potassium channels. More particularly, the invention relates to polypeptides related to ShK in which all of the amino acid residues are in the D-configuration. The invention also relates to the use of the peptides in the treatment or prevention of autoimmune diseases.
Background of the Invention
All human T lymphocytes express predominantly two types of potassium (K+) channels, Kv 1.3 and KCa3.1, which play crucial roles in human T-cell activation (1-3). The number of channels expressed by a given cell depends on its state of activation and differentiation (4). Kv 1.3 channels dominate in terminally-differentiated effector memory (TEM) cells, and Kv 1.3 blockers inhibit the activation of these cells, while KCa3.1 blockers are ineffective (4, 5). Naive and central-memory (TCM) cells are less sensitive to Kv 1.3 blockade because they rapidly up-regulate KCa3.1 channels upon activation (4).
Sea anemones contain a family of polypeptide toxins that block potassium channels, the first representative of which to be isolated and characterized was ShK, from Stichodactyla helianthus (6, 7). Its solution structure, determined by NMR spectroscopy (8, 9), consists of two short α-helices encompassing residues 14-19 and 21-24, and an N-terminus with an extended conformation up to residue 8 followed by a pair of interlocking turns that resembles a 310-helix. It contains no β-sheet and is thus distinct from the α/β fold found in scorpion K+ channel blockers such as charybdotoxin (ChTX) (10) and margatoxin (MgTX) (11), but is similar to that of BgK toxin (12).
ShK blocks K+ channels by binding to a shallow vestibule at the outer entrance to the ion conduction pathway and occluding the entrance to the pore. ShK blocks not only Kv 1.3 (Kd 11 pM) but also KvLl (Kd 16 pM), KvI.6 (Kd 165 pM) (14) and Kv3.2 (17, 18). More selective analogues have been created, such as ShK-Dap22, in which the critical Lys22 was replaced by the shorter, positively charged, non-natural residue 1,3-diaminopropionic acid (Dap) (13), ShK-FoCA, a fluorescein-labeled analogue of ShK (14), and ShK(L5), in which a pTyr residue is attached through a hydrophilic linker to Argl (15).
Kv 1.3 blockers constitute a valuable source of new therapeutics for the treatment of autoimmune diseases, particularly those mediated by TEM cells, such as multiple sclerosis (MS), rheumatoid arthritis, and type 1 diabetes mellitus (4, 5). Indeed, ShK and its analogue ShK(L5) potently inhibit proliferation and cytokine production by disease- associated autoreactive T cells from patients with autoimmune diseases (4, 5). ShK, ShK- Dap22 (16), and ShK(L5) (17, 5) have been shown to prevent and treat adoptive transfer experimental autoimmune encephalomyelitis (EAE) in rats, an animal model for MS, treat pristane-induced arthritis in rats, a model for rheumatoid arthritis, and suppress delayed- type hypersensitivity caused by skin-homing TEM cells.
However, ShK has a short half-life in vivo (~30 min) (16) as a result of proteolytic degradation and/or rapid renal clearance. There is a need for Kv 1.3 channel blockers that have selectivity and improved in vivo stability.
Summary of the Invention
The present invention is predicated in part on the surprising discovery that polypeptides related to ShK in which all of the amino acid residues are in the D-configuration, maintain the ability to bind and block the KvI.3 potassium channel and are completely resistant to proteolysis and are non-antigenic.
In one aspect of the invention there is provided a peptide or a pharmaceutically acceptable salt thereof comprising amino acid sequence 1 : R -xaa29xaa28Cixaa1xaa2xaa3xaa4xaa5xaa6xaa7xaa8C2xaa9xaai0xaa1ixaa12c3xaa13xaa14xaai5 xaai6xaai7xaa18xaa!9xaa2oxaa2ixaa22c4xaa23xaa24xaa25C5xaa26xaa27C6-R2
SEQ ID NO:!
wherein xaa17 is a positively charged D-amino acid residue; xaa18 is an aromatic D-amino acid residue; xaa! to xaa16, xaaig to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue;
R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of Ci and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both C1 and C6 are D-amino acid residues having a side chain double bond and the side chains of C1 and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to C5 through disulfide, diseleno or sulfide-selenium bonds.
As used herein the term "amino acid" refers to compounds having an amino group and a carboxylic acid group. The amino acids incorporated into the amino acid sequences of the present invention are D-amino acids and may be the D-form of naturally occurring proteogenic amino acids, or may be D-forms of non-naturally occurring amino acids. Amino acids incorporated into oligopeptides at the N- or C-terminus (R1 or R2) may be D-amino acids or naturally or non-naturally occurring L-amino acids. - A -
The naturally occurring proteogenic amino acids are shown in Table 1 together with their three letter and one letter codes. L-amino acids are referred to using capital letters or initial capital letters whereas D-amino acids are referred to using lower case letters.
TABLE 1
Figure imgf000006_0001
* Glycine does not have an asymmetric centre at the alpha carbon and therefore is not
D or L. Herein glycine may be referred to using codes designating D or L amino acids (GIy, gly, G, or g).
Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal- 5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulfhydryl groups that may occur in oligopeptides at the N- or C-terminus (R1 or R2) or are other than those present in ci to C6, may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid and 2-thienyl alanine. Examples of suitable non-proteogenic or non-naturally occurring amino acids contemplated herein is shown in Table 2.
TABLE 2
Non-conventional Code Non-conventional Code amino acid amino acid
α-aminobutyric acid Abu N-(4-aminobutyl)glycine NgIu D-ornithine Dorn N-(2-am inoethyl)glycine Naeg aminocyclopropane- Cpro N-(3-aminopropyl)glycine Norn carboxylate N-benzylglycine Nphe aminoisobutyric acid Aib N-(2-carbamylethyl)glycine NgIn aminonorbornyl- Norb N-(carbamylmethyl)glycine Nasn carboxylate N-(2-carboxyethyl)glycine NgIu cyclohexylalanine Chexa N-(carboxymethyl)glycine Nasp cyclopentylalanine Cpen N-cyclobutylglycine Ncbut
D-N-methylalanine Dnmala N-cycloheptylglycine Nchep
D-N-methylarginine Dnmarg N-cyclohexylglycine Nchex
D-N-methylasparagine Dnmasn N-cyclodecylglycine Ncdec
D-N-methylaspartate Dnmasp N-cylcododecylglycine Ncdod
D-N-methylcysteine Dnmcys N-cyclooctylglycine Ncoct
D-N-methylglutamine Dnmgln N-cyclopropylglycine Ncpro
D-N-methylglutamate Dnmglu N-cycloundecylglycine Ncund
D-N-methylhistidine Dnmhis N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylisoleucine Dnmile N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylleucine Dnmleu N-(3-guanidinopropyl)glycine Narg
D-N-methyllysine Dnmlys N-( 1 -hydroxyethyOglycine Nthr
N-methylcyclohexylalanine Nmchexa N-(hydroxyethyl))glycine Nser
D-N-methylornithine Dnmorn N-(imidazolylethyl))glycine Nhis
N-methylglycine NaIa N-(3-indolylyethyl)glycine Nhtrp
N-methylam inoisobutyrate Nmaib N-methyl-γ-aminobutyrate Nm gabu
N-( 1 -methylpropyOglycine Nile D-N-methylmethionine Dnmmet
N-(2-methylpropyl)glycine Nleu N-methylcyclopentylalanine Nmcpen
D-N-methyltryptophan Dnmtrp D-N-methylphenylalanine Dnmphe
D-N-methyltyrosine Dnmtyr D-N-methylproline Dnmpro
D-N-methylvaline Dnmval D-N-methylserine Dnmser γ-aminobutyric acid Gabu D-N-methy lthreon ine Dnmthr
D-t-butylglycine tbug N-( 1 -methylethyl)glycine Nval
D-ethylglycine etg N-methyl-napthylalanine Nmanap
D-homophenylalanine hphe N-methylpenicillamine Nmpen
N-(N-(2,2-diphenylethyl) Nnbhm N-(/?-hydroxyphenyl)glycine Nhtyr carbamylmethyl)glycine N-(thiomethyl)glycine Ncys
1 -carboxy- 1 -(2,2-diphenyl Nmbc penicillamine Pen ethylam ino)cyclopropane D-methylethylglycine metg
5-hydroxy-D-lysine Dhlys N-(2-methylthioethyl)glycine Nmet
D-pipecolic acid (D-homo Dpip D-N-methylhomophenylalanine Nmhphe proline) N-(N-(3,3-diphenylpropyl) Nnbhe
D-norlysine D-nor-Lys carbamylmethyl)glycine D-pyroglutamic acid Dpglu 1,3-diaminopropionic acid dap
4-hyroxyproline Hyp O-methyl-D-serine Omser
D-homotyrosine htyr ornithine Orn
D-β-homolysine Dbhk D-homoleucine DHIe
D-homoarginine Dharg phosphotyrosine ptyr
D-allylglycine DHag
If not specified, the non-naturally occurring amino acids listed in Table 2 are preferably in the D-conformation, although amino acids having corresponding L-conformations may be incorporated as the amino acid residue of R1 or R2 or into oligopeptides at the N- or C-terminus (R1 and R2).
Suitable β-amino acids include, but are not limited to, D-β-homoalanine, D-β-homoarginine, D-β-homoasparagine, D-β-homoaspartic acid, D-β-homoglutamic acid, D-β-homoglutamine, D-β-homoisoleucine, D-β-homoleucine, D-β-homolysine,
D-β-homomethionine, D-β-homophenylalanine, D-β-homoproline, D-β-homoserine,
D-β-homothreonine, D-β-homotryptophan, D-β-homotyrosine, D-β-homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric acid, 3-amino-nitrophenylbutyric acid, 3-amino- methylphenylbutyric acid, 3-amino-pentanoic acid, 2-amino-tetrahydroisoquinoline acetic acid, 3-amino-naphthyl-butyric acid, 3-amino-pentafluorophenyl-butyric acid, 3-amino- benzothienyl-butyric acid, 3-amino-dichlorophenyl-butyric acid, 3-amino-difluorophenyl- butyric acid, 3-amino-iodophenyl-butyric acid, 3-amino-trifluoromethylphenyl-butyric acid, 3-amino-cyanophenyl-butyric acid, 3 -amino-thienyl -butyric acid, 3-amino-5- hexanoic acid, 3-amino-furyl-butyric acid, 3-amino-diphenyl-butyric acid, 3-amino-6- phenyl-5-hexanoic acid and 3-amino-hexynoic acid.
xaaπ is a positively charged D-amino acid residue. This is an amino acid having a side chain capable of bearing a positive charge. Examples of suitable amino acid residues include, but are not limited to, D-lysine, D- 1,3-diaminopropionic acid, D-arginine, D-histidine, 5-hydroxy-D-lysine, D-norlysine, D-homolysine and D-ornithine, especially D-lysine, D-l,3-diaminopropionic acid, 5-hydroxy-D-lysine, D-norlysine and D-homolysine, more especially D-lysine and D-l,3-diaminopropionic acid.
xaais is an aromatic D-amino acid residue. Suitable aromatic D-amino acid residues include, but are not limited to, D-tyrosine, D-phenylalanine, D-tryptophan, D-homotyrosine, D-naphthylalanine and D-phosphotyrosine, especially D-tyrosine, D-phenylalanine, D-tryptophan and D-homotyrosine, more especially D-tyrosine.
R1 is selected from hydrogen, an amino acid residue, an N-terminal capping group and an oligopeptide optionally capped with an N-terminal capping group. The amino acid residue or sequence of amino acid residues in the oligopeptide may include one or more of D- and L-amino acid residues, naturally occurring amino acid residues, non-naturally occurring amino acid residues, proteogenic amino acid residues and non-proteogenic amino acid residues.
The N-terminal capping group is a group that caps the N-terminal amino group without affecting the tertiary structure of the peptide. In some cases the N-terminal capping group is an acyl group, such as acetyl or succinyl, or the N-terminal capping group may be a label such as a fluorescent biological label attached directly, or through a linker, to Xaa29 or Xaa28 or in their absence to ci. A suitable fluorescent label is fluoroscein and suitable biological labels include biotin and streptavidin. Alternatively, the N-terminal capping group is an antibody or a molecule that recognises and targets a TEM cell.
Linkers that link an N-terminal capping group to xaa29, xaa28 or cj may be any divalent linker that provides the required length and does not inhibit or prevent the binding of the peptide to the KvI .3 channel. Suitable linkers include, but are not limited to, alkylene groups -(CH2)p- where p is an integer from 1-20, preferably 1-10 and alkylene groups in which one or more -CH2- group has been replaced by a heteroatom, selected from the oxygen, sulfur or nitrogen. Each linker may also include substituents suitable for forming an attachment with the peptide and the label or other group such as an antibody. For example, the linker may contain a terminal carboxyl group that is able to form an amide bond with the amino terminus of xaa2g, xaa28 or ci or with the label or a biological molecule, or the linker may include a terminal amino group that is able to link to a label or biological molecule or to a reactive group in the side chain of xaa2g or xaa28. Suitable compounds for use as linkers include, but are not limited to, amino-ethyloxy-ethyloxy acetic acid, 5-amino-3-oxapentanoic acid, 6-aminohexanoic acid, 5-aminopentanoic acid and 8-aminooctanoic acid.
R2 is selected from hydrogen, an amino acid residue, a C-terminal capping group and an oligopeptide optionally capped with a C-terminal capping group. The amino acid residue or oligopeptide may be any amino acid residue or sequence of amino acid residues including both D- or L-amino acid residues, naturally occurring amino acid residues, non-naturally occurring amino acid residues, proteogenic amino acid residues and non-proteogenic amino acid residues.
The C-terminal capping group is a group that caps the C-terminal carboxy group without affecting the tertiary structure of the peptide. In some cases the C-terminal capping group is an amide group, such as NH2, or the C-terminal capping group may be a label, such as a fluorescent or biological label attached directly, or through a linker, to C6. A suitable fluorescent label is fluorescein and suitable biological labels include biotin and streptavidin. Alternatively, the C-terminal capping group is an antibody or a molecule that recognises and targets a TEM cell.
Suitable linkers for linking R2 to C6 are similar to those that link R1 to xaa28, xaa29 or C1 and may include suitable groups that can form an amide bond with the C-terminus, such as a terminal amino group and a group suitable for linking the linker to the biological molecule, such as a second terminal amino group or a terminal carboxylic acid. Suitable compounds for use as linkers include, but are not limited to, amino-ethyloxy-ethyloxy acetic acid,
5-amino-3-oxapentanoic acid, 6-aminohexanoic acid, 5-aminopentanoic acid and 8-aminooctanoic acid. As used herein, the term "D-amino acid residue having a side chain double bond" refers to an D-amino acid that includes a -CH=CH- functional group in its side chain. In particular embodiments, the double bond is present at the terminus of the side chain. Examples of suitable amino acid residues include, but are not limited to, D-allylglycine, D-but-3- enylglycine, 2-methylbut-3-enylglycine and the like, especially D-allylglycine.
The term "linked to form a dicarba bond" refers to where two amino acid residues both having a side chain double bond are reacted with one another to result in covalent bonding between a carbon atom from each side chain to form a -CH=CH- or -CH2-CH2- bond. In particular embodiments, the covalent bond is formed by ring closing metathesis to form a -CH=CH- bond which may then be optionally reduced to provide a single bond, -CH2-CH2-.
In particular embodiments where C1 and C6 are linked by a dicarba bond, ci and C6 are both D-allylglycine residues and the dicarba bond is a double bond formed by ring closing metathesis between the double bonds of the allyl groups.
The amino acid sequence has a minimum of 33 amino acids in the sequence. In some embodiments, the amino acid sequence is 33 to 45 amino acids in length.
The compounds of the invention may be in the form of pharmaceutically acceptable salts. It will be appreciated however that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
In some embodiments of the invention one or more of the following applies:
Each of C1 to C6 are independently selected from D-cysteine or D-selenocysteine and C1 is linked to C6, C2 is linked to C4 and C3 is linked to C5 through disulfide, diseleno or sulfide-selenium bonds. C1 is D-cysteine, D-homocysteine or D-penicillamine, especially D-cysteine. xaaj is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-isoleucine. xaa2 is a negatively charged D-amino acid (Naa), especially D-aspartic acid or D-glutamic acid, more especially D-aspartic acid; xaa3 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine. xa^ is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-isoleucine. xaa5 is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-proline. xaaό is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine. xaa7 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine. xaag is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine.
C2 is D-cysteine. xaa9 is polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine,
D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine. xaaio is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan or glycine, more especially D-alanine, D-valine, D-leucine, D-isoleucine or glycine, most especially D-alanine. xaaπ is a non-polar or aromatic D-amino acid, especially a non-polar D-amino acid (NPaa); especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine, D-tryptophan or D-tyrosine, especially a non-polar and aromatic D-amino acid such as D-phenylalanine or D-tryptophan, most especially D-phenylalanine. xaai2 is polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-glutamine or D-asparagine, most especially D-glutamine.
C3 is D-cysteine. xaa13 is a positively charged amino acid (Paa), especially D-lysine, D-arginine,
D-histidine or D-l,3-diaminopropionic acid, more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine. xaai4 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-histidine. xaa15 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine. xaai6 is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-methionine, D-alanine, D-valine, D-leucine or D-isoleucine, most especially D-methionine. xaa17 is selected from D-lysine, D-l,3-diaminopropionic acid, D-arginine, D-histidine, 5-hydroxy-D-lysine, D-norlysine, D-homolysine and D-ornithine, especially D-lysine, D-l,3-diaminopropionic acid, 5-hydroxy-D-lysine, D-norlysine, D-homolysine, more especially D-lysine or D-l,3-diaminopropionic acid. xaa18 is selected from D-tyrosine, D-phenylalanine, D-tryptophan, D-homotyrosine, D-naphthylalanine and D-phosphotyrosine, especially D-tyrosine, D-phenylalanine, D-tryptophan or D-homotyrosine, more especially D-tyrosine. Xaa^ is a positively charged amino acid (Paa), especially D-lysine, D-arginine,
D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine. xaa20 is a non-polar D-amino acid (NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine or D-tryptophan, more especially D-isoleucine, D-leucine or D-valine, most especially D-leucine. xaa21 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine,
D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine. xaa22 is a non-polar or aromatic D-amino acid, especially a non-polar D-amino acid
(NPaa), especially D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine, D-tryptophan or D-tyrosine, especially a non-polar and aromatic
D-amino acid such as D-phenylalanine and D-tryptophan, most especially
D-phenylalanine.
C4 is D-cysteine. xaa23 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine. xaa24 is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-lysine or D-l,3-diaminopropionic acid, most especially D-lysine. xaa25 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine.
C5 is D-cysteine. xaa26 is glycine or a small non-polar D-amino acid (SNPaa), especially glycine, D-alanine, D-valine, D-leucine or D-isoleucine, more especially glycine or D-alanine, most especially glycine. xaa27 is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, especially D-threonine or D-serine, more especially D-threonine.
C6 is D-cysteine, D-homocysteine or D-penicillamine, especially D-cysteine. C1 and C6 are both D-allylglycine and they are linked by a dicarba double bond. xaa28 is absent or is a polar, uncharged D-amino acid (PUaa), especially D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine or D-glutamine, more especially D-serine or D-threonine, most especially D-serine. xaa29 is absent or is a positively charged amino acid (Paa), especially D-lysine, D-arginine, D-histidine or D-l,3-diaminopropionic acid, more especially D-arginine.
R2 is hydrogen or NH2.
R1 is hydrogen, acyl, a phosphorylated amino acid residue, or a fluorescent or biological label optionally linked to C1, xaa2g or xaa28 through a linker, especially hydrogen, acetyl, phosphotyrosine or a fluoroscein or biotin label optionally linked to ci, xaa29 or xaa28 through a linker, more especially hydrogen, phosphotyrosine or a fluoroscein or biotin label linked to ci, xaa29 or xaa28 through a linker, even more especially hydrogen, D-phosphotyrosine or a fluoroscein label linked to xaa29 through an amino-ethyloxy- ethyloxy-acetyl linker.
In some embodiments, the peptide of SEQ ID NO: 1 has one of the following sequences: rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:2 rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO: 3 ptyr-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID N0:4 ptyr-rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO: 5 fluoroscein-aminoethyloxyethyloxyacetyl-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:6 fluoroscein-aminoethyloxyethyloxyacetyl-rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc
SEQ ID NO:7 sscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 8
(N-acetyl)-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 9 scidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 10 cidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 11 ascidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 12 rscadtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 13 rscadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO: 14 rscadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 15 rscidaipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 16 rscidtapksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 17 rscidtiaksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 18 rscidtipasrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 19 rscidtipesrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:20 rscidtipqsrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:21 rscidtipkarctafqckhsmkyrlsfcrktcgtc SEQ ID NO:22 rscidtipksactafqckhsmkyrlsfcrktcgtc SEQ ID NO:23 rscidtipksectafqckhsmkyrlsfcrktcgtc SEQ ID NO:24 rscidtipksqctafqckhsmkyrlsfcrktcgtc SEQ ID NO:25 rscidtipksrcaafqckhsmkyrlsfcrktcgtc SEQ ID NO:26 rscidtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO:27 rscidtipksrctawqckhsmkyrlsfcrktcgtc SEQ ID NO:28 rscidtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO:29 rscidtipksrctafackhsmkyrlsfcrktcgtc SEQ ID NO:30 rscidtipksrctafeckhsmkyrlsfcrktcgtc SEQ ID NO:31 rscidtipksrctafqcahsmkyrlsfcrktcgtc SEQ ID NO: 32 rscidtipksrctafqcehsmkyrlsfcrktcgtc SEQ ID NO:33 rscidtipksrctafqckasmkyrlsfcrktcgtc SEQ ID NO:34 rscidtipksrctafqckksmkyrlsfcrktcgtc SEQ ID NO:35 rscidtipksrctafqckhamkyrlsfcrktcgtc SEQ ID NO:36 rscidtipksrctafqckhsakyrlsfcrktcgtc SEQ ID NO:37 rscidtipksrctafqckhs(norleu)kyrlsfcrktcgtc SEQ ID NO:38 rscidtipksrctafqckhsm(orn)yrlsfcrktcgtc SEQ ID NO:39 rscidtipksrctafqckhsmkfrlsfcrktcgtc SEQ ID NO:40 rscidtipksrctafqckhsmk(nitrophe)rlsfcrktcgtc SEQ ID N0:41 rscidtipksrctafqckhsmk(aminophe)rlsfcrktcgtc SEQ ID NO:42 rscidtipksrctafqckhsmk(benzylphe)rlsfcrktcgtc SEQ ID NO:43 rscidtipksrctafqckhsmkyalsfcrktcgtc SEQ ID NO:44 rscidtipksrctafqckhsmkyelsfcrktcgtc SEQ ID NO:45 rscidtipksrctafqckhsmkyrasfcrktcgtc SEQ ID NO:46 rscidtipksrctafqckhsmkyrlafcrktcgtc SEQ ID NO:47 rscidtipksrctafqckhsmkyrlsacrktcgtc SEQ ID NO:48 rscidtipksrctafqckhsmkyrlsfcaktcgtc SEQ ID NO:49 rscidtipksrctafqckhsmkyrlsfcratcgtc SEQ ID NO:50 rscidtipksrctafqckhsmkyrlsfcrkacgtc SEQ ID NO:51 rscidtipksrctafqckhsmkyrlsfcrktcgac SEQ ID NO:52 scadtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:53 scadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO: 54 scadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 55 scidaipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 56 scidtapksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:57 scidtiaksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:58 scidtipasrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:59 scidtipesrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:60 scidtipqsrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:61 scidtipkarctafqckhsmkyrlsfcrktcgtc SEQ ID NO:62 scidtipksactafqckhsmkyrlsfcrktcgtc SEQ ID NO:63 scidtipksectafqckhsmkyrlsfcrktcgtc SEQ ID NO:64 scidtipksqctafqckhsmkyrlsfcrktcgtc SEQ ID NO:65 scidtipksrcaafqckhsmkyrlsfcrktcgtc SEQ ID NO: 66 scidtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO:67 scidtipksrctawqckhsmkyrlsfcrktcgtc SEQ ID NO:68 scidtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 69 scidtipksrctafackhsmkyrlsfcrktcgtc SEQ ID NO:70 scidtipksrctafeckhsmkyrlsfcrktcgtc SEQ ID NO:71 scidtipksrctafqcahsmkyrlsfcrktcgtc SEQ ID NO: 72 scidtipksrctafqcehsmkyrlsfcrktcgtc SEQ ID NO:73 scidtipksrctafqckasmkyrlsfcrktcgtc SEQ ID NO: 74 scidtipksrctafqckksmkyrlsfcrktcgtc SEQ ID NO:75 scidtipksrctafqckhamkyrlsfcrktcgtc SEQ ID NO: 76 scidtipksrctafqckhsakyrlsfcrktcgtc SEQ ID NO:77 scidtipksrctafqckhs(norleu)kyrlsfcrktcgtc SEQ ID NO:78 scidtipksrctafqckhsm(orn)yrlsfcrktcgtc SEQ ID NO: 79 scidtipksrctafqckhsm(diaminopropionic)yrlsfcrktcgtc SEQ ID NO:80 scidtipksrctafqckhsmkfrlsfcrktcgtc SEQ ID N0:81 scidtipksrctafqckhsmk(nitrophe)rlsfcrktcgtc SEQ ID NO:82 scidtipksrctafqckhsmk(aminophe)rlsfcrktcgtc SEQ ID NO:83 scidtipksrctafqckhsmk(benzylphe)rlsfcrktcgtc SEQ ID NO: 84 scidtipksrctafqckhsmkyalsfcrktcgtc SEQ ID NO:85 scidtipksrctafqckhsmkyelsfcrktcgtc SEQ ID NO:86 scidtipksrctafqckhsmkyrasfcrktcgtc SEQ ID NO: 87 scidtipksrctafqckhsmkyrlafcrktcgtc SEQ ID NO: 88 scidtipksrctafqckhsmkyrlsacrktcgtc SEQ ID NO:89 scidtipksrctafqckhsmkyrlsfcaktcgtc SEQ ID NO: 90 scidtipksrctafqckhsmkyrlsfcratcgtc SEQ ID N0:91 scidtipksrctafqckhsmkyrlsfcrkacgtc SEQ ID NO:92 scidtipksrctafqckhsmkyrlsfcrktcgac SEQ ID NO:93 yscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:94 kscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 95 hscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:96 qscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:97 pprscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:98 mrscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 99 grscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 1 OO rscidtipvsrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 101 rscidtipksrctdfqckhsmkyrlsfcrktcgtc SEQ ID NO: 102 scidtipksrctdfqckhsmkyrlsfcrktcgtc SEQ ID NO: 103 rscidtipksrctaiqckhsmkyrlsfcrktcgtc SEQ ID NO: 104 scidtipksrctaiqckhsmkyrlsfcrktcgtc SEQ ID NO: 105 rscidtipksrctavqckhsmkyrlsfcrktcgtc SEQ ID NO: 106 scidtipksrctavqckhsmkyrlsfcrktcgtc SEQ ID NO : 107 rscidtipksrctafrckhsmkyrlsfcrktcgtc SEQ ID NO: 108 scidtipksrctafrckhsmkyrlsfcrktcgtc SEQ ID NO: 109 rscidtipksrctafkckhsmkyrlsfcrktcgtc SEQ ID NO: 110 scidtipksrctafkckhsmkyrlsfcrktcgtc SEQ ID NO: 111 rscidtipasectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 112 scidtipasectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 113 rscidtipvsectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 114 scidtipvsectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 115 rscidtipvsactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 116 scidtipvsactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 1 17 rscidtipasactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 1 18 scidtipasactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 119 rscidtipksectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 120 scidtipksectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 121 rscidtipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 122 scidtipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 123 rscidtipvsectdiqckhsmkyrlsfcrktcgtc SEQ ID NO: 124 scidtipvsectdiqckhsmkyrlsfcrktcgtc SEQ ID NO: 125 rtckdlipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 126 tckdlipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 127 qscadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO: 128 qscadtipksrctaaqckhsm(dap)yrlsfcrktcgtc SEQ ID NO : 129 qscadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO : 130 qscadtipksrctaaqckhsm(dap)yrasfcrktcgtc SEQ ID NO : 131
The compounds of the present invention may be prepared using techniques known in the art. For example, peptides can be synthesized using various solid phase techniques (45) or using an automated synthesis, for example, using a Pioneer peptide synthesizer and standard F-moc chemistry (46). D-amino acids may be substituted for L-amino acids in such synthesis.
Once the peptides of the present invention have been prepared, they may be substantially purified by preparative FIPLC. The composition of the synthetic peptides can be confirmed by amino acid analysis or by sequencing (using the Edman degradation procedure).
Suitable protecting and deprotecting methods for reactive functional groups such as carboxylic acids and amines are known in the art, for example, in Protective Groups in Organic Synthesis, T.W. Green & P. Wutz, John Wiley & Son, 3rd Ed, 1999.
The compounds of the present invention in which C1 and C6 are linked by a dicarba bond can also be prepared by methods known in the art. For example, a linear peptide may be prepared using Fmoc-solid phase synthesis where the residues at ci and c6 both include a double bond in their side chains, for example, both C1 and C6 are D-allylglycine. Once linear peptide synthesis is complete, but before deprotection of the side chains or cleavage from the resin, the peptide is subjected to ring closing metathesis conditions to form a dicarba double bond between a carbon atom of the double bond of ci side chain and a carbon atom of the double bond of C6 side chain. In a particular embodiment, the ring closing metathesis is carried out in the presence of 5-10% second generation Grubbs catalyst in dichloromethane containing 10% 0.4M lithium chloride in dimethylfbrmamide. The metathesis reaction may be carried out under standard conditions or may be carried out using microwave irradiation at 2.45 GHz for 1-2 hours thereby maintaining a temperature of about 100°C. Once the ring closing metathesis is achieved, the peptide may be deprotected and cleaved from the resin. The disulfide, diseleno or sulphide-selenium bonds can be formed between C2 and C4 and C3 and C5 before or after cleavage from the resin. If required, selective protection and deprotection at C2 and C4 and/or C3 and C5 may be used to ensure correct connectivity of disulfide bonds.
The peptides of the invention may be tested for their ability to selectively bind to Kv 1.3 channels using assays known in the art. For example, cells stably expressing mKvl.l, rKvl.2, mKvl .3 and hKvl.5 channels may be exposed to the peptides of the invention and the channel currents measured by the patch clamp method as described in Grissmer et al. (28). Alternatively, if a fluorescent label is present on the peptide, fluorescence detection may be possible as described in Beeton et al, (14).
In another aspect of the invention there is provided a method of blocking a Kv 1.3 potassium channel comprising exposing a cell expressing the Kv 1.3 potassium channel to a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
R1-xaa29xaa28Cixaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8c2xaa9xaai0xaai1xaa12c3xaai3xaai4xaai5 xaai6xaa17xaa18xaa19xaa2oxaa21xaa22C4xaa23xaa24xaa25c5xaa26xaa27c6-R2 SEQ ID NO:!
wherein xaaπ is a positively charged D-amino acid residue; xaajs is an aromatic D-amino acid residue; xaaj to xaai6, xaai9 to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue;
R is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group; R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of Ci and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C1 is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both C1 and C6 are D-amino acid residues having a side chain double bond and the side chains of C1 and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to C5 through disulfide, diseleno or sulfide-selenium bonds.
In yet another aspect of the present invention there is provided a method of inhibiting activation of T-cells comprising exposing the T-cells to a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
R1-xaa29xaa28cjxaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8c2xaa9xaa10xaa1ixaa12C3xaa13xaa14xaai5 xaai6xaai7xaa18xaa19xaa20xaa2)xaa22C4xaa23xaa24xaa25C5xaa26xaa27c6-R
SEQ ID NO:!
wherein xaai7 is a positively charged D-amino acid residue; xaai8 is an aromatic D-amino acid residue; xaai to xaa16, xaaig to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue; R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group; R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of cj and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both C1 and C6 are D-amino acid residues having a side chain double bond and the side chains of C1 and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to c5 through disulfide, diseleno or sulfide-selenium bonds.
It should be understood that the cell expressing a KvI.3 potassium channel or T-cells which are treated according to a method of the present invention may be located ex vivo or in vivo. By "ex vivo" is meant that the cell has been removed from the body of a subject wherein the modulation of its activity will be initiated in vitro. For example, the cell may be a cell which is to be used as a model for studying any one or more aspects of the pathogenesis of conditions which are characterised by aberrant activation of T-cells. In a preferred embodiment, the subject cell is located in vivo.
In yet another aspect of the present invention there is provided a method of treatment and/or prophylaxis of a disease or disorder characterized by aberrant or abnormal activation of T-cells in a subject, comprising administering to the subject an effective amount of a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
R -xaa29xaa28Cixaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8C2xaa9xaaioxaai jxaai2C3xaai3xaai4xaai5 xaai6xaai7xaai8xaai9xaa2oxaa2ixaa22c4xaa23xaa24xaa25c5xaa26xaa27c6-R
SEQ ID NO: !
wherein xaaπ is a positively charged D-amino acid residue; xaais is an aromatic D-amino acid residue; xaai to xaai6, xaajg to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue; R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of C1 and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C1 is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both C1 and C6 are D-amino acid residues having a side chain double bond and the side chains of C1 and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to c5 through disulfide, diseleno or sulfide-selenium bonds.
In some embodiments, the T-cells are human effector memory T-cells (TEM cells).
In a further aspect of the present invention there is provided a method of treatment and/or prophylaxis of an autoimmune disease or organ or tissue transplant rejection in a subject, comprising administering to the subject an effective amount of a peptide, or pharmaceutically acceptable salt thereof, said peptide comprising amino acid sequence 1 :
R1-xaa29xaa28cixaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8C2xaa9xaa10xaanxaai2c3xaai3xaai4xaai5 xaa 16xaa 17xaa 18xaa 19xaa20xaa21 xaa22c4xaa23xaa24xaa25C5xaa26xaa27c6-R2
SEQ ID NO: !
wherein xaai7 is a positively charged D-amino acid residue; xaais is an aromatic D-amino acid residue; xaai to xaai6, xaa^ to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue; R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of C1 and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and ci is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both ci and C6 are D-amino acid residues having a side chain double bond and the side chains of cj and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and c2 is linked to C4 and c3 is linked to c5 through disulfide, diseleno or sulfide-selenium bonds.
The term "subject" as used herein refers to an organism that has cells that express KvI .3 receptors, especially T-cells that express KvI.3 receptors. In some embodiments the subject is a mammal. The term "mammal" as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer). In some embodiments, the mammal is human or a laboratory test animal, especially a human.
As used herein the term "disease or disorder characterized by aberrant or abnormal activation of T-cells" refers to diseases or disorders in which T-cells are overactivated or inappropriately activated. This may occur in autoimmune diseases an in organ or tissue transplant rejection. As used herein "autoimmune diseases" refers to diseases in which the subjects own immune system malfunctions and immune cells such as T-cells become activated resulting in an immune response against the subject's own cells and/or tissues. Autoimmune diseases that may be treated by the present invention include multiple sclerosis, rheumatoid arthritis, Type-I diabetes (IDDM), psoriasis, Hashimoto's disease, Sjogren's syndrome, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anaemia, autoimmune hepatitis, autoimmune oophoritis, Coeliac disease, Crohn's disease, gestational pemphigoid, Goodpasture's syndrome, Grave's disease, Guillian-Barre syndrome, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, polyarthritis (in dogs), primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis, warm autoimmune haemolytic anaemia and Wegener's granulomatosis. In some embodiments, the autoimmune disease is multiple sclerosis, rheumatoid arthritis, Type-I diabetes, psoriasis or lupus erythematosis.
Aberrant or abnormal activation of T-cells may also occur after organ or tissue transplants resulting in rejection of the transplanted organ or tissue. The present invention also relates to methods of treating or preventing rejection of organ or tissue transplants. Suitable transplants include organs and tissues such as heart, lungs, kidney, liver, pancreas, intestine and skin.
An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. An effective amount in relation to a human patient, for example, may lie in the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. The dosage is preferably in the range of lμg to 1 g per kg of body weight per dosage, such as is in the range of lmg to Ig per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 μg to 1 mg per kg of body weight per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.
Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.
The present invention further contemplates a combination of therapies, such as the administration of the peptides of the invention or pharmaceutically acceptable salts thereof together with the subjection of the subject to other agents or procedures which are useful in the treatment of diseases and disorders characterized by the aberrant or abnormal activation of T-cells or autoimmune diseases. For example, the compounds of the present invention may be administered in combination with, in the same composition or in separate compositions simultaneously or sequentially, other chemotherapeutic drugs used to treat the cause of or symptoms of the disease or disorder. Suitably, the peptides of the present invention may be combined with immunosuppressants, such as glucocorticoid immunosuppressants, cytostatic immunosuppressants, antibody immunosuppressants, immunosuppressants that act on immunophilins and other immunosuppressants. Immunosuppressants that are useful in combination therapies with the peptides of the present invention include, but are not limited to, Cortisol, hydrocortisol, dexamethasone, cyclophosphamide, nitrosoureas, folic acid analogues such as methotrexate, purine analogues such as azathioprine and mercaptopurine, pyrimidine analogues, protein synthesis inhibitors, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, Atgam (R), Thymoglobuline (R), OKT3 (R), basilixmab, daclizumab, cyclosporine, tacrolimus, sirolimus, interferons, infliximab, etanercept, adalimumab, mycophenolic acid and FTY720.
While it is possible that, for use in therapy, a peptide of the invention or a pharmaceutically acceptable salt thereof may be administered as a neat chemical, it is preferable to present the active ingredient as a pharmaceutical composition.
Accordingly, in a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide or pharmaceutically acceptable salt thereof comprising amino acid sequence 1 :
R1-xaa29xaa28Cixaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8c2xaa9xaai0xaaiixaa12c3xaai3xaai4xaai5 xaai6xaa17xaa]8xaai9xaa20xaa2ixaa22c4xaa23xaa24xaa25C5xaa26xaa27c6-R
SEQ ID NO:!
wherein xaa17 is a positively charged D-amino acid residue; xaa18 is an aromatic D-amino acid residue; xaai to xaai6, xaajg to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue;
R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of cj and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C1 is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both C1 and C6 are D-amino acid residues having a side chain double bond and the side chains of cj and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to C5 through disulfide, diseleno or sulfide-selenium bonds; and a pharmaceutically acceptable carrier.
Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The peptides of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to two hundred (200) milligrams, per tablet, are accordingly suitable representative unit dosage forms. The compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt or derivative of the compound of the invention. For preparing pharmaceutical compositions from the peptides of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
The peptides according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-iϊlled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents, as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump. To improve nasal delivery and retention the compounds according to the invention may be encapsulated with cyclodextrins, or formulated with their agents expected to enhance delivery and retention in the nasal mucosa.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofiuoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.
Alternatively the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 1 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
When desired, formulations adapted to give sustained release of the active ingredient may be employed.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
The invention will now be described with reference to the following examples which illustrate some preferred aspects of the present invention. However, it is to be understood that the particularity of the following description of the invention is not to supersede the generality of the preceding description of the invention.
Brief Description of Figures
Figure 1. Pharmacology of D-ShK. A, Effect of ShK (top) and D-allo-ShK (bottom) on
KvI .3 (left) and KvI .1 (right) currents in stably transfected cells. B, Dose-dependent inhibition of Kv 1.3 (open symbols) and Kv 1.1 (closed symbols) by ShK (A), ShK-amide (T) and D-allo-ShK (■). Each data point is the mean ± SD of three independent determinations. C, Selectivity of D-allo-ShK for K channels.
Figure ID. Characterization of the effects of ShK and D-alloShK on Kv 1.3 currents, ShK blocks the closed state of the channel, but D-allo-ShK does not. A Kv 1.3 control current was elicited by a 200ms depolarizing pulse to 40 mV. ShK (2OpM; middle panel; n =3) or D-allo-ShK (5OnM; bottom panel, n = 6) was applied to the bath solution whereas the membrane was held at -80 mV. After 5 minutes, consecutive 200 ms pulses were applied every 30 seconds. The dotted line shows the peak current amplitude before the addition of the blocker.
Figure 2. D-allo-ShK preferentially suppresses the proliferation of TEM cells. Dose-dependent inhibition by ShK (o) and D-allo-ShK (•) of [3H]-Tdr incorporation by rat TEM (A) and naive and TCM (B) lymphocytes. IC50S on TEM cells « 80 pM (ShK) and 10 nM (D-allo-ShK); IC50S on naϊve and TCM cells » 90 nM (ShK) and 10 μM (D-allo-ShK).
Figure 3. Circulating half-life of D-allo-ShK A, a single dose of 1 mg/kg of D-allo-ShK was injected subcutaneously into five rats. Blood was drawn at the indicated times and tested by patch-clamp to determine the amount of D-allo-ShK. B, data fit to a single exponential decay. Half-life was ~ 40 min.
Figure 3C. DTH reaction was elicited against ovalbumin and rats (n = 6/group) were treated with saline or 1 mg/kg D-allo-ShK dissolved in saline. Ear swelling was measured 24 h later. Statistical analysis was carried out using the Mann- Whitney Latest.
EXAMPLES
Example 1: Synthesis and analysis of D-allo-ShK amide
Fmoc-D-amino acids (Bachem Feinchemikalien) included D-Arg(Pmc), D-Asp(OtBu), D- Cys(Trt), D-GIn(Tn), D-His(Trt), D-Lys(Boc), D-Ser(tBu) and D-Thr(tBu). All other non- side chain protected derivatives used for synthesis were also in the D-configuration except for GIy which is achiral. Stepwise assembly was carried out on an Applied Biosystems 43 IA peptide synthesizer at the 0.25 mmol scale using Fmoc-RamageTm-amide-resin. Residues 34 through 22 were single coupled. At this point, half of the resin was removed to effect better mixing. The remainder of the peptide sequence was double coupled to the remaining resin aliquot. All couplings were mediated by dicyclohexylcarbodiimide in the presence of 2 eq of 1-hydroxybenzotriazole. Following final removal of the Fmoc-group, the peptide resin (2.42 g) was cleaved from the resin and simultaneously deprotected using reagent K (18) for 2 h at room temperature. Following cleavage, the peptide was filtered to remove the spent resin beads and precipitated with ice-cold diethyl ether. The peptide was collected on a fine filter funnel, washed with ice-cold ether and finally extracted with 20% AcOH in H2O. The peptide extract was subsequently diluted into 2 L of H2O, the pH adjusted to 8.0 with NH4OH and allowed to air oxidize at room temperature for 36 h. Following oxidation of the disulfide bonds, the peptide solution was acidified to pH 2.5 and pumped onto a Rainin Dynamax Ci8 column (5.0 x 30 cm). The sample was eluted with a linear gradient from 5-30% acetonitrile into H2O containing 0.1% trifluoroacetic acid (TFA). The resulting fractions were analyzed using two analytical RP-HPLC systems: TFA and triethylammonium phosphate (19). Pure fractions were pooled and lyophilized. Upon lyophilization, 120 mg of D-allo-ShK toxin amide was obtained, representing a theoretical yield of 21% (from the starting resin).
Peptide Analysis. Purified peptide was analysed by reverse phase HPLC (Vydac C18-silica, 5 μ, 120 angstrom, 0.46 x 250 mm column) using gradient conditions of 5 to 50% B in 20 minutes at 1.5 mL/min (A buffer is 0.1% trifluoroacetic acid (TFA) in water and B is acetonitrile containing 0.1% TFA). The peptide was detected at 215 nm. The correctly folded peptide eluted at 10.5 minutes. Synthetic peptide samples were hydrolyzed in 6 N HCl at 110°C for 22 h in vacuo. Peptide and amino acid analysis was performed on a Beckman 126AA System Gold amino acid analyzer. MALDI-ToF mass spectroscopic analysis was performed on a Kratos Kompact mass spectrometer using CCA as a matrix. The theoretical mass of the peptide is 4054. ID, the observed mass [M+H] was 4055.2D. Amino acid analysis of the purified D-allo-ShK showed the following average amino acid ratios: Asx (1) 0.97, Thr (4) 3.89, Ser (4) 4.04, GIx (1) 0.98, Pro (1) 0.93, GIy (1) 1.02, Ala (1) 1.00, Met (1) 0.88, He (2) 1.78, Leu (1) 1.01, Tyr (1) 0.99, Phe (2) 2.00, Lys (4) 3.99, His (1) 0.95, Arg (4) 3.89, and Cys (6) 5.26.
NMR Spectroscopy. Spectra were recorded on a sample of D-allo-ShK in 95% H2O/5% 2H2O at pH 4.9. Two-dimensional homonuclear total correlation (TOCSY) spectra with a spin-lock time of 60 ms and double quantum filtered correlation (DQF-COSY) NMR spectra were acquired at 500 MHz on a Bruker AMX-500 spectrometer. A two-dimensional nuclear Overhauser enhancement (NOESY) spectrum with a mixing time of 200 ms was also acquired on a Bruker AMX-500 spectrometer. NOESY spectra for ShK were acquired at 500 MHz and 600 MHz as described previously (8, 9); the 600 MHz NOESY was used in the comparison with D-allo-ShK. Water was suppressed using the WATERGATE pulse sequence (20). All spectra were collected at 20 °C unless otherwise stated and were referenced to an impurity peak at 0.15 ppm or to the water resonance.
Diffusion measurements were performed using a pulsed field gradient longitudinal eddy- current delay pulse sequence (21, 22) as implemented by Yao et al. (23).
Spectra were processed using XWINNMR (Version 3.5, Bruker Biospin) and analyzed using XEASY (Version 1.3.13) (24). Structural figures were prepared using VMD (25).
The NMR assignment is provided in Table 3.
Table 3 - 1H NMR chemical shifts (in ppm) of D-ShK in 95% H2O/5% 2H2O at 293 K and pH 4.9
Residue NU CaH CβH Others
Argl n.o.a 4.15 1.95 CyH2 1.73; CδH23.23; NεH 7.25
Ser2 8.86 4.46 3.82, 3.82
Cys3 9.01 4.83 3.03, 3.03
Ile4 7.74 4.66 1.95 CyH2 1.17, 0.97; CyH3 0.84; CδH3 0.81
Asp5 8.61 5.32 3.24, 2.69
Thr6 9.50 4.47 4.55 CyH31.24
Ile7 7.23 4.76 1.85 CyH2 1.31, 0.78; CyH30.40; CδH30.46
Pro8 — 4.26 2.41, 1.74 CyH22.05, 1.94; CδH23.80, 3.37
Lys9 8.34 3.87 2.02, 1.85 CyH21.51; CδH21.73; CsH23.07; NζH3 + SerlO 8.43 4.09 3.90, 3.90
Argi l 8.13 4.43 2.27, 1.92 CyH2 1.74; CδH23.27, 3.11; NεH 7.46
Cysl2 7.97 5.03 3.26, 2.90
Thrl3 7.25 4.36 4.75 CyH3 1.30; OH 5.84
Alal4 8.86 3.99 1.46
Phel5 8.52 4.12 3.23, 2.90 C(2,6)H 7.07, 7.07; C(3,5)H 7.05, 7.05;
C(4)H 6.84
Glnlό 7.82 4.19 1.98, 1.53 CyH22,34, 2.29; NsH26.46, 6.33
Cysl7 8.52 4.23 3.19, 2.90
Lyslδ 7.55 4.02 1.56, 1.40 CyH2 1.14; CδH20.97,0.97; CεH22.84,
2.84
Hisl9 7.77 4.46 3.08, 2.35 C(2)H 8.42;C(4)H 6.54
Ser20 8.34 5.00 4.07, 3.87
Met21 9.15 4.07 2.64, 2.12 CyH22.53, 2.53; CsH32.04
Lys 22 8.31 3.85 1.43, 1.43 CyH21.67, 1.67; CδH20.98, 0.98; CsH2
2.82, 2.82
Tyr23 8.02 4.04 3.32, 2.59 C(2,6)H 7.48, 7.48; C(3,5)H 6.93, 6.93
Arg24 8.09 3.92 2.24, 1.76 CyH21.52, 1,52; CδH23.35, 3.21; NεH 7.3(
NηH26.78, 6.52
Leu25 8.27 4.44 1.77, 1.47 CyH 1.70; CδH30.89,0.85
Ser26 7.50 4.76 3.55, 3.43
Phe27 7.52 5.30 3.18, 2.56 C (2,6)H 6.18,6.18; C(3,5)H 7.11,7.11; C(4)H7.17
Cys28 8.68 5.84 3.29, 3.17
Arg29 8.41 3.94 1.84, 1.64 CyH21.48, 1.48; CδH23.38, 3.27; NεH
7.18; NnH27.23, 6.64
Lys30 7.23 4.14 1.84, 1.84 CyH2 1.31,1.31; CδH21.64, 1.65; CsH2
3.02, 3.02
Thr31 10.87 3.85 4.07 CyH3 1.31
Cys32 9.14 4.82 3.32, 2 .88
Gly33 7.82 4.07,4.07
Thr34 8.63 4.18 4.44 CyH3 1.13
Cys35 8.08 4.53 3.31, 3 .10 NH2 7.60,7.19
Differences of > 0.03 ppm from native ShK are highlighted in bold font. a This resonance could not be observed because of fast exchange with the water.
The differences in chemical shift for Cys28, Arg29, Gly33, Thr34, and Cys35 can be attributed to the C-terminal amidation of D-allo-ShK. The chemical shift perturbation for
Ser20 NH could be indicative of a slight drift in pH and/or a difference in salt content, which is supported by the differences in chemical shift of the His 19 C(2)H and C(4)H protons, which were 8.42 ppm and 6.54 ppm in the D-allo-ShK spectrum and 8.46 ppm and 6.57 ppm in the ShK spectrum (the pKa of His 19 in ShK is 6.1) (4). The 0.04 ppm difference in NH chemical shift for Ile4 may have arisen from the stereochemistry of its asymmetric side chain, which is not an exact mirror image of ShK. However, the overall similarity of the structures, even in the immediate vicinity of Thr and He residues, is emphasized by the observation that the downfield-shifted backbone amides of Thrό and Thr31, at 9.50 and 10.87 ppm, respectively, the upfield-shifted amides of Ile7 and Thrl3, at 7.23 and 7.25 ppm, respectively, the side chain hydroxyl of Thrl3, at 5.84 ppm, and the slightly upfield-shifted methyls of Ile7, at 0.40 and 0.46 ppm, all have identical chemical shifts in both.
Example 2: Proteolytic digestion of D-allo-ShK
The stability of D-allo-ShK to proteolytic digestion was investigated under the same conditions used to determine the disulfide bridges of ShK toxin (26). D-allo-ShK (15 μg) was dissolved in 0.05 M HEPES, pH 6.5, containing 10 mM CaCl2 (30 μL) and trypsin, chymotrypsin or a mixture of trypsin and chymotrypsin (enzyme:substratel :50, w/w, 30 0C, 6 h). The digestion was terminated by acidification with 10% aqueous TFA (3 μl), the solution centrifuged (13,000 g, 5 min), and the supernatant analyzed directly by RP-HPLC.
Neither trypsin nor chymotrypsin, nor a mixture of both proteases, had any effect on D-allo- ShK, as assessed by RP-HPLC (Vydac Cis-silica, 5 μ, 120 angstroms 0.46 x 250 mm column, gradient: 5 to 50% B in 20 minutes at 1.5 mL/min (A buffer is 0.1% TFA in water and B is acetonitrile containing 0.1% TFA). The peptide eluted at 10.5 minutes showing the peptide did not undergo proteolysis. Cleavage at basic or aromatic residues was prevented by the D- stereochemistry at Cα.
Example 3: K+ Channel blocking activity of D-allo-ShK
ShK, ShK-amide and D-allo-ShK were tested on Kv 1.3 and Kv 1.1 channels stably expressed in L929 cells. Cells and Cell Lines. L929, B82 and MEL cells stably expressing mKvl.l, rKvl.2, mKvl.3, and hKvl.5 have been described previously (28) and were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, and 500 μg/ml G418 (Calbiochem). LTK cells expressing hKvl.4 were obtained from M. Tamkun (University of Colorado, Boulder), CHL cells expressing mKvl.7 from Vertex Pharmaceutical Inc (San Diego, CA), and HEK293 cells stably expressing hKCa3.1 were a kind gift from Dr. Khaled Houamed (Chicago, IL). PAS T cells, a MHC class II-restricted myelin basic protein (MBP)-specific encephalitogenic CD4+ rat T cell line (29), were a kind gift from Dr. Evelyne Beraud (Marseille, France). Mononuclear cells were isolated from Lewis rat (Harlan-Sprague Dawley, Indianapolis, IN) spleens using Histopaque-1083™ gradients (Sigma).
Electrophysiology. Cells were studied in the whole-cell configuration of the patch-clamp technique. The holding potential in all experiments was -80 mV. KvI.1, KvI .2, KvI .3, KvI.4, KvI.5, and KvI .7 currents were recorded in normal Ringer solution with a calcium-free pipette solution containing (in mM): 145 KF, 10 HEPES, 10 EGTA, 2 MgCl2, pH 7.2, 300 mOsm, as described previously (28). KCa3.1 currents were recorded as described previously (30). ^s were determined from dose-response curves shown using Microcal Origin software.
Fig. IA shows the effects of ShK and D-allo-ShK on Kvl .3 (left) and KvI.1 (right) currents elicited by 200 ms depolarizing pulses from a holding potential of -80 mV to 40 mV. Both peptides reversibly blocked Kvl.3 and Kv 1.1 in a dose-dependent manner with Hill coefficients of 1 (Fig. IB). Native ShK and ShK-amide blocked Kvl.3 with essentially identical affinities, and both displayed a two-fold selectivity for KvI .3 over KvI .1 (ShK: Ki for Kvl.3 13 ± 4 pM and for KvI.1 29 ± 3 pM; ShK-amide: Kά for Kvl.3 14 ± 3 pM and for KvI.1 31 ± 4 pM), as expected (Fig. 1). D-allo-ShK blocked Kvl.3 with a 2,800-fold lower affinity (Kd 36 ± 3 nM) than ShK but displayed the same two-fold selectivity for Kvl.3 over KvI.1 as ShK (Kd on KvI.1 83 ± 9 nM) (Fig. 1). D-allo-ShK had no effect on Kvl .2, Kvl .4, Kvl.5, Kvl .7, or KCa3.1 at concentrations up to 1 μM (Fig. 1C). Although D-allo-ShK had a lower affinity for Kvl.3 than native ShK, its selectivity profile was maintained. Both ShK and D-allo-ShK were tested on the proliferation of rat Kvl.3highKCa3.1l0W TEM and Kvl.3lowKCa3.1high naϊve/TCM lymphocytes. In keeping with its loss of affinity for Kv 1.3 channels, D-allo-ShK was 100-fold less effective than ShK in inhibiting the proliferation of TEM cells (Fig. 2A). Both ShK and D-allo-ShK inhibited the proliferation of TEM cells more potently than of naϊve/TcM lymphocytes (Fig. 2B).
To further characterize the blocking activity of ShK and D-allo-ShK on Kv 1.3, their ability to bind to the closed state of the channel was tested. Equilibration of the internal solution for 5 minutes was allowed before applying a 200 ms pulse from -80 to 40 mV to elicit a control KvI.3 current (Figure ID). ShK (2OpM) or D-allo-ShK (5OnM) was then perfused into the bath. The channel was closed for 5 minutes before pulsing again at 30 second intervals. ShK blocked 60% of the Kv 1.3 current at the first pulse after peptide incubation, and this blockade did not increase further after applying a further 10 depolarizing pulses, indicating that ShK binds to the closed state of the KvI.3 channel. In contrast, D-allo-ShK had no effect on the current at the first pulse after peptide incubation, and steady-state block was only reached after several depolarizing pulses, a phenomenon termed "use- dependent block", indicating that D-allo-ShK binds to an open or inactivated conformation of the channel.
Example 4: Modelling and docking of D-allo-ShK with Kv 1.3
An initial model of D-allo-ShK was created by inverting the structure of ShK derived by NMR (8) (PDB access code IROO, structure 1), and correcting the side-chains of threonine and isoleucine residues for the appropriate stereochemistry. Both the D-allo model and NMR-derived structures were subjected to molecular dynamics (MD) simulation using the GROMACS (v3.3.1) package of programs (33). All simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed and MD continued for a further 10 ns. MD simulations of both diastereomers of ShK were performed using the OPLS -aa force field (34). Ionizable residues were assumed to be in their standard state at neutral pH. Each peptide was placed in a 50 x 50 x 50 A3 water box with no pressure coupling. The total charge on the system was made neutral by replacing water molecules with chloride ions using the Genion program. Peptide, water and ions were coupled separately to a thermal bath at 300 K using a Berendsen thermostat (35) applied with a coupling time of 0.1 ps. All simulations were performed with a single non-bonded cutoff of 10 A, applying a neighbour-list update frequency of 10 steps (20 fs). The particle-mesh Ewald method was used to account for long-range electrostatics, applying a grid width of 1.2 A, and a fourth-order spline interpolation. Bond lengths were constrained using the LINCS algorithm (36). All simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed and MD continued for a further 10 ns.
Comparative models of the trans-membrane region (only) of the murine Kv 1.3 channel were constructed using the X-ray structure of the K+ channel from Streptomyces lividans (KcsA, PDB accession code 1BL8) as a template. The MODELLER (6v2) program (37) was used to create nine models based on the sequence alignment shown in Table S2. mKvl .3 has good sequence similarity with KcsA over the entire pore domain (32% identity), whereas there is 91% sequence identity with Kv 1.2. Despite the greater sequence similarity with KvI.2, the structure of KcsA was chosen as the template for model building because the structure of the two loops comprising the extracellular face of KcsA, the site of toxin binding, has been well characterized (38), whereas those in the more closely related channel, Kv 1.2, are disordered in the electron density (39), and are therefore less suitable for model building.
Complexes of the D-allo and L-forms of ShK with mKvl.3 were modelled using the
ZDOCK program (40). This program uses a fast Fourier transform to explore all possible binding modes of the two proteins; docking was restricted to residues on the extracellular surface of the channel to ensure the exclusion of unphysical binding predictions. The interaction is evaluated using shape complementarity, desolvation energy and electrostatics.
Models of each form of the toxin were extracted at 1 ns intervals during the MD simulation. Including the initial model, we considered eleven models of each form of the toxin. Each model of the toxin was docked with one of the nine models of the channel; thus, we considered all 99 possible combinations of toxin with channel for both D-allo and
L-forms of the toxin. The top 2,000 scoring predictions from each combination were then refined using the RDOCK program (41), in which the binding interface was refined using molecular mechanics minimization. The final docking predictions from all 198,000 complexes (for both forms of the toxin) were ranked according to the RDOCK scoring function.
The final docked complexes of both D-allo and L-forms of ShK with the channel were subjected to a short 100 ps MD simulation to permit further relaxation of the atoms at the interface. The extracellular face of the complex was capped in a sphere of water molecules, with the molecules at the surface of the sphere fixed at their originally minimized positions
(to prevent evaporation). Atoms of residues of the channel more than 8 A from the ShK peptide were held fixed during these MD calculations. The structures of the complexes were minimized without restraints at the completion of the MD simulation.
The interaction between residues at the interface between L-ShK and the channel were assessed in relation to earlier experimental mutant-cycle analysis (32). Mutant-cycle analysis had indicated that ShK was strongly coupled with His404 of the channel. In their model of ShK complexed to KvI .3, Lanigan et al. noted that the distance of closest approach of Argi l of ShK with His404 of KvI.3 was 11 A. In the current model this separation is 4.3 A (Argl 1 Nη with His404B Nε). The channel residues Asp386 and Ser379 were also implicated in coupling with Argl 1 of ShK in the complex. In the present model the distance between the Nε of Argi l and the Oδ of Asp386A is 2.6 A. Notably, the Asp380A and Ser379A that contact Argi l are on a different channel monomer from the His404β also close to Argl 1. Mutant-cycle analysis also suggested that Ser379 and His404 of the channel were close to Arg29 of ShK. In the current model the separations are 8.3 A (between Ser379β Oγ and Arg29 Nη) and 4.4 A (between His404c Nε and Arg29 Nη); these residues on the channel are on different monomers from those that interact with Argl 1. The mutant-cycle analysis indicated cooperativity between Asp386 of the channel and Arg29 of ShK. In the model, these residues are ~9 A apart. While mutant-cycle analysis did not support significant energetic coupling between His404 of the channel and Phe27 of ShK, these residues are in close contact in the current model (His404c Nδ is separated by 4.4 A from the centroid of the aromatic ring of Phe27 of ShK). In contrast, Asp386 of the channel and Ser20 of ShK are separated by almost 10 A in the model, consistent with only modest coupling observed in the mutant-cycle analysis. In the Lanigan et al. model His404A and Ser20 were separated by 11.6 A, while in the current model they are separated by 3.9 A.
While the side-chains of Tyr400 of the channel are not in close contact with the Nζ of Lys22, these groups help maintain the shape of the selectivity filter of the channel, in particular ensuring close contact with N^ of Lys22 and the backbone carbonyl of Gly399. The cooperativity observed in the mutant-cycle analysis between Tyr400 and Lys22 reflects the structural role played by the side-chains of Tyr400. Similarly, the backbone atoms of Asp402 form part of the selectivity filter; the side-chain of these residues contribute to the structure of the filter. A summary of several internuclear distances for the two diasteromeric models of ShK complexed with the channel is presented in Table 4.
Buried surface areas were calculated from the difference in surface areas of channel and toxin from the complex. Surface areas were calculated using the NACCESS program (42). Table 4 - Summary of internuclear distances for the present models of ShK complexed with Kv 1.3, and a comparison with the earlier model of Lanigan et al. (32)
interaction Lanigan L-ShK D-allo-ShK et al.
RI l Cζ ... S379 Oy 5.4A 5.6A 4.6D
RI l Cζ ... D386 Cγ 6.2A 3.8A 12.0D
RI l Cζ ... D402 Cγ 7.1A 4.1A 9.7c
RI l Cζ ... H404 Nδ. ,ε2
6.2A 5.3B 5.1D S20 oγ ... D386 Cγ 12.6D 9.8D 13.8D
S20 oγ ... H404 Nδl 11.6D 3.9A 4.5A
S20 oγ ... D402 Cγ 5.6D 5.5D
K22 Cγ ... D386 Cγ 15.4D 15.9-17.6 16.1-17.6
K22 Cγ ... Y400A-D X 8.2±0.4 8.8-9.3 8.6-9.7 K22 Nζ ... Y400A-D X 6.5±0.1 7.3-7.6 7.2-7.8
K22 σ ... D402 Cγ 8.1D 9.0-10.8 7.2-9.3
K22 Cγ ... H404 Nδl 9.9A 9.0-10.4 8.2-9.9
F27 x ... D386 Cγ 10.8A 1 1.7A 13.2c
R29 Cζ ... D386 Cγ 6.0B 10.1B 12.1A R29 Cζ ... D402 Cγ 5.1 B 8.4A
Channel subunits (A-D) are indicated by subscripts. X is the centroid of the aromatic ring. Distances are reported in Angstrom.
To compare how D-allo-ShK and native ShK could block KvI.3, models of the complexes of the toxins with the pore-vestibule region and inner helices of the channel were constructed. The models of the highest ranked predictions from the docking of ShK and the chiral analogue, D-allo-ShK, with the channel were identified. In both models, Lys22 is located in the ion-selectivity filter, blocking passage of K+ ions through the channel; its ammonium group forms hydrogen bonds with the backbone carbonyl oxygen atoms of Gly399. Phe27 packs alongside Lys22, in the space created by two Gly401 residues of the ion-selectivity filter from neighbouring channel monomers, and the side chains of Asp402 and His404. The side chain of Met21 occupies the equivalent pocket in the space diametrically opposite to the pocket filled by Phe27.
The overlay of the two complexes illustrated the approximate mirror symmetry of the binding of the two diastereomers to the channel. Residues of the toxins that contact the channel and lie along this plane share common binding interactions in the two diastereomers. Additionally, residues of the toxins that contact the channel along the channel monomer interface perpendicular to this plane also share common binding modes in the two diastereomers.
The structure of the complex with L-ShK presented here is similar to an earlier model (32). The proximity of channel and ShK residues is consistent with the mutant-cycle analysis used to derive that model. In the complex of ShK with the channel the side-chains of several residues form hydrogen bonds with the channel, including the hydroxyl of Tyr23, which hydrogen bonds with the backbone carbonyl oxygen atom of Gly40lA of the selectivity-filter, and the side-chain imidazole of His404A- Tyr23 is completely buried in the interface between channel and ShK, In contrast, Tyr23 of D-allo-ShK, while in close proximity to Asp402o, remains exposed to solvent in the complex. Similarly, the hydroxyl groups of Thr6 and Ser26 participate in hydrogen bonding interactions with the channel in the complexes of both diastereomers of ShK, yet the residues on the channel they interact with are different in the two complexes. In both complexes, Arg24 lies in close proximity to His404. The loss of solvent-accessible surface area upon complex formation of ShK is 1981 A2. This compares favourably with the earlier model of Lanigan et al. (32) of 1600 A2. The complex of D-allo-ShK with the channel buries 1987 A2 surface in the interface. Thus, the models suggest that D-allo-ShK and ShK bury roughly the same surface area upon channel binding. Example 5: [3H] -Thymidine incorporation assays.
Rat splenocytes seeded at 2x105 cells per well in RPMI culture medium in flat-bottom 96- well plates (final volume 200 μL) were pre-incubated with increasing concentrations of ShK or D-allo-ShK for 30 min and then stimulated with 2 μg/ml concanavalin A for 48 hr.
PAS T cells (2x104 cells per well) were stimulated in the presence of 2x106 irradiated
(2500 rad) Lewis rat thymocytes as antigen presenting cells with 10 μg/mL MBP isolated from guinea pig spinal cords as described previously (43). [3H] -thymidine (1 μCi per well) was added for the last 16-18 h. Cells were harvested onto glass fiber filters and radioactivity measured in a β-scintillation counter. The results are shown in Figure 2.
Example 6: Circulating half-life determination and plasma stability
A patch-clamp bioassay was used to determine the circulating half-life of D-allo-ShK.
Female inbred Lewis rats 9-11 weeks old were purchased from Harlan-Sprague Dawley (Indianapolis, IN) and housed under barrier conditions with irradiated rodent chow and acidified water ad libitum. All experiments were in accordance with NIH guidelines and approved by the University of California, Irvine, Institutional Animal Care and Use Committee.
Known amounts of D-allo-ShK were added to Lewis rat serum and the blocking activity on KvI .3 channels tested by patch-clamp to establish a standard dose-response curve. Serum samples from Lewis rats obtained from the saphenous vein at various times after a single subcutaneous injection of 1 mg/kg D-allo-ShK were tested for KvI.3 blocking activity by patch-clamp and the levels of D-allo-ShK determined from the standard curve as described (16).
Normal rat serum did not exhibit detectable blocking activity, indicating an absence of endogenous channel blockers. The spiked serum samples blocked KvI.3 currents in a dose-dependent fashion (Kd 38 ± 4 nM), similar to the effect of D-allo-ShK observed in the absence of serum (not shown). Levels of D-allo-ShK in rats following a single subcutaneous injection of 1 mg/kg were determined by comparison with the standard curve. D-allo-ShK was detectable in serum 25 min after injection (Fig. 3A). Peak levels (950 nM) were reached within 50 min and the level then fell to 15 nM within 24 h (Fig. 3A). The disappearance of D-allo-ShK from the serum could be fitted by a single exponential (Fig. 3B) and its circulating half-life was estimated to be 40 min.
Example 7: D-allo-ShK inhibits DTH response in rats
As an assessment of the immunosuppressive activity of D-allo-ShK in vivo, its ability to inhibit a delayed-type hypersensitivity (DTH) reaction to ovalbumin mediated predominantly by skin-homing TEM cells was tested (44). For DTH experiments, rats were immunized with an emulsion of ovalbumin in complete Freund's adjuvant (Difco, Detroit, MI). Seven days later they received an injection of ovalbumin dissolved in saline in the pinna of one ear and saline in the other ear. Rats then received a subcutaneous injection of D-allo -ShK (1 mg/kg) or vehicle (PBS + 2% rat serum). Ear swelling was measured 24 h later using a spring-loaded micrometer (Mitutoyo, Spokane, WA). All vehicle-treated control rats developed ear swelling 24 h after ovalbumin challenge in the ear, but the DTH reaction was significantly milder in animals treated with 1 mg/kg D-allo-ShK at the time of challenge in the ear (Fig. 3C). Thus, D-allo-ShK inhibited the TEM-mediated DTH response.
Discussion for Examples 1-7
D-allo-ShK folded in good yield and gave a high-resolution NMR spectrum essentially identical to the native (all-L) polypeptide. The most significant NMR spectral changes could be attributed to the presence of a C-terminal amide on D-allo-ShK, compared with a free C-terminal carboxylate on naturally occurring ShK. D-allo-ShK is not a perfect mirror image of ShK because the stereochemistry of the side chains was not changed in concert with the change in the backbone, but this is only significant for the two He and four Thr side chains, and the NMR data show that the local structure in the vicinity of these residues is essentially identical to that of ShK.
D-allo-ShK blocked the Kv 1.3 channel with nM affinity, making it a weaker blocker than the native toxin but still a potent inhibitor. D-allo-ShK was also able to block T-cell proliferation. It is highly unusual for an all-D analogue of a folded polypeptide or protein that acts at a specific binding site on a target protein to retain activity.
ShK has a circulating half-life of - 30 min (16). Such a rapid clearance from the blood could be due to renal elimination and/or proteolysis. The finding that D-allo-ShK has a similar half-life (~ 40 min) implies that the disappearance of ShK and D-allo-ShK is due to rapid renal clearance since endogenous proteases can only cleave L-forms of polypeptide chains and are therefore unable to proteolyse D-allo-ShK. Moreover, the kidney allows peptides up to 5 kDa to pass through without filtration and both forms of ShK are smaller than this cutoff. Even though it has a similar circulating half-life to ShK and is less potent as a blocker of KvI.3 channels, D-allo-ShK is expected not to be recognized by the immune system as it should be resistant to proteolytic processing by T-cells for presentation on MHC complexes, and therefore is not likely to be antigenic in vivo.
References
1. Price, M., Lee, S. C. & Deutsch, C. (1989) Proc Natl Acad Sci USA 86, 10171-5.
2. Leonard, R. J., Garcia, M. L., Slaughter, R. S. & Reuben, J. P. (1992) Proc Natl Acad Sci USA 89, 10094-8.
3. Lin, C. S., Boltz, R. C, Blake, J. T., Nguyen, M., Talento, A., Fischer, P. A., Springer, M. S., Sigal, N. H., Slaughter, R. S., Garcia, M. L. & et al. (1993) J Exp Med 111, 637-45.
4. Wulff, H., Calabresi, P. A., Allie, R., Yun, S., Pennington, M., Beeton, C. & Chandy, K. G. (2003) J Clin Invest 111, 1703-13.
5. Beeton, C, Wulff, H., Standifer, N. Ε., Azam, P., Mullen, K. M., Pennington, M. W., Kolski-Andreaco, A., Wei, Ε., Grino, A., Counts, D. R., Wang, P. H., Leehealey, C. J., B, S. A., Sankaranarayanan, A., Homerick, D., Roeck, W. W., Tehranzadeh, J., Stanhope, K. L., Zimin, P., Havel, P. J., Griffey, S., Knaus, H. G., Nepom, G. T., Gutman, G. A., Calabresi, P. A. & Chandy, K. G. (2006) Proc Natl
Acad Sci USA 103, 17414-9.
6. Castaneda, O., Sotolongo, V., Amor, A. M., Stocklin, R., Anderson, A. J., Harvey, A. L., Εngstrom, A., Wernstedt, C. & Karlsson, Ε. (1995) Toxicon 33, 603-13.
7. Pennington, M. W., Byrnes, M. Ε., Zaydenberg, I., Khaytin, I., de Chastonay, J., Krafte, D. S., Hill, R., Mahnir, V. M., Volberg, W. A., Gorczyca, W. & et al.
(1995) IntJPept Protein Res 46, 354-8.
8. Tudor, J. Ε., Pallaghy, P. K., Pennington, M. W. & Norton, R. S. (1996) Nat Struct Biol 3, 317-20.
9. Tudor, J. Ε., Pennington, M. W. & Norton, R. S. (1998) Eur J Biochem 251, 133- 41.
10. Bontems, F., Gilquin, B., Roumestand, C, Menez, A. & Toma, F. (1992) Biochemistry 31, 7756-64.
11. Johnson, B. A., Stevens, S. P. & Williamson, J. M. (1994) Biochemistry 33, 15061- 70. 12. Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C, Vita, C, de Medeiros, C. L., Rowan, E. G., Harvey, A. L. & Menez, A. (1997) J Biol Chem 272, 4302-9.
13. Kalman, K., Pennington, M. W., Lanigan, M. D., Nguyen, A., Rauer, H., Mahnir, V., Paschetto, K., Kern, W. R., Grissmer, S., Gutman, G. A., Christian, E. P.,
Cahalan, M. D., Norton, R. S. & Chandy, K. G. (1998) J Biol Chem 273, 32697- 707.
14. Beeton, C, Wulff, H., Singh, S., Botsko, S., Crossley, G., Gutman, G. A., Cahalan, M. D., Pennington, M. & Chandy, K. G. (2003) J Biol Chem 278, 9928-37. 15. Rauer, H., Lanigan, M. D., Pennington, M. W., Aiyar, J., Ghanshani, S., Cahalan,
M. D., Norton, R. S. & Chandy, K. G. (2000) J Biol Chem 275, 1201-8. 16. Beeton, C, Wulff, H., Barbaria, J., Clot-Faybesse, O., Pennington, M., Bernard, D.,
Cahalan, M. D., Chandy, K. G. & Beraud, E. (2001) Proc Natl Acad Sci USA 9S,
13942-7. 17. Beeton, C, Pennington, M. W., Wulff, H., Singh, S., Nugent, D., Crossley, G.,
Khaytin, I., Calabresi, P. A., Chen, C. Y., Gutman, G. A. & Chandy, K. G. (2005)
MoI Pharmacol 67, 1369-81.
18. King, D. S., Fields, C. G. & Fields, G. B. (1990) Int J Pept Protein Res 36, 255-66.
19. Rivier, J. & McClintock, R. (1983) J Chromatogr 268, 112-9. 20. Piotto, M., Saudek, V. & Sklenal, V. (1992) Journal of Biomolecular NMR 2, 661- 5.
21. Gibbs, S. J. & Johnson, C. S. (1991) J Magn Reson 93, 395-402.
22. Dingley, A. J., Mackay, J. P., Chapman, B. E., Morris, M. B., Kuchel, P. W., Hambly, B. D. & King, G. F. (1995) JBiomolNMR 6, 321-8. 23. Yao, S., Howlett, G. J. & Norton, R. S. (2000) JBiomol NMR 16, 109-19.
24. Bartels, C, Xia, T. H., Billeter, M., Gϋntert, P. & Wϋthrich, K. (1995) J Biomol NMR 6, 1-10.
25. Humphrey, W., Dalke, A. & Schulten, K. (1996) JMo/ Graph 14, 33-8, 27-8.
26. Pohl, J., Hubalek, F., Byrnes, M. E., Nielsen, K. R., Woods, A. & Pennington, M. W. (1995) Lett Pept Sci 1, 291-97.
27. Milton, R. C, Milton, S. C. & Kent, S. B. (1992) Science 256, 1445-8. 28. Grissmer, S., Nguyen, A. N., Aiyar, J., Hanson, D. C, Mather, R. J., Gutman, G. A., Karmilowicz, M. J., Auperin, D. D. & Chandy, K. G. (1994) MoI Pharmacol 45, 1227-34.
29. Beraud, E., Balzano, C, Zamora, A. J., Varriale, S., Bernard, D. & Ben-Nun, A. (1993) J Neuroimmunol 47, 41-53.
30. Ghanshani, S., Wulff, H., Miller, M. J., Rohm, H., Neben, A., Gutman, G. A., Cahalan, M. D. & Chandy, K. G. (2000) J Biol Chem 275, 37137-49.
31. Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T. & MacKinnon, R. (1998) Science 280, 69-77. 32. Lanigan, M. D., Kalman, K., Lefievre, Y., Pennington, M. W., Chandy, K. G. & Norton, R. S. (2002) Biochemistry 41, 11963-71.
33. Lindahl, E., Hess, B. & van der Spoel, D. (2001) J MoI Model 7, 306-17.
34. Jorgensen, W. L. & Tirado-Rives, J. (1988) JAm Chem Soc 110, 1657-66.
35. Berendsen, H. J. C, Postma, J. P. M., van Gunsteren, W. F., DiNoIa, A. & Haak, J. R. (1984) J Chem Phys 81, 3684-90.
36. Hess, B., Bekker, H. & Berendsen, H. J. C. (1977) J Comput Chem 18, 1463-72.
37. Fiser, A. & SaIi, A. (2003) Methods Enzymol 374, 461-91.
38. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. (2001) Nature 414, 43-8. 39. Long, S. B., Campbell, E. B. & Mackinnon, R. (2005) Science 309, 897-903.
40. Chen, R., Li, L. & Weng, Z. (2003) Proteins 52, 80-7.
41. Li, L., Chen, R. & Weng, Z. (2003) Proteins 53, 693-707.
42. Hubbard, S. J. & Thornton, J. M. (1993) Department of Biochemistry and Molecular Biology, University College, London. 43. Deibler, G. E., Martenson, R. E. & Kies, M. W. (1972) Prep Biochem 2, 139-65.
44. Soler, D., Humphreys, T. L., Spinola, S. M. & Campbell, J. J. (2003) Blood 101, 1677-82.
45. Roberge et al, Science, 269:202-204, 1995.
46. Fields, C. G., Lloyd, D. H., Macdonald, R. L., Otteson, K. M., Noble, R. L., HBTU activation for automated Fmoc solid-phase peptide synthesis, Peptide Research,
4(2), 95-101, 1991.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A peptide or a pharmaceutically acceptable salt thereof comprising amino acid sequence 1 :
R1-xaa29xaa28Cixaaixaa2xaa3xaa4xaa5xaa6xaa7xaa8C2xaa9xaa10xaaiixaa12C3xaai3xaai4xaai5 xaai6xaai7xaai8xaai9xaa20xaa2ixaa22C4xaa23xaa24xaa25C5xaa26xaa27c6-R2
SEQ ID NO:!
wherein xaa17 is a positively charged D-amino acid residue; xaais is an aromatic D-amino acid residue; xaai to xaa16, xaai9 to xaa27 are each independently selected from any D-amino acid residue; xaa28 and xaa29 are independently absent or are selected from any D-amino acid residue;
R1 is hydrogen or is an amino acid residue, an N-terminal capping group or an oligopeptide optionally capped with an N-terminal capping group;
R2 is hydrogen or is an amino acid residue, a C-terminal capping group or an oligopeptide optionally capped with a C-terminal capping group; wherein each of ci and C6 are independently selected from D-cysteine, D- homocysteine, D-penicillamine or D-selenocysteine and C1 is linked to C6 through a disulfide, diseleno or sulfide-selenium bond; or both ci and C6 are D-amino acid residues having a side chain double bond and the side chains of ci and C6 are linked to form a dicarba bond; and each of C2 to C5 are independently selected from D-cysteine and D-selenocysteine and C2 is linked to C4 and C3 is linked to c5 through disulfide, diseleno or sulfide-selenium bonds.
2. A peptide according to claim 1, wherein xaaπ is selected from D-lysine, D-l,3-diaminopropionic acid, D-arginine, D-histidine, 5-hydroxy-D-lysine, D-norlysine, D-homolysine and D-ornithine.
3. A peptide according to claim 1 or claim 2, wherein xaa17 is selected from D-lysine or D-l,3-diaminopropionic acid.
4. A peptide according to any one of claims 1 to 3, wherein xaa18 is selected from D-tyrosine, D-phenylalanine, D-tryptophan, D-homotyrosine, D-naphthylalanine and D-phosphotyrosine.
5. A peptide according to claim 4, wherein xaa18 is D-tyrosine.
6. A peptide according to any one of claims 1 to 5, wherein one or more of Ci to C6 are D-cysteine.
7. A peptide according to claim 6, wherein all of Ci to C6 are D-cysteine.
8. A peptide according to any one of claims 1 to 7, wherein at least one of xaai, xaa4, xaa5, xaaio, xaaj6 and xaa20 are independently a non-polar amino acid residue.
9. A peptide according to claim 8, wherein each xaaj, xaa4, xaa5, xaaio, xaaj6 and xaa20 is independently selected from D-alanine, D-valine, D-leucine, D-isoleucine,
D-proline, D-methionine, D-phenylalanine and D-tryptophan.
10. A peptide according to any one of claims 1 to 9, wherein xaa2 is a negatively charged amino acid residue.
11. A peptide according to claim 10, wherein xaa2 is selected from D-aspartic acid and D-glutamic acid.
12. A peptide according to any one of claims 1 to 1 1, wherein at least one of xaa3, xaa7, xaa9, xaaj2, xaai5, xaa2i, xaa25 and xaa27 are independently uncharged amino acid residues.
13. A peptide according to claim 12, wherein each of wherein each of xaa3, xaa7, xaac>, xaai2, xaa15, xaa2i, xaa25 and xaa27 are independently selected from D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine and D-glutamine.
14. A peptide according to any one of claims 1 to 13, wherein at least one of xaaό, xaag, xaao, xaa14, xaa^, xaa23, and xaa24 are independently selected from a positively charged amino acid residue.
15. A peptide according to claim 14, wherein each of xaaβ, xaa8, xaa13, xaai4, xaai9, xaa23 and xaa24 are independently selected from D-lysine, D-arginine, D-histidine and
D-l,3-diaminopropionic acid.
16. A peptide according to any one of claims 1 to 15, wherein at least one of xaan and xaa22 are independently selected from a non-polar or aromatic D-amino acid residue.
17. A peptide according to claim 16, wherein each of xaan and xaa22 are independently selected from D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, D-methionine, D-phenylalanine, D-tryptophan and D-tyrosine.
18. A peptide according to any one of claims 1 to 17, wherein xaa26 is glycine or a small non-polar D-amino acid residue.
19. A peptide according to claim 18 wherein xaa26 is selected from glycine, D-alanine, D-valine, D-leucine and D-isoleucine.
20. A peptide according to any one of claims 1 to 19, wherein xaa28 is absent or is a polar, uncharged D-amino acid residue.
21. A peptide according to claim 20, wherein xaa28 is absent or is selected from D-glycine, D-serine, D-threonine, D-cysteine, D-tyrosine, D-asparagine and D-glutamine.
22. A peptide according to any one of claims 1 to 21, wherein xaa29 is absent or is a positively charged amino acid residue.
23. A peptide according to claim 22, wherein xaa29 is absent or is selected from D-lysine, D-arginine, D-histidine and D-l,3-diaminopropionic acid.
24. A peptide according to any one of claims 1 to 23, wherein R2 is hydrogen or NH2.
25. A peptide according to any one of claims 1 to 24, wherein R1 is selected from hydrogen, acyl, a phosphorylated amino acid residue, or a fluorescent or biological label optionally linked to ci, xaa2g or xaa28 through a linker.
26. A peptide according to claim 1 having one of the following sequences: rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:2 rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO:3 ptyr-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:4 ptyr-rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc SEQ ID NO: 5 fluoroscein-aminoethyloxyethyloxyacetyl-rscidtipksrctafqckhsmkyrlsfcrktcgtc
SEQ ID NO:6 fluoroscein-aminoethyloxyethyloxyacetyl-rscidtipksrctafqckhsm-dap-yrlsfcrktcgtc
SEQ ID NO: 7 sscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 8
(N-acetyl)-rscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 9 scidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 10 cidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 11 ascidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 12 rscadtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 13 rscadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO : 14 rscadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 15 rscidaipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 16 rscidtapksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 17 rscidtiaksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 18 rscidtipasrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 19 rscidtipesrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:20 rscidtipqsrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:21 rscidtipkarctafqckhsmkyrlsfcrktcgtc SEQ ID NO:22 rscidtipksactafqckhsmkyrlsfcrktcgtc SEQ ID NO:23 rscidtipksectafqckhsmkyrlsfcrktcgtc SEQ ID NO:24 rscidtipksqctafqckhsmkyrlsfcrktcgtc SEQ ID NO:25 rscidtipksrcaafqckhsmkyrlsfcrktcgtc SEQ ID NO:26 rscidtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO:27 rscidtipksrctawqckhsmkyrlsfcrktcgtc SEQ ID NO:28 rscidtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO:29 rscidtipksrctafackhsmkyrlsfcrktcgtc SEQ ID NO:30 rscidtipksrctafeckhsmkyrlsfcrktcgtc SEQ ID N0:31 rscidtipksrctafqcahsmkyrlsfcrktcgtc SEQ ID NO:32 rscidtipksrctafqcehsmkyrlsfcrktcgtc SEQ ID NO:33 rscidtipksrctafqckasmkyrlsfcrktcgtc SEQ ID NO:34 rscidtipksrctafqckksmkyrlsfcrktcgtc SEQ ID NO:35 rscidtipksrctafqckhamkyrlsfcrktcgtc SEQ ID NO:36 rscidtipksrctafqckhsakyrlsfcrktcgtc SEQ ID NO:37 rscidtipksrctafqckhs(norleu)kyrlsfcrktcgtc SEQ ID NO:38 rscidtipksrctafqckhsm(orn)yrlsfcrktcgtc SEQ ID NO.39 rscidtipksrctafqckhsmkfrlsfcrktcgtc SEQ ID NO:40 rscidtipksrctafqckhsmk(nitrophe)rlsfcrktcgtc SEQ ID N0:41 rscidtipksrctafqckhsmk(aminophe)rlsfcrktcgtc SEQ ID NO:42 rscidtipksrctafqckhsmk(benzylphe)rlsfcrktcgtc SEQ ID NO:43 rscidtipksrctafqckhsmkyalsfcrktcgtc SEQ ID NO:44 rscidtipksrctafqckhsmkyelsfcrktcgtc SEQ ID NO:45 rscidtipksrctafqckhsmkyrasfcrktcgtc SEQ ID NO:46 rscidtipksrctafqckhsmkyrlafcrktcgtc SEQ ID NO:47 rscidtipksrctafqckhsmkyrlsacrktcgtc SEQ ID NO:48 rscidtipksrctafqckhsmkyrlsfcaktcgtc SEQ ID NO:49 rscidtipksrctafqckhsmkyrlsfcratcgtc SEQ ID NO:50 rscidtipksrctafqckhsmkyrlsfcrkacgtc SEQ ID NO:51 rscidtipksrctafqckhsmkyrlsfcrktcgac SEQ ID NO:52 scadtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:53 scadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO: 54 scadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO:55 scidaipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:56 scidtapksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 57 scidtiaksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 58 scidtipasrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:59 scidtipesrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 60 scidtipqsrctafqckhsmkyrlsfcrktcgtc SEQ ID N0:61 scidtipkarctafqckhsmkyrlsfcrktcgtc SEQ ID NO:62 scidtipksactafqckhsmkyrlsfcrktcgtc SEQ ID NO:63 scidtipksectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 64 scidtipksqctafqckhsmkyrlsfcrktcgtc SEQ ID NO:65 scidtipksrcaafqckhsmkyrlsfcrktcgtc SEQ ID NO:66 scidtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO:67 scidtipksrctawqckhsmkyrlsfcrktcgtc SEQ ID NO:68 scidtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 69 scidtipksrctafackhsmkyrlsfcrktcgtc SEQ ID NO: 70 scidtipksrctafeckhsmkyrlsfcrktcgtc SEQ ID N0:71 scidtipksrctafqcahsmkyrlsfcrktcgtc SEQ ID NO: 72 scidtipksrctafqcehsmkyrlsfcrktcgtc SEQ ID NO:73 scidtipksrctafqckasmkyrlsfcrktcgtc SEQ ID NO:74 scidtipksrctafqckksmkyrlsfcrktcgtc SEQ ID NO:75 scidtipksrctafqckhamkyrlsfcrktcgtc SEQ ID NO: 76 scidtipksrctafqckhsakyrlsfcrktcgtc SEQ ID NO:77 scidtipksrctafqckhs(norleu)kyrlsfcrktcgtc SEQ ID NO:78 scidtipksrctafqckhsm(orn)yrlsfcrktcgtc SEQ ID NO:79 scidtipksrctafqckhsm(diaminopropionic)yrlsfcrktcgtc SEQ ID NO:80 scidtipksrctafqckhsmkfrlsfcrktcgtc SEQ ID NO:81 scidtipksrctafqckhsmk(nitrophe)rlsfcrktcgtc SEQ ID NO: 82 scidtipksrctafqckhsmk(aminophe)rlsfcrktcgtc SEQ ID NO:83 scidtipksrctafqckhsmk(benzylphe)rlsfcrktcgtc SEQ ID NO: 84 scidtipksrctafqckhsmkyalsfcrktcgtc SEQ ID NO:85 scidtipksrctafqckhsmkyelsfcrktcgtc SEQ ID NO: 86 scidtipksrctafqckhsmkyrasfcrktcgtc SEQ ID NO:87 scidtipksrctafqckhsmkyrlafcrktcgtc SEQ ID NO : 88 scidtipksrctafqckhsmkyrlsacrktcgtc SEQ ID NO:89 scidtipksrctafqckhsmkyrlsfcaktcgtc SEQ ID NO: 90 scidtipksrctafqckhsmkyrlsfcratcgtc SEQ ID N0:91 scidtipksrctafqckhsmkyrlsfcrkacgtc SEQ ID NO: 92 scidtipksrctafqckhsmkyrlsfcrktcgac SEQ ID NO: 93 yscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 94 kscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 95 hscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 96 qscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 97 pprscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO:98 mrscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 99 grscidtipksrctafqckhsmkyrlsfcrktcgtc SEQ ID NO : 100 rscidtipvsrctafqckhsmkyrlsfcrktcgtc SEQ ID NO: 101 rscidtipksrctdfqckhsmkyrlsfcrktcgtc SEQ ID NO : 102 scidtipksrctdfqckhsmkyrlsfcrktcgtc SEQ ID NO: 103 rscidtipksrctaiqckhsmkyrlsfcrktcgtc SEQ ID NO: 104 scidtipksrctaiqckhsmkyrlsfcrktcgtc SEQ ID NO: 105 rscidtipksrctavqckhsmkyrlsfcrktcgtc SEQ ID NO: 106 scidtipksrctavqckhsmkyrlsfcrktcgtc SEQ ID NO: 107 rscidtipksrctafrckhsmkyrlsfcrktcgtc SEQ ID NO: 108 scidtipksrctafrckhsmkyrlsfcrktcgtc SEQ ID NO: 109 rscidtipksrctafkckhsmkyrlsfcrktcgtc SEQ ID NO: 110 scidtipksrctafkckhsmkyrlsfcrktcgtc SEQ ID NO: 111 rscidtipasectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 112 scidtipasectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 113 rscidtipvsectafqckhsmkyrlsfcrktcgtc SEQ ID NO : 114 scidtipvsectafqckhsmkyrlsfcrktcgtc SEQ ID NO: 115 rscidtipvsactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 116 scidtipvsactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 117 rscidtipasactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 118 scidtipasactafqckhsmkyrlsfcrktcgtc SEQ ID NO: 119 rscidtipksectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 120 scidtipksectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 121 rscidtipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO : 122 scidtipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 123 rscidtipvsectdiqckhsmkyrlsfcrktcgtc SEQ ID NO: 124 scidtipvsectdiqckhsmkyrlsfcrktcgtc SEQ ID NO: 125 rtckdlipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 126 tckdlipvsectdirckhsmkyrlsfcrktcgtc SEQ ID NO: 127 qscadtipksrctaaqckhsmkyrlsfcrktcgtc SEQ ID NO: 128 qscadtipksrctaaqckhsm(dap)yrlsfcrktcgtc SEQ ID NO: 129 qscadtipksrctaaqckhsmkyrasfcrktcgtc SEQ ID NO: 130 qscadtipksrctaaqckhsm(dap)yrasfcrktcgtc SEQ ID NO: 131.
27. A method of inhibiting activation of T-cells comprising exposing the T-cells to a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof.
28. A method of treatment and/or prophylaxis of a disease or disorder characterized by aberrant or abnormal activation of T-cells in a subject, comprising administering to the subject an effective amount of a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof.
29. A method of treatment and/or prophylaxis of an autoimmune disease or organ or tissue transplant rejection in a subject, comprising administering to the subject an effective amount of a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof.
30. Use of a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof in the manufacture of a medicament for treatment and/or prophylaxis of a disease or disorder characterized by aberrant or abnormal activation of T- cells in a subject.
31. Use of a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof in the manufacture of a medicament for treatment and/or prophylaxis of an autoimmune disease or organ or tissue transplant rejection in a subject.
32. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 26, or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
33. Use of a peptide according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof as a medicament.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687608A (en) * 2011-06-06 2014-03-26 奇尼塔一有限责任公司 Shk-based pharmaceutical compositions and methods of manufacturing and using the same
US8909738B2 (en) 2008-03-20 2014-12-09 Tajitshu Transfer Limited Liability Company Redundant data forwarding storage
WO2015162285A1 (en) * 2014-04-25 2015-10-29 Phi Pharma Sa C6s specific transporter molecules
US9203928B2 (en) 2008-03-20 2015-12-01 Callahan Cellular L.L.C. Data storage and retrieval

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023639A2 (en) * 1996-11-27 1998-06-04 University Of Florida ShK TOXIN COMPOSITIONS AND METHODS OF USE
WO2006042151A2 (en) * 2004-10-07 2006-04-20 The Regents Of The University Of California Analogs of shk toxin and their uses in selective inhibition of kv1.3 potassium channels

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023639A2 (en) * 1996-11-27 1998-06-04 University Of Florida ShK TOXIN COMPOSITIONS AND METHODS OF USE
WO2006042151A2 (en) * 2004-10-07 2006-04-20 The Regents Of The University Of California Analogs of shk toxin and their uses in selective inhibition of kv1.3 potassium channels

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BEETON, C. ET AL.: "The D-diastereomer of ShK toxin selectively blocks voltage- gated K+ channels and inhibits T lymphocyte proliferation", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, January 2008 (2008-01-01), pages 988 - 997 *
HARVEY, A. J. ET AL.: "A three-residue, continuous binding epitope peptidomimetic of ShK toxin as a Kvl.3 inhibitor", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 15, 2005, pages 3193 - 3196 *
KALMAN, K. ET AL.: "ShK-Dap22, a potent Kvl.3-specific immunosuppressive polypeptide", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 49, 1998, pages 32697 - 32707, XP002997411, DOI: doi:10.1074/jbc.273.49.32697 *
LANIGAN, M. D. ET AL.: "Designed peptides analogues of the potassium channel blocker ShK toxin", BIOCHEMISTRY, vol. 40, 2001, pages 15528 - 15537, XP002966782, DOI: doi:10.1021/bi011300b *
NORTON, R. S. ET AL.: "Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases", CURRENT MEDICINAL CHEMISTRY, vol. 11, 2004, pages 3041 - 3052, XP002426887 *
PENNINGTON , M. W. ET AL.: "Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin", BIOCHEMISTRY, vol. 38, 1999, pages 14549 - 14558 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8909738B2 (en) 2008-03-20 2014-12-09 Tajitshu Transfer Limited Liability Company Redundant data forwarding storage
US9203928B2 (en) 2008-03-20 2015-12-01 Callahan Cellular L.L.C. Data storage and retrieval
US9961144B2 (en) 2008-03-20 2018-05-01 Callahan Cellular L.L.C. Data storage and retrieval
CN103687608A (en) * 2011-06-06 2014-03-26 奇尼塔一有限责任公司 Shk-based pharmaceutical compositions and methods of manufacturing and using the same
US9381261B2 (en) 2011-06-06 2016-07-05 Kineta One, Llc SHK-based pharmaceutical compositions and methods of manufacturing and using the same
US9878058B2 (en) 2011-06-06 2018-01-30 Kv1.3 Therapeutics, Inc. SHK-based pharmaceutical compositions and methods of manufacturing and using the same
WO2015162285A1 (en) * 2014-04-25 2015-10-29 Phi Pharma Sa C6s specific transporter molecules
US10131690B2 (en) 2014-04-25 2018-11-20 Phi Pharma Sa C6S specific transporter molecules

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