JP2002534996A - Alpha-conotoxin peptide - Google Patents

Abstract

  (57) [Summary] The present invention relates to relatively short peptides of about 10-30 residues in length (referred to herein as α-conotoxins), which are naturally available in trace amounts in the venom of cone snails. , Or analogs of naturally available peptides, which preferably contain two disulfide bonds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to the National Institute of General Medica, Bethesda, MD.
l Grant number PO1 invested by Sciences, National Institutes of Health
It was made with government support under GM48677. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION [0002] The present invention relates to relatively short peptides of about 10-30 residues in length (referred to herein as α-conotoxins), which are present in trace amounts in the venom of cone snails. Or analogs of naturally available peptides, which preferably contain two disulfide bonds.

[0003] In describing the background of the invention and in particular providing further details regarding the implementation, the publications and other materials used herein are hereby incorporated by reference and are considered a part of this specification. For convenience, the text below is referred to by author and date, and is listed alphabetically by author in the attached source list.

[0004] Predatory mussels (toxic mussels; Conus) have developed a unique biological strategy. The venom contains relatively small peptides that target various neuromuscular receptors, which may correspond to the pharmaceutical diversity of plant alkaloids or microbial secondary metabolites. Many of these peptides are among the smallest nucleic acid-encoded translation products with a defined conformation, and as such it is somewhat unusual.
Usually, peptides in this size range equilibrate in many conformations. Generally, proteins with an immobilized conformation are much larger.

[0005] The mussels producing these peptides are a large genus of toxic gastropods, including approximately 500 species. All mussel species are predators that inject venom into captured prey, and the range of animals that can inject venom as a whole genus is wide. However, a wide variety of hunting strategies are used, and all mussel species use essentially the same basic pattern of venom infusion.

[0006] Several peptides isolated from venom of the mussel have been characterized. These include α-, μ- and ω-conotoxins, which target nicotinic acetylcholine receptors, muscle sodium channels and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressin, a vasopressin analog, has also been identified (Cruz et al., 1987). In addition, Conan Tokin (cona
ntokin) is a peptide named Conus geographus
) And Conus tulipa (Mena et al., 19
90; Haack et al., 1990).

[0007] α-conotoxins are directed against nicotinic acetylcholine receptors at neuronal muscle junctions (Gray et al., 1981; Marshall and Harvey, 1990; Blount et al., 1992; Jacobsen et al.,
1997), or against neuronal nicotinic acetylcholine receptors (Fainzilber
Et al., 1994; Johnson et al., 1995; Cartier et al., 1996; Luo et al., 1998) Very specific small peptides. Alpha-conotoxins having specificity for the neuronal muscular junction nicotinic acetylcholine receptor are disclosed in U.S. patent application Ser. Number 09 / (
As disclosed in attorney file number 2314-178.A) and International Patent Application No. PCT / US00 / filed on January 21, 2000 (attorney file number 2314-138.PCT), Used as a neuromuscular blocking agent used in combination. Additional α-conotoxins and their uses are described in U.S. Pat. Nos. 4,447,356 (Olivera et al., 1984); 5,432, each of which is hereby incorporated by reference.
No. 155; 5,514,774.

[0008] Further uses for α-conotoxins are described in US patent application Ser. No. 09 / 219,446, filed Dec. 22, 1998, which is incorporated herein by reference. In this application, α-conotoxin, which has specificity for neuronal nicotinic acetylcholine receptors, is used to treat disorders modulated at neuronal nicotinic acetylcholine receptors. Such disorders include, but are not limited to, cardiovascular disorders, gastric motility disorders, urinary incontinence, nicotine addiction, mood disorders (such as bipolar disorder, unipolar depression, dysthymia and seasonal effects disorder) and minor disorders Cellular lung cancer, as well as the localization of small cell lung cancer are included. It would be desirable to provide additional α-conotoxin peptides having the uses described herein.

SUMMARY OF THE INVENTION [0009] The present invention relates to the use of about 10-30 (referred to herein as α-conotoxin).
For peptides of relatively short residue length, they are naturally available in trace amounts in venom of the mussel or in analogs of the peptide which are naturally available, and they preferably contain two disulfide bonds.

[0010] More particularly, the present invention is directed to an α-conotoxin peptide having the following general formula I:

[0011]

Embedded image

Wherein Xaa₁Is Death-Xaa₁, Ile, Leu or Val; Xaa₂Is
Su-Xaa₂, Ala or Gly; Xaa₃Is Death-Xaa₃, Gly, Tr
p (D or L), neo-Trp, halo-Trp or any non-natural fragrance
Group amino acids; Xaa₄Is Death-Xaa₄, Asp, Phe, Gly, Ala, G
lu, γ-carboxy-Glu (Gla) or any unnatural aromatic amino
Acid; Xaa₅Are Glu, Gla, Asp, Ala, Thr, Ser, Gly, I
le, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-s
Rufo-Tyr, O-phospho-Tyr, nitro-Tyr or any non-natural
A droxy-containing amino acid; Xaa₆Is Ser, Thr, Arg, ornithine, homo
Arginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N,
N-trimethyl-Lys or any unnatural basic amino acid; Xaa₇Is A
sp, Glu, Gla, Arg, ornithine, homoarginine, Lys, N-meth
Cyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or
Is any unnatural basic amino acid; Xaa₈Is Ser, Thr, Asn, Al
a, Gly, His, halo-His, Pro or hydroxy-Pro; Xaa ₉ Are Thr, Ser, Ala, Asp, Asn, Pro, Hydroxy-Pro,
Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N-
Dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural base
Amino acid; Xaa₁₀Are Gly, Ser, Thr, Ala, Asn, Arg,
Ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl
-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino
Acid; Xaa₁₁Represents Gln, Leu, His, halo-His, Trp (D or
L), halo-Trp, neo-Trp, Tyr, nor-Trp, mono-halo-Ty
r, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-T
yr, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N
, N-Dimethyl-Lys, N, N, N-trimethyl-Lys, any unnatural salt
A basic amino acid or any unnatural aromatic amino acid; Xaa₁₂Is Asn, H
is, halo-His, Ile, Leu, Val, Gln, Arg, ornithine,
Homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N,
Xaa. N, N-trimethyl-Lys or any unnatural basic amino acid;_1
3Is Death-Xaa₁₃, Val, Ile, Leu, Arg, Ornithine, Homoia
Luginin, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N
-Trimethyl-Lys or any unnatural basic amino acid. C-terminal
May contain a free carboxyl group or an amide group. Halo is chlorine,
Bromine or iodine, preferably iodine for Tyr and His.
For Trp, it is preferably bromine. Cys residue in D or L conformation
And optionally substituted with homocysteine (D or L).
You may. Tyr residues may be 3-hydroxyl or 2-hydroxyl isomers and
May be substituted with O-sulfo- and O-phospho-derivatives corresponding to acid
The acidic amino acid residue can be any synthetic acidic bioisoteric
Amino acid surrogates, such as tetrazolyl attraction of Gly and Ala
It may be replaced by a conductor.

More particularly, the present invention is directed to an α-conotoxin peptide having the following general formula II:

[0014]

Embedded image

Wherein Xaa₁Is Death-Xaa₁, Asp, Glu or γ-carboxy-Gl
u (Gla); Xaa₂Is Death-Xaa₂, Gln, Ala, Asp, Glu,
Gla; Xaa₃Is Death-Xaa₃, Gly, Ala, Asp, Glu, Gla
Xaa, Pro or hydroxy-Pro;₄Is Death-Xaa₄, Gly, Gl
u, Gla, Gln, Asp, Asn, Pro or hydroxy-Pro; Xa
a₅Are Ser, Thr, Gly, Glu, Gla, Asn, Trp (D or
L), neo-Trp, halo-Trp, Arg, ornithine, homoarginine, L
ys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl
-Lys, any unnatural basic amino acid, Tyr, nor-Tyr, mono-ha
B-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr,
Toro-Tyr or any unnatural hydroxy-containing amino acid; Xaa₆Is As
p, Asn, His, halo-His, Thr, Ser, Tyr, nor-Tyr,
Mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-T
yr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₇ Is Pro or hydroxy-Pro; Xaa₈Is Ala, Ser, Thr, A
sp, Val, Ile, Pro, hydroxy-Pro, Tyr, nor-Tyr,
Mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-T
yr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₉ Are Gly, Ile, Leu, Val, Ala, Thr, Ser, Pro, Hid
Roxy-Pro, Phe, Trp (D or L), Neo-Trp, Halo-Tr
p, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N,
N-dimethyl-Lys, N, N, N-trimethyl-Lys, any unnatural base
Xaa or any unnatural aromatic amino acid; Xaa₁₀Is Ala, As
n, Phe, Pro, hydroxy-Pro, Glu, Gla, Gln, His,
Halo-His, Val, Ser, Thr, Arg, ornithine, homoarginine
, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trime
Chill-Lys or any unnatural basic amino acid; Xaa₁₁Is Thr, S
er, His, halo-His, Leu, Ile, Val, Asn, Met, Pr
o, hydroxy-Pro, Arg, ornithine, homoarginine, Lys, N-
Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys,
Any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-Tyr
, Di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Ty
r or any unnatural hydroxy-containing amino acid; Xaa₁₂Is Asn, Pr
o, hydroxy-Pro, Gln, Ser, Thr, Arg, ornithine, homo
Arginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N,
N-trimethyl-Lys, any unnatural basic amino acid, Tyr, nor-T
yr, mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phos
E-Tyr, nitro-Tyr or any unnatural hydroxy-containing amino acid;
Xaa₁₃Is Death-Xaa₁₃, Gly, Thr, Ser, Pro, hydroxy
-Pro, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O
-Sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any non-
Natural hydroxy-containing amino acid; Xaa₁₄Is Death-Xaa₁₄, Ile, Val,
Asp, Leu, Phe, Arg, ornithine, homoarginine, Lys, N-
Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys,
Any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-Tyr
, Di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Ty
r or any unnatural hydroxy-containing amino acid; Xaa_FifteenIs Death-Xaa _Fifteen , Gly, Ala, Met, Ser, Thr, Trp (D or L), Ne
E-Trp, halo-Trp, any unnatural aromatic amino acid, Arg, orni
Tin, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-Ly
s, N, N, N-trimethyl-Lys or any unnatural basic amino acid; X
aa₁₆Is Death-Xaa₁₆, Trp (D or L), Neo-Trp, Halo-
Trp, any unnatural aromatic amino acid, Arg, ornithine, homoarginine
, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-tri
Methyl-Lys or any unnatural basic amino acid; Xaa₁₇Is Death-X
aa₁₇, Arg, ornithine, homoarginine, Lys, N-methyl-Lys
, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any of
It is an unnatural basic amino acid. At the C-terminus, a free carboxyl group or amide
Groups may be included. Halo is preferably bromine, chlorine or iodine,
More preferably it is iodine for His or Tyr and odor for Trp.
Is prime. Cys residues may be present in the D or L conformation, and
It may be substituted with cysteine (D or L). Tyr residue is 3-hydro
Xyl or 2-hydroxyl isomers and the corresponding O-sulfo- and O-
It may be substituted with a phospho-derivative. The acidic amino acid residue is any synthetic acid
Bioisosteric amino acid surrogates, such as tetras of Gly and Ala
It may be substituted with a zolyl derivative.

More specifically, the present invention is directed to an α-conotoxin peptide having the general formula III:

[0017]

Embedded image

Wherein Xaa₁Is Death-Xaa₁, Ser or Thr; Xaa₂Is Death-Xa
a₂, Asp, Glu, γ-carboxy-Glu (Gla), Asn, Ser
Or Thr; Xaa₃Is Death-Xaa₃, Ala, Gly, Asn, Ser, T
hr, Pro, hydroxy-Pro, Arg, ornithine, homoarginine, L
ys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl
-Lys or any unnatural basic amino acid; Xaa₄Is Death-Xaa₄,
Ala, Val, Leu, Ile, Gly, Glu, Gla, Gln, Asp,
Asn, Phe, Pro, Hydroxy-Pro or any non-natural aromatic alcohol
Amino acid; Xaa₅Is Death-Xaa₅, Thr, Ser, Asp, Glu, Gla
, Gln, Gly, Val, Asp, Asn, Ala, Pro, Hydroxy-P
ro, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N
, N-Dimethyl-Lys, N, N, N-trimethyl-Lys or any non-
Natural amino acid; Xaa₆Are Thr, Ser, Asp, Asn, Met, Va
l, Ala, Gly, Leu, Ile, Phe, any non-natural aromatic amino
Acid, Pro, Hydroxy-Pro, Tyr, Nor-Tyr, Mono-halo-Tyr
, Di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Ty
r or any unnatural hydroxy-containing amino acid; Xaa₇Is Ile, Leu
, Val, Ser, Thr, Gln, Asn, Asp, Arg, His, halo-
His, Phe, any unnatural aromatic amino acid, homoarginine, ornichi
, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-tri
Methyl-Lys, any unnatural basic amino acid, Tyr, nor-Tyr,
No-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Ty
r, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa₈ Is Pro, Hydroxy-Pro, Ser, Thr, Ile, Asp, Leu, V
al, Gly, Ala, Phe, any unnatural aromatic amino acid, Arg,
Lunitine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-
Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid
Xaa₉Are Val, Ala, Gly, Ile, Leu, Asp, Ser, Th
r, Pro, hydroxy-Pro, Arg, ornithine, homoarginine, Ly
s, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-
Lys or any unnatural basic amino acid; Xaa₁₀Is His, halo-H
is, Arg, homoarginine, ornithine, Lys, N-methyl-Lys, N
, N-Dimethyl-Lys, N, N, N-trimethyl-Lys, any unnatural salt
Basic amino acids, Asn, Ala, Ser, Thr, Phe, Ile, Leu, G
ly, Trp (D or L), Neo-Trp, Halo-Trp,
Naturally aromatic amino acids, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-T
yr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any
Any hydroxy-containing amino acid; Xaa₁₁Are Leu, Gln, Val, Ile,
Gly, Met, Ala, Lys, N-methyl-Lys, N, N-dimethyl-L
ys, N, N, N-trimethyl-Lys, Ser, Thr, Arg, homoarginine
, Ornithine, any unnatural basic amino acid, Asn, Glu, Gla,
Gln, Phe, Trp (D or L), Neo-Trp, Halo-Trp or
Any unnatural aromatic amino acid; Xaa₁₂Is Glu, Gla, Gln, As
n, Asp, Pro, hydroxy-Pro, Ser, Gly, Thr, Lys,
N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Ly
s, Arg, homoarginine, ornithine, any unnatural basic amino acid,
Phe, His, halo-His, any unnatural aromatic amino acid, Leu, M
et, Gly, Ala, Tyr, nor-Tyr, mono-halo-Tyr, di-halo
-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or
Some non-natural hydroxy-containing amino acids; Xaa₁₃Is His, halo-His,
Asn, Thr, Ser, Ile, Val, Leu, Phe, any non-natural
Aromatic amino acid, Arg, homoarginine, ornithine, Lys, N-methyl-
Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys, any of
Non-natural basic amino acids, Tyr, nor-Tyr, mono-halo-Tyr, di-ha
B-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or
Any of the unnatural hydroxy-containing amino acids; Xaa₁₄Is Ser, Thr, Al
a, Gln, Pro, hydroxy-Pro, Gly, Ile, Leu, Arg,
Ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl
-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino
Acid; Xaa_FifteenIs Asn, Glu, Gla, Asp, Gly, His, halo-H
is, Ala, Leu, Gln, Arg, ornithine, homoarginine, Lys
, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-L
ys, any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-
Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro
-Tyr or any unnatural hydroxy-containing amino acid; Xaa₁₆Is Met
, Ile, Thr, Ser, Val, Leu, Pro, Hydroxy-Pro, P
he, any unnatural aromatic amino acid, Tyr, nor-Tyr, mono-halo-
Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro
-Tyr, any unnatural hydroxy-containing amino acid, Glu, Gla, Ala
, His, halo-His, Arg, ornithine, homoarginine, Lys, N-
Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
Or any unnatural basic amino acid; Xaa₁₇Is Death-Xaa₁₇, Gly
, Asp, Asn, Ala, Ile, Leu, Ser, Thr, His, halo-
His, Arg, ornithine, homoarginine, Lys, N-methyl-Lys,
N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any non-
Natural basic amino acid; Xaa₁₈Is Death-Xaa₁₈, Gly, Glu, Gla
, Gln, Trp (D or L), neo, halo-Trp, any of the non-natural
Aromatic amino acid, Arg, ornithine, homoarginine, Lys, N-methyl-L
ys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any
Non-natural basic amino acid; Xaa₁₉Is Death-Xaa₁₉, Ser, Thr,
Val, Ile, Ala, Arg, ornithine, homoarginine, Lys, N-
Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
Or any unnatural basic amino acid; Xaa₂₀Is Death-Xaa₂₀, Val
, Asp, His, halo-His, Arg, ornithine, homoarginine, Ly
s, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-
Lys or any unnatural basic amino acid; Xaa₂₁Is Death-Xaa₂₁ Xaa, Asn, Pro or hydroxy-Pro;₂₂Is Death-Xaa₂₂,
Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N-
Dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural base
Amino acid; Xaa₂₃Is Death-Xaa₂₃, Ser or Thr; Xaa₂₄ Is Death-Xaa₂₄, Leu, Ile or Val; provided that (a) Xaa₁Is death
-Xaa₁, Xaa₂Is Death-Xaa₂, Xaa₃Is Death-Xaa₃, Xaa₄ Is Death-Xaa₄, Xaa₆Is Ser, Xaa₇Is His, Xaa₈Is Pro,
Xaa₉Is Ala, Xaa₁₀Is Ser, Xaa₁₁Is Val, Xaa₁₂Is A
sn, Xaa₁₃Is Asn, Xaa₁₄Is Pro, Xaa_FifteenIs Asp, Xaa ₁₆ Is Ile, Xaa₁₇Is Death-Xaa₁₇, Xaa₁₈Is Death-Xaa₁₈ , Xaa₁₉Is Death-Xaa₁₉, Xaa₂₀Is Death-Xaa₂₀, Xaa₂₁ Is Death-Xaa₂₁, Xaa₂₂Is Death-Xaa₂₂, Xaa₂₃Is Death-Xa
a₂₃And Xaa₂₄Is Death-Xaa₂₄Xaa₅Is Gly
No. The C-terminus may contain a free carboxyl group or an amide group
. Halo is preferably bromine, chlorine or iodine, more preferably for His.
Iodine, and bromine for Trp. Cys residue is D or L
Can be in a conformation, optionally substituted by homocysteine (D or L)
May be. If the Tyr residue is a 3-hydroxyl or 2-hydroxyl isomer
And O-sulfo- and O-phospho-derivatives.
Acidic amino acid residues can be substituted for any of the synthetic acidic bioisosteric amino acid substitutes.
For example, tetralyl derivatives of Gly and Ala.

The present invention is also directed to novel specific α-conotoxins of general formula I having the following formula:

[0020]

Embedded image

Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂ is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, nor-Tyr, mono-halo-Tyr, di-halo-
Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; the C-terminus contains a carboxyl or amide group. Halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp. In addition, His residues may be substituted with halo-His; Arg residues may be Lys, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-. Lys may be substituted with Lys or any unnatural basic amino acid; Lys residue is Arg, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl -Ty, which may be substituted with Lys or any unnatural basic amino acid;
The r residue may be substituted with any unnatural hydroxy-containing amino acid;
The er residue may be substituted with Thr; the Thr residue may be substituted with Ser; and the Phe and Trp residues may be substituted with any unnatural aromatic amino acid. Cys residues may be in the D or L conformation and may be optionally substituted with homocysteine (D or L). Tyr residue is 3
-Hydroxy or 2-hydroxyl isomers and the corresponding O-sulfo- and O-phospho-derivatives may be substituted. Acidic amino acid residues can be any synthetic acidic bioisosteric amino acid surrogates, such as Gly and A
la may be substituted with a tetrazolyl derivative.

More specifically, the present invention is directed to the following α-conotoxin peptides of general formula I: Im1.1: SEQ ID NO: 4, wherein Xaa ₁ is Glu and Xaa ₂ is Lys Im1.2: SEQ ID NO: 5, wherein Xaa ₃ is Trp; Rg 1.2: SEQ ID NO: 6; Rg 1.6: SEQ ID NO: 7, where Xaa ₄ is Tyr; Rg 1.6A: SEQ ID NO: : 8, wherein Xaa ₄ is Tyr and Xaa ₅ are Pro; Rg1.7: SEQ ID NO: 9, wherein Xaa ₄ is Tyr and Xaa ₅ Pro; Rg1.9: SEQ ID NO: 10, wherein Xaa ₃ is Trp and Xaa ₅ is Pro; Rg1.10: SEQ ID NO: 11, where Xaa ₁ is Glu and Xaa ₂ is L
ys, Xaa ₃ is Trp and Xaa ₅ is Pro; and Rg1.11: SEQ ID NO: 12, where Xaa ₂ is Lys and Xaa ₅ is Pro. The C-terminus of Im1.1, Rg1.7 and Rg1.10 preferably contains a free carboxyl group. Im1.2, Rg1.2, Rg1.6, Rg1.6A, Rg
The C-terminal of 1.9 and Rg1.11 preferably contains an amide group.

The present invention further provides a novel specific α-formula of general formula II having the following formula:
Directed to conotoxin peptides:

[0024]

Embedded image

Embedded image

Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂ is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, nor-Tyr, mono-halo-Tyr, di-halo-
Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; the C-terminus contains a carboxyl or amide group. Halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp. In addition, His residues may be substituted with halo-His; Arg residues may be Lys, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-. Lys may be substituted with Lys or any unnatural basic amino acid; Lys residue is Arg, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl -Ty, which may be substituted with Lys or any unnatural basic amino acid;
The r residue may be substituted with any unnatural hydroxy-containing amino acid;
The er residue may be substituted with Thr; the Thr residue may be substituted with Ser; and the Phe and Trp residues may be substituted with any unnatural aromatic amino acid. Cys residues may be in the D or L conformation and may be optionally substituted with homocysteine (D or L). Tyr residue is 3
-Hydroxy or 2-hydroxyl isomers and the corresponding O-sulfo- and O-phospho-derivatives may be substituted. Acidic amino acid residues can be any synthetic acidic bioisosteric amino acid surrogate such as Gly and Ala.
May be substituted with a tetrazolyl derivative.

More specifically, the present invention relates to the following α-conotoxin peptides of general formula II
Directed to the tide: Sn1.1: SEQ ID NO: 13, where Xaa₂Is Lys and Xaa
₅Is Pro; Sn1.2: SEQ ID NO: 14, where Xaa₁Is Glu and Xaa
₅Is Pro; Sl1.3: SEQ ID NO: 15, wherein Xaa₃Is Trp, Xaa₄Is
Tyr and Xaa₅Is Pro; A1.2: SEQ ID NO: 16, wherein Xaa₂Is Lys and Xaa
₅Is Pro; Bu1.1: SEQ ID NO: 17, wherein Xaa₂Is Lys and Xaa
₅Is Pro; Bu1.2: SEQ ID NO: 18, where Xaa₂Is Lys and Xaa
₅Is Pro; Bu1.3: SEQ ID NO: 19, wherein Xaa₁Is Glu, Xaa₃Is
Trp and Xaa₅Is Pro; Bu1.4: SEQ ID NO: 20, where Xaa₄Is Tyr and Xaa ₅ Is Pro; Cr1.3: SEQ ID NO: 21, wherein Xaa₅Is Pro; Di1.1: SEQ ID NO: 42, wherein Xaa₅Is Pro; Ms 1.7: SEQ ID NO: 154, wherein Xaa₂Is Lys, Xaa₃ Is Trp, Xaa₄Is Tyr and Xaa₅Is Pro; P1.7: SEQ ID NO: 155, wherein Xaa₁Is Glu, Xaa₂ Is Lys, Xaa₅Is Pro and Xaa₆Is Gln; Ms1.2: SEQ ID NO: 156, wherein Xaa₅Is Pro; Ms1.3: SEQ ID NO: 157, wherein Xaa₂Is Lys, Xaa₃
Is Trp, Xaa₄Is Tyr and Xaa₅Is Pro; Ms1.4: SEQ ID NO: 158, wherein Xaa₁Is Glu, Xaa₂
Is Lys, Xaa₄Is Tyr and Xaa₅Is Pro; Ms1.5: SEQ ID NO: 159, wherein Xaa₂Is Lys and Xa
a₅Is Pro; Ms1.8: SEQ ID NO: 160, wherein Xaa₁Is Glu, Xaa₂
Is Lys, Xaa₄Is Tyr and Xaa₅Is Pro; Ms1.9: SEQ ID NO: 161 wherein Xaa₁Is Glu, Xaa₂
Is Lys, Xaa₄Is Tyr and Xaa₅Is Pro; Bt1.7: SEQ ID NO: 162, wherein Xaa₂Is Lys, Xaa₄
Is Tyr and Xaa₅Is Pro; Lv1.5: SEQ ID NO: 163, wherein Xaa₅Is Pro; Ms1.10: SEQ ID NO: 164, wherein Xaa₂Is Lys, Xaa₄
Is Tyr and Xaa₅Is Pro; Om1.1: SEQ ID NO: 165, wherein Xaa₄Is Tyr and Xa
a₅Is Pro; R1.6: SEQ ID NO: 166, wherein Xaa₄Is Tyr and Xa
a₅Is Pro; R1.7: SEQ ID NO: 167, wherein Xaa₄Is Tyr and Xa
a₅Is Pro; Vr1.1: SEQ ID NO: 168, wherein Xaa₅Is Pro; and Vr1.2: SEQ ID NO: 169, wherein Xaa₅Is Pro.

At the C-terminus, preferably the peptides Sn1.1, Sn1.2, Cr1.3, D
i1.1, Ms1.2, Ms1.4, Ms1.5, Ms1.8, Ms1.9, V
Carboxyl groups are included for r1.1 and Vr1.2. The C-terminus of the other peptide preferably contains an amide group.

The present invention also provides a novel specific α-formula represented by the general formula III having the following formula:
It is also directed to conotoxin peptides:

[0029]

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Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂ is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, nor-Tyr, mono-halo-Tyr, di-halo-
Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; Xaa ₆ is Gln or pyro-
Glu; the C-terminus contains a carboxyl or amide group. Halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp. Further, the His residue is a halo-H
Arg residue may be Lys, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-.
Lys or any unnatural basic amino acid;
The residue may be substituted with Arg, ornithine, homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid; The Tyr residue may be substituted with any unnatural hydroxy-containing amino acid; the Ser residue may be substituted with Thr; the Thr residue may be substituted with Ser; and Phe and Trp
The residue may be substituted with any unnatural aromatic amino acid. Cys residues may be in the D or L conformation and may be optionally substituted with homocysteine (D or L). Tyr residues may be substituted with 3-hydroxyl or 2-hydroxyl isomers and the corresponding O-sulfo- and O-phospho-derivatives. The acidic amino acid residue may be substituted with any synthetic acidic bioisosteric amino acid surrogate, such as the tetrazolyl derivative of Gly and Ala.

More specifically, the present invention is directed to the following α-conotoxin peptides of general formula III:

SmI: SEQ ID NO: 22, wherein Xaa₁Is Glu and Xaa₅ Is Pro; OB-29: SEQ ID NO: 23, wherein Xaa₁Is Glu, Xaa₃Is T
yr and Xaa₅Is Pro; Tx1.1: SEQ ID NO: 24, wherein Xaa₁Is Glu and Xaa₅ Is Pro; R1.1A: SEQ ID NO: 25, wherein Xaa₁Is Glu and Xaa₅ Is Pro; R1.1B: SEQ ID NO: 26, wherein Xaa₁Is Glu and Xaa₅ Is Pro; Om-9: SEQ ID NO: 27, wherein Xaa₁Is Glu and Xaa₅ Is Pro; Om-10: SEQ ID NO: 28, wherein Xaa₅Is Pro; Om-21: SEQ ID NO: 29, wherein Xaa₁Is Glu and Xaa₅ Is Pro; Om-25: SEQ ID NO: 30, where Xaa₁Is Glu and Xaa₅ Is Pro; Om-27: SEQ ID NO: 31, where Xaa₁Is Glu and Xaa₅ Is Pro; Om-28: SEQ ID NO: 32, wherein Xaa₁Is Glu and Xaa₅ Is Pro; Bt1.2: SEQ ID NO: 33, wherein Xaa₁Is Glu and Xaa₅ Is Pro; Bt1.4: SEQ ID NO: 34, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Da1.1: SEQ ID NO: 35, wherein Xaa₁Is Glu and Xaa₅
Is Pro; OB-20: SEQ ID NO: 36, wherein Xaa₁Is Glu, Xaa₂Is L
ys and Xaa₅Is Pro; TI: SEQ ID NO: 37, wherein Xaa₁Is Glu and Xaa₅
Is Pro; TIB: SEQ ID NO: 38, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Pn1.1: SEQ ID NO: 39, wherein Xaa₅Is Pro; Pn1.2: SEQ ID NO: 40, wherein Xaa₁Is Glu and Xaa₅
Is Pro; T1: SEQ ID NO: 41, wherein Xaa₂Is Lys and Xaa₅
Is Pro; TIA: SEQ ID NO: 43, wherein Xaa₅Is Pro; Da1.2: SEQ ID NO: 44, wherein Xaa₅Is Pro; Cr1.2: SEQ ID NO: 45, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Sl1.2: SEQ ID NO: 46, wherein Xaa₁Is Glu, Xaa₂Is L
ys and Xaa₅Is Pro; Tx1.3: SEQ ID NO: 47, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Da1.3: SEQ ID NO: 48, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Da1.4: SEQ ID NO: 49, wherein Xaa₁Is Glu, Xaa₅Is P
ro and Xaa₆Is Gln; Tx1.2: SEQ ID NO: 50, wherein Xaa₅Is Pro; Om-35: SEQ ID NO: 51, wherein Xaa₁Is Glu and Xaa₅
Is Pro; Sl 1.1: SEQ ID NO: 52, wherein Xaa₁Is Glu, Xaa₃Is T
rp, Xaa₄Is Tyr and Xaa₅Is Pro; Sl1.6: SEQ ID NO: 53, wherein Xaa₁Is Glu, Xaa₂Is L
ys, Xaa₄Is Tyr and Xaa₅Is Pro; Sl1.7: SEQ ID NO: 54, wherein Xaa₁Is Glu, Xaa₄Is T
yr and Xaa₅Is Pro; Bt1.1: SEQ ID NO: 55, wherein Xaa₁Is Glu, Xaa₄Is T
yr and Xaa₅Is Pro; Bt: 1.3: SEQ ID NO: 56, wherein Xaa₁Is Glu, Xaa₄Is T
yr and Xaa₅Is Pro; Bt1.5: SEQ ID NO: 57, wherein Xaa₁Is Glu, Xaa₄Is T
yr and Xaa₅Is Pro; A1.4: SEQ ID NO: 170, wherein Xaa₁Is Glu and Xaa ₅ Is Pro; A1.5: SEQ ID NO: 171 wherein Xaa₁Is Glu and Xaa
₅Is Pro; A1.6: SEQ ID NO: 172, wherein Xaa₁Is Glu and Xaa
₅Is Pro; Af1.1: SEQ ID NO: 173, wherein Xaa₁Is Glu, Xaa₄Is
Tyr, Xaa₅Is Pro and Xaa₆Is Gln; Af1.2: SEQ ID NO: 174, wherein Xaa₁Is Glu and Xaa
₅Is Pro; Ar1.2: SEQ ID NO: 175, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp, Xaa₄Is Try and Xaa₅Is Pro; Ar1.3: SEQ ID NO: 176, wherein Xaa₁Is Glu, Xaa₄Is
Tyr and Xaa₅Is Pro; Ar1.4: SEQ ID NO: 177, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp, Xaa₄Is Try and Xaa₅Is Pro; Ar1.5: SEQ ID NO: 178, wherein Xaa₁Is Glu and Xaa
₅Is Pro; Ar1.6: SEQ ID NO: 179, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp, Xaa₄Is Try and Xaa₅Is Pro; Ay1.2: SEQ ID NO: 180, wherein Xaa₄Is Tyr and Xaa
₅Is Pro; Ay1.3: SEQ ID NO: 181, wherein Xaa₅Is Pro; Bn1.4: SEQ ID NO: 182, wherein Xaa₄Is Tyr and Xaa
₅Is Pro; Bt1.8: SEQ ID NO: 183, wherein Xaa₁Is Glu and Xaa
₅Is Pro; Bt1.9: SEQ ID NO: 184, wherein Xaa₁Is Glu, Xaa₄Is
Tyr and Xaa₅Is Pro; Ca1.3: SEQ ID NO: 185, wherein Xaa₁Is Glu, Xaa₃Is
Trp, Xaa₄Is Try and Xaa₅Is Pro; Ca1.4: SEQ ID NO: 186, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp, Xaa₄Is Try, Xaa₅Is Pro and Xaa ₆ Is Gln; C1.2: SEQ ID NO: 187, wherein Xaa₁Is Glu and Xaa ₅ Is Pro; C1.3: SEQ ID NO: 188, wherein Xaa₄Is Tyr and Xaa ₅ Is Pro; Ep1.2: SEQ ID NO: 189, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp and Xaa₅Is Pro; G1.1: SEQ ID NO: 190, wherein Xaa₅Is Pro; G1.3: SEQ ID NO: 191 wherein Xaa₅Is Pro; Im1.3: SEQ ID NO: 192, wherein Xaa₅Is Pro; Lv1.2: SEQ ID NO: 193, wherein Xaa₁Is Glu and Xaa ₅ Is Pro; Lv1.3: SEQ ID NO: 194, wherein Xaa₂Is Lys and Xaa ₅ Is Pro; Lv1.4: SEQ ID NO: 195, wherein Xaa₁Is Glu and Xaa ₅ Is Pro; Lv1.6: SEQ ID NO: 196, wherein Xaa₅Is Pro; Lv1.7: SEQ ID NO: 197, wherein Xaa₁Is Glu and Xaa ₅ Is Pro; Lv1.8: SEQ ID NO: 198, wherein Xaa₅Is Pro; Lv1.9: SEQ ID NO: 199, wherein Xaa₄Is Tyr and Xaa ₅ Is Pro; Lv1.10: SEQ ID NO: 200, wherein Xaa₁Is Glu; Mr1.3: SEQ ID NO: 201, wherein Xaa₄Is Tyr and Xaa ₅ Is Pro; Mr1.4: SEQ ID NO: 202, wherein Xaa₅Is Pro; Ms1.1: SEQ ID NO: 203, wherein Xaa₂Is Lys, Xaa₄Is
Tyr and Xaa₅Is Pro; Ms 1.6: SEQ ID NO: 204, wherein Xaa₄Is Tyr and Xaa
₅Is Pro; O1.1: SEQ ID NO: 205, wherein Xaa₂Is Lys, Xaa₄Is
Tyr and Xaa₅Is Pro; O1.2: SEQ ID NO: 206, wherein Xaa₁Is Glu, Xaa₄Is
Tyr and Xaa₅Is Pro; O1.4: SEQ ID NO: 207, wherein Xaa₁Is Glu, Xaa₃Is
Trp, Xaa₄Is Tyr and Xaa₅Is Pro; O1.7: SEQ ID NO: 208, wherein Xaa₄Is Tyr and Xaa ₅ Is Pro; O1.8: SEQ ID NO: 209, wherein Xaa₁Is Glu, Xaa₄Is
Tyr and Xaa₅Is Pro; Om1.2: SEQ ID NO: 210, wherein Xaa₁Is Glu, Xaa₄Is
Tyr and Xaa₅Is Pro; Om1.3: SEQ ID NO: 211, wherein Xaa₂Is Glu, Xaa₂Is
Lys, Xaa₃Is Trp and Xaa₅Is Pro; Om1.4: SEQ ID NO: 212, wherein Xaa₅Is Pro; Oml. 5: SEQ ID NO: 213, wherein Xaa₄Is Tyr and Xaa
₅Is Pro; Oml. 6: SEQ ID NO: 214, wherein Xaa₁Is Glu and Xaa
₅Is Pro; P1.4: SEQ ID NO: 215, wherein Xaa₁Is Glu and Xaa
₅Is Pro; P1.5: SEQ ID NO: 216, wherein Xaa₅Is Pro; P1.6: SEQ ID NO: 217, wherein Xaa₃Is Trp and Xaa
₅Is Pro; P1.8: SEQ ID NO: 218, wherein Xaa₅Is Pro; Rg1.1: SEQ ID NO: 219, wherein Xaa₁Is Glu and Xaa
₅Is Pro; Rg1.3: SEQ ID NO: 220, wherein Xaa₅Is Pro; Rg1.4: SEQ ID NO: 221 where Xaa₂Is Lys, Xaa₄Is
Tyr and Xaa₅Is Pro; Rg1.5: SEQ ID NO: 222, wherein Xaa₅Is Pro; Rg1.8: SEQ ID NO: 223, wherein Xaa₂Is Lys, Xaa₄Is
Tyr and Xaa₅Is Pro; Sm1.4: SEQ ID NO: 224, wherein Xaa₂Is Lys and Xaa
₅Is Pro; Sm1.5: SEQ ID NO: 225, wherein Xaa₁Is Glu and Xaa
₅Is Pro; S1.5: SEQ ID NO: 226, wherein Xaa₅Is Pro; Tx1.5: SEQ ID NO: 227, wherein Xaa₁Is Glu, Xaa₅Is
Pro and Xaa₆Is Gln; T1.1: SEQ ID NO: 228, wherein Xaa₅Is Pro; Vr1.3: SEQ ID NO: 229, wherein Xaa₅Is Pro; and Tb: SEQ ID NO: 230, wherein Xaa₂Is Lys and Xaa
₅Is Pro.

At the C-terminus, preferably the peptides OB-29, Tx1.1, R1.1A, R
1.1B, Om-9, Om-10, Om-21, Om-25, Om-27, Om
-28, Cr1.2, Om-35, Bt1.1, Bt1.3, Bt1.5, A1
. 4, A1.6, Ar1.2, Ar1.3, Ar1.4, Ar1.5, Ar1.
6, Ca1.3, Ca1.4, Ep1.2, Lv1.9, O1.2, m1.3,
Om1.6, P1.6, Rg1.1, Rg1.3, Rg1.4, Sm1.5, T
Carboxyl groups are included for x1.5 and Vr1.3. The C-terminus of the other peptide preferably contains an amide group.

The present invention is also directed to a new specific α-conotoxin peptide having the formula:

[0035]

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Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂ is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₄ is Tyr, nor-Tyr, mono-halo-Tyr, di-
Halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr
And Xaa ₅ is Pro or hydroxy-Pro; the C-terminus contains a carboxyl or amide group. Halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr. In addition, His residues may be substituted with halo-His; Arg residues are Lys, ornithine,
Homoarginine, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-
Lys residues may be Arg, ornithine, homoarginine, N-methyl-Lys, optionally substituted with trimethyl-Lys or any unnatural basic amino acid.
, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid; the Tyr residue may be replaced with any unnatural hydroxy-containing amino acid. A Ser residue may be substituted with Thr; a Thr residue may be substituted with Ser; and Phe
The residue may be substituted with any unnatural aromatic amino acid. Cys residues may be in the D or L conformation and may be optionally substituted with homocysteine (D or L). Tyr residues may be substituted with 3-hydroxyl or 2-hydroxyl isomers and the corresponding O-sulfo- and O-phospho-derivatives. The acidic amino acid residue may be replaced by any acidic bioisosteric amino acid surrogate, for example, the tetrazolyl derivative of Gly and Ala.

More specifically, the present invention is directed to the following α-conotoxin peptides:

G1.2: SEQ ID NO: 231, wherein Xaa₁Is Glu, Xaa₂Is
Lys, Xaa₄Is Tyr and Xaa₅Is Pro; and Rg1.12: SEQ ID NO: 232, wherein Xaa₂Is Lys and Xaa ₅ Is Pro.

The C-terminus of G1.2 preferably contains a carboxyl group, and Rg1.
The 12 C-terminus preferably contains an amide group. Examples of non-natural aromatic amino acids include, but are not limited to, nitro-Phe
, 4-substituted -Phe (where the substituents are _{C 1-C 3} alkyl, carboxyl, hydroxymethyl, sulfomethyl, halo, phenyl, -CHO, -CN, -SO ₃ is H and -NHAc) but such as included. Examples of unnatural hydroxy-containing amino acids include, but are not limited to, 4-hydroxymethyl-Phe,
Hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-T
yr. Examples of non-naturally occurring basic amino acids include, but are not limited to, N-1- (2-pyrazolinyl) -Arg, 2- (4-piperinyl) -G
ly, 2- (4-piperinyl) -Ala, 2- [3- (2S) pyrrolininyl] -G
ly and 2- [3- (2S) pyrrolininyl] -Ala. These and other unnatural basic amino acids, unnatural hydroxy-containing amino acids or unnatural aromatic amino acids can be found in the Building Block Index, Version 3.0 (RPS Amino Acid Analogues, Inc., Worcester, MA) and available therefrom. 1999 Catalog,
Hydroxy-containing and aromatic amino acids are described on pages 4-47 and basic amino acids on pages 66-87;
See also tp: //www.amino-acids.com).

If desired, in the peptides of general formulas I, II and III and the specific peptides described above, the Asn residue may be modified to include N-glycans;
The er and Thr residues may be modified to include O-glycans. According to the present invention, glycans can be any N-, S- or O-linked mono-, 2-, 3-,
By poly- or oligosaccharides, they can be attached to the hydroxy, amino or thiol groups of any natural or modified amino acids by synthetic or enzymatic methods known in the art. The monosaccharides constituting glycans include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-
Contains idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, DN-acetyl-glucosamine (GlcNAc), DN-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. obtain. These sugars may include, for example, one or more O-sulfate, O-phosphate,
It may be structurally modified with an acidic group such as O-acetyl or sialic acid and combinations thereof. Glycans can also include similar polyhydroxy groups or polypropylene glycol derivatives such as D-penicillamine 2,5 and its halogenated derivatives. The glycosidic linkage is beta and is 1-4 or 1-3
, Preferably 1-3. The bond between the glycan and the amino acid may be alpha or beta, preferably alpha and 1-.

Core O-glycans are described by Van de Steen et al. (1998), which is hereby incorporated by reference. Mucin-type O-linked oligosaccharides can be Ser or Thr (or other hydroxylated residues of the peptide) depending on the GalNAc residue.
Is bound to. The monosaccharide building block and the linkage attached to this first GalNAc residue are defined as "core glycans", of which eight have been identified. For each core glycan, the type of glycosidic linkage (orientation and binding) is defined. Suitable glycans and glycan analogs are described in U.S. Ser. No. 09 / 420,797, filed Oct. 19, 1999, and US Pat. PCT Application No. PCT / US99 / 24380. A preferred glycan is Gal (β1 → 3) GalNAc (α1 →).

Optionally, in the peptides of general formulas I and II and the specific peptides described above, the pair of Cys residues is Ser / (Glu or Asp) or Lys /
(Glu or Asp) can be replaced in pairs. Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows the replacement of a natural Cys bridge with a lactam bridge.

The present invention is further directed to propeptides and nucleic acid sequences encoding the propeptides or peptides, as described in further detail herein.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to relatively short peptides of about 10-30 residues in length (referred to herein as α-conotoxins), which are naturally available in small quantities in the venom of mussels. Or similar to a naturally available peptide, which preferably contains two disulfide bonds.

In another aspect, the invention is directed to a pharmaceutical composition comprising an effective amount of an α-conotoxin peptide. Such a pharmaceutical composition has the ability to act as an antagonist for the nicotinic acetylcholine receptor. In one embodiment, an α-conotoxin having specificity for a neuronal muscular junction nicotinic acetylcholine receptor was filed January 21, 2000, which is hereby incorporated by reference. U.S. Patent Application No. 09 / (Attorney File No. 2314-178.A) and International Patent Application No. PCT / US00 / (Attorney File No. 2314-138.PCT) filed on January 21, 2000. As such, it is used as a neuromuscular blocking agent for use in conjunction with surgery. In a second embodiment, additional [alpha] -conotoxins and their uses are each incorporated by reference herein, U.S. Patent Nos. 4,447,356 (Olivera et al., 1984); 5,432,155;
No. 5,514,774.

In a third aspect, a further use for α-conotoxins is found in US Patent Application Serial No. 09 / 219,446 filed December 22, 1998, which is hereby incorporated by reference. Has been described. In this application, α-conotoxins, which have specificity for neuronal nicotinic acetylcholine receptors, are used to treat disorders modulated by neuronal nicotinic acetylcholine receptors.
Such disorders include, but are not limited to, cardiovascular disorders, gastric motility disorders, urinary incontinence, nicotine addiction, mood disorders (such as bipolar disorder, unipolar depression, dysthymia and seasonal effects disorder) and minor disorders Cellular lung cancer, as well as the localization of small cell lung cancer are included.

The α-conotoxin peptides described herein are in small quantities sufficient for chemical synthesis. A general chemical synthesis for preparing the α-conotoxin peptides described above is provided later herein. A variety of α-conotoxin peptides can be isolated from specific mussel species using the techniques described in U.S. Pat. No. 4,447,356 (Olivera et al., 1984), which is hereby incorporated by reference. It can also be obtained by separation and purification.

Although the α-conotoxin peptide of the present invention can be obtained by purification from a mussel, the amount of α-conotoxin peptide obtainable from individual shellfish is very small, so that the desired substance can be obtained. Pure α-conotoxin peptides are actually best obtained in commercially valuable quantities by chemical synthesis using a solid phase strategy. For example, the yield from a single mussel can be about 10 micrograms or less of the α-conotoxin peptide. By "substantially pure" is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably at least about 85% pure, preferably at least about 95% Present in amounts of purity. Chemical synthesis of biologically active α-conotoxin peptides relies on accurate determination of amino acid sequence.

[0049] The α-conotoxin peptides can also be produced by recombinant DNA techniques well known in the art. Such a technique is described in Sambrook et al. (1989)
). The peptide produced in this way is monosaccharide, reduced and, if necessary, oxidized to form the correct disulfide bond.

One method of forming disulfide bonds in the conantokin peptides of the invention is air oxidation of the linear peptide for extended periods at or below cold room temperature. This procedure results in the creation of substantial amounts of biological activity, disulfide-linked peptides. The oxidized peptide is fractionated using, for example, reverse phase high performance liquid chromatography (HPLC) to separate peptides having different binding configurations. These fractions are then compared to the elution of the natural substance, or by using a simple assay, the particular fraction with the correct binding of maximum biological potency is conveniently determined. However, somewhat higher doses may be required due to dilution resulting from the presence of other fractions of lower biopotency. The peptides are synthesized by any suitable method, such as by absolute solid phase techniques, partial solid phase techniques, fraction condensation or classical solution coupling.

In normal solution phase peptide synthesis, peptide chains can be prepared by a series of coupling reactions that add constituent amino acids to a growing peptide chain in the desired order. Various coupling reagents such as dicyclohexylcarbodiimide or diisopropylcarbonyldiimidazole, various active esters such as N-
Performing the reaction in solution with the subsequent isolation and purification of the intermediate using an ester of hydroxyphthalimide or N-hydroxy-succinimide and various cleavage reagents is a well-known classical peptide method. Classical solution synthesis is described in the paper "Methoden der Organischen Chemie (Houben-Weyl): Synthese von
Peptiden "(1974). Techniques for absolute solid phase synthesis are described in the textbook," Solid-Phase Peptide Synthesis "(Stewart and Young, 1969); U.S. Pat. No. 4,105,603 ( Illustrated by the disclosure of Vale et al., 1978. Fragment condensation methods of synthesis are illustrated in U.S. Patent No. 3,972,859 (1976) .Other available syntheses are described in U.S. Patent Nos. 3,842,067 (1974) and 3,862,925. (1975
). The synthesis of peptides containing gamma-carboxyglutamic acid residues is exemplified by Rivier et al. (1987), Nishiuchi et al. (1993) and Zhou et al. (1996).

Common to such chemical syntheses is the lack of a variety of amino acid moieties with suitable protecting groups that will prevent chemical reactions from taking place at that site until the group is finally removed. Protecting stable side groups. It is also common that amino groups or amino acids are reacted while the group reacts at the carboxyl group, followed by selective removal of the α-amino protecting group to allow subsequent reactions to take place at that position. The purpose is to protect the α-amino group on the fragment. Thus, as a step in such synthesis, an intermediate comprising each amino acid residue located in the desired order in a peptide chain having an appropriate side chain protecting group attached to a variety of residues having an unstable side chain. It is common to produce somatic compounds.

As far as the choice of the side chain amino protecting group is concerned, one is generally chosen that is not removed during the deprotection of the α-amino group during the synthesis. However, for some amino acids, such as His, protection is generally not required. In selecting a particular side-chain protecting group for use in peptide synthesis, the following general rules are followed: (a) the protecting group preferably retains its protecting properties under coupling conditions and does not split; ) The protecting group must be stable under the reaction conditions chosen to remove the α-amino protecting group at each step of the synthesis, and (c) the side chain protecting group
Must be removable upon completion of the synthesis containing the desired amino acid sequence under reaction conditions that do not undesirably alter the peptide chain.

Many or even all of these peptides can be prepared using recombinant DNA technology. However, if the peptide is not so prepared,
Although other equivalent chemical syntheses known in the art may be used as described above, they are preferably prepared using Merrifield solid phase synthesis. Solid phase synthesis involves coupling the protected α-amino acid to a suitable resin to form the C
-Starting from the end Such starting materials include benzhydrylamine (BHA) by coupling an α-amino-protected amino acid to a chloromethylated or hydroxymethyl resin via an ester bond or via an amide bond.
It can be prepared by coupling to a resin or paramethylbenzhydrylamine (MBHA) resin. The preparation of the hydroxymethyl resin is described by Bodansky et al. (1966). Chloromethylated resins are available from Bio Rad Laboratories (Richmond
, CA) and Lab. Systems, Inc. The preparation of such resins is
Described by Stewart and Young (1969). BHA and MBHA resin supports are commercially available and are commonly used when the polypeptide of interest to be synthesized has an unsubstituted amide at the C-terminus. Therefore, the formula -O-CH ₂ - resin support, as for those with a -NH BHA resin support or -NH-MBHA resin support, the solid resin supports are any of those known in the art You may. If unsubstituted amides are desired, the use of BHA or MBHA resins is preferred. Because the cleavage gives the amide directly. If N-methylamide is desired, it can be produced from N-methyl BHA resin. If other substituted amides are desired, the teachings of US Pat. No. 4,569,967 (Kornreich
Et al., 1986), or if still other groups than the free acid are desired at the C-terminus, synthesize the peptide group using the classical method described in the Houben-Weyl text (1974). Would be preferred.

The C-terminal amino acid protected by Boc or Fmoc and by the side-chain protecting group may suitably be added to about 60 ° C. with stirring if a peptide having a free acid at the C-terminus is first to be synthesized. For 24 hours using KF in DMF. Horiki
(1978), it can be coupled to a chloromethylated resin. After coupling the BOC-protected amino acid to the resin support, the α-amino protecting group is removed by using methylene chloride or trifluoroacetic acid (TFA) in TFA alone. Deprotection is performed at a temperature between about 0 ° C. and room temperature. Other standard cleavage reagents, such as HCl in dioxane, and conditions for removal of specific α-amino protecting groups can be used as described in Schroder and Lubke (1965).

After removal of the α-amino protecting group, the remaining α-amino- and side-chain-protected amino acids are coupled step-wise in the desired order to give the intermediate compound as hereinbefore defined. Alternatively, or separately, each amino acid may be added separately during synthesis, some of which may be coupled to each other before being added to the solid-state reactor. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N, N'-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).

Activating reagents used for solid phase synthesis of peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N, N'-diisopropylcarbodiimide and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide. Other activating reagents and their use in peptide coupling have been described by Schroder and Lubke (1965) and Kapoor (1970).

Each protected amino acid or amino acid sequence is introduced into the solid-state reactor in about a two-fold or more excess and is added to a dimethylformamide (DMF): CH ₂ Cl ₂ (1: 1) medium or DMF or CH. _The coupling can be performed in ₂ Cl ₂ alone. If intermediate coupling occurs, the coupling procedure is repeated before removal of the α-amino protecting group prior to coupling of the next amino acid. When performed manually, the success of the coupling reaction at each stage of the synthesis is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970). The coupling reaction can be performed automatically using a program such as that reported in Rivier et al. (1978), as in the Beckman 990 automated synthesizer.

After completion of the desired amino acid sequence, liquid hydrogen fluoride or TFA (Fmoc
Treatment with reagents (when using chemistry) allows the removal of the intermediate peptide from the resin support, which not only cleaves the peptide from the resin, but also removes any remaining side-chain protecting groups and previously removed If not, the N-terminal α-amino protecting group is also cleaved to give the peptide in free acid form. If Met is present in the sequence, the potential Boc protecting group is preferably first removed with trifluoroacetic acid (TFA) / ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential Soc. -Eliminate alkylation. If hydrogen fluoride or TFA is used for cleavage, one or more scavengers, such as anisole, cresol, dimethyl sulfide and methyl ethyl sulfide, are included in the reaction vessel.

[0060] In contrast to cyclization of peptides when part of a peptide-resin, it preferably affects the cyclization of linear peptides to produce bonds between Cys residues. To effect such a disulfide cyclization bond, the fully protected peptide is cleaved from the hydroxymethylated or chloromethylated resin support by ammonolysis, as is well known in the art, to provide a fully protected peptide. An amide intermediate can be obtained, which is then suitably cyclized and deprotected. Alternatively, deprotection and cleavage of the peptide from the above resin or a benzhydrylamine (BHA) resin or methylbenzhydrylamine (MBHA) may be carried out using hydrofluoric acid (
It can be carried out at 0 ° C. with HF) or TFA, and then oxidized as described above.

Also, peptides can be synthesized using an automatic synthesizer. Advanced Chemtech 357
Using an automated peptide synthesizer, the amino acids were started at the C-terminus and MBHA Link
Coupling in sequence to the resin (typically 100 mg of resin). Coupling uses 1,3-diisopropylcarbodiimide in N-methylpyrrolidinone (NMP) or 2- (1H-benzotriazol-1-yl) -1,1
, 3,3-tetramethyluronium hexafluorophosphate (HBTU)
And using diethylisopropylethylamine (DIEA). FMOC
Protecting groups are removed by treatment with a 20% solution of piperidine in dimethylformamide (DMF). Thereafter, the resin is washed with DMF (twice), followed by methanol and NMP.

A pharmaceutical composition containing the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be prepared according to a conventional pharmaceutical preparation technique. For example, Remington's Ph
armaceutical Sciences, 18th edition (1990, Mack Publishing Co., Easton, PA)
Was referred to. Typically, an antagonist amount of the active ingredient will be mixed with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, eg, intravenous, oral or parenteral. The composition may further include antioxidants, stabilizers, preservatives, and the like.

For oral administration, the compounds may be formulated in solid or liquid preparations, such as capsules, pills, tablets, troches, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, for example, for oral liquid preparations (eg, suspensions, elixirs and solutions) In the case of oral solid preparations, such as water, glycols, oils, alcohols, fragrances, preservatives, coloring agents, suspending agents, etc .; or (for example, powders, capsules and tablets) It is a carrier such as starch, sugar, diluent, granulating agent, lubricant, binder, disintegrant and the like. Because of their ease in administration, tablets and capsules represent the most advantageous dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent may be encapsulated to allow it to cross the blood-brain barrier while stably passing through the gastrointestinal tract. See, for example, WO 96/11698.

For parenteral administration, the compounds may be dissolved in a pharmaceutical carrier and administered as a solution or suspension. Examples of stable carriers are water, saline, dextrose solution, fructose solution, ethanol, or oils of animal, plant or synthetic origin.
The carrier may also include other ingredients such as preservatives, suspending agents, solubilizing agents, buffers, and the like. If the compound is administered intrathecally, it can also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeutically effective amount. The actual amount administered, and rate and time-course of administration, will depend on the nature and extent of the condition being treated.
Decisions on treatment prescriptions, eg, dosage, timing, etc., are within the responsibility of the general practitioner or spareist, and typically involve the disorder being treated, the symptoms of the individual patient, the delivery. Consider the site, method of administration, and other factors known to the practitioner. Examples of technologies and protocols can be found in Remington's Pharma
can be found in ceutical Sciences. Typically, the conopeptides of the present invention comprise from about 0.001 mg / kg to about 250 mg / kg, preferably about 0.05 m / kg.
g / kg to about 100 mg / kg of the active ingredient, more preferably about 0.1 mg / kg.
It shows its effect in the dosage range of kg to about 75 mg / kg. Suitable doses may be administered in multiple sub-doses per day. Typically, a dose or sub-dose will contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form. A more preferred dosage will contain from about 0.5 mg to about 100 mg of the active ingredient per unit dosage form. Dosages generally start at lower levels and are increased until the desired effect is achieved.

Alternatively, targeted therapies can be used to deliver an active agent more specifically to a particular type of cell by using a targeting system such as an antibody or cell-specific ligand. Targeting is desirable for a variety of reasons, such as when the agent is unacceptably toxic, otherwise requires too high a dose, or otherwise cannot enter the target cells.

An active agent that is a peptide is a cell-based delivery system in which a DNA sequence encoding the active agent has been introduced into cells designed to be implanted in the body of a patient, particularly in the spinal cord region.
The system may also administer. A preferred delivery system is U.S. Pat. No. 5,550,050
And international publication numbers WO92 / 19195, WO94 / 25503, WO95 / 01203, WO95
No. 05452, WO96 / 02286, WO96 / 02646, WO96 / 40871, WO96 / 40959 and WO97 / 12635. Suitable DNA sequences can be prepared synthetically for each active agent based on the disclosed sequences and the known genetic code.

EXAMPLES The invention is illustrated by reference to the following examples, which are provided by way of example and are not intended to limit the invention in any way. Standard techniques well known in the art or techniques specifically described below were utilized.

Example 1 Isolation of α-Conotoxin Crude venom was extracted from venom tubes (Cruz et al., 1976) and the components were purified as previously described (Cartier et al., 1996a). The crude extract from the venom tube is
Reversed phase liquid chromatography (RPLC) using a _C18 semi-preparative column (10 × 250 mm) eluting with a linear gradient of acetonitrile in 0.1% TFA.
). Further purification of the biological activity peak was achieved by using a Vydac _C18 analytical column (4.6) eluting with a gradient of acetonitrile in 0.1% TFA.
× 220 mm). The effluent was monitored at 220 mm. Peaks were collected and aliquots were assayed for activity. The activity is Xenopus (Xe
nopus) was monitored by assessing the block of α3β4 nAChR expressed in oocytes.

[0070] The amino acid sequence of the purified peptide was determined by standard methods. Purified peptides were reduced and alkylated (DNA / Peptide Facility, University of Utah) before sequencing by automated Edman degradation on an Applied Biosystems 477A protein sequencer with a 120A analyzer (Martinez et al., 1995; Shonn).
Et al., 1994). By this method, peptides MII, AuIA, AuIB, AuIC, MAR-
1, MAR-2, TI, OB-29, EpI, S1.1, Bn1.1, Bn1.
2, Ca1.1, Ca1.2, Cn1.1, Cn1.2 and Sm1.3 were obtained.

Example 2 Synthesis of Conopeptides The synthesis of conopeptides, either mature toxins or precursor peptides, was performed separately using conventional protection chemistry as described by Cartier et al. (1996). Briefly, amino acid derivatives purchased from Bachem (Torrence, CA) and ABI mode
2- (1H-benzotriol-1-yl)-using l430A peptide synthesizer
Linear chains were constructed on Rink amide resin by the Fmoc technique using 1,1,3,3-tetramethyluronium-tetrafluoroborate coupling. Orthogonal protection was used for cysteine: Cys ³ and Cys ¹⁶ were stable Cys (
Cys ² and Cys ⁸ were protected as acid-labile Cys (S-trityl). Removing the terminal Fmoc protecting group;
After cleavage of the peptide from the resin, the released peptide was precipitated by filtering the reaction mixture into methyl tert-butyl ether at -10 ° C to remove protecting groups other than on Cys ³ and Cys ¹⁶ . The peptide was combined with 0.1% TFA and 6%.
0% acetonitrile, VydacC ₁₈ preparative column (22 × 250 m
m) Purified by RPLC above and eluted with a gradient of acetonitrile in 0.1% TFA at a flow rate of 20 mL / min.

The disulfide bridge in the three conopeptides was formed as described by Cartier et al. (1996). Briefly, the disulfide bridge between Cys ² and Cys ⁸ formed by air oxidation, which was determined to be completed by analytical RPLC. The monocyclic peptide was purified by RPLC on a VydacC ₁₈ preparative column (22 × 250 mm) and eluted with a gradient of acetonitrile in 0.1% TFA. Removal of the S-acetamidomethyl group and closure of the disulfide bridge between Cys ³ and Cys ¹⁶ was achieved simultaneously by iodine oxidation. The cyclic peptide was purified by RPLC on a VydacC ₁₈ preparative column (22 × 250 mm) and eluted with a gradient of acetonitrile in 0.1% TFA.

Example 3 Isolation of DNA Encoding α-Conotoxin DNA encoding α-conotoxin was obtained by general techniques well known in the art, as described in Olivera et al. (1996). Isolated and cloned according to conventional techniques used. Alternatively, a cDNA library was prepared from mussel venom tubes using conventional techniques. DNA from a single clone was amplified by conventional techniques using primers approximately corresponding to the M13 universal priming site and the M13 reverse universal priming site. Clones having a size of approximately 300 nucleotides were sequenced and screened for similarities in sequence to known α-conotoxins. The DNA sequence and the encoded propeptide or peptide sequence are listed in Table 1-134.

[0074]

[Table 1]

[0075]

[Table 2]

[0076]

[Table 3]

[0077]

[Table 4]

[0078]

[Table 5]

[0079]

[Table 6]

[0080]

[Table 7]

[0081]

[Table 8]

[0082]

[Table 9]

[0083]

[Table 10]

[0084]

[Table 11]

[0085]

[Table 12]

[0086]

[Table 13]

[0087]

[Table 14]

[0088]

[Table 15]

[0089]

[Table 16]

[0090]

[Table 17]

[0091]

[Table 18]

[0092]

[Table 19]

[0093]

[Table 20]

[0094]

[Table 21]

[0095]

[Table 22]

[0096]

[Table 23]

[0097]

[Table 24]

[0098]

[Table 25]

[0099]

[Table 26]

[0100]

[Table 27]

[0101]

[Table 28]

[0102]

[Table 29]

[0103]

[Table 30]

[0104]

[Table 31]

[0105]

[Table 32]

[0106]

[Table 33]

[0107]

[Table 34]

[0108]

[Table 35]

[0109]

[Table 36]

[0110]

[Table 37]

[0111]

[Table 38]

[0112]

[Table 39]

[0113]

[Table 40]

[0114]

[Table 41]

[0115]

[Table 42]

[0116]

[Table 43]

[0117]

[Table 44]

[0118]

[Table 45]

[0119]

[Table 46]

[0120]

[Table 47]

[0121]

[Table 48]

[0122]

[Table 49]

[0123]

[Table 50]

[0124]

[Table 51]

[0125]

[Table 52]

[0126]

[Table 53]

[0127]

[Table 54]

[0128]

[Table 55]

[0129]

[Table 56]

[0130]

[Table 57]

[0131]

[Table 58]

[0132]

[Table 59]

[0133]

[Table 60]

[0134]

[Table 61]

[0135]

[Table 62]

[0136]

[Table 63]

[0137]

[Table 64]

[0138]

[Table 65]

[0139]

[Table 66]

[0140]

[Table 67]

[0141]

[Table 68]

[0142]

[Table 69]

[0143]

[Table 70]

[0144]

[Table 71]

[0145]

[Table 72]

[0146]

[Table 73]

[0147]

[Table 74]

[0148]

[Table 75]

[0149]

[Table 76]

[0150]

[Table 77]

[0151]

[Table 78]

[0152]

[Table 79]

[0153]

[Table 80]

[0154]

[Table 81]

[0155]

[Table 82]

[0156]

[Table 83]

[0157]

[Table 84]

[0158]

[Table 85]

[0159]

[Table 86]

[0160]

[Table 87]

[0161]

[Table 88]

[0162]

[Table 89]

[0163]

[Table 90]

[0164]

[Table 91]

[0165]

[Table 92]

[0166]

[Table 93]

[0167]

[Table 94]

[0168]

[Table 95]

[0169]

[Table 96]

[0170]

[Table 97]

[0171]

[Table 98]

[0172]

[Table 99]

[0173]

[Table 100]

[0174]

[Table 101]

[0175]

[Table 102]

[0176]

[Table 103]

[0177]

[Table 104]

[0178]

[Table 105]

[0179]

[Table 106]

[0180]

[Table 107]

[0181]

[Table 108]

[0182]

[Table 109]

[0183]

[Table 110]

[0184]

[Table 111]

[0185]

[Table 112]

[0186]

[Table 113]

[0187]

[Table 114]

[0188]

[Table 115]

[0189]

[Table 116]

[0190]

[Table 117]

[0191]

[Table 118]

[0192]

[Table 119]

[0193]

[Table 120]

[0194]

[Table 121]

[0195]

[Table 122]

[0196]

[Table 123]

[0197]

[Table 124]

[0198]

[Table 125]

[0199]

[Table 126]

[0200]

[Table 127]

[0201]

[Table 128]

[0202]

[Table 129]

[0203]

[Table 130]

[0204]

[Table 131]

[0205]

[Table 132]

[0206]

[Table 133]

[0207]

[Table 134]

It will be appreciated that the methods and compositions of the present invention may be embodied in various illustrative forms, only some of which are disclosed herein. It will be apparent to those skilled in the art that other embodiments exist and do not depart from the spirit of the invention. Therefore, the specific examples described are merely examples and should not be construed as limiting.

List of References Barnay, G. et al. (2000). J. Med. Chem. Bitan, G. et al. (1997). J. Peptide Res. 49: 421-426. Blount, K. et al. (1992). Toxicon 30: 835-842. Bodansky et al. (1966). Chem. Ind. 38: 1597-98. Cartier, GE et al. (1996). J. Biol. Chem. 271: 7522-7528. Cruz, LJ at al. (1976) .Verliger 18: 302-308. Cruz, LJ et al. (1987). J. Biol. Chem. 260: 9280-9288. Fainzilber, M. et al. (1994) .Biochemistry 33: 9523-9529. Gray, WR. (1981). J. Biol. Chem. 256: 4734-4740. Haack, JA et al. (1990). J. Biol. Chem. 265: 6025-6029. Horiki, K. et al. (1978). Chemistry Letters 165-. 68. Hubry, V. et al. (1994). Reactive Polymers 22: 231-241. Jacobsen, R. et al. (1997). J. Biol. Chem. 272: 22531-22537. Johnson, DS et al. (1995). Mol. Pharmacol. 48: 194-199. Kapoor (1970). J. Pharm. Sci. 59: 1-27. Kornreich, WD et al. (1986). US Patent No. 4,569,967. Luo, S. et al. (1998). Neurosci. 18: 8571-8679. Marshall, IG and Harvey, AL (1990). Toxicon 28: 231-234. Martinez, JS et al. (1995). Biochem. 34: 14519-14526. McIntosh, JM et al. (1982). Arch. Bioch em. Biophys. 218: 329-334. Mena, EE et al. (1990). Neurosci. Lett. 118: 241-244.Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden, E
Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974). Myers, RA et al. (1991). Biochemistry 30: 9370-9377. Nishiuchi, Y. et al. (1993). Int. J. Pept. Protein. Res. 42: 533-538. Nowak, L. et al. (1984) .Nature 307: 462-465. Olivera, BM et al. (1984). U.S. Patent No. 4,447,356. Olivera, BM et al. (1985). Olivera, BM et al. (1996). U.S. Patent No. 5,514,774. Rivier, JR et al. (1978). Biopolymers 17: 1927-38. Rivier, JR et al. (1987). Biochem. 26: 8508-8512. Sambrook, J. et al. (1989) .Molecular Cloning: A Laboratory Manual, 2nd edition., Co.
ld Spring Harbor Laboratory, Cold Spring Harbor, NY. Schroder and Lubke (1965). The Peptides 1: 72-75, Academic Press, NY. Shon, K.-J. et al. (1994) .Biochemistry 33: 11420-11425. Stewart and Young, Solid-Phase Peptide Synthesis, Freeman & Co., San Fr
Ancisco, CA (1969). Vale et al. (1978). U.S. Patent No. 4,105,603. Van de Steen, P. et al. (1998). Critical Rev. in Biochem. and Mol. Biol. 33: 1.
51-208. Zafaralla, GC et al. (1988). Biochemistry 27: 7102-7105. Zhou, LM et al. (1996). J. Neurochem. 66: 620-628. US Patent 3,972,859 US Patent 3,842,067 US Patent 3,862,925 U.S. Pat.No. 5,550,050 International Publication Number WO 92/19195 International Publication Number WO 94/25503 International Publication Number WO 95/01203 International Publication Number WO 95/05452 International Publication Number WO 96/02286 International Publication Number WO 96/02646 International Publication number WO 96/11698 International publication number WO 96/40871 International publication number WO 96/40959 International publication number WO 97/12635

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, (72) Invention NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW Mullen Watkins 84105 East Garfield Ave, Salt Lake City, Utah 84105 Inventor Bardello Eme Olivera Inventor 84370 Brian Avenue, Salt Lake City, Utah 84108 Utah Inventor David Earl Hilliard United States 84124 Salt Lake City, Utah, Juneau Circle No. 3685 (72) Inventor Jay Michael McIntosh United States 84108 Salt Lake City, Utah South 2000 East 1151 (72) Inventor Robert M. Jones United States 84101 Salt Lake City, Utah West Broadway Number 2103 South 44 F term (reference) 4B024 AA01 BA38 CA04 HA01 4H045 AA10 BA17 CA51 DA83 FA58 GA25

Claims (39)

[Claims] 1. A compound of the general formula I:[Wherein Xaa₁Is Death-Xaa₁, Ile, Leu or Val; Xaa₂Is
Death-Xaa₂, Ala or Gly; Xaa₃Is Death-Xaa₃, Gly, T
rp (D or L), neo-Trp, halo-Trp or any non-natural
Aromatic amino acid; Xaa₄Is Death-Xaa₄, Asp, Phe, Gly, Ala,
Glu, γ-carboxy-Glu (Gla) or any non-natural aromatic amino acid
Noic acid; Xaa₅Are Glu, Gla, Asp, Ala, Thr, Ser, Gly,
Ile, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-
Sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any non-natural
Hydroxy-containing amino acids; Xaa₆Are Ser, Thr, Arg, ornithine, e
Moarginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N
, N-trimethyl-Lys or any unnatural basic amino acid; Xaa₇Is
Asp, Glu, Gla, Arg, ornithine, homoarginine, Lys, N-
Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
Or any unnatural basic amino acid; Xaa₈Is Ser, Thr, Asn, A
la, Gly, His, halo-His, Pro or hydroxy-Pro; Xa
a₉Is Thr, Ser, Ala, Asp, Asn, Pro, Hydroxy-Pro
, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N
-Dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural salt
Basic amino acid; Xaa₁₀Is Gly, Ser, Thr, Ala, Asn, Arg
, Ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl
Le-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid
Noic acid; Xaa₁₁Represents Gln, Leu, His, halo-His, Trp (D or
L), halo-Trp, neo-Trp, Tyr, nor-Trp, mono-halo-T
yr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-
Tyr, Arg, ornithine, homoarginine, Lys, N-methyl-Lys,
N, N-dimethyl-Lys, N, N, N-trimethyl-Lys, any non-natural
A basic amino acid or any unnatural aromatic amino acid; Xaa₁₂Is Asn,
His, halo-His, Ile, Leu, Val, Gln, Arg, ornithine
, Homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys,
X, N, N, N-trimethyl-Lys or any unnatural basic amino acid; ₁₃ Is Death-Xaa₁₃, Val, Ile, Leu, Arg, ornithine, homo
Arginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N,
N-trimethyl-Lys or any unnatural basic amino acid;
Containing free carboxyl or amide groups at the end]
Α-conotoxin peptide. 2. The following: Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂
Is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, Nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; A carboxyl or amide group] or a substantially pure α-conotoxin peptide of the general formula I or a derivative thereof. 3. The substantially pure α- according to claim 2, wherein Xaa ₁ is Glu.
Conotoxin peptides. 4. The substantially pure α- according to claim 2, wherein Xaa ₂ is Lys.
Conotoxin peptides. 5. The substantially pure α- according to claim 2, wherein Xaa ₄ is Tyr.
Conotoxin peptides. 6. The substantially pure α-conotoxin peptide of claim 2, wherein Xaa ₄ is mono-iodo-Tyr. 7. The substantially pure α-conotoxin peptide of claim 2, wherein Xaa _{4 is} di-iodo-Tyr. 8. The substantially pure α-conotoxin peptide of claim 1, wherein the peptide is modified to include O-glycans, S-glycans or N-glycans. 9. The substantially pure α-conotoxin peptide of claim 2, wherein the peptide is modified to include O-glycans, S-glycans, or N-glycans. 10. A compound of the general formula II: Wherein Xaa ₁ is des-Xaa ₁ , Asp, Glu or γ-carboxy-G
lu (Gla); Xaa ₂ is Death-Xaa ₂ , Gln, Ala, Asp, Glu
Xaa ₃ is Death-Xaa ₃ , Gly, Ala, Asp, Glu, Gl
a, Pro or hydroxy-Pro; Xaa ₄ is Des-Xaa ₄ , Gly, G
lu, Gla, Gln, Asp, Asn, Pro or hydroxy-Pro; X
aa ₅ is Ser, Thr, Gly, Glu, Gla, Asn, Trp (D or L), neo-Trp, halo-Trp, Arg, ornithine, homoarginine,
Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys, any unnatural basic amino acid, Tyr, nor-Tyr, mono-
Halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr,
Nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₆ is A
sp, Asn, His, halo-His, Thr, Ser, Tyr, nor-Tyr
, Mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-
Tyr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xa
a ₇ is Pro or hydroxy -Pro; Xaa ₈ is Ala, Ser, Thr,
Asp, Val, Ile, Pro, hydroxy-Pro, Tyr, nor-Tyr
, Mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-
Tyr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xa
a ₉ is Gly, Ile, Leu, Val, Ala, Thr, Ser, Pro, hydroxy -Pro, Phe, Trp (D or L), neo -Trp, halo -T
rp, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N
, N-dimethyl-Lys, N, N, N-trimethyl-Lys, any unnatural basic amino acid or any unnatural aromatic amino acid; Xaa ₁₀ is Ala, A
sn, Phe, Pro, hydroxy-Pro, Glu, Gla, Gln, His
, Halo-His, Val, Ser, Thr, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural base Xaa ₁₁ is Thr,
Ser, His, Halo-His, Leu, Ile, Val, Asn, Met, P
ro, hydroxy-Pro, Arg, ornithine, homoarginine, Lys, N
-Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
, Any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-Ty
r, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-T
yr or any unnatural hydroxy-containing amino acid; Xaa ₁₂ is Asn, P
ro, hydroxy-Pro, Gln, Ser, Thr, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N
, N-trimethyl-Lys, any unnatural basic amino acid, Tyr, nor-
Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₁₃ is des-Xaa ₁₃ , Gly, Thr , Ser, Pro, hydroxy-Pro, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr,
O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₁₄ is des-Xaa ₁₄ , Ile, Val
, Asp, Leu, Phe, Arg, ornithine, homoarginine, Lys, N
-Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
, Any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-Ty
r, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-T
yr or any unnatural hydroxy-containing amino acid; Xaa ₁₅ is Des-Xa
a ₁₅ , Gly, Ala, Met, Ser, Thr, Trp (D or L),
Neo-Trp, halo-Trp, any unnatural aromatic amino acid, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl-L
ys, N, N, N-trimethyl-Lys or any unnatural basic amino acid;
Xaa ₁₆ is des-Xaa ₁₆ , Trp (D or L), neo-Trp, halo-Trp, any unnatural aromatic amino acid, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N -Dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid; Xaa ₁₇ is des-
Xaa ₁₇ , Arg, ornithine, homoarginine, Lys, N-methyl-Ly
s, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid; the C-terminus contains a free carboxyl or amide group] A substantially pure α-conotoxin peptide. 11. The following: Embedded image Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂
Is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, Nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; A carboxyl or amide group is included], and a substantially pure α-conotoxin peptide of the general formula II or a derivative thereof. 12. Substantially pure α- conotoxin peptide according to claim 11, wherein wherein Xaa ₂ is Lys. 13. The substantially pure α-conotoxin peptide of claim 11, wherein Xaa ₁ is Glu. 14. The substantially pure α-conotoxin peptide of claim 11, wherein Xaa ₃ is Trp. 15. The substantially pure α-conotoxin peptide of claim 11, wherein Xaa ₄ is Tyr. 16. The substantially pure α-conotoxin peptide of claim 11, wherein Xaa ₄ is mono-iodo-Tyr. 17. The substantially pure α-conotoxin peptide of claim 11, wherein Xaa _{4 is} di-iodo-Tyr. 18. The substantially pure α-conotoxin peptide of claim 10, wherein the peptide is modified to include O-glycans, S-glycans or N-glycans. 19. The substantially pure α-conotoxin peptide of claim 11, wherein the peptide is modified to include O-glycans, S-glycans, or N-glycans. 20. The general formula III:[Wherein Xaa₁Is Death-Xaa₁, Ser or Thr; Xaa₂Is Death-X
aa₂, Asp, Glu, γ-carboxy-Glu (Gla), Asn, Ser
Or Thr; Xaa₃Is Death-Xaa₃, Ala, Gly, Asn, Ser,
Thr, Pro, hydroxy-Pro, Arg, ornithine, homoarginine,
Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl
Le-Lys or any unnatural basic amino acid; Xaa₄Is Death-Xaa₄ , Ala, Val, Leu, Ile, Gly, Glu, Gla, Gln, Asp
, Asn, Phe, Pro, hydroxy-Pro or any non-natural aromatic
Amino acid; Xaa₅Is Death-Xaa₅, Thr, Ser, Asp, Glu, Gl
a, Gln, Gly, Val, Asp, Asn, Ala, Pro, hydroxy-
Pro, Arg, ornithine, homoarginine, Lys, N-methyl-Lys,
N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any non-
Natural basic amino acid; Xaa₆Is Thr, Ser, Asp, Asn, Met, V
al, Ala, Gly, Leu, Ile, Phe, any of non-natural aromatic amino acids
Acid, Pro, hydroxy-Pro, Tyr, nor-Tyr, mono-halo-Ty
r, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-T
yr or any unnatural hydroxy-containing amino acid; Xaa₇Is Ile, Le
u, Val, Ser, Thr, Gln, Asn, Asp, Arg, His, halo
-His, Phe, any unnatural aromatic amino acid, homoarginine, orni
Tin, Lys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-to
Limethyl-Lys, any unnatural basic amino acid, Tyr, nor-Tyr,
Mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-T
yr, nitro-Tyr or any unnatural hydroxy-containing amino acid; Xaa ₈ Are Pro, Hydroxy-Pro, Ser, Thr, Ile, Asp, Leu,
Val, Gly, Ala, Phe, any unnatural aromatic amino acid, Arg,
Ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl
-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino
Acid; Xaa₉Are Val, Ala, Gly, Ile, Leu, Asp, Ser, T
hr, Pro, hydroxy-Pro, Arg, ornithine, homoarginine, L
ys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl
-Lys or any unnatural basic amino acid; Xaa₁₀Is His, halo-
His, Arg, homoarginine, ornithine, Lys, N-methyl-Lys,
N, N-dimethyl-Lys, N, N, N-trimethyl-Lys, any non-natural
Basic amino acids, Asn, Ala, Ser, Thr, Phe, Ile, Leu,
Gly, Trp (D or L), Neo-Trp, Halo-Trp,
Natural aromatic amino acids, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-
Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or any
Xaa₁₁Are Leu, Gln, Val, Ile
, Gly, Met, Ala, Lys, N-methyl-Lys, N, N-dimethyl-
Lys, N, N, N-trimethyl-Lys, Ser, Thr, Arg, homoargi
Nin, ornithine, any unnatural basic amino acid, Asn, Glu, Gla
, Gln, Phe, Trp (D or L), Neo-Trp, Halo-Trp or
Is any unnatural aromatic amino acid; Xaa₁₂Is Glu, Gla, Gln, A
sn, Asp, Pro, hydroxy-Pro, Ser, Gly, Thr, Lys
, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-L
ys, Arg, homoarginine, ornithine, any unnatural basic amino acid
, Phe, His, halo-His, any unnatural aromatic amino acid, Leu,
Met, Gly, Ala, Tyr, nor-Tyr, mono-halo-Tyr, di-ha
B-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or
Any of the unnatural hydroxy-containing amino acids; Xaa₁₃Is His, halo-His
, Asn, Thr, Ser, Ile, Val, Leu, Phe
Natural aromatic amino acids, Arg, homoarginine, ornithine, Lys, N-methyl
-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys,
Unnatural basic amino acids, Tyr, nor-Tyr, mono-halo-Tyr, di-
Halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro-Tyr or
Is any unnatural hydroxy-containing amino acid; Xaa₁₄Is Ser, Thr, A
la, Gln, Pro, hydroxy-Pro, Gly, Ile, Leu, Arg
, Ornithine, homoarginine, Lys, N-methyl-Lys, N, N-dimethyl
Le-Lys, N, N, N-trimethyl-Lys or any unnatural basic amino acid
Noic acid; Xaa_FifteenRepresents Asn, Glu, Gla, Asp, Gly, His, halo-
His, Ala, Leu, Gln, Arg, ornithine, homoarginine, Ly
s, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-
Lys, any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo
-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro
B-Tyr or any unnatural hydroxy-containing amino acid; Xaa₁₆Is Me
t, Ile, Thr, Ser, Val, Leu, Pro, hydroxy-Pro,
Phe, any unnatural aromatic amino acid, Tyr, nor-Tyr, mono-halo
-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr, nitro
B-Tyr, any unnatural hydroxy-containing amino acid, Glu, Gla, Al
a, His, halo-His, Arg, ornithine, homoarginine, Lys, N
-Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
Or any unnatural basic amino acid; Xaa₁₇Is Death-Xaa₁₇, Gl
y, Asp, Asn, Ala, Ile, Leu, Ser, Thr, His, halo
-His, Arg, ornithine, homoarginine, Lys, N-methyl-Lys
, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any of
Unnatural basic amino acid; Xaa₁₈Is Death-Xaa₁₈, Gly, Glu, Gl
a, Gln, Trp (D or L), Neo, Halo-Trp, any non-natural
Aromatic amino acid, Arg, ornithine, homoarginine, Lys, N-methyl-
Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys or any
Xaa₁₉Is Death-Xaa₁₉, Ser, Thr
, Val, Ile, Ala, Arg, ornithine, homoarginine, Lys, N
-Methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl-Lys
Or any unnatural basic amino acid; Xaa₂₀Is Death-Xaa₂₀, Va
1, Asp, His, halo-His, Arg, ornithine, homoarginine, L
ys, N-methyl-Lys, N, N-dimethyl-Lys, N, N, N-trimethyl
-Lys or any unnatural basic amino acid; Xaa₂₁Is Death-Xaa_{2 1} Xaa, Asn, Pro or hydroxy-Pro;₂₂Is Death-Xaa₂₂ , Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N, N
-Dimethyl-Lys, N, N, N-trimethyl-Lys or any unnatural salt
Basic amino acid; Xaa₂₃Is Death-Xaa₂₃, Ser or Thr; Xaa_{2 4} Is Death-Xaa₂₄, Leu, Ile or Val; free at the C-terminus
A carboxyl group or an amide group, provided that (a) Xaa₁Is Death-Xa
a₁, Xaa₂Is Death-Xaa₂, Xaa₃Is Death-Xaa₃, Xaa₄Is death
-Xaa₄, Xaa₆Is Ser, Xaa₇Is His, Xaa₈Is Pro, Xaa ₉ Is Ala, Xaa₁₀Is Ser, Xaa₁₁Is Val, Xaa₁₂Is Asn,
Xaa₁₃Is Asn, Xaa₁₄Is Pro, Xaa_FifteenIs Asp, Xaa₁₆But
Ile, Xaa₁₇Is Death-Xaa₁₇, Xaa₁₈Is Death-Xaa₁₈, Xa
a₁₉Is Death-Xaa₁₉, Xaa₂₀Is Death-Xaa₂₀, Xaa₂₁Is death
-Xaa₂₁, Xaa₂₂Is Death-Xaa₂₂, Xaa₂₃Is Death-Xaa₂₃ And Xaa₂₄Is Death-Xaa₂₄Xaa₅Does not become Gly
Substantially pure α-conotoxin peptide having the formula: 21. The following: Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂
Is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₃ is Trp (D or L), halo-Trp or neo-Trp; Xaa ₄ is Tyr, Nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulfo-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa ₅ is Pro or hydroxy-Pro; Xaa ₆ is Gln or pyro- Glu; a carboxyl or amide group is included at the C-terminus.]. A substantially pure α-conotoxin peptide or a derivative thereof represented by the general formula III selected from the group consisting of: 22. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa ₂ is Lys. 23. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa ₁ is Glu. 24. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa ₃ is Trp. 25. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa ₄ is Tyr. 26. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa ₄ is mono-iodo-Tyr. 27. The substantially pure α-conotoxin peptide of claim 21, wherein Xaa _{4 is} di-iodo-Tyr. 28. The substantially pure α-conotoxin peptide of claim 20, which has been modified to include O-glycans, S-glycans or N-glycans. 29. The substantially pure α-conotoxin peptide of claim 21, which has been modified to include O-glycans, S-glycans or N-glycans. 30. The following: Wherein Xaa ₁ is Glu or γ-carboxy-Glu (Gla); Xaa ₂
Is Lys, N-methyl-Lys, N, N-dimethyl-Lys or N, N, N-trimethyl-Lys; Xaa ₄ is Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O- Sulfo-Tyr, O-phospho-Tyr or nitro-Ty
and Xaa ₅ is Pro or hydroxy-Pro; the C-terminus contains a carboxyl or amide group], or a substantially pure α-conotoxin peptide or derivative thereof. 31. Substantially pure α- conotoxin peptide according to claim 30, wherein wherein Xaa ₂ is Lys. 32. The substantially pure α-conotoxin peptide of claim 30, wherein Xaa ₁ is Glu. 33. The substantially pure α-conotoxin peptide of claim 30, wherein Xaa ₄ is Tyr. 34. The substantially pure α-conotoxin peptide of claim 30, wherein Xaa ₄ is mono-iodo-Tyr. 35. The substantially pure α-conotoxin peptide of claim 30, wherein Xaa _{4 is} di-iodo-Tyr. 36. The substantially pure α-conotoxin peptide of claim 30, which has been modified to include O-glycans, S-glycans or N-glycans. 37. An isolated nucleic acid comprising a nucleic acid encoding an α-conotoxin precursor comprising an amino acid sequence selected from the group of amino acid sequences listed in Table 1-134. 38. The nucleic acid of claim 37, comprising a nucleotide sequence selected from the group of nucleotide sequences listed in Table 1-134 or their complements. 39. A substantially pure α-conotoxin protein precursor comprising an amino acid sequence selected from the group of amino acid sequences listed in Table 1-134.