Contulakin-G, analogs thereof and uses therefor

Details Contulakin-G, analogs thereof and uses therefor Contulakin-G, analogs thereof and uses therefor

2000-06-30

2003-02-25

Romeo, David S. (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C514S015800, C530S326000

Reexamination Certificate

active

06525021

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is directed to contulakin-G (which is the native glycosylated peptide), a des-glycosylated contulakin-G (termed Thr
10
-contulakin-G), and derivatives thereof, to a cDNA clone encoding a precursor of this mature peptide and to a precursor peptide. The invention is further directed to the use of this peptide as a therapeutic for anti-seizure, anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson's disease, gastrointestinal disorders, depressive states, cognitive dysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack, mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis, vascular and vasodilation disorders, as well as neurological, neuropharmalogical and neuropsychopharmacological disorders.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are numerically referenced in the following text and respectively grouped in the appended bibliography.
Mollusks of the genus Conus produce a venom that enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom that is injected by means of a highly specialized venom apparatus, a disposable hollow tooth that functions both in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. Many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
Several peptides isolated from Conus venoms have been characterized. These include the &agr;-, &mgr;- and &ohgr;-conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al. 1987). In addition, peptides named conantokins have been isolated from
Conus geographus
and
Conus tulipa
(Mena et al., 1990; Haack et al., 1990). These peptides have unusual age-dependent physiological effects: they induce a sleep-like state in mice younger than two weeks and hyperactive behavior in mice older than 3 weeks (Haack et al., 1990). The isolation, structure and activity of &kgr;-conotoxins are described in U.S. Pat. No. 5,633,347. Recently, peptides named contryphans containing D-tryptophan residues have been isolated from
Conus radiatus
(U.S. Ser. No. 09/061,026), and bromo-tryptophan conopeptides have been isolated from
Conus imperialis
and
Conus radiatus
(U.S. Ser. No. 08/785,534).
It is desired to identify additional conopeptides having activities of the above conopeptides, as well as conotoxin peptides having additional activities.
SUMMARY OF THE INVENTION
The present invention is directed to contulakin-G (which is the native glycosylated peptide), a des-glycosylated contulakin-G (termed Thr
10
-contulakin-G), and derivatives thereof, to a cDNA clone encoding a precursor of this mature peptide and to a precursor peptide. The invention is further directed to the use of this peptide as a therapeutic for anti-seizure, anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson's disease, gastrointestinal disorders, depressive states, cognitive dysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack, mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis, vascular and vasodilation disorders, as well as neurological, neuropharmacological and neuropsychopharmacological disorders.
In one embodiment, the present invention is directed to contulakin-G, contulakin-G propeptide and nucleic acids encoding this peptide. The contulakin-G has the following formula:
Xaa
1
-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa
2
-Tyr-Ile-Leu (SEQ ID NO:1)
where Xaa
1
is pyro-Glu, Xaa
2
is proline or hydroxyproline and Thr
10
is modified to contain an O-glycan. X
2
is preferably proline. In accordance with the present invention, a glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art. The monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. These saccharides may be structurally modified as described herein, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof. The gylcan may also include similar polyhydroxy groups, such as D-penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives. The glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-. Preferred glycans are described further herein, with the most preferred glycan being Gal(p1→3)GalNAc(&agr;1→).
In a second embodiment, the present invention is directed to a generic contulakin-G having the following general formula,
Xaa
1
-Xaa
2
-Xaa
3
-Xaa
3
-Gly-Gly-Xaa
2
-Xaa
4
-Xaa
5
-Xaa
6
-Xaa
7
-Xaa
8
-Xaa
9
-Xaa
10
-Ile-Leu (SEQ ID NO:2),
wherein Xaa
1
is pyro-Glu, Glu, Gln or &ggr;-carboxy-Glu; Xaa
2
is Ser, Thr or S-glycan modified Cys; Xaa
3
is Glu or &ggr;-carboxy-Glu; Xaa
4
is Asn, N-glycan modified Asn or S-glycan modified Cys; Xaa
5
is Ala or Gly; Xaa
6
is Thr, Ser, S-glycan modified Cys, Tyr or any synthetic hydroxy containing amino acid (such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr, 3-nitro-Tyr and 5-amino-Tyr); Xaa
7
is Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, omithine, homoarginine or any synthetic basic amino acid (such as N-1-(2-pyrazolinyl)-Arg); Xaa
8
is Ala, Gly, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, any synthetic basic amino acid (such as N-1-(2-pyrazolinyl)-Arg) or X-Lys where X is (CH
2
)
n
, phenyl, —(CH
2
)
m
—(CH═CH)—(CH
2
)
m
H or —(CH
2
)
m
—(C≡C)—(CH
2
)
m
H in which n is 1-4 and m is 0-2; Xaa
9
is Pro or hydroxy-Pro; and Xaa
10
is Tyr, mono-iodo-Tyr, di-iodo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Trp, D-Trp, bromo-Trp, bromo-D-Trp, chloro-Trp, chloro-D-Trp, Phe, L-neo-Trp, any snthetic aromatic amino acid (such as nitro-Phe, 4-substituted-Phe wherein the substituent is C
1
-C
3
alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, —CHO, —CN, —SO
3
H and —NHAc, 2,6-dimethyl-Tyr and 5-amino-Tyr). The C-terminus contains a free carboxyl group, is amidated, is acylated, contains a glycan or contains an aldehyde. It is preferred that the C-terminus contains a free carboxyl. This peptide may further contain one or more glycans as described above. The glycans may occur at residues 2, 7, 8, 10 and 16. The Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
Examples of synthetic aromatic amino acid include, but are not limited to, such as nitro-Phe, 4-substituted-Phe wherein the substituent is C
1
-C
3
alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, —CHO, —CN, —SO
3
H and —NHAc. Examples of synthetic hydroxy containing amino acid, include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples of

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