Contulakin-G, analogs thereof and uses therefor

Abstract

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.

1 . An isolated conopeptide selected from the group consisting of: 
 (a) contulakin-G comprising the amino acid sequence 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, wherein said glycan is not Gal(β1→3)GalNAc(α1→);    (b) 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), where Xaa 1  is pyro-Glu, Glu, Gln or γ-carboxy-Glu; Xaa 2  is Ser, Thr or S-glycan modified Cys; Xaa 3  is Glu or γ-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, O-glycan modified Thr, O-glycan modified Ser, S-glycan modified Cys, Tyr or any hydroxy containing unnatural amino acid; Xaa 7  is Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine or any unnatural basic amino acid; Xaa 8  is Ala, Gly, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, any unnatural basic amino acid 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, or any unnatural aromatic amino acid,with the proviso that the generic contulakin-G is not a peptide of the formula Xaa 1 -Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr -Lys-Lys-Xaa 2 -Tyr-Ile-Leu (SEQ ID NO:1), wherein Xaa 1  is pyro-Glu, Xaa 2  is proline or hydroxyproline and Thr 10  is unmodified or modified to contain an O-glycan;    (c) a generic contulakin-G of (b) which is modified to contain an O-glycan, an S-glycan or an N-glycan;    (d) a contulakin-G analog which comprises an N-terminal truncation of from 1 to 9 amino acids of the generic contulakin-G of (b);    (e) a contulakin-G analog of (d), wherein an Ser-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted for the amino acid residue at the truncated N-terminus;    (f) a contulakin-G analog, wherein an Ser-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted for a residue at positions 1-9 of the generic contulakin-G of (b); and    (g) a contulakin-G analog which comprises an N-terminal truncation of 10 amino acids of the generic contulakin-G of (b) which is further modified to contain a Lys-N-glycan at residue 11 of the generic contulakin-G.    
     
     
         2 . The isolated conopeptide of  claim 1 , wherein the conopeptide is Contulakin-G of (a) and Xaa 2  is proline.  
     
     
         3 . The isolated conopeptide of  claim 1 , wherein the conopeptide is Contulakin-G of (a) and Xaa 2  is hydroxy-proline.  
     
     
         4 . The isolated conopeptide of  claim 1 , wherein the conopeptide is Contulakin-G of (a) and the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr; X is O; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acety or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         5 . The isolated conopeptide of  claim 4 , wherein the conopeptide is Contulakin-G of (a) and Xaa 2  is proline.  
     
     
         6 . The substantially isolated conopeptide of  claim 4 , wherein the conopeptide is Contulakin-G of (a) and Xaa 2  is hydroxy-proline.  
     
     
         7 . The isolated conopeptide of  claim 1 , wherein the conopeptide is selected from the group consisting of the generic Contulakin-G of (b)-(c) and the Contulakin-G analog of (d)-(g) and the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         8 . The isolated conopeptide of  claim 1 , wherein the conopeptide is selected from the group consisting of the generic Contulakin-G of (b)-(c) and the Contulakin-G analog of (d)-(g) and the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R, is Thr, Ser, Cys or Lys; X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn or Lys; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         9 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the generic Contulakin-G of (b).  
     
     
         10 . The isolated conopeptide of  claim 9 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         11 . The isolated conopeptide of  claim 9 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr, Ser or Cys; X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         12 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the generic Contulakin-G of (c).  
     
     
         13 . The isolated conopeptide of  claim 12 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         14 . The isolated conopeptide of  claim 12 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr, Ser or Cys; X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         15 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the Contulakin-G analog of (d).  
     
     
         16 . The isolated conopeptide of  claim 15 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         17 . The isolated conopeptide of  claim 15 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr, Ser or Cys; X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         18 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the Contulakin-G analog of (e).  
     
     
         19 . The isolated conopeptide of  claim 18 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         20 . The isolated conopeptide of  claim 18 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr, Ser or Cys; X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn; R 2  is is an amino acid capable of being derivatized with a glycan either chemically or enzymatically; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         21 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the Contulakin-G analog of (f).  
     
     
         22 . The isolated conopeptide of  claim 21 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         23 . The isolated conopeptide of  claim 21 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Thr, Ser or Cys, X is O when R 1  is Thr or Ser, or X is S when R 1  is Cys or X is N when R 1  is Asn; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         24 . The isolated conopeptide of  claim 1 , wherein the conopeptide is the Contulakin-G analog of (g).  
     
     
         25 . The isolated conopeptide of  claim 24 , wherein the glycan is Gal(β1→3)GalNAc(α1→).  
     
     
         26 . The isolated conopeptide of  claim 24 , wherein the glycan has the structure  
       
         
           
           
               
               
           
         
         wherein R 1  is Lys; X is N; R 2  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, or O-glycan; R 3  is H, SO 3 , PO 3 , acetyl, sialic acid or monosaccharide; R 4  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 5  is OH, NH 2 , NHSO 3 Na, NHAc, O-sulphate, O-phosphate, O-monosaccharide or, O-acetyl; R 6  is H, SO 3 , PO 3 , acetyl or monosaccharide; R 7  is H, SO 3 , PO 3 , acety or monosaccharide; R 8  is H, SO 3 , PO 3 , acetyl or monosaccharide; n is 0-4 and m is 1-4.  
       
     
     
         27 . The isolated conopeptide of  claim 1  which is chemically synthesized.  
     
     
         28 . The isolated conopeptide of  claim 4  which is chemically synthesized.  
     
     
         29 . The isolated conopeptide of  claim 7  which is chemically synthesized.  
     
     
         30 . The isolated conopeptide of  claim 8  which is chemically synthesized.  
     
     
         31 . The isolated conopeptide of  claim 9  which is chemically synthesized.  
     
     
         32 . The isolated conopeptide of  claim 10  which is chemically synthesized.  
     
     
         33 . The isolated conopeptide of  claim 11  which is chemically synthesized.  
     
     
         34 . The isolated conopeptide of  claim 12  which is chemically synthesized.  
     
     
         35 . The isolated conopeptide of  claim 13  which is chemically synthesized.  
     
     
         36 . The isolated conopeptide of  claim 14  which is chemically synthesized.  
     
     
         37 . The isolated conopeptide of  claim 15  which is chemically synthesized.  
     
     
         38 . The isolated conopeptide of  claim 16  which is chemically synthesized.  
     
     
         39 . The isolated conopeptide of  claim 17  which is chemically synthesized.  
     
     
         40 . The isolated conopeptide of  claim 18  which is chemically synthesized.  
     
     
         41 . The isolated conopeptide of  claim 19  which is chemically synthesized.  
     
     
         42 . The isolated conopeptide of  claim 20  which is chemically synthesized.  
     
     
         43 . The isolated conopeptide of  claim 21  which is chemically synthesized.  
     
     
         44 . The isolated conopeptide of  claim 22  which is chemically synthesized.  
     
     
         45 . The isolated conopeptide of  claim 23  which is chemically synthesized.  
     
     
         46 . The isolated conopeptide of  claim 24  which is chemically synthesized.  
     
     
         47 . The isolated conopeptide of  claim 25  which is chemically synthesized.  
     
     
         48 . The isolated conopeptide of  claim 26  which is chemically synthesized.