Preparation and use of ginsentides and ginsentide-like peptides

Abstract

The present invention relates to the methods of solid-phase peptide synthesis or recombinant production of ginsentide or ginsentide-like peptides or salts thereof. Further provided are uses of the ginsentide or ginsentide-like peptides or salts thereof as al-adrenergic receptor antagonists and vasorelaxants, nitric oxide-boosting agents, anti-thrombotic agents, anti-atherosclerotic agents, as protective agents against doxorubicin-induced cardiotoxicity, anti-ageing and adaptogenic agents, nutraceuticals, health supplements, or cosmetic ingredients.

1 . A method of solid-phase peptide synthesis of a ginsentide or ginsentide-like peptide or a pharmaceutically acceptable salt thereof, said ginsentide or ginsentide-like peptide having (i) the amino acid sequence set forth in any one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing at least 65% sequence identity with the peptide of (i) over its entire sequence, wherein the method comprises the steps of:
 (a) synthesizing the ginsentide or ginsentide-like peptide on a solid support by stepwise coupling of Fmoc-protected;   (b) cleaving the ginsentide or ginsentide-like peptide from the solid support and deprotecting the peptide;   and subsequently,   (c) performing organic oxidative folding of the ginsentide or ginsentide-like peptide to form native disulfide connections.   
     
     
         2 . The method of  claim 1 , wherein step (a) comprises the steps of:
 (i) deprotecting a first amino acid linked to the solid support by removing protective chemical groups from the first amino acid;   (ii) activating chemical groups on a second amino acid to prepare the second amino acid for coupling with the first amino acid;   (iii) coupling the activated second amino acid to the deprotected first amino acid to form a peptide from the first and second amino acids; and   (iv) successively deprotecting, and coupling a plurality of amino acids into the growing peptide chain until the ginsentide or ginsentide-like peptide is synthesized.   
     
     
         3 . The method of  claim 2 , wherein the method comprises accelerating at least one of the deprotecting, activating, and coupling steps by applying microwave energy. 
     
     
         4 . The method of  claim 1 , wherein the organic oxidative folding is performed in non-aqueous conditions and performed in organic solvents. 
     
     
         5 . The method of  claim 1 , wherein the organic oxidative folding comprises folding the ginsentide or ginsentide-like peptide in an organic solvent comprising cysteamine and/or morpholine. 
     
     
         6 . The method of  claim 1 , wherein the organic oxidative folding is performed at 4° C. 
     
     
         7 . The method of  claim 1 , wherein the method comprises purifying the folded ginsentide or ginsentide-like peptide or salt thereof. 
     
     
         8 . A method of recombinantly preparing a ginsentide or ginsentide-like peptide or a pharmaceutically acceptable salt thereof, said ginsentide or ginsentide-like peptide having (i) the amino acid sequence set forth in any one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing at least 65% sequence identity with the peptide of (i) over its entire sequence, wherein the method comprises the steps of:
 (a) providing a host cell comprising a polynucleotide, wherein the polynucleotide encodes a polypeptide comprising Maltose-binding protein having the amino acid sequence set forth in SEQ ID NO:65, a enterokinase cleavage sequence having the amino acid sequence set forth in SEQ ID NO:66, and the ginsentide or ginsentide-like peptide or salt thereof;   (b) culturing the host cell in a growth medium under conditions allowing production of the polypeptide, and recovering the polypeptide from the medium; and   (c) generating the ginsentide or ginsentide-like peptide or salt thereof by cleaving the polypeptide using enterokinase.   
     
     
         9 . The method of  claim 8 , wherein the polypeptide has the amino acid sequence set forth in any one of SEQ ID NOs: 67-80. 
     
     
         10 . The method of  claim 8 , wherein the method further comprises purification of the recombinantly produced polypeptide prior to the enterokinase cleavage. 
     
     
         11 . The method of  claim 8 , wherein the method further comprises purification of the ginsentide or ginsentide-like peptide or salt thereof by anion-exchange chromatography and/or reverse-phase chromatography after the enterokinase cleavage. 
     
     
         12 . A pharmaceutical composition comprising a ginsentide or ginsentide-like peptide or a pharmaceutically acceptable salt, said ginsentide or ginsentide-like peptide having (i) the amino acid sequence set forth in any one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing at least 65% sequence identity with the peptide of (i) over its entire sequence. 
     
     
         13 . A method of treating and/or preventing thrombosis in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of  claim 12 .