Antimicrobial peptide, process to obtain a peptide and uses therefor

Abstract

The invention refers to small peptides with low hemolytic activity, presenting equal antiparasitic, antifungal and antibacterial activities. More specifically, it refers to a peptide called gomesin, with 18 amino acid residues, configured as a clamp-like structure consisting of two anti-parallel beta-folded sheets joined by a beta turn, containing four invariable residues of cystein forming two disulphide bridges, optionally configurable as a cyclic chain with closed edges.

1 . Peptide of the general formula:  
       
         
           
           
               
               
           
         
       
       in which: 
 Z 1  is: 
 in the absence of X 19 , a free amino end residue or a residue with a blocked end amino group, providing resistance against protease activity;  
 when X 19  is present, a basic, hydrophobic, polar/large or small residue, with a free amino end group available to close the edges of the molecule.  
 
 C 2,  C 6,  C 11  and C 15  are independently cystein, homocystein or pennicilamine;  
 P 1  and P 2  are disulphide bridges from C 2  to C 15  and from C 6  to C 11 , respectively, either one or both bridges being present;  
 A 3 , A 4 , A 5 , A 12 , A 13 , A 14 , A 16 , A 17  are independently basic, hydrophobic, polar/large or small residues;  
 A 7  to A 10  are residues which are able to effect a beta turn and can be independently basic, hydrophobic, polar/large or small, with at least one of them being basic and the other hydrophobic;  
 A 18  is: 
 in the absence of X 19 : a basic, hydrophobic, polar/large or small residue, bearing a free carboxyl group end or forming an acceptable salt, or amidated with an amine of the formula NH 3  or RNH 2  or R 2 NH, in which R is independently a saturated or insaturated hydrocarbyl with one to six carbons;  
 when X 19  is present: a basic, hydrophobic, polar/large or small residue, which free carboxyl end is involved with the closing of peptide molecule edges;  
 
 X 19  may be absent or present; if present, it is a chemical link between Z 1  and A 18  or a chemical structure or molecule present between Z 1  and A 18  and linked to both, closing the amino acid chain edges of the peptide of the invention;  
 from about 15% to about 50% of all amino acid residues are basic;  
 the peptides present a positive charge of at least +1 at physiologic pH.  
 
     
     
         2 . Peptide of  claim 1  in which, in the absence of X 19 , Z 1  is pyroglutamic acid.  
     
     
         3 . Peptide of  claim 1  in which, when X 19  is present, Z 1  is a glutamine residue.  
     
     
         4 . Peptide of  claim 1 , in which A 3 , A 4 , A 8 , A 10  and A 16  are basic amino acid residues.  
     
     
         5 . Peptide of  claim 1 , in which A 5 , A 7 , A 12  and A 14  are hydrophobic amino acid residues.  
     
     
         6 . Peptide of  claim 1 , in which A 13  and A 17  are small amino acid residues.  
     
     
         7 . Peptide of  claim 1 , in which A 8  and A 10  are both basic amino acid residues.  
     
     
         8 . Peptide of  claim 1  in which, in the absence of X 19 , A 18  is a basic amino acid residue.  
     
     
         9 . Peptide of  claim 1  in which, when X 19  is present, A 18  is a basic amino acid residue which free carboxyl group end is involved in closing the edges of the peptide molecule.  
     
     
         10 . Peptide of  claim 4 , in which the basic amino acid residue is chosen from the group consisting of arginine, lysine and histidine.  
     
     
         11 . Peptide of  claim 5 , in which the hydrophobic amino acid residue is chosen from the group consisting of leucine, valine, isoleucine, methyonine, phenylalanine, tryptophan and tyrosine.  
     
     
         12 . Peptide of  claim 1 , in which the A 9  residue is polar, preferably chosen from the group consisting of glutamine and asparagine.  
     
     
         13 . Peptide of  claim 1 , in which the small amino acid residues are chosen from the group consisting of threonine, glycine, serine and alanine.  
     
     
         14 . Peptide of  claim 1 , in which: 
 A 3 , A 4 , A 10 , A 16  and A 18  are arginine;    A 5  is leucine;    A 7  and A 14  are tyrosine;    A 8  is lysine;    A 9  is glutamine;    A 12  is valine;    A 13  is threonine;    A 17  is glycine;    A 18  is amidated arginine;    Z 1  is pyroglutamic acid;    X 9  is not present.    
     
     
         15 . Peptide of  claim 1 , which is a cyclic chain with closed edges, in which: 
 A 3 , A 4 , A 10 , A 16  and A 18  are arginine;    A 5  is leucine;    A 7  and A 14  are tyrosine;    A 8  is lysine;    A 9  is glutamine;    A 12  is valine;    A 13  is threonine;    A 17  is glycine;    A 18  is arginine;    Z 1  is glutamic acid;    X 9  is a link or chemical structure linking Z 1  to A 18 .    
     
     
         16 . PROCESS TO OBTAIN A PEPTIDE by means of extraction from arachnids, consisting of the stages of: 
 collection of hemolymph;    separation of hemocytes from plasma;    obtaining an acid extract from hemocytes;    isolation of the peptide, preferably by HPLC;    
     
     
         17 . PROCESS TO OBTAIN PEPTIDE of  claim 16 , wherein said spider is an  Acanthoscurria gomesiana.    
     
     
         18 . PROCESS TO OBTAIN A PEPTIDE of the general formula  
       
         
           
           
               
               
           
         
       
       of  claim 1 , in which the Fmoc chemical synthesis techniques is used.  
     
     
         19 . FORMULATION CONSISTING OF A PEPTIDE for the combat against infection in human beings, animals, and plants, especially for the combat against bacteria, fungi and parasites, consisting, among other constituents, of the peptide of the general formula:  
       
         
           
           
               
               
           
         
       
       of  claim 1 .  
     
     
         20 . RECOMBINANT EXPRESSION SYSTEM for the production of a peptide of the general formula  
       
         
           
           
               
               
           
         
       
       of  claim 1 , in which said expression system consists of the nucleotide sequence coding the said peptide as linked to a controlling sequence regulating said expression.  
     
     
         21 . METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA, in which said parasites, fungi and bacteria are contacted with an effective amount of a peptide of  claim 1 .  
     
     
         22 . METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA of  claim 21 , in which the parasite is preferably a protozoary.  
     
     
         23 . METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA of  claim 21 , in which the parasite is preferably a Leishmania.  
     
     
         24 . METHOD TO INACTIVATE THE ENDOTOXIN FROM GRAM-NEGATIVE BACTERIA, in which said endotoxin is contacted by an amount of a peptide of  claim 1  which is substantially effective to inactivate said endotoxin.  
     
     
         25 . ANTIBODY which is substantially especifically reactive to the peptide of  claim 1.