Antimicrobial theta defensins and methods of using same

Abstract

The present invention relates to an isolated cyclic peptide, theta defensin, having antimicrobial activity, and to theta defensin analogs. A theta defensin can have the amino acid sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa1-Xaa6-Xaa4-Xaa4-Xaa1-Xaa1-Xaa6-Xaa4-Xaa5-Xaa1-Xaa3-Xaa7-Xaa5, wherein Xaa1 to Xaa8 are defined; wherein Xaa1 can be linked through a peptide bond to Xaa8; and wherein crosslinks can be formed between Xaa3 and Xaa3, between Xaa5 and Xaa5, and between Xaa7 and Xaa7. For example, the invention provides a theta defensin having the amino acid sequence Gly-Phe-Cy-Arg-Cys-Leu-Cys-Arg-Arg-Gly-Val-Cys-Arg-Cys-Ile-Cys-Thr-Arg (SEQ ID NO:1), wherein the Gly at position 1 (Gly-1) is linked through a peptide bond to Arg-18, and wherein disulfide bonds are present between Cys-3 and Cys-16, between Cys-5 and Cys-14, and between Cys-7 and Cys-12. The invention also relates to antibodies that specifically bind a theta defensin and to isolated nucleic acid molecules encoding a theta defensin. In addition, the invention relates to methods of using theta defensin or a theta defensin analog to reduce or inhibit microbial growth or survival in an environment capable of sustaining microbial growth or survival by contacting the environment with the theta defensin.

1 - 57 . (canceled)  
     
     
         58 . A method of preparing theta defensin comprising, 
 (a) synthesizing a linear peptide of an amino acid sequence corresponding to the amino acid sequence of theta defensin,    (b) forming one or more crosslink bonds within said linear peptide, and    (c) cyclizing said peptide by linking the carboxyl and amino termini to form a cyclic peptide.    
     
     
         59 . The method of  claim 58 , wherein said crosslink is selected from the group consisting of disulfide, lanthionine, lactam and lactone.  
     
     
         60 . The method of  claim 58 , wherein the cysteine residues used in said linear peptide are in a pre-formed activated ester form.  
     
     
         61 . The method of  claim 59 , wherein the carboxyl terminus and amino terminus of said linear peptide are each approximately the same number of amino acids from the nearest cysteine.  
     
     
         62 . The method of  claim 61 , wherein said disulfide bonds are formed by oxidation.  
     
     
         63 . The method of  claim 62 , wherein said cyclizing is done with ethylenediaminecarbodiimide and N-hydroxybenzotriazole in a solvent.  
     
     
         64 . The method of  claim 63 , where approximately 60 equivalents of ethylenediaminecarbodiumide and approximately 20 equivalents of N-hydroxybenzotriazole are used.  
     
     
         65 . The method of  claim 64 , where the dimethylsulfoxide is the solvent.  
     
     
         66 . The method of  claim 58 , wherein said cyclized peptide is resistant to exo-peptidases.  
     
     
         67 . A method of enhancing protease resistance of a peptide, comprising synthesizing a peptide, wherein the amino-terminal amino acid and carboxyl-terminal amino acid of said peptide are positioned by intrachain crosslinks, whereby a peptide bond is formed between said amino-terminal and carboxyl-terminal amino acids.  
     
     
         68 - 70 . (canceled)  
     
     
         71 . An isolated peptide ligase, comprising an activity capable of forming a peptide bond between two polypeptides.  
     
     
         72 . The isolated peptide ligase of  claim 59 , wherein said polypeptides are theta defensin peptides.  
     
     
         73 . A method of reducing or inhibiting growth or survival of a microorganism in an individual, comprising administering a molecule, wherein said molecule increases expression of a theta defensin.