Previns as specific inhibitors and therapeutic agents for Botulinum toxin B and Tetanus neurotoxins

Abstract

The compounds of the invention are generally described by the formula: B 1 Z* 2 B 3 Z* 4 X* 5 Q 6 F 7 X 8 X 9 X 10 X 11   (1), B 1 X 2 X 3 X 4 X 5 Q 6 F 7 X 8 X 9 X 10 X 11   (2), or B 1 Z 2 B 3 X 4 Z 5 Q 6 F 7 Z 8 X 9 X 10 X 11   (3) and the salts, esters, amides, and acyl forms thereof. Each position represented by a letter indicates a single amino acid residue: B is a basic of polar/large amino acid or a modified form thereof; X is a small or hydrophobic amino acid or a modified form thereof; X* is a small or polar/large amino acid or a modified form thereof; Z is a polar/large or hydrophobic amino acid or a modified form thereof; Z* is Proline or a polar/large of hydrophobic amino acid or a modified form thereof. As described below, one or more of the peptide linkages between the amino acid residues may be replaced by a peptide linkage mimic. These compounds may be used as molecular building blocks to create compounds that are optimized for inhibiting the protease activity of Botulinum b and tetanus toxins.

1 - 7 . (canceled)  
     
     
         8 . A recombinant expression system for production of a peptide of  claim 29  capable of inhibiting the protease activity of Botulinum toxin B or tetanus toxin which expression system comprises a nucleotide sequence encoding said peptide operably linked to a control sequence for effecting expression.  
     
     
         9 . The recombinant expression system of  claim 8  wherein the nucleotide sequence encoding said peptide encodes a precursor peptide.  
     
     
         10 . A recombinant host cell modified to contain the expression system of  claim 8 .  
     
     
         11 . A method to produce a peptide capable of inhibiting the protease activity of Botulinum toxin B or tetanus toxin which method comprises culturing the modified host cells of  claim 10  under conditions wherein said peptide is produced.  
     
     
         12 - 15 . (canceled)  
     
     
         16 . A pharmaceutical composition for treating Botulinum or tetanus intoxication which comprises a protease inhibitory amount of a peptide selected from the group consisting of  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   RPKPWWFFGLM, 
                   (SEQ ID NO: 1) 
                     
                 
                     
                     
                 
                     
                   TRSSRAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                   TRAARAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                   TRLLRAGLQFPVGRVHRLLRK, 
                   (SEQ ID No: 4) 
                 
                     
                     
                 
                     
                   RAKAQQFFGLM, 
                   (SEQ ID NO: 6) 
                 
                     
                     
                 
                     
                   RAKAQQFPGLM, 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                   RAKLQQFPGLM, 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   RAKGLQFPGLM, 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                   RAGLGQFFGLM, 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLMAKLK, 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLLAKLK, 
                   (SEQ ID NO: 12) 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   TRSRAKGLQFPGLMVHRL, 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         17 . Antibodies specifically immunoreactive with the peptide of  claim 29 .  
     
     
         18 . An assay comprising contacting a sample suspected of containing a Previn with the antibodies of  claim 17  under conditions where the antibody would bind the Previn and detecting the bound Previn.  
     
     
         19 - 23 . (canceled)  
     
     
         24 . A method for treating Botulinum or tetanus intoxication comprising administering to a subject, suspected of having Botulinum or tetanus intoxication or being in the presence of a causative agent thereof a protease inhibitory amount of a peptide or salts, esters, amides, and acyl forms thereof, wherein said peptide is selected from the group consisting of  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   RPKPQQFFGLM, 
                   (SEQ ID NO:1) 
                     
                 
                     
                     
                 
                     
                   TRSSRAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:2) 
                 
                     
                     
                 
                     
                   TRAARAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:3) 
                 
                     
                     
                 
                     
                   TRLLRAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:4) 
                 
                     
                     
                 
                     
                   RAKAQQFFGLM, 
                   (SEQ ID NO:6) 
                 
                     
                     
                 
                     
                   RAKAQQFPGLM, 
                   (SEQ ID NO:7) 
                 
                     
                     
                 
                     
                   RAKLQQFPGLM, 
                   (SEQ ID NO:8) 
                 
                     
                     
                 
                     
                   RAKGLQFPGLM, 
                   (SEQ ID NO:9) 
                 
                     
                     
                 
                     
                   RAGLGQFFGLM, 
                   (SEQ ID NO:10) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLMAKLK, 
                   (SEQ ID NO:11) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLLAKLK, 
                   (SEQ ID NO:12) 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   TRSRAKGLQFPGLMVHRL, 
                   (SEQ ID NO:13) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         25 . A kit for treating or preventing Botulinum toxin B or Tetanus toxin intoxication comprising at least one peptide of  claim 29 .  
     
     
         26 . A kit for determining whether a sample contains a Previn comprising antibodies of  claim 17 .  
     
     
         27 . (canceled)  
     
     
         28 . (canceled)  
     
     
         29 . A peptide in purified and isolated form, or salts, esters, amides and acyl forms thereof, selected from the group consisting of  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   RPKPQQFFGLM, 
                   (SEQ ID NO:1) 
                     
                 
                     
                     
                 
                     
                   TRSSRAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:2) 
                 
                     
                     
                 
                     
                   TRAARAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:3) 
                 
                     
                     
                 
                     
                   TRLLRAGLQFPVGRVHRLLRK, 
                   (SEQ ID NO:4) 
                 
                     
                     
                 
                     
                   RAKAQQFFGLM, 
                   (SEQ ID NO:6) 
                 
                     
                     
                 
                     
                   RAKAQQFPGLM, 
                   (SEQ ID NO:7) 
                 
                     
                     
                 
                     
                   RAKLQQFPGLM, 
                   (SEQ ID NO:8) 
                 
                     
                     
                 
                     
                   RAKGLQFPGLM, 
                   (SEQ ID NO:9) 
                 
                     
                     
                 
                     
                   RAGLGQFFGLM, 
                   (SEQ ID NO:10) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLMAKLK, 
                   (SEQ ID NO:11) 
                 
                     
                     
                 
                     
                   DAARAKGLQFPGLLAKLK, 
                   (SEQ ID NO:12) 
                 
                     
                     
                 
                     
                   TRSRAKGLQFPGLMVHRL. 
                   (SEQ ID NO:13) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         30 . The peptide of  claim 29 , wherein the salt is a phosphate.  
     
     
         31 . The peptide of  claim 30 , wherein the phosphate salt is formed by phosphorylation of S, T, and/or Y.  
     
     
         32 . The peptide of  claim 31 , wherein the peptide is characterized as having an improved circulatory half-life, solubility, resistance to degradation, and interaction with the active site of the toxin.  
     
     
         33 . The pharmaceutical composition of  claim 16 , wherein the salt is a phosphate.  
     
     
         34 . The pharmaceutical composition  claim 33 , wherein the phosphate salt is formed by phosphorylation of S, T and/or Y.  
     
     
         35 . The pharmaceutical composition of  claim 34 , wherein the compound is characterized as having an improved circulatory half-life, solubility, resistance to degradation, and interaction with the active site of the toxin.  
     
     
         36 . The pharmaceutical composition of  claim 16  further comprising tris-(2-carboxyethyl)phosphine (TCEP).  
     
     
         37 . The pharmaceutical composition of  claim 36  further comprising biocompatable chaotropes.  
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein the biocompatible chaotropes is hydroxyurea or 2-oxo-1 pyridine acetamide.  
     
     
         39 . The method of  claim 24 , wherein the composition is administered to the subject prior to the subjects contact with Botulinum or tetanus intoxication.  
     
     
         40 . The method of  claim 39 , wherein the contact is through aerosol contamination.  
     
     
         41 . The method of  claim 40 , wherein the administration involves impregnation of a filter with the compound.  
     
     
         42 . The method of  claim 41 , wherein the filter is a breathing filter affixed to the subject after impregnation of the filter.  
     
     
         43 . The method of  claim 24 , wherein the peptide is administered directly to a wound on the subject.  
     
     
         44 . The method of  claim 24 , wherein the peptide is conjugated to Bttx-HC which directs the compounds to the desired site upon administration.