Catalysis of the cis/trans-isomerisation of secondary amide peptide compounds

Abstract

The present invention is based on the finding that the cis/trans isomerisation of secondary amide peptide bonds in oligo- and polypeptides can be catalytically promoted. This catalysis is effected by enzymes which are hereinafter called “secondary amide peptide bond cis/trans isomerases (APIases). It can be assumed that the APIase activity plays a central role in a number of pathophysiological processes. Thus, the invention relates to pharmaceutical compositions comprising substances which inhibit APIase activity.

1 . A method for identifying an inhibitor of a secondary amide peptide bond-specific cis/trans isomerase (APIase) comprising steps of:
 incubating a secondary amide peptide bond-specific cis/trans isomerase (APIase) with a peptide substrate of the APIase, and a possible inhibitor molecule of the APIase, wherein incubation is performed under conditions which allow the APIase to catalyse cis/trans isomerisation of the peptide substrate; and   determining if the possible inhibitor molecule of the APIase inhibits the cis/trans isomerisation of the peptide substrate and provides an inhibitor constant (K i ) of 100 micromolar or less.   
     
     
         2 . The method of  claim 1  where, in the step of incubating, the APIase is less than 0.01% of the concentration of the peptide substrate. 
     
     
         3 . The method of  claim 1  where the step of determining comprises spectrophotometrically determining isomerisation of the peptide substrate. 
     
     
         4 . The method of  claim 3  where the step of determining comprises spectrophotometrically determining isomerisation of the peptide substrate in the range of 222-228 nm. 
     
     
         5 . The method of  claim 1  where the step of determining comprises using nuclear magnetic resonance to determine isomerisation of the peptide substrate. 
     
     
         6 . The method of  claim 1  where the step of determining comprises using an indirect method which detects a downstream biochemical reaction of APIase activity and that are selected from group consisting of the detection of protein folding, hydrolysis, and isomer-specific chemical reactions. 
     
     
         7 . The method of  claim 1  where, in the step of incubating, the peptide substrate comprises Ala-Leu. 
     
     
         8 . The method of  claim 1  where, in the step of incubating, the APIase comprises DnaK. 
     
     
         9 . A method for identifying a peptide inhibitor of a secondary amide peptide bond-specific cis/trans isomerase (APIase) comprising steps of:
 preparing a composition comprising a library of peptides;   adding an APIase to the composition for a corresponding time and under such conditions which are sufficient to provide a peptide bound to the APIase;   determining an amino acid sequence of the peptide identified as bound to the APIase; and   determining the inhibitor constant of the peptide identified as bound to the APIase, wherein an inhibition constant of 100 micromolar or less shows an inhibitor of this APIase.   
     
     
         10 . The method of  claim 9  further comprising a step of synthesizing the peptide identified as bound to the APIase. 
     
     
         11 . The method of  claim 9  wherein the library of peptides is formed from all natural amino acids with the exception of cysteine. 
     
     
         12 . The method of  claim 9  wherein the library of peptides comprises peptides having 6 amino acids. 
     
     
         13 . The method of  claim 9  wherein the APIase comprises DnaK. 
     
     
         14 . The method of  claim 1  wherein the APIase is coupled to a matrix.