Identification and characterization of racemases, definition of protein signatures, and a test for detecting d-amino acid and for screening molecules capable of inhibiting the activity of racemase, especially proline racemase

Abstract

This invention relates to the identification and characterization of racemases and definition of protein signatures of these racemases. More particularly, this invention relates to the identification of nucleic acid molecules encoding a peptide consisting of a motif characteristic of the protein signatures, and to the peptides consisting of these motifs and more specifically SEQ ID NOS:1-4. This invention also relates to antibodies specific for the peptides and to immune complexes of these antibodies with the peptides. Further, the invention relates to methods and kits for detecting racemases using the nucleic acid molecules of the invention, as well as the peptides consisting of the motifs and antibodies to these peptides.

1 - 54 . (canceled)  
     
     
         55 . A method for detecting a D-amino acid, wherein the method comprises: 
 (A) providing a reaction medium containing the D-amino acid;    (B) reacting a primary or secondary amine on the D-amino acid with a D-amino oxidase and a prosthetic group to form a reduced prosthetic group by oxidative deamination of the D-amino acid;    (C) reacting the reduced prosthetic group with oxygen to form hydrogen peroxide; and    (D) detecting the hydrogen peroxide thus formed.    
     
     
         56 . The method of  claim 55 , wherein the prosthetic group is flavin-adenin-dinucleotide (FAD) or flavin-mononucleotide (FMN).  
     
     
         57 . The method of  claim 56 , wherein the hydrogen peroxide is detected by reaction with a catalase.  
     
     
         58 . The method of  claim 55 , wherein the D-amino acid is a D-Proline, D-Tyrosine, D-Valine, D-Threonine, D-Glutamic acid, D-Lysine, or D-Tryptophane.  
     
     
         59 . The method of  claim 56 , wherein the hydrogen peroxide is detected by reaction with a peroxidase.  
     
     
         60 . The method of  claim 55 , further comprising quantifying the D-amino acid in the reaction medium after the formation of the hydrogen peroxide.  
     
     
         61 . The method of  claim 55 , wherein the reaction medium comprises a biological sample from a subject afflicted with Alzheimer's disease, Parkinson's disease, renal disease, or schizophrenia.  
     
     
         62 . The method of  claim 61 , wherein the biological sample comprises a fluid or tissue sample from the subject.  
     
     
         63 . The method of  claim 61 , wherein the biological sample comprises cells from the subject.  
     
     
         64 . A method for detecting racemase activity in a reaction medium, wherein the method comprises: 
 (A) providing a reaction medium containing a D-amino acid specific to the racemase to be detected;    (B) reacting the D-amino acid with a D-amino oxidase and a prosthetic group to form a reduced prosthetic group by oxidation of the D-amino acid;    (C) reacting the reduced prosthetic group with oxygen to form hydrogen peroxide; and    (D) detecting the hydrogen peroxide thus formed;    wherein the detection of hydrogen peroxide indicates racemase activity in the reaction medium.    
     
     
         65 . The method of  claim 64 , wherein the hydrogen peroxide is detected by reaction with catalase.  
     
     
         66 . The method of  claim 64 , wherein the hydrogen peroxide is detected with a chromogenic reagent.  
     
     
         67 . The method of  claim 66 , wherein the chromogenic reagent is orthophenyalaninediamine (OPD), 3,3′,5,5′-tetrimethylbenzadine (TMB), or 5-aminosalicyclic acid (ASA).  
     
     
         68 . A kit for screening for inhibitors of TcPRAC, wherein the kit comprises: 
 (A) L-proline, D-proline, and a proline-racemase;    (B) a peroxidase and a substrate of a peroxidase, or a catalase and a reagent sensitive to oxygen; and    (C) a D-amino acid oxidase.    
     
     
         69 . The kit of  claim 68 , further comprising one or more molecules to be screened for inhibitory activity of TcPRAC.  
     
     
         70 . A kit for detecting a D-amino acid in a sample, wherein the kit comprises: 
 (A) a D-amino acid;    (B) a peroxidase and a substrate of a peroxidase; and    (C) a D-amino acid oxidase.    
     
     
         71 . The kit according to  claim 70 , further comprising a L-amino acid enantiomer as control.  
     
     
         72 . A method for detecting a D-amino acid in a sample, wherein the method comprises: 
 (A) oxidatively deaminating a D-amino acid by reaction with a D-amino acid oxidase and a prosthetic group; and    (B) detecting the hydrogen peroxide generated by the oxidative deamination;    wherein the presence of hydrogen peroxide is indicative of the presence of a D-amino acid in the sample.    
     
     
         73 . The method of  claim 72 , wherein the D-amino acid is D-Proline, D-Tyrosine, D-Valine, D-Threonine, D-Glutamic acid, D-Lysine, or D-Tryptophane.  
     
     
         74 . A method for screening a molecule, which can modulate a racemase activity, wherein the method comprises: 
 (A) modulating a racemase activity by means of a molecule being tested in the presence of an equimolar mixture of a L- and D-amino acid and of a racemase to be modulated;    (B) oxidatively deaminating the D-amino acid generated in step (A) by means of a D-amino oxidase and a prosthetic group; and    (C) detecting the hydrogen peroxide generated by the oxidative deamination;    wherein modulation of the hydrogen peroxide is indicative of the capability of the tested molecule to modulate racemase activity.    
     
     
         75 . The method of  claim 74 , wherein said molecule inhibits said racemase activity.  
     
     
         76 . The method of  claim 74 , wherein said racemase is a proline racemase.  
     
     
         77 . The method of  claim 74 , wherein said proline racemase is  Trypanosoma cruzi  proline racemase.  
     
     
         78 . A molecule identified by the method of  claim 74.