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 provides identification and characterization of racemases and definition of protein signatures of these racemases. This invention also provides 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. Antibodies specific for the peptides and to immune complexes of these antibodies with the peptides are also provided. 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 . A purified nucleic acid molecule encoding a peptide consisting of a motif selected from SEQ ID NOS: 1, 2, 3, or 4. 
     
     
         2 . A purified nucleic acid molecule that hybridizes to either strand of a denatured, double-stranded DNA comprising the nucleic acid molecule of  claim 1  under conditions of moderate stringency. 
     
     
         3 . A recombinant vector that directs the expression of a nucleic acid molecule selected from the group consisting of the purified nucleic acid molecules of  claims 1  or  2 . 
     
     
         4 . A purified polypeptide encoded by a nucleic acid molecule selected form the group consisting of the purified nucleic acid molecules of  claims 1  or  2 . 
     
     
         5 . A purified polypeptide consisting of Motif I (SEQ ID NO:1). 
     
     
         6 . A purified polypeptide consisting of Motif II (SEQ ID NO:2). 
     
     
         7 . A purified polypeptide consisting of Motif III (SEQ ID NO:3). 
     
     
         8 . A purified polypeptide consisting of Motif III* (SEQ ID NO:4). 
     
     
         9 . Purified antibodies that bind to a polypeptide of  claim 4 . 
     
     
         10 . Purified antibodies according to  claim 9 , wherein the antibodies are monoclonal antibodies. 
     
     
         11 . Purified antibodies that bind to a polypeptide of  claim 5 . 
     
     
         12 . Purified antibodies according to  claim 11 , wherein the antibodies are monoclonal antibodies. 
     
     
         13 . Purified antibodies that bind to a polypeptide of  claim 6 . 
     
     
         14 . Purified antibodies according to  claim 13 , wherein the antibodies are monoclonal antibodies. 
     
     
         15 . Purified antibodies that bind to a polypeptide of  claim 7 . 
     
     
         16 . Purified antibodies according to  claim 15 , wherein the antibodies are monoclonal antibodies. 
     
     
         17 . Purified antibodies that bind to a polypeptide of  claim 8 . 
     
     
         18 . Purified antibodies according to  claim 17 , wherein the antibodies are monoclonal antibodies. 
     
     
         19 . A host cell transfected or transduced with the recombinant vector of  claim 3 . 
     
     
         20 . A method for the production of a polypeptide consisting of SEQ ID NOS: 1, 2, 3, or 4, comprising culturing a host cell of  claim 17  under conditions promoting expression, and recovering the polypeptide from the host cell or the culture medium. 
     
     
         21 . The method of  claim 20 , wherein the host cell is selected from the group consisting of bacterial cells, parasite cells, and eukaryotic cells. 
     
     
         22 . An immunological complex comprising a polypeptide of  claim 4  and an antibody that specifically recognizes said polypeptide. 
     
     
         23 . A method of detecting a racemase encoded by a nucleotide sequence containing a subsequence encoding a peptide selected form SEQ ID NO: 1, 2, 3, or 4, said method comprising:
 (a) contacting the nucleotide sequence with a primer or a probe, which hybridizes with the nucleic acid molecule of  claim 1 ;   (b) amplifying the nucleotide sequence using said primer or said probe; and   (c) detecting a hybridized complex formed between said primer or probe and the nucleotide sequence.   
     
     
         24 . A method of detecting a racemase encoded by a nucleotide sequence containing a subsequence encoding a peptide selected from SEQ ID NO: 1, 2, 3, or 4, said method comprising:
 (a) contacting the product encoded by a nucleotide sequence racemase with antibodies according to any one of  claims 9  to  18 ; and   (b) detecting the resulting immunocomplex.   
     
     
         25 . A kit for detecting a racemase encoded by a nucleotide sequence containing a subsequence encoding a peptide selected form SEQ ID NO: 1, 2, 3, or 4, said kit comprising:
 (a) a polynucleotide primer or probe, which hybridizes with the polynucleotide sequence of  claim 1 ; and   (b) reagents to perform a nucleic acid hybridization reaction.   
     
     
         26 . A kit for detecting a racemase encoded by a nucleotide sequence containing a subsequence encoding a peptide selected form SEQ ID NO: 1, 2, 3, or 4, said kit comprising:
 (a) purified antibodies according to any one of  claims 9  or  10 ;   (b) standard reagents in a purified form; and   (c) detection reagents.   
     
     
         27 . An in vitro method of screening for an active molecule capable of inhibiting a racemase encoded by a nucleotide sequence containing a subsequence encoding a peptide selected form SEQ ID NO: 1, 2, 3, OR 4, said method comprising:
 (a) contacting the active molecule with said racemase;   (b) testing the capacity of the active molecule, at various concentrations, to inhibit the activity of the racemase; and   (c) choosing the active molecule that provides an inhibitory effect of at least 80% on the activity of the racemase.   
     
     
         28 . An immunizing composition containing at least a purified polypeptide according to  claim 4 , capable of inducing an immune response in vivo, and a pharmaceutical carrier. 
     
     
         29 . A method for detecting a D-amino acid, wherein the method comprises:
 (A) providing a reaction medium containing the D-amino acid;   (B) reacting the D-amino acid with a D-amino oxidase with a prosthetic group to form a reduced prosthetic group by oxidative deamination of the D-amino acid with a primary amine or oxidation of the D-amino acid with a secondary amine;   (C) reacting the reduced prosthetic group with oxygen to form hydrogen peroxide; and   (D) detecting the hydrogen peroxide thus formed.   
     
     
         30 . The method as claimed in  claim 29 , wherein the prosthetic group is flavin-adenin-dinucleotide (FAD) or flavin-mononucleotide (FMN). 
     
     
         31 . The method as claimed in  claim 30 , wherein the hydrogen peroxide is detected by reaction with a catalase. 
     
     
         32 . The method as claimed in  claim 29 , wherein the D-amino acid is a D-Proline, D-Tyrosine, D-Valine, D-Threonine, D-Glutamic acid, D-Lysine, or D-Tryptophane. 
     
     
         33 . The method as claimed in  claim 30 , wherein the hydrogen peroxide is detected by reaction with a peroxidase. 
     
     
         34 . The method as claimed in any one of  claims 29 - 33 , comprising quantifying the D-amino acid in the reaction medium after the formation of the hydrogen peroxide. 
     
     
         35 . The method as claimed in  claim 29 , wherein the reaction medium comprises a biological sample from a subject afflicted with Alzheimer's disease, Parkinson's disease, renal disease, or schizophrenia. 
     
     
         36 . The method as claimed in  claim 34 , wherein the biological sample comprises a fluid or tissue sample from the subject. 
     
     
         37 . The method as claimed in  claim 34 , wherein the biological sample comprises cells from the subject. 
     
     
         38 . 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 with 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 tin the reaction medium.   
     
     
         39 . The method as claimed in  claim 38  wherein the hydrogen peroxide is detected by reaction with catalase. 
     
     
         40 . The method as claimed in  claim 38 , wherein the hydrogen peroxide is detected with a chromogenic reagent. 
     
     
         41 . The method as claimed in  claim 39 , wherein the chromogenic reagent is orthophenylalaninediamine (OPD), 3,3′,5,5′-tetrimethylbenzadine (TMB), or 5-aminosalicyclic acid (ASA). 
     
     
         42 . 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;   (C) a D-amino acid oxidase; and   (D) optionally, one or more molecules to be screened for inhibitory activity of TcPRAC.   
     
     
         43 . 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;   (C) a D-amino acid oxidase; and   (D) optionally, a L-amino acid enantiomer as control.   
     
     
         44 . 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 in 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.   
     
     
         45 . The method as claimed in  claim 43 , wherein the D-amino acid is D-Proline, D-Tyrosine, D-Valine, D-Threonine, D-Glutamic acid, D-Lysine, or D-Tryptophane. 
     
     
         46 . 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 an of a racemase to be modulated;   (B) oxidatively deaminating the D-amino acid generated in step (A) by means of a D-amino oxidase in 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.   
     
     
         47 . The method as claimed in  claim 46 , wherein said molecule inhibits said racemase activity. 
     
     
         48 . The method as claimed in  claim 46 , wherein said racemase is a proline racemase. 
     
     
         49 . The method as claimed in  claim 47 , wherein said proline racemase is  Tripanosoma cruzi  proline racemase. 
     
     
         50 . A molecule identified by a method as claimed in  claims 45  to  48 . 
     
     
         51 . A technological platform and all reagents and devices necessary to perform the method of  claims 29  to  41  and  44  to  49 . 
     
     
         52 . The technological platform as claimed in  claim 50 , comprising
 a) L-amino acid, D-amino acid, and a racemase;   b) a peroxydase and a substrate of a peroxydase, or a catalase and a reagent sensitive to oxygen;   c) a D-amino acid oxidase; and   d) optionally, one or more molecules to be screened for inhibitory activity of said racemase.   
     
     
         53 . The technological platform as claimed in  claim 51 , wherein said racemase is a proline racemase and said L-amino acid and D-amino acid are L-proline and D-proline, respectively. 
     
     
         54 . A molecule inhibiting a proline racemase containing a subsequence selected form the SEQ ID NO: 1, 2, 3, or 4. 
     
     
         55 . A purified nucleic acid molecule encoding a polypeptide consisting of an amino acid sequence that is substantially similar to SEQ ID NO: 1, 2, 3, or 4. 
     
     
         56 . The purified nucleic acid molecule of  claim 55 , wherein the purified nucleic acid molecule hybridizes to either strand of a denatured, double-stranded nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, 2, 3, or 4, wherein the hybridization conditions comprise incubating the nucleic acids at 50° C. in a solution containing 5×SSC and 1×Denhardt's solution and three washes for twenty minutes each in 2×SSC at 60° C. 
     
     
         57 . A recombinant vector that directs the expression of the nucleic acid molecule as claimed in  claim 55 . 
     
     
         58 . An isolated host cell transfected or transduced with the recombinant vector as claimed in  claim 57 . 
     
     
         59 . A method for the production of a polypeptide consisting of an amino acid sequence substantially similar to SEQ ID NO: 1, 2, 3, or 4, comprising culturing the host cell of  claim 58  under conditions promoting expression, and recovering the polypeptide from the host cell or the culture medium. 
     
     
         60 . The method of  claim 59 , wherein the host cell is selected from the group consisting of bacterial cells, parasite cells, and eukaryotic cells. 
     
     
         61 . A purified nucleic acid molecule consisting of nucleotides encoding a polypeptides consisting of an amino acid sequence that is substantially similar to SEQ ID NO: 1, 2, 3, or 4. 
     
     
         62 . A duplex nucleic acid molecule comprising a single strand of a denatured, double-stranded DNA hybridized to either strand of a denatured, double-stranded DNA comprising the nucleic acid molecule of  claim 55  or  61 , wherein the hybridization conditions comprise incubating the nucleic acids at 50° C. in a solution containing 5×SSC and 1×Denhardt's solution and three washes for twenty minutes each in 2×SSC at 60° C. 
     
     
         63 . The purified nucleic acid molecule of  claim 61 , wherein the purified nucleic acid molecule hybridizes to either strand of a denatured, double-stranded nucleic acid molecule consisting of nucleotides encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, wherein the hybridization conditions comprise incubating the nucleic acids at 50° C. in a solution containing 5×SSC and 1×Denhardt's solution and three washes for twenty minutes each in 2×SSC at 60° C. 
     
     
         64 . A recombinant vector that directs the expression of the nucleic acid molecule as claimed in  claim 61 . 
     
     
         65 . An isolated host cell transfected or transduced with the recombinant vector of  claim 64 . 
     
     
         66 . A method for the production of a polypeptide consisting of an the amino acid sequence substantially similar to SEQ ID NO: 1, 2, 3, or 4, comprising culturing the host cell of  claim 65  under conditions promoting expression, and recovering the polypeptide from the host cell or the culture medium. 
     
     
         67 . The method of  claim 66 , wherein the host cell is selected from the group consisting of bacterial cells, parasite cells, and eukaryotic cells. 
     
     
         68 . A kit for detecting a racemase encoded by a nucleotide sequence having a subsequence encoding the peptide SEQ ID NO: 1, 2, 3, or 4, said kit comprising:
 (a) a polynucleotide primer or probe consisting of the polynucleotide sequence of  claim 55  or  61 ; and   (b) reagents to perform a nucleic acid hybridization reaction.