Gtp cyclohydrolase II as as target for fungicides

Abstract

The present invention relates to the identification of fungal GTP cyclohydrolase II as a target for fungicides, to a method for identifying antifungal agents based on fungal GTP cyclohydrolase II, and also to the use of compounds identified as fungicides via the abovementioned method.

1 - 19 . (canceled)  
     
     
         20 . A method for identifying antifungal agents comprising 
 i. incubating, with at least one candidate compound, a fungal GTP cyclohydrolase II polypeptide under conditions allowing the binding of the candidate compound to the fungal GTP cyclohydrolase II; and    ii. selecting, by step ii), at least a candidate compound which binds to the fungal GTP cyclohydrolase II of step i); or    iii. selecting, by step iii), at least one candidate compound which reduces or blocks the activity of the fungal GTP cyclohydrolase II of step i); or    iv. selecting, by step iv), at least one candidate compound which inhibits or decreases transcription, translation or expression of the fungal GTP cyclohydrolase II of step i),    wherein the GTP cyclohydrolase II activity in steps ii to iv is determined by    a) adding GTP or GTP analog, NAD +  and formate dehydrogenase to a sample comprising GTP cyclohydrolase II or I; and    b) determining the NADH content.    
     
     
         21 . The method of  claim 20 , wherein the fungal GTP cyclohydrolase II is encoded by a nucleic acid sequence comprising 
 a) a nucleic acid sequence shown in SEQ ID No: 1; or    b) a nucleic acid sequence which, owing to the degeneracy of the genetic code, can be deduced from the amino acid sequence shown in SEQ ID No: 2 by back translation; or    c) a nucleic acid sequence which, owing to the degeneracy of the genetic code, can be deduced from a functional equivalent of the amino acid sequence shown in SEQ ID No: 2, which has an identity with SEQ ID No: 2 of at least 49%, by back translation.    
     
     
         22 . A nucleic acid having a sequence comprising 
 a) a nucleic acid sequence shown in SEQ ID No: 4; or    b) a nucleic acid sequence which, owing to the degeneracy of the genetic code, can be deduced from the amino acid sequence shown in SEQ ID No: 5 by back translation; or    c) a nucleic acid sequence which, owing to the degeneracy of the genetic code, can be deduced from a functional equivalent of the amino acid sequence shown in SEQ ID No: 5, which has an identity with SEQ ID No: 5 of at least 66%, by back translation.    
     
     
         23 . The method of  claim 20 , wherein the method comprises testing a candidate compound in a fungal GTP cyclohydrolase II inhibition assay.  
     
     
         24 . The method of  claim 23 , wherein the method comprises 
 a) incubating, with a candidate compound, a fungal GTP cyclohydrolase II in a cell free system;    b) selecting, by step b), a candidate compound which decreases the activity of the fungal GTP cyclohydrolase II.    
     
     
         25 . The method of  claim 24 , wherein the enzymatic activity of the fungal GTP cyclohydrolase II is determined in comparison to the activity of a fungal GTP cyclohydrolase II not incubated with the candidate compound.  
     
     
         26 . A method for determination of GTP cyclohydrolase I or II activity comprising the steps of 
 a) adding GTP or GTP analog, NAD+ and formate dehydrogenase to a sample comprising GTP cyclohydrolase II or I; and    b) determining the NADH content.    
     
     
         27 . A method for identification of inhibitors of GTP cyclohydrolase I or II comprising the following steps: 
 a) adding GTP or GTP analog, NAD+ and formate dehydrogenase to a sample comprising GTP cyclohydrolase I or II;    b) adding formate, NAD+ and formate dehydrogenase to a second sample comprising GTP cyclohydrolase I or II;    c) adding to the sample of step a) and step b) a candidate compound;    d) determining the activity of both samples;    e) selecting candidate compounds that show inhibition in the presence of GTP and no inhibition in the presence of formic acid.    
     
     
         28 . The method of  claim 24 , wherein inhibitors of fungal GTP cyclohydrolase II are identified in an inhibition assay comprising the following steps: 
 a) adding GTP or GTP analog, NAD+ and formate dehydrogenase to a sample comprising GTP cyclohydrolase I or II;    b) adding formate, NAD+ and formate dehydrogenase to a second sample comprising GTP cyclohydrolase I or II;    c) adding to the sample of step a) and step b) a candidate compound;    d) determining the activity of both samples;    e) selecting candidate compounds that show inhibition in the presence of GTP and no inhibition in the presence of formic acid.    
     
     
         29 . The method of  claim 24 , wherein GTP is used as substrate and the NADH content is determined by monitoring the increase in the absorption at 340 nm.  
     
     
         30 . The method of  claim 28 , wherein GTP is used as substrate and the NADH content is determined by monitoring the increase in the absorption at 340 nm.  
     
     
         31 . The method of claim  1 , wherein the method comprises the following steps: 
 a) generating organisms which, following transformation with a nucleic acid sequence encoding GTP cyclohydrolase II are capable of overexpressing polypeptide with GTP cyclohydrolase II activity;    b) applying to the organism of step a) and to an analogous, untransformed organism, a candidate compound;    c) determining the growth, the viability or infectivity of the transgenic and the untransformed organism following application of the substance of step b);    d) selecting candidate compounds, which reduces growth, viability or infectivity of the transgenic and the untransformed organism following application of the substance of step b).    
     
     
         32 . The method of  claim 31 , wherein the transgenic organism and the untransformed organism are fungi.  
     
     
         33 . The method of  claim 20 , wherein the substances are identified in a high-throughput screening.  
     
     
         34 . The method of  claim 20 , wherein the antifungal agent identified via the method is applied to a phytopathogenic fungus in order to verify the fungicidal activity.  
     
     
         35 . A process for the preparation of a fungicidal composition, which comprises 
 a) identifying an antifungal agent via a method for identifying antifungal agents comprising 
 i. incubating, with at least-one candidate compound, a fungal GTP cyclohydrolase II polypeptide under conditions allowing the binding of the candidate compound to the fungal GTP cyclohydrolase II; and  
 ii. selecting, by step ii), at least a candidate compound which binds to the fungal GTP cyclohydrolase II of step i) ; or  
 iii. selecting, by step iii), at least one candidate compound which reduces or blocks the activity of the fungal GTP cyclohydrolase II of step i); or  
 iv. selecting, by step iv), at least one candidate compound which inhibits or decreases transcription, translation or expression of the fungal GTP cyclohydrolase II of step i),  
 wherein the GTP cyclohydrolase II activity in steps ii to iv is determined by  
 1) adding GTP or GTP analog, NAD+ and formate dehydrogenase to a sample comprising GTP cyclohydrolase II or I; and  
 2) determining the NADH content; and  
   b) formulating the antifungal agent identified via (a), or an agriculturally useful salt of the active ingredient identified via (a), with suitable adjuvants.    
     
     
         36 . A process for the preparation of a pharmaceutical fungicidal composition, which comprises 
 a) identifying an antifungal agent via a method for identifying antifungal agents comprising 
 i. incubating, with at least-one candidate compound, a fungal GTP cyclohydrolase II polypeptide under conditions allowing the binding of the candidate compound to the fungal GTP cyclohydrolase II; and  
 ii. selecting, by step ii), at least a candidate compound which binds to the fungal GTP cyclohydrolase II of step i) ; or  
 iii. selecting, by step iii), at least one candidate compound which reduces or blocks the activity of the fungal GTP cyclohydrolase II of step i); or  
 iv. selecting, by step iv), at least one candidate compound which inhibits or decreases transcription, translation or expression of the fungal GTP cyclohydrolase II of step i),  
 wherein the GTP cyclohydrolase II activity in steps ii to iv is determined by  
 1) adding GTP or GTP analog, NAD +  and formate dehydrogenase to a sample comprising GTP cyclohydrolase II or I; and  
 2) determining the NADH content; and  
   b) formulating the antifungal agent identified via (a), or a pharmaceutically useful salt of the active ingredient identified via (a), with suitable excipients.    
     
     
         37 . A method for culturing plants or plant cells or plant tissues thereby controlling fungal growth comprising treating said culture with a fungicide, wherein said fungicide is a compound which is an inhibitor of fungal GTP cyclohydrolase II.