Novel Assay for Inositol Phosphorylceramide Synthase Activity

Abstract

Disclosed is a simple and reproducible method for assaying inositol phosphorylceramide synthase activity that employs a fluorescence resonance energy transfer pair for measuring enzyme activity. The invention also includes a novel method for identifying IPC synthase inhibitors.

1 . A method for measuring inositol phosphorylceramide (IPC) synthase activity comprising:
 a) providing a sample containing inositol phosphorylceramide synthase;   b) providing a fluorescent labeled IPC synthase substrate;   c) providing a phosphate binding compound conjugated to a fluorophor, wherein the phosphate binding compound conjugated to a fluorophor forms a fluorescence resonance energy transfer (FRET) pair with the fluorescent labeled IPC synthase substrate; and   d) measuring fluorescence.   
   
   
       2 . The method of  claim 1 , wherein the fluorescent labeled IPC synthase substrate is a fluorescent labeled ceramide. 
   
   
       3 . The method of  claim 1 , wherein the phosphate binding compound comprises a peptide that includes at least one positively charged amino acid. 
   
   
       4 . The method of  claim 1 , wherein the phosphate binding compound comprises a peptide that includes one or more of arginine, histidine and lysine. 
   
   
       5 . The method of  claim 3 , wherein the phosphate binding compound comprises a hexapeptide. 
   
   
       6 . The method of  claim 2 , wherein the fluorescent labeled ceramide is selected from the group consisting of C5-NBD-ceramide, C6-NBD-ceramide, C12-NBD-ceramide, BODIPY-FL-C5-ceramide, BODIPY-FL-C6-ceramide and BODIPY-TR-C6-ceramide. 
   
   
       7 . The method of  claim 2  wherein the fluorescent labeled ceramide is C6-NBD-ceramide. 
   
   
       8 . The method of  claim 1  wherein the fluorophor conjugated to the phosphate binding compound is selected from the group consisting of Alexa Fluor 350, anilinonapthalene, dansyl, dapoxyl, dibromobimane, diethylaminocoumarin, dimethylaminocoumarin, dimethylaminonapthalene, monobromobimane, monochlorobimane, napthanlene, pyrene, stilbene and D-346. 
   
   
       9 . The method of  claim 1  wherein the fluorophor conjugated to the phosphate binding compound is D-346. 
   
   
       10 . The method of  claim 1  wherein the fluorescent labeled substrate is C6-NBD-ceramide and the fluorophor conjugated to the phosphate binding compound is D-346. 
   
   
       11 . The method of  claim 1  wherein the sample containing inositol phosphorylceramide synthase is derived from fungal cells. 
   
   
       12 . A method for identifying inositol phosphorylceramide synthase inhibitors comprising
 a) providing a sample containing inositol phosphorylceramide synthase;   b) providing a test compound to the sample;   c) providing a fluorescent labeled IPC synthase substrate;   d) providing a phosphate binding compound conjugated to a fluorophor, wherein the phosphate binding compound conjugated to a fluororphor forms a fluorescence resonance energy transfer (FRET) pair with the fluorescent labeled IPC synthase substrate; and   e) measuring fluorescence.   
   
   
       13 . A kit comprising
 a) a fluorescent labeled IPC synthase substrate and   b) a phosphate binding compound conjugated to a fluorophor, wherein the phosphate binding compound conjugated to a fluororphor forms a fluorescence resonance energy transfer (FRET) pair with the fluorescent labeled IPC synthase substrate.   
   
   
       14 . The kit of  claim 13  further comprising a sample containing inositol phosphorylceramide synthase. 
   
   
       15 . The kit of  claim 13  further comprising phosphatidyl inositol. 
   
   
       16 . The kit of  claim 14  further comprising a known modulator of IPC synthase.