Identification and use of compounds that affect the fidelity of eukaryotic translation initiation codon selection

Abstract

A screening method for identifying compounds that alter the fidelity with which the initiation codon in mRNAs is recognized by the translational apparatus in eukaryotes is disclosed. This screening method was used to identify compounds having such activity. Methods of altering the fidelity of initiation codon selection are also disclosed. Methods of treating disorders characterized by single nucleotide mutations in initiation codons using compounds identified by the screening method, as well as methods of treating fungal and parasitic infections and hyperproliferative disorders using compounds identified by the screening method are also disclosed.

1 . A method for identifying an active compound affecting fidelity of eukaryotic translation initiation codon selection comprising:
 introducing a compound to eukaryotic cells in culture, wherein the cells comprise a DNA sequence encoding a first reporter protein, and the mRNA from the first reporter protein has an initiation codon that is a near-cognate of AUG;   measuring an amount of the first reporter protein;   determining a change in the amount of first reporter protein by comparing the amount of the first reporter protein to a baseline amount of the first reporter protein measured in the absence of the compound; and   selecting the compound as the active compound for affecting fidelity of eukaryotic translation initiation codon of the near-cognate of AUG initiation codons when the change in the amount of the first reporter protein meets or exceeds a predetermined threshold.   
     
     
         2 . The method of  claim 1 , wherein the predetermined threshold is an increase or decrease of 50%. 
     
     
         3 . The method of  claim 1 , further comprising measuring the baseline amount of the first reporter protein. 
     
     
         4 . The method of  claim 1 , further comprising counter-screening the active compound to determine non-specific activity and eliminating the compound as the active compound if it exhibits non-specific activity. 
     
     
         5 . The method of  claim 4 , wherein the counter-screening comprises measuring a second amount of the first reporter protein wherein the second amount of the first reporter protein is an amount produced by introducing the compound to eukaryotic cell in culture, wherein the cells comprise a DNA sequence encoding the first reporter protein, and the mRNA from the first reporter protein has an AUG initiation codon. 
     
     
         6 . The method of  claim 1 , wherein the cells are yeast cells. 
     
     
         7 . The method of  claim 1 , wherein the cells are mammalian cells. 
     
     
         8 . The method of  claim 1 , wherein the first reporter protein is a luciferase protein. 
     
     
         9 . The method of  claim 8 , wherein the luciferase protein is a firefly luciferase protein. 
     
     
         10 . The method of  claim 1 , wherein the cells further comprise a DNA sequence encoding a second reporter protein, and the mRNA from the second reporter protein has an AUG initiation codon;
 the method further comprising measuring the amount of the second reporter protein.   
     
     
         11 . The method of  claim 10 , further comprising measuring the ratio between the amount of first reporter protein and the amount of the second reporter protein. 
     
     
         12 . The method of  claim 10 , where the second reporter protein is a luciferase protein. 
     
     
         13 . The method of  claim 10 , wherein the second reporter protein is selected from the group consisting of a luciferase protein, a fluorescent protein, or a colorimetric protein. 
     
     
         14 . The method of  claim 10 , wherein the first reporter protein is a firefly luciferase protein and the second reporter is a Renilla luciferase protein. 
     
     
         15 . The method of  claim 10 , wherein the first and/or the second reporter protein is quantified by a method selected from the group consisting of colorimetric, luminescence, fluorescence, absorbance, immunogenically, level of cell growth in a selected media, and measuring protein expression. 
     
     
         16 . A method of treating a disorder treatable by a compound affecting fidelity of eukaryotic translation initiation codon selection comprising administrating a therapeutically effective amount of an active compound selected by the method of  claim 1  to a subject in need thereof. 
     
     
         17 . The method of  claim 16 , wherein the disorder is selected from the group consisting of a genetic disorder, a fungal infection, a parasitic infection, or a hyperproliferative disorder. 
     
     
         18 . The method of  claim 17 , wherein the disorder is a genetic disorder characterized by a single nucleotide mutation in the initiation codon selected from the group consisting of beta-thalassemia, alpha-thalassemia, hemoglobin H disease, phenylketonuria, congenital adrenal hyperplasia, Smith-Lemli-Opitz Syndrome, and Refsum disease. 
     
     
         19 . The method of  claim 17 , wherein the disorder is a fungal infection. 
     
     
         20 . The method of  claim 17 , wherein the disorder is a hyperproliferative disorder.