Identification and use of compounds that affect the fidelity of eukaryotic translation initiation codon selection
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 of altering the fidelity of eukaryotic translation initiation codon selection, the method comprising administering to a cell a compound of Formula I-V:
where n is an integer from 1 to 4; R 1 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 2 is H or alkyl; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 1 is Br or I; R 6 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 2 is F, Cl, Br, or I; R 7 is H or alkyl;
where X 3 is F, Cl, Br, or I; R 8 is H or alkyl; R 9 is H or alkyl;
where R 10 is H or alkyl and R H is H or alkyl;
and pharmaceutically acceptable salts thereof.
2. The method of claim 1 , wherein the compound is selected from the group consisting of:
3. The method of claim 1 , wherein the compound is
where n is an integer from 1 to 4; R 1 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 2 is H or alkyl; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl; or
where X 1 is Br or I; R 6 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl.
4. The method of claim 3 , wherein the compound is selected from
5. A method of screening a test compound for identifying activity of the test compound in altering the fidelity of translation initiation codon selection, comprising
introducing a test compound according to Formula I-V to eukaryotic cells in a culture wherein:
Formula I-V are defined as:
where n is an integer from 1 to 4; R 1 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 2 is H or alkyl; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 1 is Br or I; R 6 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 2 is F, Cl, Br, or I; R 7 is H or alkyl;
where X 3 is F, Cl, Br, or I; R 8 is H or alkyl; R 9 is H or alkyl;
where R 10 is H or alkyl and R 11 is H or alkyl; and pharmaceutically acceptable salts thereof; and
the cells comprise a DNA sequence encoding a first reporter protein, where the mRNA from the first reporter protein has an initiation codon that is a near-cognate of AUG, and
measuring the change in the amount of reporter protein to identify the activity of the test compound in altering the fidelity of translation initiation codon selection.
6. The method of claim 5 wherein the cell further comprises DNA sequence encoding a second reporter protein, where the mRNA from the second reporter protein has an AUG initiation codon.
7. The method of claim 6 , wherein said measuring step comprises measuring the ratio between the amount of first reporter protein and the second reporter protein.
8. The method of claim 5 , where the cells are yeast cells.
9. The method of claim 5 , where the cells are mammalian cells.
10. The method of claim 5 , where the first reporter protein is a luciferase protein.
11. The method of claim 5 , where the first reporter protein is a firefly luciferase protein.
12. The method of claim 6 , where the second reporter protein is a luciferase protein.
13. The method of claim 6 , where the first reporter protein is a firefly luciferase protein and the second reporter is a Renilla luciferase protein.
14. A method of treating a disorder comprising administering to a subject in need of treatment an effective amount of a compound selected from the group consisting of
where n is an integer from 1 to 4; R 1 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 2 is H or alkyl; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 1 is Br or I; R 6 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl;
where X 2 is F, Cl, Br, or I; R 7 is H or alkyl;
where X 3 is F, Cl, Br, or I; R 8 is H or alkyl; R 9 is H or alkyl; and
or pharmaceutically acceptable salt thereof, wherein the disorder is: a disorder characterized by a non-AUG initiation codon.
15. The method of claim 14 , wherein the compound is selected from the group consisting of:
16. The method of claim 14 wherein the compound is
where n is an integer from 1 to 4; R 1 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 2 is H or alkyl; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl; or
where X 1 is Br or I; R 6 is H, halogen, alkyl, NO 2 , OR 3 , N(R 4 ) 2 , or SR 5 ; R 3 is H or alkyl, and R 4 is H or alkyl and R 5 is H or alkyl.
17. The method of claim 16 , wherein the compound is
18. The method of claim 14 , 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 Syndrome, and Refsum disease.
19. The method of claim 14 , wherein the disorder is a fungal infection.