SCREEN FOR ANTI-INFECTIVE COMPOUNDS
The present invention provides methods and compositions for identifying compounds useful as therapeutics and disinfectants. Such compounds would be particularly useful in treating Gram-positive bacterial infections. Such therapeutics would target the interaction of a tRNA molecule with an mRNA molecule, interrupting translation of the mRNA molecule and thus interfering with gene expression for a target gene critical to the survival of these organisms.
1 . A method for identifying a compound that interferes with the binding of a tRNA molecule comprising an anticodon to a nascent mRNA molecule comprising a complementary codon in the absence of protein, comprising:
(a) contacting the compound with an RNA reporter construct comprising (i) a reporter molecule and (ii) an RNA molecule comprising the complementary codon and representing a 5′ UTR of the nascent mRNA molecule; and
(b) measuring the binding of the RNA reporter construct and the tRNA molecule in the presence of the compound,
wherein a decrease of binding identifies a compound that interferes with the binding of the tRNA molecule comprising the anticodon to the nascent mRNA molecule comprising the complementary codon.
2 . The method of claim 1 , wherein said 5′ UTR contains a T box transcription termination control system.
3 . The method of claim 2 , wherein said 5′ UTR belongs to a gene for an amino acid metabolic enzyme.
4 . The method of claim 3 , wherein said amino acid metabolic enzyme is a gram positive bacteria amino acid metabolic enzyme.
5 . The method of claim 4 , wherein said gram positive bacteria amino acid metabolic enzyme is selected from the group consisting of aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transporter proteins.
6 . The method of claim 1 , said reporter construct consisting essentially of: (i) a reporter molecule and (ii) an RNA molecule comprising the complementary codon and representing a 5′ UTR of the nascent mRNA molecule, wherein said RNA molecule is not more than 100 nucleotides in length.
7 . The method of claim 1 , wherein said compound is a member of a compound library.
8 . The method of claim 1 , wherein said compound is a candidate anti-infective therapeutic and/or disinfecting agent for Gram-positive bacteria.
9 . The method of claim 1 , wherein:
said 5′ UTR contains a T box transcription termination control system that belongs to a gene for a gram negative bacteria amino acid metabolic enzyme selected from the group consisting of aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transporter proteins; and
said reporter construct consists essentially of: (i) a reporter molecule and (ii) an RNA molecule comprising the complementary codon and representing a 5′ UTR of the nascent mRNA molecule, wherein said RNA molecule is not more than 100 nucleotides in length.
10 . An anti-infective and/or disinfecting compound for Gram-positive bacteria identified by the method of claim 1 .
11 . An RNA reporter construct useful for measuring the binding of a tRNA molecule to a nascent mRNA molecule of a target gene, comprising:
(a) a reporter molecule; and
(b) an RNA molecule comprising a complementary codon and representing the 5′ UTR of the nascent mRNA molecule of the target gene.
12 . The construct of claim 11 , wherein said target gene is a gene having a T box transcription termination control system.
13 . The construct of claim 11 , wherein said construct is unimolecular.
14 . The construct of claim 11 , wherein said construct comprises at least two molecules.
15 . The construct of claim 11 , wherein said reporter molecule is a fluorescent reporter molecule.
16 . The construct of claim 15 , wherein said fluorescent reporter molecule is 2-aminopurine ribonucleoside.
17 . The construct of claim 11 , wherein said 5′ UTR contains at least the specifier segment of a T box transcription termination control system.
18 . The construct of claim 11 , wherein said 5′ UTR belongs to a gene for an amino acid metabolic enzyme.
19 . The construct of claim 18 , wherein said amino acid metabolic enzyme is a gram positive bacteria amino acid metabolic enzyme.
20 . The construct of claim 19 , wherein said gram positive bacteria amino acid metabolic enzyme is selected from the group consisting of aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transporter proteins.
21 . The construct of claim 11 , said reporter construct consisting essentially of: (i) a reporter molecule and (ii) an RNA molecule comprising the complementary codon and representing a 5′ UTR of the nascent mRNA molecule, wherein said RNA molecule is not more than 100 nucleotides in length.