Compositions that bind antiterminator RNA and assay for screening for such compositions
AssigneeOhio University
InventorsJennifer V. Hines, Stephen C. Bergmeier
Life of this patent
Estimating the term…
Abstract
Compounds that are selective binding agents specific for RNA secondary and tertiary structures and a fluorescence assay for detecting the binding of small molecules to T-box antiterminator model mRNAs. The compounds include novel tri-substituted oxazolidinones and amino alcohols. The assay provides fluorescently labeled model mRNAs that include conserved sequence information typical of antiterminator bulge regions found in the mRNA of T-box gene regulatory systems.
Claims27
A compound of formula I:
- 5
- wherein
- R1 is selected from the group consisting of alkyl, aryl, alkylaryl, and arylalkyl;
- R2 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, alkoxy, ester, alcohol, and amide;
- and
- R3 is aliphatic or substituted aliphatic, wherein the substituent is selected from the group consisting of alkyl, aryl, and halo.
A compound of formula II:
- 6
- wherein
- R4 is selected from the group consisting of alkyl, aryl, alkylaryl, and arylalkyl;
- R5 is selected from the group consisting of H, alkyl, aryl, alkylaryl, arylalkyl, alkoxy, ester, alcohol, and amide;
- and
- R6 is selected from an alcohol radical, an aliphatic radical, and substituted aliphatic radical, wherein the substituent is selected from the group consisting of alkyl, aryl, and halo.
A method of inhibiting replication of microorganisms in a subject, comprising administering a therapeutically effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, to the subject in need of such treatment, wherein the microorganism is selected from the group consisting of bacteria, viruses, fungi, and parasites.
A method of disinfecting a surface comprising applying an effective amount of a compound of Formula I or II or combinations thereof to the surface in need of disinfection.
A method of preparing a trisubstituted oxazolidinone compound of Formula I:
- 7
- wherein
- R1 is selected from the group consisting of alkyl, aryl, alkylaryl, and arylalkyl;
- R2 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, alkoxy, ester, alcohol, and amide;
- and
- R3 is aliphatic or substituted aliphatic, wherein the substituent is selected from the group consisting of alkyl, aryl, and halo;
- comprising the steps of:
- (a) providing a substituted allylic alcohol of Formula A:
- 8
- wherein R7 is selected from the group consisting of alkyl, aryl, and ester;
- (b) converting compound 1 to an azidoformate;
- (c) heating the resulting azidoformate for a sufficient time at a sufficient temperature to provide an aziridine;
- (d) adding R1 to the aziridine;
- (e) adding R2-X to the resulting compound, wherein X is a halide, to provide substitution R2 on the oxazoline N atom;
- and
- (f) optionally, adding an R3-acid chloride to produce the trisubstituted oxazolidinone compound of Formula I.
An assay for detecting compounds that bind to RNA and inhibit or disrupt the function of antiterminator regulatory elements, comprising:
- (a) a model RNA molecule, wherein said model RNA includes conserved nucleotide sequences typical of antiterminator regulatory elements occurring in T-box gene systems, and forms an antiterminator bulge structure in vitro;
- (b) at least one fluorescent indicator bound to said model RNA;
- and
- (c) a means for measuring change in the fluorescence of said at least one fluorescent indicator when said compound binds to said model RNA.
A method for detecting the binding of compounds to RNA molecules, comprising the steps of:
- (a) preparing a solution of labeled model RNA;
- (b) adding said labeled RNA solution to a buffer solution;
- (c) preparing stock solutions of test ligand;
- (d) for RNA control fluorescence measurements, adding buffer to the labeled RNA solution from step (b);
- (e) mixing the sample from step (d) gently and adding said sample to the cuvette on a SPEX Fluoromax-2-spectrofluorometer with excitation at 310 nm and collecting emission spectra from 330-450 nm;
- (f) measuring the fluorescence of the buffer minus said labeled RNA;
- (g) calculating the fluorescence of the labeled RNA using the formula:
- F370(labeled RNA)−F370(buffer);
- (h) adding stock ligand solution to the RNA sample prepared in step (b);
- (i) adding said test sample to the cuvette on a SPEX Fluoromax-2-spectrofluorometer with excitation at 310 nm and collecting emission spectra from 330-450 nm;
- (j) measuring the fluorescence of an identical concentration of said test ligand in buffer;
- and
- (k) calculating the fluorescence of said labeled RNA with said test ligand using the formula:
- F370(ligand+labeled RNA)−F370(ligand in buffer).
Dependencies are inferred from the claim text.