Inhibitors of Rv0256c
Described herein are methods for identifying inhibitors of Rv0256c functions, e.g., inhibitors of Rv0256c expression, DNA binding, nuclear localization and iNOS inhibition.
1. A method of identifying a Rv0256c inhibitor comprising:
contacting a test agent with a eukaryotic cell expressing a Rv0256c polypeptide, wherein the Rv0256c polypeptide has a polypeptide sequence with at least about 75% sequence identity with the polypeptide sequence of SEQ ID NO:1; and
measuring one or more of:
(i) the level of Rv0256c mRNA or polypeptide expressed by the eukaryotic cell;
(ii) the level of nuclear localization of the Rv0256c polypeptide in the eukaryotic cell;
(iii) the level of nitric oxide produced by the eukaryotic cell; and
(iv) the level of inducible nitric oxide synthase (iNOS) expressed by the eukaryotic cell;
wherein a statistically significant decrease in (i) and/or (ii), or a statistically significant increase in (iii) and/or (iv), or any combination thereof, compared to a control eukaryotic cell not contacted with the test agent indicates that the test agent is an inhibitor of Rv0256c expression or function.
2. The method of claim 1 , wherein the expression level of the Rv0256c mRNA polypeptide is measured and a statistically significant decrease in the expression level of the Rv0256c polypeptide compared to a reference level of the Rv0256c polypeptide indicates that the test agent is a Rv0256c inhibitor.
3. The method of claim 2 , wherein the expression level of Rv0256c mRNA is measured using a quantitative northern blot, quantitative reverse transcription PCR (RT-PCR), or DNA microarray technologies.
4. The method of claim 2 , wherein the expression level of Rv0256c polypeptide is measured using a quantitative Western blot or fusing the Rv0256c polypeptide to a reporter gene.
5. The method of claim 1 , wherein the level of nuclear localization of the Rv0256c polypeptide is measured and a statistically significant decrease in the level of nuclear localization compared to a reference level of nuclear localization indicates that the test agent is a Rv0256c inhibitor.
6. The method of claim 1 , wherein the level of expression of inducible nitric oxide synthase (iNOS) is measured and an increase in the level of iNOS expression compared to a reference level of iNOS expression indicates that the test agent is a Rv0256c inhibitor.
7. The method of claim 1 , wherein the Rv0256c polypeptide comprises the polypeptide sequence of SEQ ID NO:1.
8. The method of claim 1 , wherein the test agent is selected from the group consisting of: a small molecule; a nucleic acid; a polypeptide; a lipid, and an antibody, or a fragment thereof.
9. The method of claim 1 , wherein measuring the level of nitric oxide produced by the cell comprises the use of Griess reagent.
10. The method of claim 1 , wherein the eukaryotic cell expresses the Rv0256c polypeptide as a result of transient transfection of the cell.
11. The method of claim 1 , wherein the eukaryotic cell is a macrophage.