Novel Screening Methods
The present invention includes a luciferase-based high-throughput screening assay that identifies compounds that are inhibitors of cellular metabolism. This assay is applicable to all bacterial and eukaryotic membranes.
1 . A method of identifying a compound that inhibits cellular metabolism, the method comprising the steps of:
(a) isolating a membrane from a cell having a functional oxidative phosphorylation pathway;
(b) contacting an aliquot of the isolated membrane with a test compound to yield a first mixture;
(c) contacting an aliquot of the isolated membrane with a control compound to yield a second mixture;
(d) adding luciferase to the first mixture and to the second mixture;
(e) measuring the level of luminescence emitted from the first and second mixtures,
wherein, if the level of luminescence in the first mixture is lower than the level of luminescence in the second mixture, the test compound is identified as inhibiting cellular metabolism.
2 . The method of claim 1 , wherein the level of luminescence in the first mixture is less than about 70% of the level of luminescence in the second mixture.
3 . The method of claim 1 , wherein the test compound inhibits or disrupts the activity of at least one enzyme in the oxidative phosphorylation pathway in the cell.
4 . The method of claim 1 , wherein the test compound inhibits or disrupts the proton motive force (H + gradient) altering the electrochemical gradient across the cellular membrane affecting the proton concentration across the membrane.
5 . The method of claim 1 , wherein the test compound inhibits or disrupts the difference in electric potential across the cellular membrane.
6 . The method of claim 1 , wherein the cell is eukaryotic.
7 . The method of claim 6 , wherein the eukaryotic cell is mammalian.
8 . The method of claim 1 , wherein the cell is bacterial.
9 . The method of claim 8 , wherein the bacterium is Gram positive.
10 . The method of claim 9 , wherein the Gram positive bacterium is selected from the group consisting of Staphylococcus aureus, Mycobacterium smegmatis, Bacillus thuringiensis, Enterococcus , and combinations thereof.
11 . The method of claim 8 , wherein the bacterium is Gram negative.
12 . The method of claim 11 , wherein the Gram negative bacterium is selected from the group consisting of Acinetobacter baumannii, Pseudomonas aeruginosa, Yersinia enterocolitica, Escherichia coli, Klebsiella pneumonia , and combinations thereof.
13 . The method of claim 1 , wherein the luciferase is added at the beginning of the assay.
14 . The method of claim 1 , wherein the assay is carried out over a period of time of two hours or less.
15 . The method of claim 1 , wherein the volume of assay reactions ranges from about 5 μL to about 20 μL.