Inhibitors of bacterial nitric oxide synthase, and related screening methods
AssigneeMassachusetts Institute of Technology
InventorsStephen J. Lippard, Lindsey E. McQuade, Evgeny A. Nudler
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Abstract
The invention relates in part to compounds that act as highly nitric oxide (NO)-specific turn-on fluorescent probes. The present invention also relates to the use of these fluorescein-based sensors to screen selectively for inhibitors of bacterial nitric oxide synthase (bNOS). Compounds of the present invention readily detect nitric oxide produced in vivo. Therefore they can be used in an assay that measures NO production by bNOS. Using a sensor of the invention one can screen libraries of small molecules for inhibitors of bNOS.
Claims24
A method of determining if a small molecule is a bacterial nitric oxide synthase (bNOS) inhibitor, comprising the steps of:
- a) preparing a first solution comprising bacteria taken up by macrophages, wherein said bacteria expresses bNOS;
- b) adding to the first solution a transition metal-containing fluorescein-based sensor, thereby forming a first mixture;
- c) incubating the first mixture for a first period of time, thereby forming a first sample;
- d) measuring the fluorescence of the first sample;
- e) preparing a second solution comprising the first solution and a small molecule;
- f) adding to the second solution the transition metal-containing fluorescein-based sensor, thereby forming a second mixture;
- g) incubating the second mixture for a second period of time, thereby forming a second sample;
- h) measuring the fluorescence of the second sample;
- i) comparing the fluorescence of the first sample with the fluorescence of the second sample;
- and
- k) identifying the small molecule as a bNOS inhibitor when the fluorescence of the second sample is reduced compared to the fluorescence of the first sample;
- wherein steps (d) and (h) are both completed within less than about 8 hours after the bacteria are taken up by the macrophages.
A method of determining if a small molecule is a selective bacterial nitric oxide synthase (bNOS) inhibitor, comprising the steps of:
- a) preparing a first solution comprising bacteria taken up by macrophages, wherein said bacteria expresses bNOS;
- b) adding to the first solution a transition metal-containing fluorescein-based sensor, thereby forming a first mixture;
- c) incubating the first mixture for a first period of time, thereby forming a first sample;
- d) measuring the fluorescence of the first sample;
- e) incubating the first sample for a second time, thereby forming a second sample;
- f) measuring the fluorescence of the second sample;
- g) preparing a second solution comprising the first solution and a small molecule;
- h) adding to the second solution the transition metal-containing fluorescein-based sensor, thereby forming a second mixture;
- i) incubating the second mixture for a third time, thereby forming a third sample;
- j) measuring the fluorescence of the third sample;
- k) incubating the third sample for a fourth time, thereby forming a fourth sample;
- l) measuring the fluorescence of the fourth sample;
- m) comparing the fluorescence of the first sample with the fluorescence of the third sample, and comparing the fluorescence of the second sample with the fluorescence of the fourth sample;
- and
- n) identifying the small molecule as a selective bNOS inhibitor when both (1) fluorescence of the third sample is reduced compared to the fluorescence of the first sample, and (2) fluorescence of the fourth sample is reduced compared to the fluorescence of the second sample;
- wherein steps (d) and (j) are both completed within less than about 8 hours after the bacteria are taken up by the macrophages;
- and steps (f) and (l) are both completed greater than about 8 hours after the bacteria are taken up by the macrophages.
Dependencies are inferred from the claim text.