Class- and isoform-specific HDAC inhibitors and uses thereof
View Patent ↗The present invention relates to compounds of the following formula wherein R 3 is a fluorescent tag. Another aspect of the invention provides an assay for determining the inhibitory effect of a test compound on an HDAC protein comprising: incubating the HDAC protein with a substrate of the above formula in the presence of a test compound; and determining the activity of the HDAC protein.
1. A compound of formula:
wherein R 3 is a fluorescent tag;
or salt thereof.
2. The compound of claim 1 , wherein R 3 comprises coumarin.
3. The compound of claim 1 , wherein the compound is:
4. The compound of claim 1 , wherein the fluorescent tag of R 3 can be cleaved by an enzyme.
5. An assay for determining the inhibitory effect of a test compound on an HDAC protein comprising: incubating the HDAC protein with a substrate of claim 1 in the presence of a test compound; and determining the activity of the HDAC protein.
6. An assay for determining the inhibitory effect of a test compound on an HDAC protein comprising incubating the HDAC protein with a substrate of formula:
in the presence of a test compound; and determining the activity of the HDAC protein by monitoring the release of 7-amino-4-methylcoumarin after cleavage by trypsin.
7. The compound of claim 1 , wherein the fluorescent tag is selected from a group consisting of brodifacoum, bromadiolone, coumafuryl, difenacoum, auraptene, ensaculin, phenprocoumon, warfarin, and derivatives thereof.
8. The compound of claim 4 , wherein the enzyme is esterase.
9. The compound of claim 4 , wherein the enzyme is protease.
10. The compound of claim 4 , wherein the enzyme is serine protease.
11. The compound of claim 4 , wherein the enzyme is trypsin.
12. The compound of claim 1 , wherein the fluorescent tag of R 3 can be removed by a mechanical process.
13. The compound of claim 1 , wherein R 3 comprises a coumarin derivative thereof.
14. The assay of claim 5 , wherein the step of determining the activity of the HDAC protein comprises monitoring the release of the fluorescent tag from the substrate.
15. The assay of claim 5 , wherein the fluorescent tag comprises coumarin or a derivative thereof.
16. The assay of claim 5 , wherein the assay is carried out at a concentration of the substrate greater than the substrate K m .
17. The assay of claim 5 , wherein the assay is carried out at a concentration of the substrate approximately equivalent to the substrate K m .
18. The assay of claim 5 , wherein the concentration of the substrate is 1-20 μM.
19. The assay of claim 5 , wherein the HDAC protein is a Class II HDAC.
20. The assay of claim 5 , wherein the HDAC protein is 0.01-5 ng/μL.
21. The assay of claim 5 , wherein the HDAC protein is 0.1-0.5 ng/μL.