Compounds and methods for detection of enzymes that remove formyl, succinyl, methyl succinyl or myristoyl groups from epsilon-amino lysine moieties
Provided is a compound that comprises the structure: where SIG is a signaling molecule and R 3 is a formyl, a succinyl, a methyl succinyl, or a myristoyl. Also provided is a kit is provided that comprises the above compound, with instructions for determining the presence of the enzyme. Additionally, a method is provided for determining whether a sample has an enzyme that removes a succinyl, a methyl succinyl, a formyl, or a myristoyl moiety from an ε-amino of a lysine. Also provided is a method of determining whether a molecule inhibits an enzyme that removes a succinyl, a methyl succinyl, a formyl, or a myristoyl moiety from an ε-amino of a lysine.
1. A method of determining whether at least one molecule inhibits an enzyme that removes a formyl moiety from an ε-amino of a lysine, the method comprising:
(a) mixing the enzyme and a molecule with a compound having the formula
wherein
SIG is a fluorophore or luminescent moiety, and
m is an integer from 1 to 10;
(b) incubating the enzyme-molecule-compound mixture to allow the enzyme to remove the formyl moiety in the absence of the molecule; and
(c) determining whether the formyl moiety is removed from the compound to an equivalent degree that the formyl moiety would be removed from the compound in the absence of the molecule,
wherein the failure of the removal of the formyl moiety from the compound to an equivalent degree as in the absence of the molecule indicates that the molecule is an inhibitor of the enzyme.
2. The method of claim 1 , wherein the enzyme is a histone deacetylase (HDAC).
3. The method of claim 1 , wherein the compound is a substrate for a peptidase after the enzyme cleaves the formyl moiety from the compound but not if the formyl moiety is not removed from the compound;
wherein the determining step further comprises
(i) adding the peptidase to the mixture to allow the peptidase to cleave the resulting molecule between the nitrogen bound to SIG and the carbonyl carbon bound to said nitrogen, such that SIG generates an increased signal relative to the signal generated with the compound; and
(ii) determining whether SIG generates an increased signal relative to the signal generated with the compound,
wherein an increased signal indicates the removal of the formyl moiety from the compound.
4. The method of claim 3 , wherein the peptidase is a trypsin.
5. The method of claim 3 , wherein the enzyme is a histone deacetylase (HDAC).
6. The method of claim 5 , wherein the peptidase is a trypsin.
7. The method of claim 1 , wherein SIG is a fluorophore.
8. The method of claim 1 , wherein SIG is a luminescent moiety.
9. The method of claim 2 , wherein SIG is a fluorophore.
10. The method of claim 2 , wherein SIG is a luminescent moiety.
11. The method of claim 3 , wherein SIG is a fluorophore.
12. The method of claim 3 , wherein SIG is a luminescent moiety.
13. The method of claim 1 , wherein the compound is
14. The method of claim 2 , wherein the compound is
15. The method of claim 3 , wherein the compound is
16. The method of claim 1 , wherein a plurality of different molecules are subjected to the method simultaneously.
17. The method of claim 2 , wherein a plurality of different molecules are subjected to the method simultaneously.
18. The method of claim 3 , wherein a plurality of different molecules are subjected to the method simultaneously.