Compounds and methods for detection of enzymes that remove methyl succinyl 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 methyl succinyl 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,
m is an integer from 1 to 10,
R 1 is NH, O, S or SO 2 ,
R 2 is a hydrogen, a halogen, an isothiocyano group (SNC), an azido group (N 3 ), a sulfonate group (SO 3 R 4 ), a sulfate group (OSO 3 R 4 ), a carboxyl group (CO 2 H), a carbonyl group (COR 4 ), an amido group (CONR 4 2 or NR 3 COR 4 ), a carbamate group (NR 4 CO 2 R 4 ), a phosphate group (OPO 3 R 4 3 ), a phosphonate group (PO 3 R 4 2 ), an amino group (NR 4 2 ), an alkoxy group (OR 4 ), a thiol group (SR 4 ), a sulfoxy group (SOR 4 ), a sulfone group (SO 2 R 4 ), a sulfonamide group (SO 2 NR 4 2 ), a phosphino group (PR 4 2 ), a silane group (SiR 4 3 ), an oligopeptide sequence of 1-20 modified or unmodified amino acids or amino acid substitutes, a protein, a glycoprotein or a lipoprotein,
each R 4 is independently a hydrogen, 1 to 3 halogen atoms, a substituted or unsubstituted C 1 -C 10 straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups may be substituted with an O atom, N atom, S atom, or NH group, an unsubstituted or substituted aromatic group wherein one or more C, CH or CH 2 groups may be substituted with an O atom, N atom, S atom, or NH group, and
R 3 is a methyl succinyl moiety;
(b) incubating the enzyme-molecule-compound mixture to allow the enzyme to remove the methyl succinyl moiety in the absence of the molecule; and
(c) determining whether the methyl succinyl moiety is removed from the compound to an equivalent degree that the methyl succinyl moiety would be removed from the compound in the absence of the molecule,
wherein the failure of the removal of the methyl succinyl 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 methyl succinyl moiety from the compound but not if the methyl succinyl 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 methyl succinyl 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
wherein m is an integer from 1 to 10.
14. The method of claim 13 , wherein m=1.
15. The method of claim 13 , wherein m is an integer from 2 to 10.
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.