# US 10,308,974 B2 · Compounds and methods for detection of enzymes that remove formyl, succinyl, methyl succinyl or myristoyl groups from epsilon-amino lysine moieties

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- Number: US 10,308,974 B2
- Document ID: US10308974B2
- Status: Granted
- Kind: B2
- Application number: 15/944,336
- Filed: 2018-04-03
- Published: 2018-08-09
- Granted: 2019-06-04
- Earliest claimed: 2011-01-13
- Assignees: Enzo Life Sciences, Inc.
- Applicants: Enzo Life Sciences, Inc.
- Inventors: Konrad T. Howitz; Zhongda Zhang; Anne Kisielewski; Elizabeth Dale; Wayne Forrest Patton
- Web page: https://patentgrep.com/patent/US10308974B2

## CPC

- C12Q 1/34: involving hydrolase
- C07D 311/16: substituted in position 7
- C07D 493/10: Spiro-condensed systems
- C12Y 305/01098: Histone deacetylase (3.5.1.98), i.e. sirtuin deacetylase
- G01N 2333/98: acting on amide bonds in linear amides (3.5.1)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided is a compound that comprises the structure: where SIG is a signaling molecule and R3 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.

## Claims

### Claim 1

A method of determining whether at least one molecule inhibits an enzyme that removes a myristoyl 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 myristoyl moiety in the absence of the molecule; and
(c) determining whether the myristoyl moiety is removed from the compound to an equivalent degree that the myristoyl moiety would be removed from the compound in the absence of the molecule,
wherein the failure of the removal of the myristoyl 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the enzyme is a histone deacetylase (HDAC).

### Claim 3 (depends on 2)

The method of claim 2, wherein the histone deacetylase is HDAC1, HDAC2, HDAC3, or HDAC3/NCOR1 complex.

### Claim 4 (depends on 2)

The method of claim 2, wherein the histone deacetylase is a sirtuin.

### Claim 5 (depends on 4)

The method of claim 4, wherein the sirtuin is SIRT1, SIRT3 or SIRT6.

### Claim 6 (depends on 1)

The method of claim 1, wherein the compound is a substrate for a peptidase after the enzyme cleaves the myristoyl moiety from the compound but not if the myristoyl 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 myristoyl moiety from the compound.

### Claim 7 (depends on 6)

The method of claim 6, wherein the peptidase is a trypsin.

### Claim 8 (depends on 6)

The method of claim 6, wherein the enzyme is a histone deacetylase (HDAC).

### Claim 9 (depends on 8)

The method of claim 8, wherein the histone deacetylase is HDAC1, HDAC2, HDAC3, or HDAC3/NCOR1 complex.

### Claim 10 (depends on 8)

The method of claim 8, wherein the histone deacetylase is a sirtuin.

### Claim 11 (depends on 10)

The method of claim 10, wherein the sirtuin is SIRT1, SIRT3 or SIRT6.

### Claim 12 (depends on 8)

The method of claim 8, wherein the peptidase is a trypsin.

### Claim 13 (depends on 1)

The method of claim 1, wherein SIG is a fluorophore.

### Claim 14 (depends on 1)

The method of claim 1, wherein SIG is a luminescent moiety.

### Claim 15 (depends on 2)

The method of claim 2, wherein SIG is a fluorophore.

### Claim 16 (depends on 2)

The method of claim 2, wherein SIG is a luminescent moiety.

### Claim 17 (depends on 1)

The method of claim 1, wherein the compound is

### Claim 18 (depends on 2)

The method of claim 2, wherein the compound is

### Claim 19 (depends on 1)

The method of claim 1, wherein a plurality of different molecules are subjected to the method simultaneously.

### Claim 20 (depends on 2)

The method of claim 2, wherein a plurality of different molecules are subjected to the method simultaneously.