# US 7,608,407 B2 · Fluorescence polarization assay for determining histidine decarboxylase activity

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- Number: US 7,608,407 B2
- Document ID: US7608407B2
- Status: Granted
- Kind: B2
- Application number: 11/273,526
- Filed: 2005-11-14
- Published: 2006-05-18
- Granted: 2009-10-27
- Earliest claimed: 2004-11-16
- Assignees: Boehringer Ingelheim Pharmaceuticals, Inc.
- Inventors: E. Michael August; Daniel Rajotte
- Web page: https://patentgrep.com/patent/US7608407B2

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C12Q 1/34: involving hydrolase
- G01N 33/573: for enzymes or isoenzymes
- G01N 2333/988: Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- 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)
- 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

## Abstract

The present invention relates to fluorescence polarization assays for determining the HDC modulating activity of a candidate compound including providing a reaction mixture comprising a HDC, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine: incubating the reaction mixture; and determining whether inhibition of HDC has occurred in the presence of the test compound, wherein an increase in fluorescence signal is an indication that the test compound inhibits the activity of the HDC.

## Claims

### Claim 1

A fluorescence polarization assay for determining the histidine decarboxylase (HDC) inhibiting activity of a candidate compound comprising the steps of:
a) providing a reaction mixture comprising a HDC, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine;
b) incubating the reaction mixture;
c) determining whether inhibition of HDC has occurred in the presence of the candidate compound, wherein an increase in fluorescence polarization signal relative to a reference fluorescence polarization signal is an indication that the candidate compound inhibits the activity of the HDC,
wherein said HDC comprises the polypeptide of SEQ ID. No. 1.

### Claim 2 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the anti histamine antibody has a selectivity for histamine of at least 100 fold greater than histidine.

### Claim 3 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the reaction mixture is incubated for 15 to 180 minutes.

### Claim 4 (depends on 3)

The fluorescence polarization assay of claim 3, wherein the reaction mixture is incubated for between 60 and 120 minutes.

### Claim 5 (depends on 4)

The fluorescence polarization assay of claim 4, wherein the reaction mixture is incubated for about 80 to 100 minutes.

### Claim 6 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the HDC is a recombinant enzyme.

### Claim 7 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the HDC is partially purified.

### Claim 8 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the histamine probe has a greater affinity than 1 μm for the anti histamine antibody.

### Claim 9 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the antihistamine antibody used is generated by immunizing mice with a histamine bridged by a linker region to a carrier and where said linker is structurally homologous to the fluorescein probe.

### Claim 10 (depends on 9)

The fluorescence polarization assay of claim 9, wherein the linker region is 1,4-benzoquinone coupled to a carrier.

### Claim 11 (depends on 9)

The fluorescence polarization assay of claim 9, wherein said carrier is albumin.

### Claim 12 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the fluorescently labeled histamine probe is chosen from the group consisting of FITC, rhodamine, tetramethylrhodamine and Cy5.

### Claim 13 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the histidine concentration is between 10 μM to 5 mM.

### Claim 14 (depends on 9)

The fluorescence polarization assay of claim 9, wherein histidine concentration is between 100 μM and 1 mM.

### Claim 15 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the reference fluorescence polarization signal is determined by:
d) providing a reference mixture comprising the fluorescently labeled histamine probe and the anti histamine antibody;
e) incubating the reference mixture; and
f) measuring the fluorescence polarization signal of the reference mixture, wherein the measured signal is the reference fluorescence polarization signal.

### Claim 16

A fluorescence polarization (FP) assay for determining the histidine decarboxylase (HDC) inhibiting activity of a candidate compound comprising the steps of:
a) providing a reaction mixture comprising a HDC comprising the polypeptide of SEQ ID No. 1, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine;
b) incubating the reaction mixture for a time sufficient to produce histamine but no longer than about 180 minutes;
c) determining the FP signal of the incubated reaction mixture; and
d) comparing the FP signal of the incubated reaction mixture with a reference EP signal, wherein if the FP signal of the incubated reaction mixture is about the same as or is greater than the reference FP signal, the candidate compound is an inhibitor of HDC.

### Claim 17 (depends on 1)

The fluorescence polarization assay of claim 1, wherein the reaction mixture is incubated for about 30 minutes.