# US 9,040,249 B2 · Pan-kinase activation and evaluation of signaling pathways

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- Number: US 9,040,249 B2
- Document ID: US9040249B2
- Status: Granted
- Kind: B2
- Application number: 13/665,776
- Filed: 2012-10-31
- Published: 2013-11-07
- Granted: 2015-05-26
- Earliest claimed: 2008-09-04
- Assignees: Beckman Coulter, Inc.; University Health Network
- Applicants: Beckman Coulter, Inc.; University Health Network
- Inventors: David Hedley; Sue Chow; T. Vincent Shankey
- Web page: https://patentgrep.com/patent/US9040249B2

## CPC

- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/5041: {involving analysis of members of signalling pathways}
- G01N 33/5047: {Cells of the immune system}
- G01N 33/56972: {White blood cells}
- G01N 33/573: for enzymes or isoenzymes
- 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

Methods and reagents are provided for determining the activation state of a signal transduction pathway signaling protein. There exists a need in the art for methods that can monitor the efficacy of a signal transduction inhibitor in a patient. Other needs exist for detecting and monitoring certain disease or disorders that are associated with aberrant activation of a signal transduction pathway signaling protein. The present assay provides a highly sensitive assay that is also useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates.

## Claims

### Claim 1

A method of identifying and assessing the efficacy of an inhibitor of ribosomal S6 pathway activation, said method comprising the steps of:
a) contacting a population of cells comprising a protein activated by the ribosomal S6 pathway with a first inhibitor, wherein said first inhibitor is a putative inhibitor of extracellular signal-related kinase (ERK) or phosphoinositide 3-kinase (PI3K);
b) contacting said population of cells with a second inhibitor known to inhibit lipopolysaccharide activation of the ribosomal S6 pathway wherein said second inhibitor is an ERK inhibitor when said first inhibitor is a PI3K inhibitor and said second inhibitor is a PI3K inhibitor when said first inhibitor is an ERK inhibitor, wherein complete inhibition of LPS induced activation of ribosomal S6 pathway requires both of the ERK inhibitor and the PI3K inhibitor;
c) contacting said population of cells with a lipopolysaccharide to activate said protein;
d) contacting said activated population of cells with a preservative;
e) unmasking intracellular epitopes of said preserved population of cells;
f) contacting said preserved and unmasked population of cells with a fluorescently labeled capture molecule being specific for the activated form of said protein;
g) detecting the fluorescence of the first putative inhibitor exposed cells indicative of binding of said labeled capture molecule to the activated form of said protein; and
h) comparing the detected fluorescence of the first putative inhibitor exposed cells to detected fluorescence of a control cell sample comprising the protein that was not exposed to the first putative inhibitor but subjected to steps b) to f),
wherein said first putative inhibitor is assessed to be an effective inhibitor of ribosomal S6 pathway activation if the detected fluorescence of the first putative inhibitor exposed cells is decreased relative to the detected fluorescence of the control sample not exposed to the first putative inhibitor.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said first inhibitor is an ERK inhibitor and said second inhibitor is a PI3K inhibitor.

### Claim 3 (depends on 2)

The method according to claim 2, wherein said PI3K inhibitor is LY294002.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said first inhibitor is a PI3K inhibitor and said second inhibitor is an ERK inhibitor.

### Claim 5 (depends on 4)

The method according to claim 4, wherein said ERK inhibitor is UO126.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said protein is a ribosomal S6 protein.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the activated form of said ribosomal S6 protein comprises phosphorylation at amino acids Ser235, Ser240, Ser244, or Ser247.

### Claim 8 (depends on 7)

The method according to claim 7, wherein said second inhibitor is a known PI3K pathway inhibitor and wherein said first inhibitor is a putative ERK pathway inhibitor, and the capture molecule is an antibody specific for phosphorylated ribosomal 6 protein.

### Claim 9 (depends on 7)

The method according to claim 7, wherein said second inhibitor is a known ERK pathway inhibitor and wherein said first inhibitor is a putative PI3K pathway inhibitor, and the capture molecule is an antibody specific for phosphorylated ribosomal 6 protein.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said second inhibitor is a known PI3K pathway inhibitor and said first inhibitor is a putative ERK pathway inhibitor, and wherein an amount of activation of said protein as measured by detected fluorescence is negatively correlated with efficacy of said first inhibitor.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said second inhibitor is a known ERK pathway inhibitor and said first inhibitor is a putative PI3K pathway inhibitor, and wherein an amount of activation of said protein as measured by the detected fluorescence is negatively correlated with the efficacy of said first inhibitor.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said first inhibitor is an inhibitor which has been or shall be administered to a patient.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said population of cells comprises monocytes.

### Claim 14 (depends on 1)

The method according to claim 1, further comprising the step of permeabilizing said population of cells following the contacting said activated population of cells with a preservative.

### Claim 15 (depends on 1)

The method according to claim 1, wherein efficacy of said first inhibitor is assessed by administration in vivo or in vitro.

### Claim 16 (depends on 1)

The method according to claim 1, wherein flow cytometry is used to detect binding of said labeled capture molecule to said protein.

### Claim 17 (depends on 1)

The method according to claim 1, wherein efficacy of said first inhibitor is assessed at multiple time points.

### Claim 18 (depends on 1)

The method according to claim 1, wherein said labeled capture molecule is a labeled antibody.

### Claim 19 (depends on 1)

The method according to claim 1, wherein unmasking comprises contacting the population of cells with an alcohol and a detergent.

### Claim 20 (depends on 19)

The method according to claim 19, wherein said alcohol is at a concentration of about 25 percent to about 90 percent.

### Claim 21 (depends on 19)

The method according to claim 19, wherein said alcohol comprises ethanol or methanol.

### Claim 22 (depends on 19)

The method according to claim 19, wherein said detergent is at a concentration of about 0.1 percent to about 10 percent.

### Claim 23 (depends on 22)

The method according to claim 22, wherein said detergent comprises ethyoxylated octyphenol, octylphenoxypoly(ethyleneoxy)ethanol, or a linear alcohol alkoxylate.

### Claim 24 (depends on 1)

The method according to claim 1, wherein the preservative comprises aldehyde, paraformaldehyde, or formaldehyde.