Method of identifying a compound which affects the multienzyme metabolic assembly of glucose metabolism and its association with cell cycle progression in cancer cells

Patent metadata

Field	Value
Publication number	US-11474097-B2
Application number	US-201916452767-A
Country	US
Kind code	B2
Filing date	Jun 26, 2019
Priority date	Jun 26, 2018
Publication date	Oct 18, 2022
Grant date	Oct 18, 2022

Abstract

A cell-based quantitative high-throughput screening assay to monitor the formation of PFK1-mEGFP clusters by the action of small molecules to identify small molecules that promote intracellular PFK1 clustering in a cell cycle-dependent manner and may be used to treat cancer.

First claim

What is claimed is:

1. An assay method to identify an active testing compound which induces clustering in a host cell, the method comprising:
providing a medium comprising host cells that express phosphofructokinase 1 (PFK1) fused to a fluorescent protein (PFK1-FP);
introducing at least two concentrations of a testing compound into individual aliquots of the medium and incubating same;
imaging the host cells of the incubated medium to identify testing compounds inducing clusters having at least a minimum area and sensitivity; and
plotting a quantitative high-throughput screening (qHTS) titration curve of the percent of host cells showing PFK1-FP clustering versus the log compound concentration for the testing compound,
wherein the qHTS titration curve of the testing compound includes the titration curve of a positive control compound, wherein the testing compound is considered active when the qHTS titration curve is a full titration curve or a partial titration curve.

2. The assay method of claim 1 , wherein the host cells comprise a species selected from the group consisting of Chinese hamster ovary (CHO) cells, HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, human mammary gland/breast carcinoma cells, Madin-Darby bovine kidney (“MDBK”) cells, and NOS cells derived from carcinoma cells.

3. The assay method of claim 1 , wherein the host cells comprise a HeLa-T-PFK1G cell line.

4. The assay method of claim 1 , wherein the host cells are harvested cells from a specific individual for personalized medicine.

5. The assay method of claim 1 , wherein the fluorescent protein is a green fluorescent protein (GFP).

6. The assay method of claim 5 , wherein the GFP is a monomeric form of enhanced green fluorescent protein (mEGFP).

7. The assay method of claim 1 , wherein a qHTS titration curve class of 1.1, 1.2, 2.1, and 2.2 is indicative of an active testing compound capable of inducing medium- or large-sized PFK1-mEGFP clusters.

8. The assay method of claim 1 , wherein the positive control compound comprises a species selected from the group consisting of resveratrol, SU9516, kenpaullone, and olomoucine.

9. The assay of claim 1 , wherein the medium is plated in wells of microtiter plates.

10. The assay method of claim 1 , wherein clustering is associated with changes in cell cycle progression within the host cell.

11. The assay method of claim 1 , wherein the active testing compound may be used to treat cancer.

Assignees

• Univ Maryland
• Nat Institutes Of Health

Inventors

• An Songon
• Schmitt Danielle L
• Inglese James
• Dranchak Patricia

Classifications

• C12Y207/01011 Primary

  6-Phosphofructokinase (2.7.1.11) · CPC title

• G01N2333/91215

  with a definite EC number (2.7.1.-) · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N33/5035 Primary

  on sub-cellular localization · CPC title

• G01N33/5011

  for testing antineoplastic activity · CPC title