Method of identifying a compound which affects the multienzyme metabolic assembly of glucose metabolism and its association with cell cycle progression in cancer cells
Inventors
An, Songon • SCHMITT, Danielle L. • Inglese, James • Dranchak, Patricia
Assignees
National Institutes of Health NIH • University of Maryland Baltimore County UMBC
Publication Number
US-11474097-B2
Publication Date
2022-10-18
Expiration Date
2039-06-26
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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.
Core Innovation
The invention describes a cell-based quantitative high-throughput screening (qHTS) assay and related methods to identify compounds that induce clustering of phosphofructokinase 1 (PFK1) fused with a fluorescent protein (PFK1-FP) inside host cells. The assay involves exposing host cells expressing PFK1-FP to multiple concentrations of testing compounds, imaging the cells to detect clusters of PFK1-FP with at least a minimum area and sensitivity, and plotting a qHTS titration curve showing the percentage of cells with PFK1-FP clustering against the log concentration of the testing compound. Testing compounds that produce full or partial titration curves consistent with those of positive control compounds are considered active.
The background establishes that enzymes involved in glycolysis, including PFK1, form multienzyme complexes called glucosomes that spatially organize glucose metabolism within human cells, including cancer cells. Glycolytic activity and the expression of metabolic regulators oscillate throughout the cell cycle, particularly during the G1/S transition, suggesting intimate regulation of glucose metabolism with cell cycle progression. The cell cycle itself is tightly regulated by cyclin-dependent kinases (CDKs), cyclins, and transcription factors. Existing high-throughput screening methods are limited by single-concentration assays and may miss biologically relevant phenotypes; thus, quantitative high-throughput screening (qHTS) employing multi-point titrations and imaging are advantageous for dissecting cellular mechanisms such as reversible compartmentalization of enzymes involved in glucose metabolism.
The problem addressed is the need for a cell-based qHTS assay capable of monitoring PFK1 clustering inside host cells, which can identify small molecules that promote intracellular PFK1 clustering in a cell cycle-dependent manner. Such an assay can be used to discover compounds that affect cell cycle progression in cancer cells and potentially be developed as cancer therapeutics. Prior to this invention, there was no robust high-throughput cell-based assay to quantitatively monitor PFK1 multienzyme complex formation and its association with cell cycle progression.
Claims Coverage
The patent comprises one independent claim describing an assay method with multiple inventive features for identifying compounds that induce clustering in a host cell expressing PFK1 fused to a fluorescent protein.
Quantitative high-throughput screening of PFK1 clustering
An assay method providing a medium containing host cells expressing phosphofructokinase 1 fused to a fluorescent protein (PFK1-FP), introducing at least two concentrations of a testing compound into aliquots of the medium, incubating, imaging cells to detect clusters meeting minimum area and sensitivity criteria, and plotting a qHTS titration curve of the percentage of cells showing PFK1-FP clusters versus log compound concentration.
Use of positive control compound titration curve for activity determination
Including the titration curve of a positive control compound in the qHTS titration curve analysis and considering a testing compound active when the qHTS curve is either a full or partial titration curve similar to the positive control.
Host cells expressing PFK1 fused to fluorescent proteins suitable for screening
Host cells can be of various species including HeLa cells and derivatives, and express PFK1 fused to fluorescent proteins such as green fluorescent protein (GFP), specifically monomeric enhanced GFP (mEGFP). The method facilitates detection of medium- or large-sized PFK1-mEGFP clusters associated with compound activity.
Microtiter plate format with imaging and analysis
The assay is performed with cells plated in wells of microtiter plates at various densities, and imaging is analyzed with algorithm-based software to identify phenotypic responses characterized by PFK1 clustering within cells.
Association of compound-induced clustering with cell cycle progression and potential cancer treatment
Testing compounds that induce PFK1 clustering are linked with changes in cell cycle progression in the host cells and may be used as candidates for treating cancer due to their ability to promote apoptosis.
The independent claim covers an assay method enabling quantitative high-throughput screening of compounds inducing PFK1 clustering in host cells expressing PFK1 fused to fluorescent proteins, incorporating positive controls, imaging-based detection of clustering, and potential use in identifying cancer therapeutics by linking clustering with cell cycle progression.
Stated Advantages
The assay permits rapid identification of small molecules that promote intracellular PFK1 clustering in a cell cycle-dependent manner, aiding efficient discovery of cancer therapeutics.
Quantitative high-throughput screening using multi-point titrations reduces false negatives common in single-concentration high-throughput assays, increasing biological relevance.
The assay uses a live-cell intracellular reporter system, providing mechanistic insight into enzyme assembly and cell cycle association in human cancer cells.
Validated positive controls allow robust identification of active compounds, and subsequent assays can elucidate off-target effects for identified molecular targets.
Documented Applications
The assay is used for screening libraries of small molecules to identify compounds that induce clustering of PFK1 in cancer cells in a cell cycle-dependent manner.
Identifying active compounds that affect cell cycle progression and potentially induce apoptosis, with applications toward developing cancer treatments.
Analyzing cell cycle progression in host cells following compound treatment via flow cytometry, utilizing PFK1-mEGFP clustering as a marker for changes in cell cycle phases.
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