Assays for apotosis modulators
OwnerKathryn J. Elliott, Maria Z. Kounnas +3inventors
InventorsKATHRYN J. ELLIOTT, MARIA Z. KOUNNAS, REBECCA J. DYER, BENITO MUNOZ, STEVEN L. WAGNER
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Abstract
Recombinant cells expressing fluorescence resonance energy transfer reporter polypeptides and cell-based assays for apoptosis; screening assays for identifying and selecting candidate compounds modulating apoptosis.
Claims59
A stable recombinant apoptosis reporter cell line expressing a FRET apoptosis reporter polypeptide at a level effective to detect induction of apoptosis by an apoptosis inducer agent, wherein said FRET apoptosis reporter polypeptide comprises an exciter fluorophore peptide bonded with a linker polypeptide that is in turn peptide bonded with an emitter fluorophore, wherein said linker polypeptide comprises a length that is within the Förster radius of said exciter and emitter fluorophores, and wherein addition of said apoptosis inducer agent to a plurality of cells comprising said recombinant apoptosis reporter cell line effects a change in said linker polypeptide that is effective to produce a change in resonance energy transfer from said exciter to said emitter fluorophore.
A recombinant FRET apoptosis reporter polypeptide comprising an exciter fluorophore polypeptide peptide bonded to a linker polypeptide which is in turn peptide bonded to an emitter fluorophore, wherein said linker polypeptide comprises an amino acid sequence having all of the following in serial array:
- (i) a first end-cap amino acid domain;
- (ii) an acidic domain;
- (iii) a substrate domain;
- (iv) a non-interferring domain;
- and (v) a second end-cap amino acid domain.
A method for preparing a stable FRET apoptosis reporter cell line capable of reporting intracellular apoptotic changes resulting from addition of an extracellular apoptosis inducer agent to a plurality of cells comprising the cell line, wherein said method comprises:
- introducing into a plurality of cells a plasmid capable of expressing both a FRET apoptosis reporter polypeptide and a selectable marker,
- selecting within the plurality of cells for a cell expressing the selectable marker;
- testing within the selected cells for a reporter cell expressing the FRET apoptosis reporter polypeptide at a level of about 1% to about 3% of total cell protein;
- adding an apoptosis inducer to a first plurality of said reporter cells and determining that apoptosis is induced in more than 50% of said plurality of reporter cells;
- adding an apoptosis inducer agent to a second plurality of said reporter cells, measuring a FRET signal produced by the FRET apoptosis reporter polypeptide in the selected cell and determining that the selected cell is a FRET apoptosis reporter cell if the FRET signal is changed by at least about 14% after addition of the apoptosis inducer agent.
A cell-based assay method for identifying and selecting a candidate compound capable of modulating apoptosis induced by an extracellular apoptosis inducer agent, said cell-based assay method comprising the steps of measuring a FRET signal produced by the FRET reporter polypeptide expressed in a recombinant FRET apoptosis reporter cell in the presence and absence of said apoptosis inducer agent and in the presence of said candidate compound, and determining that said candidate compound is effective to modulate apoptosis if:
- (i) the FRET signal produced in the presence of said apoptosis inducer agent is different than in the absence of said apoptosis inducer agent;
- and, (ii) in the presence of the combination of said apoptosis inducer agent and said candidate compound the FRET signal produced is substantially the same as the FRET signal produced in the absence of the apoptosis inducer agent.
The cell-based assay method of claim 35, wherein, said exciter fluorophore comprises a fluorophore having an emission wavelength maximum;
- said fluorescence emitter fluorophore comprises a fluorophore having an excitation wavelength maximum that is about 10 nm to about 20 nm less than or greater than said emission wavelength maximum of said exciter fluorophore and said linker polypeptide comprising a peptide having about 18 to about 40 amino acid residues.
Dependencies are inferred from the claim text.