Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1999-06-22
2001-11-27
Brusca, John S. (Department: 1653)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C436S002000, C548S405000, C548S110000, C548S417000
Reexamination Certificate
active
06323039
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to biological assays for detecting physiological conditions within cells. More specifically, the invention relates to monitoring molecular interactions in subcellular compartments based on energy transfer from a first compound (the energy transfer donor) to a second compound (the energy transfer acceptor).
BACKGROUND OF THE INVENTION
The cell is the basic unit of life and comprises a variety of subcellular compartments including, for example, the organelles. An organelle is a structural component of a cell that is physically separated, typically by one or more membranes, from other cellular components, and which carries out specialized cellular functions. Organelles and other subcellular compartments vary in terms of, inter alia, their composition and number in cells derived from different tissues, among normal and abnormal cells, and in cells derived from different species. Accordingly, organelles and other subcellular compartments, and macromolecules specifically associated therewith, represent novel targets for the development of agents that specifically impact, respectively, a particular tissue within an animal, abnormal (diseased) but not normal (healthy) cells, or cells from an undesired species but not cells from a desirable species.
For example, members of the Bcl-2 family of proteins (discussed in more detail infra) associate with the outer membranes of mitochondria and with other cellular membranes. The translocation of Bcl-2 proteins from one intracellular position to another occurs during apoptosis, a process by which some abnormal (e.g., pre-cancerous) cells are directed to undergo programmed cell death (PCD), thus eliminating their threat to their host organism. Means for monitoring the accumulation of Bcl-2 proteins in various subcellular compartments, or their translocation from one intracellular location to another, would allow identification of agents designed to impact apoptosis, and to assay the effects of such agents in cells.
As another example, cytoplasmic cellular hybrids (cybrids) comprising the nucleus of one cell type and organelles (mitochondria) from another cell type have been prepared. Experiments with such cybrids have demonstrated that cellular defects associated with diseased cells are transferred with cytoplasmic elements (mitochondria) from diseased cells to cybrids. Diseases that have been demonstrated to have a cytoplasmic component in this manner include Alzheimer's disease and Parkinson's disease (Swerdlow et al.,
Neurology
49:918-925, 1997; Swerdlow et al.,
Annals of Neurology
40:663-671, 1996). Means for monitoring intracellular processes during the formation of cybrids, or for comparing intracellular processes between cybrids that have a common nuclear background but that differ according to the sources of donor cytoplasm as their sources of mitochondria, would allow one to study the mechanisms of such processes and to identify agents that impact such processes.
By way of further example, it is possible to develop antibacterial agents by taking advantage of the fact that bacterial cells comprise structures (e.g., cell walls) that are not present in eukaryotic cells, and by developing agents that specifically impact these structures. In contrast, it has been more difficult to develop agents to treat diseases and disorders resulting from eukaryotic parasites of mammals including humans, in part because of the fact that many cellular features of such parasites have structural similarities to homologous structures found in the host's cells; as a result, any agent that negatively impacts a cellular component of such a parasite is also likely to have a negative effect on the analogous component of the eukaryotic host cells.
There is thus a need for methods and compositions that allow for the rapid and detailed monitoring of processes within subcellular compartments and macromolecules associated therewith. Further, there is a need for methods and compositions for identifying and screening for agents that impact such processes in specific instances.
One objective of the present invention is to provide methods and compositions for monitoring and assaying processes within subcellular compartments and macromolecules associated therewith. When such processes are associated with particular diseases and/or disorders, the invention may be used in a predicative, diagnostic or prognostic modality.
Another objective of the present invention is to provide methods for screening for and identifying agents that impact organelles and other subcellular compartments in specific ways. When such agents are specific for undesirable abnormal cells, or for the cells of an undesirable parasites, they are expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients comprising such undesirable cells.
The present invention fulfils these needs and realizes these and other objectives. Other advantages of the invention are apparent from the disclosure.
SUMMARY OF THE INVENTION
The present invention is directed in part to methods and compositions for monitoring cellular processes, conditions and molecules using energy transfer (ET) techniques. Such ET-based methods and compositions further provide means to screen for and identify agents that alter (e.g., increase or decrease) such processes, conditions and molecules. Accordingly, in one aspect the invention provides a method for assaying mitochondrial membrane potential, comprising the steps of contacting a sample comprising one or more mitochondria, simultaneously or sequentially and in either order, with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites that are mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and wherein the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule; and detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential.
In certain embodiments of this aspect of the invention the excited energy donor molecule transfers energy to the energy acceptor molecule to produce an excited energy acceptor molecule, and the signal detected results from energy released by the excited energy acceptor molecule. In certain embodiments energy transfer from the first energy transfer molecule to the second energy transfer molecule results in a decrease in the detectable signal. In certain further embodiments the method comprises contacting the mitochondria with an agent that induces dissipation of mitochondrial membrane potential. In certain other embodiments the agent that induces dissipation of mitochondrial membrane potential is an ionophore. In certain further embodiments the method comprises contacting the mitochondria with an agent that induces collapse of mitochondrial membrane potential. In another embodiment the agent that induces collapse of mitochondrial membrane potential is CCCP or FCCP. In certain embodiments the sample is washed prior to the step of detecting a signal, and in other embodiments the signal detected is compared with a reference signal. In certain further embodiments the reference signal is generated by an indicator of cell number, an indicator of mitochondrial mass, an indicator of cellular protein, an indicator of cellular DNA, an indicator of mitochondrial DNA, an indicator of mitochondrial protein and an indicator of fluid volume.
In other embodiments of the invention, the sample comprises one o
Dykens James A.
Ghosh Soumitra S.
Velicelebi Gonul
Brusca John S.
Lundgren Jeffrey S.
Mitokor
Seed Intellectual Property Law Group PLLC
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